СПОСОБ ОЧИСТКИ БОТУЛИНИЧЕСКОГО ТОКСИНА

Изобретение относится к области биотехнологии. Описан способ очистки ботулинического токсина, включающий в себя (а) предварительную обработку культурального раствора, содержащего ботулинический токсин, (b) очистку предварительно обработанного ботулинического токсина с использованием анионообменной хроматографии и (с) очистку ботулинического токсина с использованием катионообменной хроматографии. Изобретение расширяет арсенал способов получения очищенного ботулинического токсина, обладающего высокой чистотой и активностью, при использовании простого процесса, состоящего из анионообменной хроматографии и катионообменной хроматографии. 3 з.п. ф-лы, 6 ил., 10 табл., 5 пр.

СПОСОБ ОЧИСТКИ БЕЛКА РЕКОМБИНАНТНОЙ СТАФИЛОКИНАЗЫ

Изобретение относится к области биотехнологии, а именно к способу очистки белка рекомбинантной стафилокиназы. Предложенное изобретение может быть использовано в фармацевтической промышленности. Способ включает суспендирование клеток штамма Е.coli MZ08 - продуцента рекомбинантной стафилокиназы, характеризующейся аминокислотной SEQ ID NO:1, в фосфатном буферном растворе. Проводят гомогенизацию и центрифугирование полученного супернатанта, затем хроматографию в два этапа, на первом этапе осуществляют ионообменную хроматографию на колонке, заполненной DE-целлюлозой с получением фракции, обогащенной стафилокиназой. Затем полученную фракцию разбавляют раствором хлорида натрия и осуществляют второй этап с помощью металлхелатирующей аффинной хроматографии. Предложенное изобретение позволяет осуществлять очистку белка рекомбинантной стафилокиназы с высокой чистотой.

СПОСОБЫ ОЧИСТКИ ПОЛИПЕПТИДОВ

Изобретение относится к способам очистки антитела или иммуноадгезина из композиции, содержащей антитело или иммуноадгезин и по меньшей мере одно загрязняющее вещество. Способы очистки включают очистку на катионообменном материале, представляющем собой частицы смолы, и материале смешанного типа, где плотность загрузки на катионообменном материале составляет более 150 г/л. Способ позволяет осуществлять очистку антитела или иммуноадгезина с высоким выходом. 28 з.п. ф-лы, 34 ил., 17 табл., 5 пр.

ПОЛИПЕПТИДЫ ДЛЯ ЛЕЧЕНИЯ ЗАБОЛЕВАНИЙ

Настоящее изобретение относится к области биотехнологии, в частности к новым пептидам для лечения рака. Изобретение раскрывает пептид, эффективный в блокировании CXC-хемокинового рецептора 4 (CXCR4). Изобретение может быть использовано в медицинской практике для контроля пролиферации раковых клеток при лечении злокачественных новообразований и ассоциированных с ними заболеваний. 3 н. и 6 з.п. ф-лы, 2 табл., 1 пр.

СПОСОБ ПОЛУЧЕНИЯ РЕКОМБИНАНТНОГО БЕЛКА SAV-RGD, СПЕЦИФИЧЕСКИ УЗНАЮЩЕГО КЛЕТКИ МЕЛАНОМЫ

Изобретение относится к биотехнологии. Способ получения рекомбинантного белка SAV-RGD, специфически узнающего клетки меланомы человека, предусматривает культивирование в питательной среде штамма Escherichia coli MG1655/pSAV-RGD, получение периплазматической фракции клеток указанного штамма, очистку рекомбинантного белка ионообменной хроматографией с последующей ультрафильтрацией и лиофилизацией. Штамм E. coli MG1655/pSAV-RGD получают трансформацией штамма E. coli MG1655 плазмидой pSAV-RGD, полученной на базе вектора pUC18 и включающей последовательность, кодирующую слитый пробелок SAV-RGD (pro-sav-rgd) под контролем промотора уридинфосфорилазы (Pudp) Е. coli. В процессе культивирования в первые 10 ч осуществляют подпитку 40% раствором глюкозы при скорости подачи 25 мл/час и поддержание глюкозы на уровне 1 г/л, дробно добавляют стерильный раствор пептона 10 г/л, дрожжевого экстракта 5 г/л и ампициллина 100 мг/л, продолжая культивирование в течение 8 ч. Периплазматическую фракцию клеток получают ...

СПОСОБЫ ОЧИСТКИ ОДНОДОМЕННЫХ АНТИГЕНСВЯЗЫВАЮЩИХ МОЛЕКУЛ

Изобретение относится к области получения и выделения однодоменных молекул (SDAB). Описан способ выделения или очистки SDAB молекулы, которая представляет собой трехвалентную молекулу нанотела ATN-103, направленную на TNFα и HAS, из смеси, содержащей указанную SDAB молекулу и одно или более загрязняющих веществ. Приводят смесь в контакт с катионообменным носителем в условиях, которые позволяют SDAB молекуле связываться с носителем или абсорбироваться на носителе. Удаляют одно или более загрязняющих веществ и селективно элюируют SDAB с носителя. При этом проводимость среды для кондиционирования (СМ), используемой для загрузки носителя, составляет от примерно 12 до 9 мСм/см и рН в условиях загрузки корректируют до величины от 4,0 до 4,3. Буфер для элюирования соответствует примерно 50 мМ хлорида натрия или меньше и имеет рН от примерно 5,5 до 7,2. Раскрыт способ или процесс получения рекомбинантного SDAB ATN-103. Поддерживают клетку-хозяина в условиях, при которых экспрессируется рекомбинантная ...

СПОСОБ ОЧИСТКИ ИНТЕРФЕРОНОВЫХ БЕЛКОВ С ПОМОЩЬЮ КАТИОНООБМЕННОЙ ХРОМАТОГРАФИИ

Изобретение относится к препаративной биохимии, фармакологии, медицине. Способ очистки интерфероновых белков основан на применении катионообменной хроматографии на твердой матрице, осуществляемый при более основном рН, то есть более высоком относительно рН, соответствующего изоэлектрической точке, pI, белков, предназначенных для очистки, однако при данном рН указанные белки все еще остаются абсорбированными, при этом используют буферные растворы органических или неорганических солей, способных модифицировать ионную силу раствора. Изобретение обеспечивает простоту промышленного осуществления способа и экономическую доступность. 2 н. и 6 з.п. ф-лы, 1 табл.

СПОСОБ ВЫДЕЛЕНИЯ И ОЧИСТКИ РЕКОМБИНАНТНЫХ ВАРИАНТОВ Bet v 1

Изобретение относится к биотехнологии. Предложенный способ основывается на преобразовании телец включения в растворимую форму при использовании органических денатурирующих реагентов, а также при использовании хроматографических методов. При хроматографии выбирают неорганические, щелочные, солесодержащие элюенты. После окончания очистки получают рекомбинантные варианты Bet v 1 аллергена в форме, которая может непосредственно использоваться для медицинских целей. Преимущество способа состоит в минимальной обработке образца, использовании фармакологически совместимых веществ, кроме этого в процессе очистки раствора целевого белка эффективно удаляются эндотоксины. 11 з.п. ф-лы, 2 табл.

СПОСОБ ОЧИСТКИ ЛИПОПЕПТИДОВ

Изобретение относится к способу очистки даптомицина, включающий стадии а) загрузки частично очищенного даптомицина в анионообменную хроматографическую колонку и последующие стадии очистки б) и в) в обращено-фазовых хроматографических колонках, где элюирующий буфер на стадии а) представляет собой раствор одновалентной соли и элюирующий буфер на стадии б) и в) представляет собой водный спирт. 3 н. и 13 з.п. ф-лы, 2 пр.

ПРОИЗВОДСТВО 2S-БЕЛКА КАНОЛЫ С ПРИМЕНЕНИЕМ ИОННОГО ОБМЕНА

Изобретение относится к способу производства 2S-белка канолы, предусматривающий солюбилизацию белков канолы из муки из масличных семян канолы с применением раствора хлорида натрия, имеющего концентрацию соли от 0,25М до 0,35М и pH от 5 до 6 с образованием раствора белков канолы. Отделяют раствор белков канолы от остаточной муки из масличных семян канолы. Осуществляют контактирование раствора белков канолы с катионообменной средой при pH от 5 до 6 для связывания с катионообменной средой предпочтительно 2S-белка канолы, а не других белков канолы. Отделяют связанный 2S-белок канолы от несвязанных белков канолы и примесей и извлекают связанный 2S-белка канолы из катионообменной среды с помощью водного раствора хлорида натрия, с концентрацией соли от 0,55 до 0,7 М. Способ обеспечивает получение 2S-белка канолы чистотой 99,9%. 5 з.п. ф-лы, 9 табл., 2 пр.

СПОСОБ ВЫДЕЛЕНИЯ И ОЧИСТКИ ЦЕЛЕВОГО БЕЛКА БЕЗ ПРИМЕСИ ПРИОНОВОГО БЕЛКА PrP

Группа изобретений относится к области биохимии. Предложен способ выделения и очистки целевого белка посредством хроматографии, в котором хроматография удаляет или уменьшает содержание прионов (PrP). Осуществляют взаимодействие потенциально PrP-загрязненного образца, содержащего целевой белок, с комбинированным хроматографическим материалом, содержащим лиганд. Лиганд выбирают из положительно заряженного N-бензил-N-метилэтаноламинового лиганда; отрицательно заряженного лиганда 2-(бензоиламино)бутановой кислоты; фенилпропилового лиганда; N-гексилового лиганда; 4-меркаптоэтилпиридинового лиганда; лиганда 3-[{3-метил-5-((тетрагидрофуран-2-илметил)амино)-фенил}амино]бензойной кислоты. Затем создают буферные условия в хроматографических условиях, таких, чтобы целевой белок связывался с комбинированным хроматографическим материалом, а PrPне связывался с комбинированным хроматографическим материалом. Затем элюируют целевой белок. Также предложена фракция фармацевтически применимого целевого белка ...

СПОСОБ АНАЛИЗА ПРОЦЕССА Арг-Х ПРОТЕОЛИЗА В ПОЛОЖИТЕЛЬНО ЗАРЯЖЕННЫХ ФРАКЦИЯХ БЕЛКОВ СУПРАМОЛЕКУЛЯРНЫХ СТРУКТУР В РАСТУЩЕЙ ПОПУЛЯЦИИ ESCHERICHIA COLI

Изобретение относится к области биохимии и молекулярной биологии и касается способа анализа процесса Арг-Х протеолиза в положительно заряженных фракциях белков супрамолекулярных структур в растущей популяции Escherichia coli. Представленный способ включает следующие стадии: консервация клеток в присутствии забуференного 80-90% глицерина, снятие клеточных оболочек 3% тритоном Х-100, экстракция возрастающими концентрациями солей: 0,14 М, 0,35 М; 2 М NaCl, 6 М гуанидин гидрохлоридом с 0,1% β-меркаптоэтанолом, выделение из вышеперечисленных фракций положительно заряженных белков с помощью ионообменной хроматографии с амберлитом ИРЦ-50 в прерывистом градиенте гуанидин гидрохлорида: 6%, 8,9%, 10,6%, 13% на 0,1 М калий-фосфатном буфере рН 6,8 и определение в них сайтов чувствительности к Арг-Х протеолизу. Представленное изобретение может быть использовано при анализе молекулярно-генетических механизмов формирования структуры клетки прокариот и роли белковых компонентов в их организации, а также ...

БЕЛОК, ОБЛАДАЮЩИЙ СПОСОБНОСТЬЮ ИНГИБИРОВАТЬ СТИМУЛИРОВАННУЮ КОЛЛАГЕНОМ АГРЕГАЦИЮ ТРОМБОЦИТОВ И ПРЕДОТВРАЩАТЬ ВЗАИМОДЕЙСТВИЕ МЕЖДУ КОЛЛАГЕНОМ И vWF, СПОСОБ ЕГО ПОЛУЧЕНИЯ, ФАРМКОМПОЗИЦИЯ НА ЕГО ОСНОВЕ

Изобретение относится к биотехнологии и медицине и может быть использовано для получения белка, способного ингибировать стимулированную коллагеном агрегацию тромбоцитов. Белок (Брандинин) имеет молекулярную массу около 15 кДа, связывается с коллагеном. Белок ингибирует связывание коллагена с тромбоцитами и их последующую активацию, которая ведет к агрегированию тромбоцитов и образованию тромбов. Белок предотвращает связывание фактора фон Виллебранда с коллагеном. Белок выделяют из неочищенных экстрактов пиявки Hirudo medicinalis путем электрофореза в полиакриламидном геле. Белок пригоден для профилактики и лечения тромболитических заболеваний. 3 с. и 1 з.п. ф-лы, 2 табл., 6 ил.

СПОСОБ ОЧИСТКИ КОНЪЮГАТОВ НА ОСНОВЕ IL-15/IL-15R-АЛЬФА

Группа изобретений относится к улучшенному способу биохимического извлечения конъюгата на основе IL-15/IL-15Rα в мономерной форме и фармацевтической композиции, включающей эффективное количество композиции, содержащей мономерные конъюгаты IL-15/IL-15Rα. Способ предусматривает очистку осветленного супернатанта из культуральной средытрансформированной клетки-хозяина, экспрессирующей конъюгат, имеющий аминокислотную последовательность SEQ ID NO:16 или SEQ ID NO:17. Очистку проводят посредством анионообменной хроматографии (AEX) при pH, равном или выше чем 7,0, предпочтительно равном или выше чем 7,5. Далее полученный элюат очищают хроматографией гидрофобного взаимодействия (HIC), проводимой в буферном растворе, содержащем 0,75 М - 2,5 М сульфата аммония, предпочтительно 1 М - 2 М сульфата аммония. Элюируют конъюгаты в градиенте от 2 М до 0 M, предпочтительно от 1,5 до 0 M сульфата аммония. Полученная композиция содержит по меньшей мере 80% конъюгатов в мономерной форме, удовлетворяющей их ...

Способ очистки дигликозилированного белка интерферон-бета

Изобретение относится к способу очистки дигликозилированного белка интерферон-бета и, более конкретно, к способу очистки дигликозилированного белка интерферон-бета, согласно которому (а) получают культуральный раствор, содержащий интерферон-бета, экспрессированный в клетке-хозяине; (b) проводят аффинную хроматографию в отношении культурального раствора, полученного на стадии (а); (c) инактивацию низким рН полученного на стадии (b) раствора, где указанный низкий рН представляет собой рН 4,0 или меньше; (d) проводят хроматографию гидрофобного взаимодействия в отношении полученного на стадии (c) раствора; (e) проводят анионообменную хроматографию в отношении полученного на стадии (d) раствора; и (f) проводят катионообменную хроматографию в отношении полученного на стадии (е) раствора. Способ обеспечивает достаточное количество белка и высокую чистоту и снижает возникновение модификации гликозилирования или других модификаций интерферон-бета. 8 з.п. ф-лы, 2 ил., 3 табл., 2 пр.

СПОСОБ ПОЛУЧЕНИЯ ФЕРМЕНТА ДЕСТАБИЛАЗЫ, ОБЛАДАЮЩЕГО ФИБРИНОЛИТИЧЕСКОЙ, ТРОМБОЛИТИЧЕСКОЙ, ЭНДО- И ЭКЗО- ε(γ GLY)-LYS-ИЗОПЕПТИДАЗНОЙ И АМИДОЛИТИЧЕСКОЙ АКТИВНОСТЬЮ

Изобретение относится к медицине, точнее к способам получения биологически активных веществ из животного сырья, и может найти применение в терапии тромбозов и в экспериментальных областях медицины. Изобретение позволяет повысить новый тромболитический фермент, дестабилазу, эндо-ε(γ-Glu)-Lys-изопептидазу, обладающую экзо-ε (γ-Glu)-Lys-изопептидазной и амидолитической активностью. Установлена частичная аминокислотная последовательность фермента. Для этого из пиявки семейства Hirudinidae получают экстракт, подвергают его аффинной хроматографии, дополнительно очищают на анионо- или катионообменнике, собирают целевой продукт, затем обессоливают гель-фильтрацией, собирают белок с мол.м. 126000±300 Дальтон. 2 ил.

ФАРМАКОЛОГИЧЕСКИ АКТИВНАЯ СУБСТАНЦИЯ ЗАЩИТНОГО СОЕДИНЕНИЯ ОРГАНИЗМА 3СО, СПОСОБ ЕЕ ПОЛУЧЕНИЯ И ТЕРАПЕВТИЧЕСКОЕ ПРИМЕНЕНИЕ

Изобретение относится к новой субстанции защитного соединения организма, способу ее получения и применения в целях. Изобретение включает: способ получения нового вещества из желудочного сока человека и животных и имеет следующие стадии очистки: центрифугирование, диализ, ионообменную хроматографию и лиофилизацию, а также лекарственное средство для применения в медицине и ветеринарии и способ его использования при лечении заболеваний и расстройств, индуцированных стрессорными воздействиями, воспалительных процессов, отечности, заболеваний и расстройств допаминэргической этиологии, сахарного диабета, заболеваний и нарушений функции органов желудочно-кишечного тракта, сердца, почек, поджелудочной железы, печени, мужских половых желез, кроветворных органов, скелета и нервной системы, старческого слабоумия, ран, ожогов, переломов, вирусных заболеваний, злокачественных новообразований, в хирургической практике, некоторых расстройств репродуктивной функции и иммунной системы; защиты от ионизирующей ...

Способ производства иммуноглобулина для внутривенного введения

Изобретение относится к области биотехнологии, конкретно к способу получения иммуноглобулинов, и может быть использовано в медицине. Способ, включающий разделение на фракции плазмы здорового человека с получением фракции и последующее пропускание последовательно через анионную хроматографию А, анионную хроматографию В и аффинную хроматографию C, позволяет получать иммуноглобулин, пригодный для внутривенного введения. 16 з.п. ф-лы, 1 ил., 2 табл., 18 пр.

СПОСОБЫ ПОВЫШЕНИЯ ЭФФЕКТИВНОСТИ ЭТАПОВ ОЧИСТКИ БЕЛКА, НАХОДЯЩИХСЯ НИЖЕ ПО ПОТОКУ, С ИСПОЛЬЗОВАНИЕМ МЕМБРАННОЙ ИОНООБМЕННОЙ ХРОМАТОГРАФИИ

... 1. Способ усиления эффективности этапов хроматографии для очистки белков ниже по потоку, включающий:a. пропускание композиции, содержащей представляющий интерес полипептид и различные загрязняющие вещества сквозь ионообменную мембрану, отличающуюся тем, что полипептид и мембрана имеют противоположный заряд, при рабочих условиях, которые состоят из буфера, имеющего pH, достаточно отличающийся от pI полипептида с целью повышения заряда полипептида, и низкой ионной силы, эффективной для предотвращения экранирования зарядов буферных ионов, которые приводят к тому, что мембрана связывает полипептид и по крайней мере одно загрязняющее вещество;b. перегрузку ионообменной мембраны таким образом, что по меньшей мере одно загрязняющее вещество остается связанным с мембраной, тогда как представляющий интерес полипептид находится, главным образом, в элюате;c. улавливание элюата из ионообменной мембраны, которая содержит представляющий интерес полипептид;d. этап ионообменной хроматографии, которая имеет ...

БЕЛКОВЫЕ КОМПОЗИЦИИ ПРОТИВ VEGF И СПОСОБЫ ИХ ПОЛУЧЕНИЯ

СПОСОБ ПОЛУЧЕНИЯ ПЕПТИДОВ

Изобретение относится к области биотехнологии. Предложен способ получения пептидов. Проводят автолиз дрожжей. Отделяют клеточные оболочки центрифугированием. Очищают автолизат на гелевом сульфокатионите в водородной форме, содержащем 12-16% дивинилбензола. Полученный водный раствор пептидов пропускают последовательно через гелевый анионит до получения раствора с рН 2,0-2,6, а затем через гелевый катионит с содержанием дивинилбензола 1-2% или макропористый катионит. Преимуществом заявленного способа является получение пептидов высокой степени очистки, которые обладают биологической активностью. 11 пр.

СПОСОБ РАЗДЕЛЕНИЯ БЕЛКОВ МОЛОКА НА ФРАКЦИИ

Способ разделения белка молока на фракции, предусматривающий подготовку образцов молока, отличающийся тем, что разделение проводят путем ионообменной хроматографии на колонке с анионитом АВ-17 в линейном градиенте NaCl (0-0,5 М) в 0,05 М трис - HCl, рН 8,6.

СПОСОБ ВЫДЕЛЕНИЯ ПРЕДВАРИТЕЛЬНО ОТОБРАННОГО ГЕМОГЛОБИНА И ОЧИЩЕННЫЙ ПРЕПАРАТ ГЕМОГЛОБИНА

Изобретение относится к способам хроматографического выделения гемоглобина и к составу самого гемоглобина. Способ выделения гемоглобина из водного раствора включает в себя пропускание раствора на первой хроматографической стадии и проведение хроматографии либо в условиях анионообмена, либо в условиях катионообмена, при этом непрерывное пропускание осуществляют до почти полного насыщения колонки гемоглобинами и компонентами с более высоким средством и производят дальнейшее непрерывное пропускание раствора до сверхнагрузки колонки с последующим замещением на ней гемоглобинов. Способ имеет также вторую хроматографическую стадию.

СПОСОБ ИЗГОТОВЛЕНИЯ

Способ получения препарата для парентерального белкового питания

FIBRINOGENKONZENTRATE AUS BLUTPLASMA, VERFAHREN UND ANLAGE FÜR IHRE HERSTELLUNG

Gereinigte DNAse Formen

FÜR CHITIN-DEACETYLASE KODIERENDE DNA

METHODE ZUR TRENNUNG VON GLYKOSYLIERTEN UND UNGLYKOSYLIERTEN PROTEINEN

METHODE ZUR CHROMATOGRAPHISCHEN ENTFERNUNG VON PRIONEN

TACHYKININPEPTIDE, IHRE PRÄPEPTIDE UND SIE KODIERENDE GENE

VERFAHREN ZUR REKOMBINANTEN HERSTELLUNG VON PLAZENTA WACHSTUMSFAKTOR

Neue Proteine mit TNF-Hemmender wirkung und ihre Herstellung.

PURIFICATION METHOD AND APPARATUS

Interleukin-13 antibody composition

Method of changing a globular protein structure into a fibrillar protein structure.

The method comprising the steps of providing a globular protein, forming a solution containing the globular protein, adding a detergent to the solution containing the globular protein, and applying the solution to a molecular sizing column with a pore size of at least 70 kDa. The column is preferably selected from a SuperdexRTM200 or a HW555, and the detergent is preferably selected from SDS or Zwittergent 3-14. The globular protein is preferably selected from albumin, fibronectin or recombinant capsid proteins VP1, VP2, or VP3 of the foot and mouth disease virus, or precursor or chimeric proteins thereof. The proteins may have a use in medicine, particularly in the treatment of cancer, or they may be used as vaccine adjuvants.

Process for purifying synthetically prepared peptides

... 1,132,264. Purification of synthetic peptides. FARBWERKE HOECHST A.G. 24 Jan., 1966 [23 Jan., 1965], No. 3106/66. Heading C2C. Synthetically prepared polypeptides having a corticotropin-sequence of amino acid residues within the range from 1-18 to 1-25 or an analogous synthetic peptide in which one or more of the individual amino acids are replaced by the same number of other amino acids having a similar structure provided that the ACTH activity is maintained are purified by precipitating a tert.-butyl and tert.-butyloxycarbonyl protected peptide from a solution in aq. alc. (containing 1-3 C) by means of AcOH containing NH 4 OAc, removing the protecting groups with CF 3 COOH and chromatographing the free peptide dissolved in aq. NH 4 OAc solution on carboxy methyl cellulose.

PURIFICATION OF PROTEINS

Purified forms of dnase

INTRODUCING STACKS OF PAPER LAYERS INTO CARTONS

CONTROL OF MILK SECRETION

Method for highly expressing recombinant protein of engineering bacteria and use thereof

Water-soluble polymer alkanals.

Water-soluble polymer alkanals.

Water-soluble polymer alkanals

Method for highly expressing recombinant protein of engineering bacteria and use thereof

Obese proteins (ob) recombining

Method of purifying protein

Water-soluble polymer alkanals

Method for highly expressing recombinant protein of engineering bacteria and use thereof

Water-soluble polymer alkanals

Water-soluble polymer alkanals.

VERFAHREN ZUR GEWINNUNG VON GEREINIGTEM FAKTOR VIII:C/VWF-KOMPLEX

The invention relates to a method for the production of factor VIII:C/von Willebrand factor complex from plasma or a plasma fraction by chromatography in a cation exchanger, wherein the factor VIII:C/von Willebrand factor complex is obtained with at least 300 times the purity of the plasma and the yield of factor VIII:C and the von Willebrand factor is at least 50 % in relation to cryoprecipitates or analogous plasma fractions.

VERFAHREN ZUR HERSTELLUNG EINES KONZENTRATES VON STANDARDISIERTEM, MENSCHLICHEM VON WILLEBRAND-FAKTOR

PROCEDURE FOR THE PRODUCTION OF A DESIRED PROFILE OF ERYTHROPOIETIN GLYKO ISOFORMEN

PROTEIN CLEANING OF MEANS CHROMATOGRAPHY AND FOLLOWING FILTRATION ON LOADED LAYER

CLEANING OF OF WILL FIRE FACTOR BY CATION EXCHANGER CHROMATOGRAPHY

USE FROM PROTEIN KINASES TO THE DIAGNOSIS AND TREATMENT OF THE ALZHEIMER'S ILLNESS

CLEANING OF ANTIBODIES BY ION EXCHANGE CHROMATOGRAPHY

SEPARATION FROM POLYPEPTIDEN WITH A RACEMISIERTEN AMINO ACID

PROCEDURE FOR THE REMOVAL OF VIRUSES AND PRIONEN FROM IMMUNGLOBULINZUSAMMENSETZUNGEN

CLEANED RHIGF-I/RHIGFBP-3-KOMPLEXE AND MANUFACTURING PROCESS FOR IT

SEPARATION OF LIPOPOLYSACCHARIDEN FROM LIPOPOLYSACCHARIDKOMPLEXEN BY NON ENTZUNDBAREN LÍSUNGSMITTELN

PROCEDURE FOR THE PREVENTION OF POLYMERS OF THE HUMAN SERUM ALBUMIN

PROCEDURE FOR THE PRODUCTION OF A PEPTIDS WITH A PI LARGE AS 8 OR SMALL THAN 5

VERFAHREN ZUR HERSTELLUNG EINES ANTITHROMBIN III-HEPARIN- BZW. ANTITHROMBIN III-HEPARINOIDKONZENTRATES

PROCEDURE FOR THE PRODUCTION OF CLEANED FACTOR VIII: C/VWF COMPLEX

PROCEDURE FOR THE PRODUCTION OF A CONCENTRATE OF STANDARDISATION, HUMAN ONE OF WILL FIRE FACTOR

INHIBITOR OF THE BLOOD PANEL AGGREGATION

PRODUCTION OF A REKOMBINANTEN HUMAN INTERLEUKIN-1-HEMMERS

PROCEDURE FOR THE PRODUCTION OF FACTOR VIII/VWF COMPLEX

PROCEDURE FOR THE CLEANING OF HOEHERMOLEKULAREN PEPTIDEN

Process for preparing IG G products

A process for preparing immunoglobulin G preparations is described and involves - mixing an immunoglobulin G-containing biological material which potentially contains pathogens with an organic solvent, - bringing the immunoglobulin G-containing biological material mixed with solvent in contact with an anion exchanger, whereupon the pathogens which are potentially present are adsorbed onto the anion exchange material, and immunoglobulin G, which enters into no or only a slight interaction with the ion exchanger, is not bound, and - separating the anion exchanger with the potentially adsorbed pathogens from the immunoglobulin G-containing biological material, resulting in a purified and virus- depleted immunoglobulin G preparation.

ION EXCHANGE CHROMATOGRAPHI SEPARATION FROM GLP-1 AND USED PEPTIDEN

PROCEDURE FOR THE CLEANING OF FACTOR VIII/VWF COMPLEX OF MEANS CATION EXCHANGER CHROMATOGRAPHY

CLEANING METHOD OF TRANSFORMING GROWTH FACTOR (TGF)

POLYPEPTIDE INDUCING FACTORS IN BONES AND CARTILAGES.

STABILIZATION OF HIGHLY PURIFIED PROTEINS

PROCEDURE FOR THE PROTEIN CLEANING.

DISTANCE OF PROTEIN A FROM ANTI-BODY PREPARATIONS.

VERFAHREN ZUR HERSTELLUNG EINES KONZENTRATES VON STANDARDISIERTEM, MENSCHLICHEM VON WILLEBRAND-FAKTOR

CLEANED PROTEIN A-COMPOSITIONS AND PROCEDURE FOR YOUR PRODUCTION.

ION EXCHANGE AND SEPARATION METHOD.

PROCEDURE FOR THE SEPARATION OF TOXINEN FROM PROTEIN SOLUTIONS.

PROCEDURE FOR THE CLEANING OF IGG MONOCLONAL ANTIBODIES

PROCEDURE FOR THE PRODUCTION OF A ANTITHROMBIN [...] HEPARIN AND/OR ANTITHROMBIN [...] HEPARINOIDKONZENTRATES ONE.

CLEANING, PRODUCTION AND USE OF TUMOR NEKROSISFAKTOREN.

T-CADHERIN-BINDUNGSMOLEKÜL

CLEANING OF ALPHA L PROTEINASEINHIBITOR MEANS NEW ONE CHROMATOGRAPHI SEPARATION CONDITIONS

CLEANING OF BIOLOGICAL SUBSTANCES

HIGHLY PURE LIPOPEPTIDE, LIPOPEPTID MIZELLEN, THEIR PRODUCTION AND DRUG

PROCEDURE FOR the ISOLATION AND CLEANING VITAMIN k of DEPENDENT PROTEINS

METHOD FOR CLEANING AND ISOLATION OF TWO IONI FORMS OF SOMATOMEDIN.

PROTEIN CLEANING

RIBOSOMEN INACTIVATING PROTEINS CONTAINING MATERIALS, PROCEDURES FOR YOUR PRODUCTION AND YOUR USE

PROCEDURE FOR THE PRODUCTION OF MOLECULAR-UNIFORM OF HYPER+POLYMERE HÄMOGLOBINE

PROCEDURE FOR THE PRODUCTION OF GROWTH FACTORS FROM MILK OR MILK DERIVATIVES

PROCEDURE FOR THE PRODUCTION OF IMMUNOGLOBULIN G CONCENTRATE

PROCEDURE FOR THE PRODUCTION OF A HIGHLY PURE FACTOR VIII/OF WILL FIRE FACTOR OF COMPLEX BY ANION EXCHANGE CHROMATOGRAPHY

Preparative chromatography column and methods

A chromatography column that captures components in a process liquid in a free flow state and allows elution in steps is described.

METHODS OF PURIFYING POLYPEPTIDES

The present invention provides methods for purifying a polypeptide from a composition comprising the polypeptide and at least one contaminant and formulations comprising the polypeptide purified by the methods. The methods for purifying include cation exchange material and/or mixed mode material. 1. A method for purifying a polypeptide from a composition comprising the polypeptide and at least one contaminant , wherein the method comprises either (i) or (ii):(i) sequential steps of (a) loading the composition onto a cation exchange material at a loading density of greater than about 150 g/L of cation exchange material; and (b) loading a composition recovered from the cation exchange material onto a mixed mode material; or(ii) sequential steps of (a) loading the composition onto a mixed mode material; and (b) loading a composition recovered from mixed mode material onto a cation exchange material at a loading density of greater than about 150 g/L of cation exchange material.2. The method of claim 1 , wherein the polypeptide has a pI of between about 6 and about 10.3. The method of claim 2 , wherein the polypeptide has a pI of between about 7 and about 9.4. The method of claim 1 , wherein the polypeptide is an antibody or an immunoadhesin.5. The method of claim 4 , wherein the polypeptide is an immunoadhesin.6. The method of claim 4 , wherein the polypeptide is an antibody.7. The method of claim 6 , wherein the antibody is a monoclonal antibody.8. The method of claim 7 , wherein the monoclonal antibody is a chimeric antibody claim 7 , humanized antibody claim 7 , or human antibody.9. The method of claim 7 , wherein the monoclonal antibody is an IgG monoclonal antibody.10. The method of claim 6 , wherein the antibody is an antigen binding fragment.11. The method of claim 10 , wherein the antigen binding fragment is selected from the group consisting of a Fab fragment claim 10 , a Fab′ fragment claim 10 , a F(ab′)fragment claim 10 , a scFv claim 10 , a Fv claim 10 , and ...

PROCESS FOR THE PURIFICATION OF A GROWTH FACTOR PROTEIN

A process of purifying a Growth Factor Protein in a purification sequence employing chromatography characterized in that 1. A process of purifying a Growth Factor Protein selected from the group consisting of Colony Stimulating Factor (CSF) , interleukin 3 (IL-3) , Hepatocyte growth factor , Epidermal growth factor and fibroblast growth factor (acid) in a purification sequence employing chromatography comprisingbinding the Growth Factor Protein to a multimodal resin at a pH between 4 to 6.2, andeluting the Growth Factor Protein at a pH>6.3.2. The process of wherein the multimodal resin comprises moieties bound to a matrix and the moieties are able to interact with the Growth Factor Protein in a mixture by ionic interactions claim 1 , hydrogen bonding claim 1 , and/or hydrophobic interaction.3. The process of wherein the mixture comprising the Growth Factor Protein is a solution.4. The process of wherein the Growth Factor Protein is a recombinant Growth Factor Protein.5. The process of wherein the Growth Factor Protein is eluted by a pH change>pH 6.3.6. The process of wherein the elution is performed with a change of the pH or with an elution agent comprising an amino acid having a basic side chain and/or high ionic strength in an elution buffer.7. The process according to wherein the concentration of the elution agent is in the range of from about 0.1 M to about 2.0 M.8. The process of wherein the Growth Factor Protein binds to the multimodal resin at pH about 4.0 to pH about 6.0 claim 1 , and the Growth Factor Protein is eluted from the multimodal resin at pH 6.5 or higher.9. The process of comprising using a buffering substance comprising sodium citrate claim 1 , sodium acetate claim 1 , or HEPES.10. The process of wherein a non-ionic detergent is present in any of the buffers used.11. The process of wherein the amino acid having a basic side chain is arginine claim 8 , lysine and/or histidine.12. The process of wherein buffers comprising about 0.1 M-2 M sodium ...

METHOD FOR PURIFYING PROTEIN USING AMINO ACID

In large-scale purification of proteins such as antibodies, an economic high-purity purification method is required. The present invention relates to a method for purifying a protein, including one or more chromatographic processes, in which an amino acid; or a dipeptide, an oligopeptide, or a polyamino acid thereof is included in a buffer solution used in at least one chromatographic processes (equilibration buffer, wash buffer, and elution buffer), thereby purifying a high-purity protein with a very small quantity of the impurity (e.g., polymers or host cell proteins). 1. A method for purifying a protein , comprising one or more chromatographic processes , wherein an amino acid is included as an ingredient of a buffer solution or a part thereof used in at least one chromatographic process.2. The purification method according to claim 1 , wherein an amino acid is included as an ingredient of a buffer solution or a part thereof used in a cation exchange chromatographic process.3. The purification method according to claim 2 , which further comprises claim 2 , as the chromatographic process claim 2 , a Protein A affinity chromatographic process and one or more chromatographic processes selected from an anion exchange chromatographic process claim 2 , a gel filtration chromatographic process claim 2 , a hydrophobic chromatographic process claim 2 , a hydroxyapatite chromatographic process claim 2 , and a mixed mode chromatographic process.4. The purification method according to claim 2 , wherein the cation exchange chromatographic process is carried out subsequent to the Protein A affinity chromatographic process.5. The purification method according to claim 4 , wherein the anion exchange chromatographic process is further subsequently carried out.6. The purification method according to claim 3 , wherein the cation exchange chromatographic process carried out subsequent to the Protein A affinity chromatographic process and the anion exchange chromatographic process.7. ...

METHOD TO PRODUCE AN IMMUNOGLOBULIN PREPARATION WITH IMPROVED YIELD

The present invention provides improved methods for the manufacturing of IVIG products. These methods offer various advantages such as reduced loss of IgG during purification and improved quality of final products. In other aspects, the present invention provides aqueous and pharmaceutical compositions suitable for intravenous, subcutaneous, and/or intramuscular administration. In yet other embodiments, the present invention provides methods of treating a disease or condition comprising administration of an IgG composition provided herein. 1. A method for preparing an enriched IgG composition from plasma , the method comprising the steps of:(a) precipitating a cryo-poor plasma fraction, in a first precipitation step, with from about 6% to about 10% alcohol at a pH of from about 7.0 to about 7.5 to obtain a first precipitate and a first supernatant;(b) precipitating IgG from the first supernatant, in a second precipitation step, with from about 23% to about 27% alcohol at a pH of from about 6.7 to about 7.1 at a temperature of from about −7° C., to about −9° C. to form a second precipitate;(c) suspending the second precipitate to form a suspension;(d) precipitating IgG from the suspension formed in step (c), in a third precipitation step, with from about 22% to about 28% alcohol at a pH of from about 6.7 to about 7.3 to form a third precipitate;(e) re-suspending the third precipitate to form a suspension; and(f) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition.258-. (canceled)59. The method of claim 1 , wherein the temperature of precipitation step (b) is about −7° C.60. The method of claim 1 , wherein the alcohol concentration of precipitation step (b) is about 25%.61. The method of claim 59 , wherein the alcohol concentration of precipitation step (b) is about 25%.62. The method of claim 1 , wherein at least one of the first precipitation step claim 1 , second precipitation step claim 1 , or third ...

MANUFACTURE OF FACTOR H (FH) AND FH-DERIVATIVES FROM PLASMA

The present invention provides compositions and pharmaceutical formulations of Factor H derived from plasma. Also provided are methods for the manufacture of the Factor H compositions and formulations, as well as methods for the treatment of diseases associated with Factor H dysfunction. 114-. (canceled)15. A method for preparing an enriched Factor H composition from plasma , the method comprising the steps of:(a) extracting Factor H from a Fraction I precipitate and/or a Fraction II+III precipitate, to form a Factor H extract; and(b) precipitating an impurity from the Factor H extract to form a supernatant containing Factor H, thereby preparing an enriched Factor H composition.1627-. (canceled)28. The method of claim 15 , wherein extraction step (a) comprises re-suspending the Fraction II+III precipitate with a re-suspension buffer having a pH of from 4.0 to 5.5.29. The method of claim 15 , wherein precipitation step (b) comprises precipitating an impurity from the Factor extract using a final alcohol concentration of from 10% to 20% at a pH of from 7.0 to 9.0.30. The method of claim 15 , wherein precipitation step (b) comprises precipitating an impurity from the Factor H extract using a final alcohol concentration of from 14% to 16% at a pH of from 7.5 to 8.5.31. The method of claim 15 , wherein the method further comprises the step of:(c) precipitating Factor H from the supernatant formed in step (h) using a final alcohol concentration of from 20% to 30% at a pH of from 5.0 to 7.0.32. The method of claim 15 , wherein the method further comprises the step of:(c) precipitating Factor H from the supernatant formed in step (b) using a final alcohol concentration of from 24% to 26% at a pH of from 5.8 to 6.2.33. The method of claim 28 , wherein the method further comprises the step of:(c) precipitating Factor H from the supernatant formed in step (b) using a final alcohol concentration of from 20% to 30% at a pH of from 5.0 to 7.0.34. The method of claim 28 , wherein ...

PURIFICATION OF PROTEINS

The invention describes a method for protein purification. More particularly, the invention relates to a purification process comprising protein A chromatography and anion exchange chromatography wherein protein A chromatography eluate is further purified by anion exchange chromatography at similar pH or at a pH less than or equal to 6. 1. A process for purifying an antibody , comprising:a) purifying using protein A chromatography, wherein the antibody is eluted at low pH; andb) purifying using anion exchange chromatography performed in the flow-through mode, wherein eluate obtained from step a) is loaded onto an anion exchange resin at pH values less than or equal to 6.2. A process according to claim 1 ,wherein the antibody is eluted in step a) at pH values about 3.3 to about 4.5 and loaded onto the anion exchange resin at pH values about 3.3 to about 6.3. A process according to claim 1 , wherein the antibody is eluted in step a) at pH values about 3.5 and loaded onto the anion exchange resin at pH values about 3.5 to about 6.4. A process according to claim 1 , wherein the antibody is loaded onto the anion exchange resin at pH values about 4.5. A process according to claim 1 , wherein the antibody is loaded onto the anion exchange resin at pH values about 6.6. A process according to claim 1 , wherein the anion exchange chromatography is followed by a cation exchange chromatography step in a bind-elute mode claim 1 , and wherein the flow-through from the anion exchange chromatography step is loaded onto a cation exchange resin at pH values less than or equal to 6.7. A process for purifying an antibody claim 1 , comprising:a) purifying using protein A chromatography, wherein the antibody is eluted at low pH values; andb) purifying using anion exchange chromatography, performed in the flow-through mode, wherein eluate obtained from step a) is loaded on to the anion exchange resin without substantial adjustment of pH. Aspects of this application relate to purification ...

METHOD FOR EXTRACTING UNDENATURED TYPE II COLLAGEN HAVING ACTIVE EPITOPE

A method for extracting water-soluble undenatured type II collagen having an active epitope comprises a first step for extracting animal-derived cartilage using an acidic solution at 50° C. or less, and a second step for adding pepsin to the acidic solution and performing extraction at 40° C. or less. This allows the epitope of undenatured type II collagen to be extracted in large quantities and with high efficiency without any loss of activity, and makes it possible to provide a food or beverage product obtained using the water-soluble undenatured type II collagen extracted by the extraction method. 111-. (canceled)12. An extraction method wherein water-soluble undenatured type II collagen is extracted and an active epitope contained in the water-soluble undenatured type II collagen is extracted , comprising the steps of: (1) temperature: 5 to 35° C.,', '(2) pH: 3.4 or less, and', '(3) extraction time period: 12 to 36 hours; and, 'extracting animal-derived cartilage with an acidic solution under the following conditions (1) temperature: 5 to 35° C.,', '(2) pH: 3.4 or less, and', '(3) extraction time period: 12 to 36 hours., 'adding pepsin to the acidic solution and extracting an active epitope with the acidic solution under the following conditions13. The extraction method according to claim 12 , wherein the step of extracting animal-derived cartilage with the acidic solution is a first step and the step of adding pepsin to the acidic solution and extracting an active epitope with the acidic solution is a second step claim 12 , and the total extraction time for both the first step and the second step is 48 hours or less.14. The extraction method according to claim 13 , wherein the second step is performed at lower temperature than the first step.15. The extraction method according to claim 13 , wherein elastase is further added in the second step of extraction.16. The extraction method according to claim 14 , wherein elastase is further added in the second step of ...

PREPARATION OF CANOLA PROTEIN ISOLATE FROM CANOLA OIL SEEDS ("BLENDERTEIN")

Canola protein isolate is recovered from canola oil seeds by crushing the oil seeds and extracting the crushed canola oil seeds. Fat co-extracted from the crushed oil seeds is removed from the aqueous canola protein solution which then is processed by the micellar route to obtain the canola protein isolate. 1. A process for the preparation of a canola protein isolate , which comprises:grinding canola oil seeds,extracting the ground canola oil seeds with an aqueous extracting medium to solubilize canola protein and fats in the ground canola oil seeds to form an aqueous canola protein solution,separating the aqueous canola protein solution from residual ground canola oil seeds,defatting the aqueous canola protein solution,clarifying the defatted aqueous canola protein solution,concentrating the clarified aqueous canola protein solution while maintaining the ionic strength substantially constant to form a concentrated canola protein solution,diluting the concentrated protein solution into chilled water to cause the formation of canola protein micelles,collecting the canola protein micelles as a protein micellar mass, anddrying the protein micellar mass to form a canola protein isolate having a protein content of at least about 90 wt % (N×6.25) d.b.2. The process of claim 1 , wherein said aqueous extracting medium is an aqueous salt solution having an ionic strength of at least about 0.05 M with a pH of about 5 to about 6.8 to form a canola protein solution having a concentration of about 3 to about 40 g/L.3. The process of wherein an antioxidant is present in the aqueous extracting medium.4. The process of wherein said defatting step is effected by chilling the canola protein solution to a temperature of about 3° to about 7° C. and removing fat that separates from the canola protein solution.5. The process of wherein claim 4 , following the defatting step claim 4 , the separated aqueous canola protein solution is subjected to a colour removal step.6. The process of ...

METHOD FOR THE PURIFICATION OF RECOMBINANT BLOOD COAGULATION FACTOR IX ENRICHED IN SULFATED AND/OR PHOSPHORYLATED MOLECULES

The present invention relates to a method for the purification of rFIX using anion exchange chromatography in the pseudo-affinity mode, wherein said method comprises a wash step with a wash buffer having a salt concentration of more than 200 mM. The purification according to the invention provides a method to enrich rFIX molecules which have been posttranslationally modified by sulfation and/or phosphorylation. The present invention further relates to purified rFIX compositions enriched in monosulfated and/or monophosphorylated rFIX molecules. 17-. (canceled)8. A purified composition comprising rFIX obtained by a method , the method for obtaining the composition comprising the steps:a) loading a starting composition comprising rFIX onto an anion exchange column.,b) washing the anion exchange material using a wash buffer which has a salt concentration of more than 200 mM (High Salt Wash Buffer), andc) eluting the rFIX from the anion exchange material using an elution buffer comprising divalent cations, andd) collecting the eluate.9. The purified composition according to claim 8 , wherein said purified composition comprises an increased relative amount of monosulfated and/or monophosphorylated rFIX molecules compared to the relative amount of monosulfated and/or monophosphorylated rFIX present in the High Salt Wash Buffer fraction.10. The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of monosulfated and monophosphorylated rFIX molecules which is increased at least 3 fold compared to the relative amount of monosulfated and monophosphorylated rFIX present in the High Salt Wash Buffer fraction.11. The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of at least 30% of monosulfated and/or monophosphorylated rFIX molecules.12. The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of at least 50% of ...

PROTEIN SEPARATION VIA ION-EXCHANGE CHROMATOGRAPHY AND ASSOCIATED METHODS, SYSTEMS, AND DEVICES

The present invention provides methods for separating proteins from a protein mixture. In one aspect, a method for separating a high concentration protein mixture into a bound protein fraction and a flow-through protein fraction can include delivering a protein mixture through an ion exchange column at a fixed pH and a fixed salt concentration. The fixed pH and the fixed salt concentration have been preselected to cause separation of the protein mixture into a bound protein fraction and a flow-through protein fraction. In this case, the bound protein fraction binds to the ion exchange column and the flow-though protein fraction flows though the ion exchange column. The method can further include receiving the flow-through protein fraction from the ion exchange column separate from the bound protein fraction, wherein either the bound protein fraction or the flow-through fraction contains a protein of interest. 1. A method for separating a high concentration protein mixture into a bound protein fraction and a flow-through protein fraction , comprising:delivering a high concentration protein mixture through an ion exchange column at a fixed pH and a fixed salt concentration, wherein the fixed pH and the fixed salt concentration have been preselected to cause separation of the high concentration protein mixture into a bound protein fraction and a flow-through protein fraction, wherein the bound protein fraction binds to the ion exchange column and the flow-though protein fraction flows though the ion exchange column; andreceiving the flow-through protein fraction from the ion exchange column separate from the bound protein fraction, and wherein either the bound protein fraction or the flow-through fraction contains a protein of interest.2. The method of claim 1 , wherein the protein of interest is in the flow-through protein fraction.3. The method of claim 1 , wherein the protein of interest is in the bound protein fraction.4. The method of claim 1 , wherein the high ...

Intravenous Cytomegalovirus Human Immune Globulin and Manufacturing Method Thereof

The present invention discloses an intravenous cytomegalovirus human immune globulin and a manufacturing method thereof, wherein the technical problem to be solved is to improve the purity, yield, and safety of the product. The intravenous cytomegalovirus human immune globulin of the present invention has a specific activity of no less than 2.5 PEI-U/mg, an anti-CMV titer of no less than 100 PEI-U/ml, a purity of greater than 98.2%, and a protein content of 51˜55 mg/ml. The present invention employs caprylic acid precipitation and anion exchange chromatography for replacing the step of ethanol precipitation in the conventional cold ethanol method to keep IgG in the supernatant and maintain the activity of the IgG; the present invention employing processes of caprylic acid inactivation of virus and nanometer film virus removal can effectively protect the safety of the product, and studies show that the preparing method of the present invention not only improves the purity, yield, and safety of the product; but also saves energy and reduces the cost of production. 1. An intravenous cytomegalovirus human immune globulin having a specific activity of no less than 2.5 PEI-U/mg , an anti-CMV titer of no less than 100 PEI-U/ml , a purity of greater than 98.2% , and a protein content of 51˜55 mg/ml.2. A method for preparing intravenous cytomegalovirus human immune globulin , comprising the steps of:(a) preparing FI+II+III and FII+III deposits:preparing human plasma measured by enzyme linked immunosorbent assay, dissolving said human plasma at 2˜30° C., and mixing said human plasma, wherein said human plasma has a high titer of anti-CMV;preparing FI+II+III deposit;adjusting the protein content of said human plasma to 45˜55 mg/ml with saline, adjusting the pH of said human plasma to 6.0˜6.5 with glacial acetic acid, and adding 95% ethanol or ethanol to adjust the concentration of ethanol of said human plasma to 20˜25%, wherein the reaction temperature is −5.5˜−4.5° C., and ...

PROTEIN PURIFICATION BY ION EXCHANGE

The application describes an antibody purification method comprising multiple chromatographic steps wherein the low pH eluate from a protein A chromatography is further purified without the need of substantial pH adjustment. 1. A process for purifying antibodies comprising:a) purifying using protein A chromatography, wherein the antibody is eluted at a particular pH value; andb) purifying using cation exchange chromatography in the bind-elute mode, wherein an eluate from step a) is loaded onto a cation exchange resin without substantial adjustment of pH.2. A process according to claim 1 , wherein the antibody is eluted in step a) at pH values about 3.3 to about 4.5.3. A process according to claim 1 , wherein the antibody is eluted in step a) at a pH value about 3.5.4. A process for purifying antibodies claim 1 , comprising:a) purifying using protein A chromatography, wherein an antibody is eluted at a first pH value;b) purifying using cation exchange chromatography performed in the bind-elute mode, wherein eluate obtained from step a) is loaded onto a cation exchange resin at a second pH value, and elution is carried out at a third pH value; andc) purifying using anion exchange chromatography performed in the flow-through mode, wherein eluate from step b) is loaded on to an anion exchange resin without substantial adjustment of pH.5. A process according to claim 4 , wherein the first and second pH values are similar.6. A process according to claim 4 , wherein the first and second values are different.7. A process according to claim 4 , wherein the second and third pH values are similar.8. A process according to claim 4 , wherein the first pH value is about 3.59. A process according to claim 4 , wherein the second pH value is about 6 to about 8.10. A process according to claim 4 , wherein the second pH value is about 6.11. A process according to claim 4 , wherein the third pH value is about 6 to about 8.12. A process according to claim 4 , wherein the third pH value ...

METHOD FOR ISOLATING A CYCLOHEXAPEPTIDE

The present invention relates to a method for isolating acyclohexapeptide and to a novel crystalline form of caspofungin diacetate thus obtained. 1. Method for isolating a cyclohexapeptide comprising the subsequent steps of:(a) Contacting a solution comprising a cyclohexapeptide and water with a resin;(b) Removing the liquid phase from the mixture obtained in step (a);(c) Eluting said cyclohexapeptide by contacting the material retained after removal of the liquid phase in step (b) by addition of an organic solvent;(d) Separating resin from the mixture obtained in step (c);(e) Crystallizing said cyclohexapeptide from the solution retained after removal of said resin(f) Isolating said cyclohexapeptide from the mixture obtained in step (e)2. Method according to wherein said resin is contained in a chromatography column claim 1 , wherein step (a) is carried out by introducing said solution comprising a cyclohexapeptide and water onto said chromatography column and wherein step (b) is carried out by removing said liquid phase from said chromatography column.3. Method according to wherein said resin is washed with an aqueous mixture after step (a) and before step (c).4. Method according to wherein said resin is hydrophobic interaction chromatography material claim 1 , reversed phase chromatography material claim 1 , ion-exchange chromatography material or a mixture of two or more of these materials.5. Method according to wherein said organic solvent in step (c) is an alcohol.6. Method according to wherein said cyclohexapeptide is anidulafungin or a salt thereof or caspofungin or a salt thereof or micafungin or a salt thereof.7. Method according to wherein the concentration of said cyclohexapeptide in said solution of step (a) is from 2 to 50 times lower than the concentration of said cyclohexapeptide in the solution retained after removal of said resin in step (d).8. Method according to wherein the concentration of said cyclohexapeptide in said solution of step (a) is ...

PROTEIN PURIFICATION

The present invention relates to a process for the purification of an antibody fragment from a periplasmic cell extract comprising a first cation exchange chromatography step and a second anion exchange chromatography step. 120-. (canceled)21. A process for the purification of an antibody fragment from periplasmic cell extract comprising:a) a first chromatography step to capture the antibody fragment wherein a mixture containing an antibody fragment at a concentration of at least 1.5 g/L is subjected to cation exchange chromatography and subsequently eluted to produce a first eluate containing the antibody fragment; andb) a second chromatography step wherein the first eluate is subjected to anion exchange chromatography to capture impurities and produce a flow through containing the antibody fragment.22. The process according to claim 21 , wherein the process comprises not more than two chromatography steps.23. The process according to claim 21 , wherein all chromatography steps are performed on a chromatography column.24. The process according to claim 21 , wherein the cation exchange chromatography is performed in elution mode.25. The process according to claim 21 , wherein the first cation chromatography step comprises the following steps in sequential order:a) loading a mixture containing an antibody fragment onto the cation exchange column,b) washing the cation exchange column with a wash buffer wherein during the washing the conductivity, pH and salt concentration of the buffer remains essentially unchanged, andc) eluting the antibody fragment with an elution buffer.26. The process according to claim 25 , wherein the pH of the washing buffer is identical to the pH of the mixture containing an antibody fragment prior to first chromatography step.27. The process according to claim 25 , wherein the mixture containing an antibody fragment claim 25 , prior to the first chromatography step has a pH of between 4.0 to 5.0.28. The process according to claim 21 , ...

METHOD FOR PRODUCING SURFACTIN AND SALT THEREOF

The method for producing a surfactin or a salt thereof according to the present invention comprises the steps of adding an organic solvent containing a branched alkyl alcohol to a culture fluid containing the surfactin or the salt thereof, or to a solution obtained by removing an insoluble component from the culture fluid, and extracting the surfactin or the salt thereof with the organic solvent, 2. The method according to claim 1 , wherein an aqueous layer during the extraction is acidic.3. The method according to claim 2 , wherein a pH value of the aqueous layer during the extraction is not less than 1 and not more than 5.4. The method according to claim 1 , wherein a carbon number of the branched alkyl alcohol is not less than 3 and not more than 10.5. The method according to claim 1 , wherein a ratio of the branched alkyl alcohol in the organic solvent is not less than 30 wt %.6. The method according to claim 1 , wherein the organic solvent further contains an auxiliary solvent claim 1 , and a total ratio of the branched alkyl alcohol and the auxiliary solvent is not less than 0.6 parts by weight and not more than 1.5 parts by weight relative to 1 part by weight of the culture fluid.7. The method according to claim 1 , further comprising the steps of:a) obtaining an aqueous solution containing the surfactin salt by mixing the extract containing the surfactin with a basic aqueous solution and then removing an organic layer; andb) mixing the aqueous solution containing the surfactin salt with an inorganic acid to precipitate the surfactin in a solid form from the obtained mixture.8. The method according to claim 1 , further comprising the step of mixing the extract containing the surfactin with a base to obtain the surfactin salt obtained in a solid form from the obtained mixture.9. A method for producing the surfactin salt claim 1 , comprising the step of obtaining the surfactin salt or a solution thereof by mixing the surfactin obtained by the method according ...

METHODS OF REDUCING LEVEL OF ONE OF MORE IMPURITIES IN A SAMPLE DURING PROTEIN PURIFICATION

The present invention provides novel and improved protein purification processes which incorporate certain types of carbonaceous materials and result in effective and selective removal of certain undesirable impurities without adversely effecting the yield of the desired protein product. 1. A flow-through process for purifying a target molecule from a Protein A eluate comprising the steps of:(i) contacting the eluate recovered from a Protein A chromatography column with activated carbon;(ii) contacting the flow-through sample from step (i) with an anion exchange chromatography media;(iii) contacting the flow-through sample from step (ii) with a cation exchange chromatography media; and(iv) obtaining the flow-through sample from step (iii) comprising the target molecule,wherein the eluate flows continuously through steps (i)-(iii) and wherein level of one or more impurities in the flow-through sample after step (iii) is lower than the level in the eluate in step (i).2. The flow-through process of claim 1 , further comprising subjecting the flow-through sample from step (iii) to virus filtration.3. The flow-through process of claim 1 , further comprising use of an in-line static mixer and/or a surge tank between steps (ii) and (iii) to change pH.4. The flow-through process of claim 1 , wherein the process employs a single skid.5. The flow-through process of claim 1 , wherein the eluate from the Protein A chromatography column is subjected to virus inactivation prior to contacting with activated carbon.6. The process of claim 1 , wherein steps (i)-(iii) may be performed in any order.7. A flow-through purification process for purifying a target molecule from a Protein A eluate comprising contacting the eluate with two or more matrices selected from activated carbon claim 1 , anion exchange media claim 1 , cation exchange media and virus filtration media claim 1 , wherein the flow of the eluate is continuous.8. A method for reducing the burden on one or more ...

METHOD FOR PURIFYING ACTIVE POLYPEPTIDES OR IMMUNOCONJUGATES

The present invention provides methods for isolating an active polypeptide or immunoconjugate by purification of a solution containing both the active polypeptide or immunoconjugate and an acidic variant thereof, such as a deamidated variant, using anion exchange chromatography. 1. (canceled)2. A method of producing a purified polypeptide from a solution comprising the polypeptide and an acidic variant of the polypeptide , wherein said acidic variant of the polypeptide results in an inhibition of potency of said polypeptide , the method comprising: (a) contacting the polypeptide with an anion exchange (AIEX) chromatography matrix; and (b) eluting the bound polypeptide from the AIEX chromatography matrix with a high salt buffer , thereby separating said polypeptide from the acidic variant and producing a purified polypeptide;{'i': 'Pseudomonas', 'wherein the polypeptide comprises an antibody or antigen binding fragment thereof that binds the cell surface receptor CD22 and a exotoxin, or variant thereof.'}3. (canceled)4. The method of claim 1 , wherein the AIEX matrix contains quaternary amine and tertiary amine ion exchange groups.5. The method of claim 4 , wherein the AIEX matrix contains a quaternary amino (Q) group.6. The method of claim 5 , wherein the AIEX matrix is Q sepharose.7. The method of any of claim 1 , wherein the polypeptide is eluted with a linear or step salt gradient.8. The method of claim 7 , wherein the polypeptide is eluted with a linear salt gradient that is from about 150 mM NaCl in Tris/HCl claim 7 , pH 8.0 to about 300 mM NaCl in Tris/HCl claim 7 , pH 8.0 claim 7 , from about 175 mM NaCl in Tris/HCl claim 7 , pH 8.0 to about 275 mM NaCl in Tris/HCl claim 7 , pH 8.0 claim 7 , or from about 192 mM NaCl in Tris/HCl claim 7 , pH 8.0 to about 245 mM NaCl in Tris/HCl claim 7 , pH 8.0.911-. (canceled)12. The method of claim 1 , wherein between about 75 to about 99% claim 1 , about 80% claim 1 , about 85% claim 1 , about 90% claim 1 , about 95% claim ...

METHOD FOR PURIFICATION OF COMPLEMENT FACTOR H

A method for purification of complement Factor H from a complement Factor H containing source such as blood or blood plasma, in particular a caprylate precipitate of a Factor H containing source, which is e.g. obtained by addition of caprylate ions to fractions of blood or plasma, comprising the steps of: 1. A method for purification of complement Factor H from a complement Factor H containing source , comprising the steps of:a. Providing a Factor containing source;b. performing a cation exchange chromatography;c. performing an anion exchange chromatography;d. performing a hydroxyl apatite chromatography;e. followed by ultra/diafiltration to obtain a complement Factor H concentrate.2. The method according to wherein a heparin affinity chromatography is performed.3. The method according to wherein the process comprises at least one of the following methods for pathogen removal and/or inactivation:a. solvent/detergent treatment;b. pasteurization,c. vapor heat treatment;d. dry heat treatment; ore. nanofiltration.4. The method according to wherein the complement Factor H concentrate is lyophilized.5. A complement Factor H obtainable by a method according to .6. The complement Factor H of obtainable by a method comprisinga. reconstitution of caprylate precipitate to provide a complement Factor H containing solution;b. performing virus inactivation by solvent/detergent treatment (S/D treatment).c. performing a cation exchange chromatography chromatographic step under the following conditions:d. binding of complement Factor H to a strong cation exchange resin of the sulphopropyl type, washing with a buffer comprising 20 mM tri-sodium citrate adjusted to pH 6.0 and elution of complement Factor H with an elution buffe r comprising 20 mM tri-sodium citrate and 0.2 M NaCl adjusted to pH 6.0; 'i. applying of the complement Factor H containing solution (conductivity 0.1-0.5 mS/cm) to a strong anion exchange resin of the quaternary ammonium type, washing with a buffer comprising ...

METHOD FOR PURIFYING HUMAN GRANULOCYTE-COLONY STIMULATING FACTOR FROM RECOMBINANT E. COLI

The present invention provides a method for purifying a large amount of human granulocyte-colony stimulating factors (hG-CSFs) from a recombinant with high yield and purity. According to the method of the present invention, human granulocyte-colony stimulating factor, identical to the native form expressed in the human body, can be easily purified with high yield and purity without an additional activation process. In particular, according to the purification method of the present invention, hG-CSF variants expressed in are efficiently removed to obtain physiologically active hG-CSFs with high purity. 1E. coli. A method for purifying human granulocyte-colony stimulating factors (hG-CSFs) from a recombinant in a high yield , comprising the steps of:{'i': 'E. coli', '(a) culturing an hG-CSF-expressing recombinant to obtain a cell pellet by centrifugation;'}(b) separating an hG-CSF-containing supernatant from the cell pellet obtained in step (a);(c) treating the supernatant obtained in step (b) with an acid to separate the resulting precipitate by filtration;(d) applying a filtrate obtained in step (c) to cation exchange chromatography;(e) applying an eluate obtained in step (d) to hydrophobic-interaction chromatography; and(f) applying an eluate obtained in step (e) to anion exchange chromatography.2E. coli.. The method according to claim 1 , wherein in step (a) claim 1 , the hG-CSFs are expressed into the periplasm of recombinant3E. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coli. The method according to claim 2 , wherein the recombinant is one or more selected from the group consisting of BL21(DE3)/pT14SS1SG(HM 10310) claim 2 , BL21(DE3)/pT14SS1S17SEG (HM 10311; Accession No. KCCM-10154) claim 2 , BL21(DE3)/pTO1SG (HM 10409) claim 2 , BL21(DE3)/pTO1S17SG (HM 10410; Accession No. KCCM-10151) claim 2 , BL21(DE3)/pTO17SG (HM 10411; Accession No. KCCM-10152) claim 2 , BL21(DE3)/pTO17TG (HM 10413) claim 2 , BL21(DE3)/pTO17AG(HM 10414) ...

METHODS FOR EXPRESSION AND PURIFICATION OF IMMUNOTOXINS

The present invention relates to a method of expressing an immunotoxin in strain mutated to toxin resistance comprising a) growing the in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaining a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/IO L of initial volume during induction along with a continuous infusion of yeast extract at a temperature below 17.5° C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h. 126-. (canceled)27. A method of purifying a non-glycosylated immunotoxin comprisinga) loading a solution containing the non-glycosylated immunotoxin onto a hydrophobic interaction column;b) obtaining a first non-glycosylated immunotoxin containing eluant from the hydrophobic interaction column;c) loading the non-glycosylated immunotoxin containing eluant from step (b) onto an anion exchange column;d) obtaining a second non-glycosylated immunotoxin containing eluant from the anion exchange column by eluting the non-glycosylated immunotoxin with a sodium borate solution;e) diluting the concentration of sodium borate in the second non-glycosylated immunotoxin containing eluant from step (d) to about 50 ruM or less;f) concentrating the diluted non-glycosylated immunotoxin containing eluant from step (e) over an anion exchange column; andg) obtaining a purified non-glycosylated immunotoxin from the anion exchange column.28. The method of claim 27 , wherein the non-glycosylated immunotoxin is expressed in yeast.29Pichia pastoris.. The method of claim 28 , wherein the yeast is30. The method of claim 27 , wherein the immunotoxin is a fusion protein.31. The method of claim 27 , wherein the immunotoxin comprises a diphtheria toxin moiety.32. The method of claim 31 , wherein the diphtheria toxin moiety is truncated.33. The method of claim 32 , farther comprising a CD3 antibody ...

OPTIMIZED METHOD FOR ANTIBODY CAPTURING BY MIXED MODE CHROMATOGRAPHY

Herein is reported a method for the purification of an antibody directly captured from clarified cell culture supernatants using Streamline CST and/or Capto MMC, wherein especially product related (aggregates and fragments) and process related impurities (host cell protein, media components) could efficiently be removed, resulting in a preparation with a purity comparable to classical protein A affinity chromatography. 1. A method for producing an anti-IGF-1R antibody comprising the following steps:a) applying a crude mammalian cell culture cultivation supernatant to a multimodal weak cation exchange chromatography material,b) recovering the anti-IGF-1R antibody by applying a buffered solution comprising ethylene glycol and an inorganic salt to the multimodal weak cation exchange chromatography material and thereby producing an anti-IGF-1R antibody.2. The method according to claim 1 , wherein the method comprises the following additional step a-1) prior to step a):a-1) applying a buffered solution comprising an inorganic salt to the multimodal weak cation exchange chromatography material.3. The method according to wherein the method comprises the following additional step a-b) after step a) and prior to step b):a-b) applying a buffered solution to the multimodal weak cation exchange chromatography material, whereby the anti-IGF-1R antibody is not recovered from the multimodal weak cation exchange chromatography material.4. The method according to claim 3 , wherein the step a-b) comprises two steps a-b 1) and a-b2):a-b1) applying a buffered solution comprising an inorganic salt to the multimodal weak cation exchange chromatography material, anda-b2) applying a buffered solution comprising a denaturant to the multimodal weak cation exchange chromatography material,whereby the anti-IGF-1R antibody is not recovered from the multimodal weak cation exchange chromatography material.5. The method according to wherein the denaturant is selected from guanidinium hydrochloride ...

METHOD FOR PRODUCING HIGH-PURITY SOLUBLE THROMBOMODULIN

The aim of the present invention is to obtain soluble thrombomodulin substantially not containing a denatured product of soluble thrombomodulin that is generated under acidic conditions. A method is provided for producing soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin that is generated under acidic conditions, which comprises; subjecting the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite; and carrying out linear gradient elution, stepwise gradient elution, or gradient elution in which linear gradient elution is combined with stepwise gradient elution under separation conditions in which the denatured product of the soluble thrombomodulin can be separated, wherein said gradient is a salt concentration gradient, so as to obtain an elution fraction containing soluble thrombomodulin that does not substantially contain the denatured product of the soluble thrombomodulin, either (a) after the position of a fraction has previously been confirmed, or (b) while confirming the elution fraction. 1. A method for producing soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin that may be generated from the soluble thrombomodulin under acidic conditions , which comprises: (0) a step of leaving the soluble thrombomodulin under acidic conditions of pH 5 or less; (1) a step of subjecting a soluble thrombomodulin-containing material that contains or is suspected to contain a denatured product of the soluble thrombomodulin , which is obtained by step (0) , to an anion exchanger or hydroxyapatite; and (2) a step of obtaining a soluble thrombomodulin-containing fraction that does not substantially contain a denatured product of the soluble thrombomodulin under separation conditions in which the soluble thrombomodulin can be separated from a denatured product of the soluble thrombomodulin , wherein the anion exchanger comprises a buffer solution of pH ...

REMOVAL OF VIRUCIDAL AGENTS FROM BIOMOLECULE PREPARATIONS

Methods, compositions and kits for chromatography purification of antibodies are provided. In some embodiments, antibodies are purified by hydroxyapatite (HT) or fluorapatite (FT) that is treated with a polycationic agent. In some embodiments, the antibodies are treated with a polycationic agent that is also a virucidal agent prior to purification. 1. A two-stage viral inactivation method , comprising ,incubating a biological sample comprising a target molecule with a positively-charged or neutral virucidal agent under conditions to inactivate viruses in the sample, if present;subsequently contacting the target molecule to an apatite support under conditions resulting in binding of the target biomolecule to the support such that the target biomolecule binds to the apatite and a majority of the virucidal agent flows past the support;washing the support binding the target molecule with a first wash buffer, wherein the first wash buffer comprises at least a second virucidal agent, wherein the second virucidal agent is in sufficient concentration to inactivate viruses, if present, and to dissociate complexes of the positively-charged or neutral virucidal agent and the target molecule, thereby removing at least some residual virucidal agent, if present; andeluting the target biomolecule from the support such that the target biomolecule is substantially free of the positively-charged or neutral virucidal agent.2. The method of claim 1 , wherein the positively-charged or neutral virucidal agent is selected the group consisting of polyethyleneimine claim 1 , ethacridine claim 1 , chlorhexidine claim 1 , benzalkonium chloride claim 1 , tri(n-butyl)phosphate claim 1 , and methylene blue.3. The method of claim 1 , further comprising claim 1 , between the washing and eluting claim 1 , contacting the support with a second wash buffer.4. The method of claim 3 , wherein the second wash buffer has a lower conductivity than the first wash buffer and no chaotropic agents.5. The ...

PROCESS FOR PRODUCTION OF FIBRINOGEN

The present invention relates to a method or process for the manufacture of a virus and prion save native fibrinogen concentrate of high purity and low amounts of fibrinopeptide A and fibronectin. 1. A process for purifying fibrinogen from a fibrinogen containing source , comprising subjecting a fibrinogen containing solution to chromatography on anion exchange resins , wherein the anion exchange resins are selected from the group consisting of a support material comprising a hydroxylated polymer with grafted tertiary or quaternary amino groups.2. The process of claim 1 , wherein the support material is composed of cellulose claim 1 , agarose claim 1 , silica claim 1 , polymeric or ceramic material.3. The process of claim 1 , wherein the anion-exchange resin is trimethyl-amino groups grafted to a hydroxylated methacrylic polymer via a linking group claim 1 , such as GigaCap Q-650M®.4. The process of claim 1 , wherein the fibrinogen containing source is cryoprecipitate claim 1 , preferably solubilised at about neutral pH .5. The process of claim 4 , wherein the solution is treated with Al(OH)and the resulting gel is removed.6. The process of wherein a virus inactivation is performed by employing a solvent/detergent (S/D) treatment.7. The process of wherein an extraction of S/D reagents with vegetable oil and contacting the water-phase with a TMAE resin at a pH-value of 6.9-7.1 and an osmolality of 570-610 mosmol/l is performed.8. The process of claim 6 , wherein fibrinogen is precipitated by glycine claim 6 , in particular about 1 M glycine claim 6 , and separation of the formed fibrinogen paste.9. The process of wherein the fibrinogen paste is resuspended claim 8 , preferably in about 20 mM TRIS buffer at a pH of about 8.0.10. The process of wherein after filtration the obtained fraction is loaded onto a strong anion exchange resin comprising trimethyl-amino groups grafted to a hydroxylated methacrylic polymer backbone via linking groups and washing off loosely ...

METHODS FOR PURIFICATION OF ALPHA-1-ANTITRYPSIN AND APOLIPOPROTEIN A-1

This invention relates to protein separation and purification methods for both alpha-1-antitrypsin (AAT, also known as alpha-1 proteinase inhibitor, API, and A.sub.1-PI) and Apolipoprotein A-I (ApoA-1) from, for example, a fraction of human blood plasma. In certain embodiments, the invention provides methods for separating AAT from ApoA-1 at the initial stage of purification, so that the same starting material can be used as a source for both proteins. The methods further pertain to providing compositions of AAT and of ApoA-1 suitable for pharmaceutical use and are suitable for large-scale purification. 125.-. (canceled)26. A method for purifying Apolipoprotein A-I (ApoA-I) and Alpha-1-Antitrypsin (AAT) from a single starting human plasma fraction containing both proteins comprising: (a) treating the starting human plasma fraction which is used as the starting material such that ApoA-I and AAT are solubilized;', '(b) adding an ApoA-I binding agent and ensuring an appropriate pH so that ApoA-I binds to the added ApoA-I binding agent, wherein the binding agent is fumed silica;', '(c) separating ApoA-I bound to said fumed silica from the solution containing AAT;', '(d) eluting ApoA-I from said fumed silica; and, '(i) treating a starting human plasma fraction comprising ApoA-I and AAT to separate an ApoA-I containing fraction from an AAT containing fraction, comprising'}(ii) separately purifying ApoA-I and AAT in one or more processing steps.27. The method according to claim 26 , wherein the starting human plasma fraction is selected from one or more of Cohn fractions IV claim 26 , precipitates from Kistler-Nitschmann supernatants A and A+I claim 26 , and ammonium sulfate precipitates.28. The method according to claim 27 , wherein the one or more Cohn fractions IV is Cohn fraction IV.29. The method according to claim 26 , wherein AAT and ApoA-I are not exposed to a pH of 13.69 or above.30. The method according to claim 29 , wherein AAT and ApoA-I are not exposed to a pH ...

SINGLE UNIT ION EXCHANGE CHROMATOGRAPHY ANTIBODY PURIFICATION

The present invention relates to a method for the purification of antibodies from a protein mixture produced in a bioreactor, at least comprising the steps of intermediate purification and polishing, wherein the intermediate and polishing step comprises in either order in-line anion exchange chromatography (AEX) chromatography and cation exchange chromatography (CEX) chromatography steps in flow-through mode. The present invention further relates to a single operational unit comprising both an anion exchange chromatography part and a cation exchange chromatography part in either order, which are serially connected, wherein the unit comprises an inlet at the upstream end of the first ion exchange chromatography part and an outlet at the downstream end of the second ion exchange chromatography part and wherein the unit also comprises an inlet between the first ion exchange chromatography part and the second ion exchange chromatography part. 111-. (canceled)12. A method for the purification of antibodies from a cell broth produced in a bioreactor , at least comprising the steps of intermediate purification and polishing , wherein the intermediate purification step comprises combined serially connected in-line anion exchange (AEX) and cation exchange (CEX) chromatography steps in either order , both in flow-through and together operating as one single unit operation , wherein the first chromatography step yields as a flow-through fraction a separation mixture , which is directly subjected to the second chromatography step wherein the second step yields as a flow through fraction a purified antibody preparation , and wherein the purified antibody preparation is subjected to at least one further purification step.13. A method according to claim 12 , wherein the AEX chromatography step is carried out first claim 12 , yielding as a flow-through fraction a separation mixture claim 12 , serial in-line followed by a CEX chromatography step yielding as a flow-through fraction a ...

METHOD FOR EXTRACTING A PROTEIN FROM MILK

The invention relates to a method for extracting a protein from milk, having at least one hydrophobic pocket and a negative charge to the natural pH of milk, that comprises the following steps: a) skimming and delipidation of the milk; b) passing the delipidated and skimmed fraction containing the protein on a chromatographic substrate on which is grafted a ligand having both a hydrophobic characteristic and an ionic characteristic in pH conditions enabling the protein to be retained on the substrate, the pH being higher than 4.6; c) elution of the protein; d) purification of the eluted fraction by removing the milk proteins from the eluted fraction; and e) recovering the protein. 1. A process for the extraction of a protein present in milk , wherein said protein has at least one hydrophobic patch and a negative charge at the natural pH of milk , comprising:a) skimming and defatting said milk,b) transferring said defatted and skimmed fraction containing said protein to a chromatographic support onto which is grafted a ligand that is simultaneously hydrophobic and ionic, under pH conditions allowing said protein to be captured on said support, said pH being higher than 4.6,c) eluting said protein,d) purifying said eluted fraction by removing milk proteins from said eluted fraction, ande) recovering said protein.2. The process according to claim 1 , wherein after said skimming and defatting step (step a) and prior to step b) claim 1 , a milk clarification step takes place.3. The process according to claim 2 , wherein said clarification step takes place by adding a chelating agent at a concentration such that claim 2 , after mixing with said milk claim 2 , the micellar structure of said milk disappears claim 2 , yielding clarified milk (caseins in solution or lactoserum).4. The process according to claim 1 , wherein after said skimming and defatting step (step a) and prior to step b) claim 1 , casein subunit clusters are precipitated.5. The process according to claim 1 ...

TEMPERATURE RESPONSIVE ADSORBENT HAVING A STRONG CATION EXCHANGE GROUP AND METHOD FOR PRODUCING THE SAME

Provided is a temperature responsive adsorbent prepared by immobilizing a copolymer containing at least N-isopropylacrylamide to a base material surface. The copolymer has at least a strong cation exchange group. In addition, the copolymer contains the strong cation exchange group in an amount of 0.01 to 5 mol % relative to N-isopropylacrylamide in terms of monomer. 1. A temperature responsive adsorbent in which a copolymer containing at least N-isopropylacrylamide is immobilized to a base material surface , wherein the copolymer has at least a strong cation exchange group , and contains the strong cation exchange group in an amount of 0.01 to 5 mol % relative to the N-isopropylacrylamide in terms of monomer.2. The temperature responsive adsorbent according to claim 1 , wherein at least a portion of monomer units of the copolymer having the strong cation exchange group is an acrylic acid derivative or a methacrylic acid derivative and has a group represented by the following chemical formula (1) or (2):{'br': None, 'sub': 2', '3, '—CH(—OH)—CH—SOH\u2003\u2003(1)'}{'br': None, 'sub': 3', '2, '—CH(—SOH)—CH—OH\u2003\u2003(2)'}3. The temperature responsive adsorbent according to claim 1 , wherein at least a portion of monomer units of the copolymer having the strong cation exchange group is derived from a vinyl monomer having a sulfonic acid group.4. The temperature responsive adsorbent according to claim 3 , wherein at least a portion of the monomer units of the copolymer having the strong cation exchange group is represented by the following chemical formula (3):{'br': None, 'sub': 1', '2', '3', '3, '—CRR—CR(—SOH)—\u2003\u2003(3)'}{'sub': 1', '2', '3, 'where R, R, Rare each independently H or Me.'}5. A temperature responsive adsorbent in which a copolymer containing at least N-isopropylacrylamide is immobilized to a base material surface claim 3 , wherein the copolymer has at least a strong cation exchange group claim 3 , and wherein the temperature responsive ...

METHODS FOR CAPTURING VIRUS LIKE PARTICLES FROM PLANTS USING EXPANDED BED CHROMATOGRPAHY

The present invention relates to a method for capturing virus-like particles of interest from a mixture comprising the use of an expanded bed of adsorbent; suitably wherein said method comprises the steps of: (a) providing an expanded bed of adsorbent; (b) contacting the mixture with the adsorbent such that the constituents of the mixture contact the expanded bed of adsorbent; (c) optionally washing the adsorbent; and (d) optionally eluting the particle of interest from the adsorbent. 1. A method for capturing virus like particles of interest from a mixture comprising the use of an expanded bed of adsorbent , wherein said method comprises the steps of: (a) providing an expanded bed of adsorbent; (b) contacting the mixture with the adsorbent such that the virus like particles in the mixture bind the adsorbent; (c) optionally washing the adsorbent; and (d) optionally eluting the virus like particles from the adsorbent.2. The method according to claim 1 , wherein the virus like particles comprises a protein of an influenza virus.316-. (canceled)17. The method of claim 1 , wherein the adsorbent comprises beads that comprise claim 1 , consist or consist essentially of a polysaccharide selected from the group consisting of cellulose claim 1 , agarose and dextran and derivatives thereof or a combination of two or more thereof.1820-. (canceled)21. The method according to claim 1 , wherein the virus like particles comprise a haemagglutinin; suitably a haemagglutinin subtype selected from the group consisting of H1 claim 1 , H2 claim 1 , H3 claim 1 , H4 claim 1 , H5 claim 1 , H6 claim 1 , H7 claim 1 , H8 claim 1 , H9 claim 1 , H10 claim 1 , H11 claim 1 , H12 claim 1 , H13 claim 1 , H14 claim 1 , H15 or H16 or a combination of two or more thereof.22. The method claim 1 , wherein the mixture comprises disrupted cells of a plant; suitably wherein the plant is a tobacco plant infiltrated with nucleic acid molecules that express a protein transiently in the plant claim 1 , said ...

PROTEIN PURIFICATION METHODS TO REDUCE ACIDIC SPECIES

The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for controlling the amount of charge variants, aggregates, and fragments of a protein of interest, as well as host cell proteins, present in purified preparations by applying particular chromatography conditions during such protein purification. 2. The method of claim 1 , wherein the chromatography media is selected from the group consisting of anion exchange claim 1 , cation exchange claim 1 , and mixed mode media.3. The method of claim 1 , wherein the chromatography media is a mixed mode adsorbent material comprising cation exchange and hydrophobic interaction functional groups.4. The method of claim 3 , wherein the mixed mode resin is Capto MMC resin.5. The method of claim 2 , wherein the cation exchange (CEX) adsorbent material is selected from the group consisting of a CEX resin and a CEX membrane adsorber.6. The method of claim 5 , wherein the CEX resin is the Poros XS resin.7. The method of claim 1 , wherein the pH of the loading and wash buffers are lower than the isoelectric point of the protein of interest.8. The method of claim 1 , wherein the chromatography sample contains a reduced level of acidic species.9. The method of claim 1 , wherein the chromatography sample contains a reduced level of protein fragments.10. The method of claim 1 , wherein the chromatographic samples contains a reduced level of host cell proteins.11. The method of claim 1 , wherein the chromatographic sample contains levels of basic variants that differ from the starting load material.12. The method of claim 1 , wherein the protein of interest is an anti-TNFα antibody.13. A pharmaceutical composition comprising a process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest and a pharmaceutically acceptable carrier.14. The pharmaceutical composition of claim 14 , wherein said composition is ...

BUFFER SYSTEM FOR PROTEIN PURIFICATION

The invention is directed to a method for producing a polypeptide composition comprising: combining a polypeptide with a volatile additive to form a liquid mixture and lyophilizing the liquid mixture to obtain a lyophilized polypeptide composition. 1. A multi component buffer system for the purification of proteins by a series of chromatography steps , where the modes of chromatography are selected from the group consisting of affinity chromatography , anion exchange chromatography , cation exchange chromatography , and mixed-mode chromatography , wherein the modes of chromatography are operated in either bind-elute mode or flow-through mode , where the multi component buffer system comprises an organic acid , an alkaline metal or ammonium salt of the conjugate base of the organic acid , and an organic base and wherein the modes of chromatography are performed using buffers that are made without the addition of NaCl.2. The system of claim 1 , wherein the affinity chromatography is performed using a superantigen immobilized on a solid phase.3. The system of wherein the superantigen is selected from the group consisting of Protein A claim 2 , Protein G claim 2 , and Protein L.4. The system of claim 1 , wherein the organic acid is selected from the group consisting of formic acid claim 1 , acetic acid claim 1 , acid claim 1 , citric acid claim 1 , malic acid claim 1 , maleic acid claim 1 , glycine claim 1 , glycylclycine claim 1 , succinic acid claim 1 , TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid) claim 1 , MOPS (3-(N-morpholino)propanesulfonic acid) claim 1 , PIPES (piperazine-N claim 1 ,N′-bis(2-ethanesulfonic acid)) claim 1 , and MES (2-(N-morpholino)ethanesulfonic acid).5. The system of claim 1 , wherein the organic base is selected from the group consisting of tris base claim 1 , Bis-Tris claim 1 , Bis-Tris-Propane claim 1 , Bicine (N claim 1 ,N-bis(2-hydroxyethyl)glycine) claim 1 , HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid) claim 1 ...

Method for preparing a depleted plasma material consisting of one or more thrombogenic factors

The invention concerns a method for preparing a plasma product depleted of one or more thrombogenic factors, comprising the combination of at least two steps chosen from among an ethanol fractionation step, a filtration-adsorption step, a precipitation step with caprylic acid and a chromatography step on ion exchange resin.

DRUG SUBSTANCES, PHARMACEUTICAL COMPOSITIONS AND METHODS FOR PREPARING THE SAME

Drug substances, which comprise a solid amorphous forms of a compound of structural formula I and have a BET specific surface area of up to about 94 m/g, pharmaceutical compositions comprising such drug substances, processes for preparing such drug substances and uses of such drug substances and pharmaceutical compositions are disclosed. 2. The drug substance according to claim 1 , wherein said drug substance has a BET specific surface area of from about 2.9 m/g to about 9.6 m/g.3. The drug substance according to claim 1 , wherein said drug substance has a BET specific surface area of from about 2.9 m/g to about 9.4 m/g.4. A pharmaceutical composition comprising at least one drug substance according to and at least one pharmaceutically acceptable carrier.5. The pharmaceutical composition according to claim 4 , further comprising at least one excipient.7. The process according to claim 6 , wherein said distilling step b) is conducted.8. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about −15.0° C. to about 35.0° C.9. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about 15.0° C. to about 30.1° C.10. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about 15.0° C. to about 24.6° C.11. The process according to claim 10 , wherein said distilling step b) is conducted at a temperature in a range from about 15.1° C. to about 24.6° C. for the first 10 hours of the distilling.12. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about −15.0° C. to about 15.0° C.13. The process according to claim 7 , wherein said distilling step b) is conducted over a total of 20 to 30 hours.14. The process according to claim 6 , wherein said filtering step c) is conducted at a temperature in a range from about −15.0° C. to about 15.0° C. ...

METHOD OF PURIFYING PROTEIN

The present invention relates to a method for purifying a protein by separating the protein from impurities in a non-adsorption mode using an activated carbon. In particular, the present invention relates to a method for purifying an antibody using the activated carbon instead of protein A affinity chromatography. 1. A method for purifying a protein , wherein the protein is separated from impurities using an activated carbon to obtain the protein with a low content of impurities.2. The purification method according to claim 1 , wherein the protein has a molecular weight of 30000 or more.3. The purification method according to claim 1 , wherein the protein is a glycoprotein.4. The purification method according to claim 3 , wherein the glycoprotein is an antibody.5. The purification method according to claim 1 , wherein the protein is a genetically modified protein.6. The purification method according to claim 1 , wherein the impurities are any one of host cell proteins claim 1 , protein-derived polymers claim 1 , protein-derived degradation products claim 1 , or DNAs.7. The purification method according to claim 1 , wherein the method is carried out in a non-adsorption mode.8. The purification method according to claim 1 , wherein the separation is carried out at pH 3 to 8.9. The purification method according to claim 1 , wherein the activated carbon is an activated carbon from wood.10. The purification method according to claim 1 , wherein the activated carbon has an average micropore diameter of 0.5 to 5 nm.11. A method for preparing a protein claim 1 , comprising the purification method of .12. The preparation method according to claim 11 , wherein protein A chromatography is not used.13. The preparation method according to claim 11 , comprising any one of anion exchange chromatography claim 11 , cation exchange chromatography claim 11 , hydrophobic interaction chromatography claim 11 , and multimodal chromatography.14. The preparation method according to claim 11 ...

POLYNUCLEOTIDE AND POLYPEPTIDE SEQUENCE AND METHODS THEREOF

The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably . The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins. 1) A polynucleotide sequence as set forth in SEQ ID NO: 2.2) The sequence as claimed in claim 1 , wherein the polynucleotide encodes a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.3) A polypeptide sequence as set forth in SEQ ID NO: 1.4) The sequence as claimed in claim 3 , wherein the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.5) The sequence as claimed in claim 3 , wherein the polypeptide sequence corresponds to polynucleotide sequence set forth in SEQ ID NO: 2 claim 3 , wherein the polynucleotide is subjected to post-transcriptional modification and codon optimization to obtain corresponding polypeptide of SEQ ID NO: 1.6) A method for obtaining recombinant insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1 claim 3 , said method comprising steps of:a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, and inserting the synthesized sequence in a vector,b) transforming a host cell with said vector followed by antibiotic screening host selection, andc) fermenting the selected transformed host cell and in-situ capturing of the insulin precursor molecule to obtain said precursor having polypeptide sequence as set forth in SEQ ID NO: 1.7) The method as claimed in claim 6 , wherein the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.8) ...

Polynucleotide and polypeptide sequence and methods thereof

The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably Pichia pastoris. The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins.

METHODS FOR PURIFYING IgY ANTIBODIES

A method for purifying IgY antibodies is provided. The method comprises precipitating IgY antibodies from a sample by contacting the sample with a precipitating agent; and differentially precipitating the IgY antibodies obtained in step (a) using the same or different precipitating agent to separate IgY(Fc) and IgY(ΔFc) antibodies.

TUMOUR MARKER PROTEINS AND USES THEREOF

Tumour marker proteins and their preparation from fluids from one or more cancer patients, wherein said fluids are those which collect in a body cavity or space which is naturally occurring or which is the result of cancer or medical intervention for cancer. The present application also relates to preparation of tumour marker proteins from excretions taken from patients with cancer. The tumour marker proteins are useful as immunoassay reagents in the detection of cancer-associated anti-tumour marker autoantibodies. 1. A method of detecting cancer-associated anti-tumor autoantibodies in a sample from an individual , comprising:contacting the sample with an immunoassay reagent; and detecting a presence of complexes formed by specific binding of the immunoassay reagent to any cancer-associated anti-tumor autoantibodies present in the sample,wherein the immunoassay reagent comprises two or more tumor marker proteins, one or more of which having been prepared from a tumor-induced bodily fluid produced in a body cavity or space in the presence of the tumor of one or more cancer patients, wherein the bodily fluid contains more cancer-associated forms of the tumor marker protein than a non-tumor-induced bodily fluid in the same patient, and the bodily fluid is not a fluid from the systemic circulation,wherein the one or more tumor marker proteins prepared from a bodily fluid exhibit selective reactivity with cancer-associated anti-tumor autoantibodies, wherein the tumor marker proteins are over-expressed or altered forms of wild-type proteins, andwherein detection of complexes indicates the presence of cancer-associated anti-tumor autoantibodies in the individual.2. The method of claim 1 , further comprising detecting and/or quantitatively measuring the presence of two or more types of autoantibodies claim 1 , wherein each one of the two or more types of the autoantibodies is immunologically specific to a different tumor marker protein or to different epitopes of the same ...

Ion Exchange Chromatography with Improved Selectivity for the Separation of Polypeptide Monomers, Aggregates and Fragments by Modulation of the Mobile Phase

Herein is reported a method for producing a polypeptide in monomeric form comprising the following step: recovering the polypeptide in monomeric form from an ion exchange chromatography material by applying a solution comprising a non-ionic polymer and an additive. 1. A method for producing an antibody of the IgG class in monomeric form comprising the following steps:applying a first solution that optionally comprises poly (ethylene glycol) and sorbitol to a cation exchange chromatographic material and thereby equilibrating the material,applying the solution comprising the antibody of the IgG class to the equilibrated chromatography material and thereby loading the chromatography material,producing the antibody of the IgG class in monomeric form by applying a solution to the chromatographic material comprising poly (ethylene glycol) and sorbitol, and thereby desorbing/eluting the antibody of the IgG class in monomeric form from the chromatographic material,whereby the poly (ethylene glycol) polymer has a concentration of about 10% by weight and the sorbitol has a concentration of from 5% to 20% by weight.2. A method for producing an antibody of the IgG class preparation with reduced host cell protein content comprising the following steps:applying a first solution that optionally comprises poly (ethylene glycol) and sorbitol to a cation exchange chromatographic material and thereby equilibrating the materialapplying the solution comprising the antibody of the IgG class to the equilibrated chromatography material and thereby loading the chromatography material,producing the antibody of the IgG class in monomeric form by applying a solution to the chromatographic material comprising poly (ethylene glycol) and sorbitol and thereby desorbing/eluting the antibody of the IgG class in monomeric form from the chromatographic material,whereby the poly (ethylene glycol) has a concentration of about 10% by weight and the sorbitol has a concentration of from 5% to 20% by weight ...

METHOD OF PURIFYING AN ANTIBODY

A method of purifying an antibody composition comprises application of anion exchange chromatography late in the purification process. An ultrafiltration/diafiltration-purified antibody composition is subjected to anion exchange chromatography (AEX) to form a pharmaceutically-pure antibody composition. 1. A method of purifying an antibody composition , which comprises subjecting a UF/DF-purified antibody composition to anion exchange chromatography (AEX) to form a pharmaceutically pure antibody composition.2. The method of claim 1 , wherein said UF/DF-purified antibody composition has an antibody concentration of at least 1 mg/ml.3. The method of claim 2 , wherein said UF/DF-purified antibody composition has an antibody concentration of at least 10 mg/ml.4. The method of claim 3 , wherein said UF/DF-purified antibody composition has an antibody concentration of not greater than 250 mg/ml.5. The method of claim 4 , wherein said UF/DF-purified antibody composition has an antibody concentration of 20 to 50 mg/ml.6. The method of claim 1 , which further comprises filling said pharmaceutically pure antibody composition into vials.7. The method according to claim 6 , which further comprises aseptically filtering said pharmaceutically pure antibody composition prior to said filling step.8. The method according to claim 7 , which further comprises at least one of the following steps prior to said filling step: (a) adding an excipient to said pharmaceutically-pure antibody composition; (b) concentrating said pharmaceutically-pure antibody composition; (c) diluting said pharmaceutically-pure antibody composition; and/or (d) adjusting the pH of said pharmaceutically-pure antibody composition.9. The method of claim 1 , which further comprises subjecting a partially-purified antibody composition to UF/DF to form said UF/DF-purified antibody composition.10. The method of claim 9 , which further comprises subjecting an antibody cell culture harvest to an antibody capture step and ...

MATERIALS AND METHODS FOR REMOVING ENDOTOXINS FROM PROTEIN PREPARATIONS

A method includes (i) adding allantoin in a supersaturating amount to a protein preparation including a desired protein and at least one endotoxin as a contaminant, (ii) removing solids after the adding step to provide a sample for further purification by void exclusion chromatography on a packed particle bed of electropositive particles in a column, the packed particle bed having an interparticle volume, (iii) applying a sample volume to the packed particle bed, wherein the electropositive particles support void exclusion chromatography, and wherein the sample volume is not greater than the interparticle volume, and (iv) eluting a purified sample including the desired protein and a reduced amount of the endotoxin. The method is optionally carried out with only the allantoin treatment or only the void exclusion chromatography. 116.-. (canceled)17. A method comprising: (i) adding allantoin in a supersaturating amount to a protein preparation comprising a desired protein and an amount of at least one endotoxin as a contaminant; (ii) removing solids from the protein preparation to provide a sample for further purification by void exclusion chromatography using a packed particle bed of electropositive particles in a column , the packed particle bed having an interparticle volume; (iii) applying a sample volume of the sample to the packed particle bed , wherein the electropositive particles support void exclusion chromatography , and wherein the sample volume is not greater than the interparticle volume , and (iv) eluting a purified sample comprising the desired protein and a reduced amount of the at least one endotoxin , wherein the eluted desired protein resides in a buffer to which the column was equilibrated , independently from a buffer content of the sample applied to the column.18. The method of claim 17 , wherein the supersaturating amount of allantoin comprises an amount selected from the group consisting of: (i) about 10% claim 17 , (ii) about 5% claim 17 , (ii ...

IMPROVED PROCESS FOR THE PREPARATION OF A DODECAPEPTIDE

The invention concerns an improved process for the preparation of the dodecapeptide NX210 of SEQ ID NO: 1 in which the formation of side products is minimized. 115-. (canceled)16. A process for preparing a peptide of SEQ ID NO: 1 , wherein the peptide of SEQ ID NO: 1 is constantly maintained in an aqueous solution comprising an organic solvent throughout the steps of purification and drying , of the peptide of SEQ ID NO: 1.17. A process for preparing a peptide of SEQ ID NO: 1 , wherein the steps of purification and/or drying of the peptide of SEQ ID NO: 1 are carried out in an aqueous solution comprising an organic solvent.18. The process according to claim 16 , wherein the organic solvent is acetonitrile.19. The process according to claim 17 , wherein the organic solvent is acetonitrile.20. The process according to claim 16 , wherein the volume of the organic solvent in the aqueous solution is maintained and/or adjusted in such a way that it represents in proportion from 1 to 90% of the total volume of the aqueous solution during the steps of purification.21. The process according to claim 20 , wherein said proportion is from 1 to 10%.22. The process according to claim 16 , wherein the volume of the organic solvent in the aqueous solution to be dried is maintained and/or adjusted in such a way that it represents in proportion from 1 to 30% of the total volume of the aqueous solution.23. The process according to claim 16 , comprising the following steps:a) grafting of a Fmoc-Gly residue to a resin,b) elongation of the peptide chain with Fmoc-protected amino-acids in which the side chain is protected,c) simultaneous deprotection of the side chains of the amino-acids and cleavage of the polypeptide from the resin, to obtain the polypeptide of SEQ ID NO: 1,d) purification and optional concentration of the peptide of SEQ ID NO: 1,e) drying of the peptide of the peptide of SEQ ID NO: 1 obtained in step e).24. The process according to claim 23 , further comprising a ...

METHOD FOR REMOVING N-TERMINAL TRUNCATED AND ABNORMAL VARIANTS IN RHNGF

A method for removing an N-terminal truncated variant and an abnormal variant in recombinant human nerve growth factor (rhNGF) is provided. An rhNGF raw material loaded on a cation-exchange material is washed with a washing liquid to obtain a washed raw material from which an N-terminal truncated variant and an abnormal variant have been removed, where the washing liquid has higher electrical conductivity than the rhNGF raw material. Cation-exchange chromatography (CEC) elution is then performed on the washed raw material with an elution buffer having higher electrical conductivity than the washing liquid. A purified rhNGF product is obtained from the eluate. 1. A method for removing an N-terminal truncated variant and an abnormal variant in recombinant human nerve growth factor (rhNGF) , comprising:1) washing with a washing liquid an rhNGF raw material loaded on a cation-exchange material, thereby obtaining a washed raw material from which an N-terminal truncated variant and an abnormal variant have been removed, wherein said washing liquid is a washing buffer having higher electrical conductivity than the rhNGF raw material; and2) performing cation-exchange chromatography (CEC) elution on the washed raw material of step 1) with an elution buffer having higher electrical conductivity than the washing liquid in step 1), and collecting an eluate from which a purified rhNGF product is obtained.2. The method of claim 1 , wherein the electrical conductivity of said washing liquid in step 1) is 20˜30 mS/cm.3. The method of claim 1 , wherein said washing liquid in step 1) is an NaCl-containing buffer with an NaCl content of 200˜300 mM.4. The method of claim 1 , wherein step 1) comprises: loading the cation-exchange material with said rhNGF raw material claim 1 , washing with the washing liquid claim 1 , and discarding a resulting outflowing liquid.5. The method of claim 1 , wherein said rhNGF raw material in step 1) is a preliminarily purified product obtained by ...

METHODS FOR EXTRACTING PROTEINS FROM BLOOD PLASMA

A method of producing protein products including alpha-1-proteinase inhibitor, gamma globulin, albumin, and other proteins from plasma includes steps of: (1) adding a salt to the blood product to produce a first intermediate, wherein the salt comprises between 11-13 wt % of the first intermediate; (2) separating the first intermediate to produce a first supernatant and a first paste; (3) adding a salt to the first intermediate to produce a second intermediate, wherein the salt comprises between 21-23 wt % of the second intermediate; (4) separating the second intermediate to produce a second supernatant and a second paste; (5) separating a third intermediate from the second supernatant by affinity chromatography; and (6) separating the third intermediate by ion exchange chromatography to produce an eluate containing the protein product. Advantageously, the inventive methods are simple and produce alpha-1-proteinase inhibitor, gamma globulin, albumin, and other proteins in high yields. 1. A method of separating A1P1 from a blood plasma containing product , the method comprising:thawing a frozen blood plasma product followed by stirring at a temperature suitable for dissolving a cryoprecipitate to generate the blood plasma containing product;adding a salt to the blood plasma containing product to produce a first intermediate, wherein the salt comprises between 11-20 wt % of the first intermediate;separating the first intermediate to produce a first supernatant and a first paste;adding a salt to the first supernatant to produce a second intermediate, wherein the salt comprises between 15-30 wt % of the second intermediate;separating the second intermediate to produce a second supernatant and a second paste;separating the second supernatant by affinity chromatography using an A1P1-specific affinity media to generate a flow-through fraction and a first eluate, wherein the first eluate comprises A1P1.2. The method of claim 1 , wherein the step of adding the salt to the ...

CHROMATOGRAPHIC PURIFICATION OF ANTIBODIES FROM CHROMATIN-DEFICIENT CELL CULTURE HARVESTS

Methods for the improved purification of antibodies and other proteins from protein preparations including the steps of conditioning the protein preparation by contacting it with multivalent organic ions, then applying the conditioned preparation to an adsorptive chromatography medium. 126.-. (canceled)27. A method for the purification of at least one desired protein from a protein preparation comprising the steps of (a) conditioning an impure protein preparation by contacting it with at least one species of soluble or insoluble multivalent organic ion , subsequently removing solids with the result of removing at least 95% of chromatin , thereby providing a conditioned protein preparation; then (b) applying the conditioned protein preparation , optionally after a buffer equilibration step , to an adsorptive chromatography medium for purification of the desired protein.28. The method of claim 27 , step (a) claim 27 , wherein the conditioning of the protein preparation with at least one species of organic multivalent ion comprises contacting the protein preparation with an electropositive organic additive.29. The method of claim 28 , wherein the electropositive organic additive comprises a soluble electropositive organic additive.30. The method of claim 28 , wherein the electropositive organic additive comprises an electropositive organic cation selected from the group consisting of methylene blue claim 28 , ethacridine claim 28 , chlorhexidine claim 28 , benzalkonium chloride claim 28 , and cetyl trimethyl ammonium bromide.31. The method of claim 28 , wherein the electropositive organic additive is present at a concentration of 0.001% to 2% claim 28 , 0.005% to 1% claim 28 , 0.01% to 0.05% or 0.020% to 0.025%.32. The method of claim 27 , step (a) claim 27 , wherein the conditioning of the protein preparation with at least one species of organic multivalent ions comprises contacting the protein preparation with an insoluble electropositive organic additive in the form ...

METHOD OF ISOLATING BOTULINUM TOXIN FROM BOTULINUM TOXIN-CONTAINING SOLUTION

Provided is a method of isolating a toxin type A macro complex from a toxin-containing solution, the method including performing anion exchange chromatography and cation exchange chromatography. 1botulinumbotulinum. A method of isolating a toxin type A macro complex from a toxin-containing solution , the method comprising:{'i': botulinum', 'botulinum, 'contacting the toxin-containing solution with an anion exchange chromatography medium at a pH lower than an isoelectric point (PI) of toxin;'}{'i': 'botulinum', 'contacting the solution, which is not bound to the anion exchange chromatography medium, with a cation exchange chromatography medium at a pH lower than the PI of toxin; and'}{'i': 'botulinum', 'separating the toxin type A macro complex from the cation exchange chromatography medium; or'}{'i': botulinum', 'botulinum, 'contacting the toxin-containing solution with the cation exchange chromatography medium at a pH lower than the PI of toxin;'}{'i': 'botulinum', 'separating toxin from the cation exchange chromatography medium;'}contacting the solution containing the toxin separated from the cation exchange chromatography medium, with the anion exchange chromatography medium at a pH lower than the PI; and{'i': 'botulinum', 'separating the toxin type A macro complex from the solution which is not bound to the anion exchange chromatography medium.'}2botulinum. The method of claim 1 , wherein the pH lower than the PI of toxin is pH 3.5 to 6.0.3botulinumbotulinumbotulinum. The method of claim 1 , further comprising adjusting the pH of the toxin or the toxin-containing solution to a pH lower than the PI of toxin claim 1 , before the contacting of the solution with the anion exchange chromatography medium claim 1 , before the contacting of the solution with the cation exchange chromatography medium claim 1 , or before each of these two processes.4botulinumbotulinumbotulinum. The method of claim 3 , wherein the adjusting comprises mixing the toxin or the toxin- ...

MULTISTEP FINAL FILTRATION

Herein is reported a method for the final filtration of concentrated polypeptide solutions comprising the combination of two immediately consecutive filtration steps with a first filter of 3.0 μm and 0.8 μm pore size and a second filter of 0.45 μm and 0.22 μm pore size. 1. A method for producing an immunoglobulin solution comprisinga) providing an immunoglobulin solution with a concentration of at least 100 g/l, andb) applying the immunoglobulin solution to a combination of a first and second filter unit, whereby the first filter unit comprises a pre-filter with a pore size of 3.0 μm and a main-filter with a pore size of 0.8 μm and the second filter unit comprises a pre-filter with a pore size of 0.45 μm and a main-filter with a pore size of 0.22 μm with a pressure of from 0.1 to 4.0 bar, and thereby producing an immunoglobulin solution.2. A method for producing an immunoglobulin comprising the following stepsa) cultivating a cell comprising a nucleic acid encoding an immunoglobulin,b) recovering the immunoglobulin from the cell or the cultivation medium,c) purifying the immunoglobulin with one or more chromatography steps, and providing an immunoglobulin solution,d) optionally adding a sugar, an amino acid and/or a detergent to the solution,e) optionally concentrating the immunoglobulin solution to a concentration of 100 g/l or more with a method selected from diafiltration or tangential-flow filtration, andf) applying the immunoglobulin solution of the previous step to a combination of a first and second filter unit, whereby the first filter unit comprises a pre-filter with a pore size of 3.0 μm and a main-filter with a pore size of 0.8 μm and the second filter unit comprises a pre-filter with a pore size of 0.45 μm and a main-filter with a pore size of 0.22 μm with a pressure of from 0.1 to 4.0 bar, and thereby producing an immunoglobulin.3. The method of or wherein the filter in the first and second filter unit have about the same filter area.4. The method of or ...

METHODS OF PURIFYING MONOMERIC MONOCLONAL ANTIBODIES

In certain embodiments, the present invention provides a method of purifying a monomeric monoclonal antibody from a mixture which comprises the monomeric monoclonal antibody and one or more contaminants, comprising: a) subjecting the mixture to cation exchange chromatography (CEX) matrix, wherein the monomeric monoclonal antibody binds to the CEX matrix; b) contacting the CEX matrix with a wash solution at a pH which is between about 7 and about 7.8; c) eluting the monomeric monoclonal antibody from the CEX matrix into an elution solution, thereby purifying the monomeric monoclonal antibody. 1. A method of purifying a monomeric monoclonal antibody from a mixture which comprises the monomeric monoclonal antibody and one or more contaminants , comprising:a) subjecting the mixture to cation exchange chromatography (CEX) matrix, wherein the monomeric monoclonal antibody binds to the CEX matrix;b) contacting the CEX matrix with a wash solution at a pH which is between about 7 and about 7.8; andc) eluting the monomeric monoclonal antibody from the CEX matrix into an elution solution, thereby purifying the monomeric monoclonal antibody.2. The method of claim 1 , wherein the contaminants are selected from aggregates of the monoclonal antibody claim 1 , host cell proteins claim 1 , host cell metabolites claim 1 , host cell constitutive proteins claim 1 , nucleic acids claim 1 , endotoxins claim 1 , viruses claim 1 , product related contaminants claim 1 , lipids claim 1 , media additives and media derivatives.3. The method of claim 1 , wherein aggregates of the monoclonal antibody comprise dimers claim 1 , multimers claim 1 , and an intermediate aggregate species.4. The method of claim 1 , wherein the mixture has been obtained by an affinity chromatography.5. The method of claim 1 , wherein the elution solution is not subjected to a second chromatography step.6. The method of claim 1 , wherein the elution solution is further subjected to a second chromatography step.7. The ...

METHOD FOR PURIFYING COMPOSITION COMPRISING ANTIBODIES WITH ANIONIC POLYMER

A composition containing an antibody is prepared in such a state that the composition contains an anionic polymer at pH lower than the pI of the antibody, and impurities insolubilized by the anionic polymer are removed. More preferably, the composition is prepared in such a state that the composition contains an anionic polymer at pH lower than or equal to the pI of the antibody minus one, and impurities insolubilized by the anionic polymer are removed. 1. A method for purifying a composition comprising an antibody , which comprises the steps of:(a) preparing a composition comprising an antibody in such a state that the composition comprises an anionic polymer at pH lower than the pI of the antibody; and(b) removing an impurity insolubilized by the anionic polymer from the composition.2. The method of claim 1 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH lower than or equal to the pI of the antibody minus one.3. The method of claim 1 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH of 3.5 to less than the pI of the antibody.4. The method of claim 1 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH of 3.5 to the pI of the antibody minus one or lower.5. A method for purifying a composition comprising an antibody claim 1 , which comprises the steps of:(a) preparing a composition comprising an antibody in such a state that the composition comprises an anionic polymer at pH of 3.5 to 5.0; and(b) removing an impurity insolubilized by the anionic polymer from the composition.6. The method of claim 5 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH of 3.8 to 5.0.7. The method of claim 1 , wherein the pI of the antibody is 3.0 to 8.0.8. The method of claim 1 , wherein the pI of the antibody is ...

PURIFYING INSULIN USING CATION EXCHANGE AND REVERSE PHASE CHROMATOGRAPHY IN THE PRESENCE OF AN ORGANIC MODIFIER AND ELEVATED TEMPERATURE

A process is described for purifying insulin and insulin analogs that comprises high-pressure liquid chromatography with an acidic cation exchange medium performed in the presence of a water miscible organic modifier and at an elevated temperature followed by reverse phase chromatography performed in the presence of a water miscible organic modifier and at an elevated temperature. 1. A process for isolating properly folded insulin or insulin analog from an aqueous mixture comprising the insulin analog and related impurities , wherein the process comprises:(a) performing an acid-stable cation exchange chromatography with the aqueous mixture in the presence of a first water miscible organic modifier and at an elevated temperature to yield a first insulin or insulin analog mixture; and(b) performing a reverse phase high performance liquid chromatography on the first insulin or insulin analog mixture in the presence of a second water miscible organic modifier and at an elevated temperature to provide a second mixture comprising the isolated properly folded insulin or insulin analog.2. The process of claim 1 , wherein the insulin analog is an acid-stable insulin analog or pI-shifted insulin analog.3. The process of claim 1 , wherein the acid-stable insulin analog has a pI between 5.8 and 8.0.4. The process of claim 1 , wherein the acid-stable insulin analog is insulin glargine.5. The process of claim 1 , wherein the first water miscible organic modifier is hexylene glycol.6. The process of claim 1 , wherein the first water miscible organic modifier is at a concentration between 20 to 50 percent by volume.7. The process of claim 1 , wherein the second water miscible organic modifier is isopropanol.810-. (canceled)11. The process of claim 1 , wherein the acidic cation exchange chromatography is performed with a differential pressure of less than 1.0 MPa claim 1 , or less than 0.5 MPa claim 1 , or about 0.276 MPa.12. The process of claim 1 , wherein the acid-stable cation ...

LIQUID FORMULATION OF LONG ACTING INSULINOTROPIC PEPTIDE CONJUGATE

Provided are a liquid formulation of long-acting insulinotropic peptide conjugate, containing a pharmaceutically effective amount of long-acting insulinotropic peptide conjugate consisting of a physiologically active peptide, insulinotropic peptide, and an immunoglobulin Fc region; and an albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, and a non-ionic surfactant, and a method for preparing the formulation. For the purpose of preventing microbial contamination, a preservative may be added. The liquid formulation of the present invention is free of human serum albumin and other potentially hazardous factors to body, having no risk of viral contamination, and thus can provide excellent storage stability for insulinotropic peptide conjugates at high concentration. 1. A liquid formulation of a long-acting insulinotropic peptide conjugate , comprising:a pharmaceutically effective amount of the long-acting insulinotropic peptide conjugate wherein an insulinotropic peptide is linked to an immunoglobulin Fc region; andan albumin-free stabilizer, wherein the stabilizer comprises a buffer, a sugar alcohol, and a non-ionic surfactant,wherein the insulinotropic peptide is glucagon-like peptide-1, glucagon-like peptide-2, exendin-3, exendin-4, or imidazo-acetyl exendin-4;wherein the formulation has a pH of about 5.2 to about 7.0; andwherein the non-ionic surfactant has a concentration of about 0.001% (w/v) to about 0.05% (w/v).2. The liquid formulation according to claim 1 , wherein the immunoglobulin Fc region is an Fc region derived from IgG claim 1 , IgA claim 1 , IgD claim 1 , IgE claim 1 , or IgM.3. The liquid formulation according to claim 2 , wherein the immunoglobulin Fc region is a hybrid of domains of different origins derived from immunoglobulins selected from the group consisting of IgG claim 2 , IgA claim 2 , IgD claim 2 , IgE claim 2 , and IgM.4. The liquid formulation according to claim 2 , wherein the immunoglobulin Fc region ...

OPTIMIZED METHOD FOR ANTIBODY CAPTURING BY MIXED MODE CHROMATOGRAPHY

Herein is reported a method for the purification of an antibody directly captured from clarified cell culture supernatants using Streamline CST and/or Capto MMC, wherein especially product related (aggregates and fragments) and process related impurities (host cell protein, media components) could efficiently be removed, resulting in a preparation with a purity comparable to classical protein A affinity chromatography. 1. A method for producing an anti-IGF-1R antibody comprising the following steps:a) applying a crude mammalian cell culture cultivation supernatant to a multimodal weak cation exchange chromatography material,b) recovering the anti-IGF-1R antibody by applying a buffered solution comprising ethylene glycol and an inorganic salt to the multimodal weak cation exchange chromatography material and thereby producing an anti-IGF-1R antibody.2. The method according to claim 1 , characterized in that the method comprises the following additional step a-1) prior to step a):a-1) applying a buffered solution comprising an inorganic salt to the multimodal weak cation exchange chromatography material.3. The method according to any one of the preceding claims claim 1 , characterized in that the method comprises the following additional step a-b) after step a) and prior to step b):a-b) applying a buffered solution to the multimodal weak cation exchange chromatography material, whereby the anti-IGF-1R antibody is not recovered from the multimodal weak cation exchange chromatography material.4. The method according to claim 3 , characterized in that the step a-b) comprises two steps a-b1) and a-b2):a-b1) applying a buffered solution comprising an inorganic salt to the multimodal weak cation exchange chromatography material, anda-b2) applying a buffered solution comprising a denaturant to the multimodal weak cation exchange chromatography material,whereby the anti-IGF-1R antibody is not recovered from the multimodal weak cation exchange chromatography material.5. The ...

PURIFYING INSULIN USING CATION EXCHANGE AND REVERSE PHASE CHROMATOGRAPHY IN THE PRESENCE OF AN ORGANIC MODIFIER AND ELEVATED TEMPERATURE

A process is described for purifying insulin and insulin analogs that comprises high-pressure liquid chromatography with an acidic cation exchange medium performed in the presence of a water miscible organic modifier and at an elevated temperature followed by reverse phase chromatography performed in the presence of a water miscible organic modifier and at an elevated temperature. 1. A process for isolating properly folded insulin or insulin analog from an aqueous mixture comprising the insulin analog and related impurities , wherein the process comprises:(a) performing an acid-stable cation exchange chromatography with the aqueous mixture in the presence of a first water miscible organic modifier and at an elevated temperature to yield a first insulin or insulin analog mixture; and(b) performing a reverse phase high performance liquid chromatography on the first insulin or insulin analog mixture in the presence of a second water miscible organic modifier and at an elevated temperature to provide a second mixture comprising the isolated properly folded insulin or insulin analog.2. The process of claim 1 , wherein the insulin analog is an acid-stable insulin analog or pI-shifted insulin analog.3. The process of claim 1 , wherein the acid-stable insulin analog has a pI between 5.8 and 8.0.4. The process of claim 1 , wherein the acid-stable insulin analog is insulin glargine.5. The process of claim 1 , wherein the first water miscible organic modifier is hexylene glycol.6. The process of claim 1 , wherein the first water miscible organic modifier is at a concentration between 20 to 50 percent by volume.7. The process of claim 1 , wherein the second water miscible organic modifier is isopropanol.810-. (canceled)11. The process of claim 1 , wherein the acidic cation exchange chromatography is performed with a differential pressure of less than 1.0 MPa claim 1 , or less than 0.5 MPa claim 1 , or about 0.276 MPa.12. The process of claim 1 , wherein the acid-stable cation ...

METHOD FOR PURIFYING IMMUNOGLOBULIN

The present invention relates to a method for purifying an immunoglobulin, and more particularly, to a method for purifying an immunoglobulin, which comprises: dissolving immunoglobulin-containing plasma protein fraction I+II+III or fraction II+III; adding caprylate to the solution to cause precipitation; performing dialysis and concentration after removal of the precipitate; performing anion exchange resin and ceramic cation exchange resin purification processes to effectively remove a solvent and detergent added to inactivate viruses; and performing elution while maintaining salt concentration at a constant level to maintain the immunoglobulin polymer content at a low level. According to the method for preparing the intravenous immunoglobulin according to the present invention, a precipitation step of preparing fraction II from fraction I+II+III or fraction II+III as a starting material can be omitted, and problems, including a complicated process and a low yield, which occur in the conventional preparation process employing the polyethylene glycol treatment process, can be solved by use of first sodium caprylate precipitation, anion exchange chromatography and cation exchange chromatography. In addition, when the immunoglobulin purification method according to the present invention is used, the efficiency with which impurities and thrombotic substances are removed can be increased and the immunoglobulin polymer content can be maintained, and thus a stable immunoglobulin with increased quality can be produced. 1. A method for purifying an immunoglobulin , comprising the steps of:(a) dissolving immunoglobulin-containing plasma protein fraction I+II+III or fraction II+III, followed by performing a precipitation reaction by adding a precipitant;(b) removing a precipitate produced from (a), followed by filtering a supernatant comprising immunoglobulin, concentrating a filtrate, subjecting a concentrate to an anion exchange chromatography, and recovering a fraction not ...

METHODS FOR SEPARATING ISOFORMS OF MONOCLONAL ANTIBODIES

Charge variants of a recombinantly expressed antibody population may be separated both from the main antibody molecule and from each other. Separation and isolation of charge variants may proceed via a combined modulation of salt concentration and pH during charge variant elution from a cation exchange support. Isolated charge variants may be assessed for their contribution to the potency of the overall antibody preparation. The make-up of an antibody preparation, at least in terms of the proportion of charge variants and of the main antibody can thus be controlled, for example, for biosimilar matching or for improving potency of the preparation. 1. A method for separating isoforms of recombinantly expressed trastuzumab , comprisingloading a recombinantly expressed trastuzumab preparation comprising trastuzumab and a plurality of charge variants of trastuzumab onto a cation exchange chromatography support comprising a ligand capable of capturing the trastuzumab and the charge variants, andfractionating the charge variants comprising passing a first mobile phase buffer comprising from about 20 mM to about 30 mM MES and having a pH of about 6.1 through the support, while the first mobile phase buffer is being passed through the support, adding a second mobile phase buffer comprising from about 40 mM sodium phosphate to about 60 mM sodium phosphate and about 95 mM sodium chloride and having a pH of about 8.0 to the first mobile phase buffer to achieve a mixture of about 90% by volume of the first mobile phase buffer and about 10% by volume of the second mobile phase buffer, gradient eluting one or more of the charge variants from the ligand by gradually increasing the amount of the second mobile phase buffer in the mixture to achieve about 55% by volume of the first mobile phase buffer and about 45% by volume of the second mobile phase buffer, and collecting the one or more charge variants into separate fractions.2. The method according to claim 1 , wherein the first ...

PROCESS FOR PRODUCTION OF FIBRINOGEN AND FIBRINOGEN PRODUCED THEREBY

A process for purifying fibrinogen from a fibrinogen containing source by precipitation of fibrinogen by a precipitating agent from a fibrinogen containing solution in the presence of one or more chelating agent(s) and removal of the supernatant from the fibrinogen paste, characterised in that fibrinogen is extracted from the paste forming a liquid fraction containing fibrinogen, and an undissolved residue, which is separated from the liquid. 1. A fibrinogen product obtained by a process comprising a precipitation of fibrinogen by a precipitating agent from a fibrinogen containing solution in the presence of one or more chelating agent(s) and removal of the supernatant from the fibrinogen paste , characterised in that fibrinogen is extracted from the paste forming a liquid fraction containing fibrinogen , and an undissolved residue , which is separated from the liquid , while addition of one or more protease inhibitor(s) is omitted ,wherein the fibrinogen product has less than 11% of compounds of higher molecular weight than fibrinogen, determined as % of total area by size exclusion chromatography at 280 nm.2. The fibrinogen product according to claim 1 , wherein the process comprises precipitating the fibrinogen with the precipitating agent from the fibrinogen containing source in the presence of the one or more chelating agent(s) to form the fibrinogen paste claim 1 , removing the supernatant from the fibrinogen paste claim 1 , extracting the fibrinogen from the fibrinogen paste in an aqueous medium void of the chelating agent(s) for a suitable extraction time thereby forming the liquid fraction containing fibrinogen and the undissolved residue claim 1 , and separating the undissolved residue from the liquid fraction containing fibrinogen claim 1 , whereinaddition of one or more protease inhibitor(s) is omitted in all steps of the process,wherein the fibrinogen is precipitated in a temperature range of from 4.1° C. to 40° C., andthe one or more protease inhibitor ...

Methods and Reagents for Purification of Proteins

The present disclosure provides compositions and methods for purifying a protein such as DNase using a chromatographic process. The methods include a single chromatographic step and the use of high concentration salt buffers. The present disclosure provides methods for purification of a protein from a sample comprising loading the sample onto a chromatography column and washing the column with at least one buffer having very high conductivity. In some embodiments, the wash buffer has a conductivity of about 50 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, or higher. In further embodiments, the protein is eluted from the column with an elution buffer following the wash with the very high conductivity buffer. 1. A method for purification of a protein from a sample comprising: loading the sample onto a chromatography column ,washing the column with a wash buffer, wherein the wash buffer has a conductivity of 80 mS/cm or higher, andeluting the protein from the column with an elution buffer.2. The method of claim 1 , wherein the wash buffer comprises a salt selected from the group consisting of (NH)SO claim 1 , NaSO claim 1 , and CHCOONa.3. The method of claim 2 , wherein the wash buffer comprises a concentration of about 0.8 M (NH)SO.4. The method of claim 1 , wherein the elution buffer has a conductivity of 80 mS/cm or higher.5. The method of claim 1 , wherein the elution buffer comprises a salt selected from the group consisting of NaCl claim 1 , KCl claim 1 , and NaOAc.6. The method of claim 5 , wherein the elution buffer comprises a concentration of about 1 M NaCl.7. The method of claim 1 , wherein the method comprises at least two washing steps.8. The method of claim 7 , wherein a first washing step comprises washing the column with a buffer having a conductivity of 2 mS/cm or less.9. The method of claim 8 , further comprising a third washing step claim 8 , wherein the third washing step comprises washing the column with a buffer having a conductivity of 2 ...

Separation Method

The invention relates to a method of separating immunoglobulin variants, comprising the steps of: a) providing a column packed with an Fc-binding affinity chromatography resin; b) loading a sample comprising at least two Fc-comprising immunoglobulin variants onto the column; c) optionally washing the column with a washing liquid; and d) conveying an eluent through said column to elute at least a target immunoglobulin variant from said column and recovering one or more eluate fractions comprising the target immunoglobulin variant in enriched form. 1. A method of separating immunoglobulin variants , comprising the steps of:a) providing a column packed with an Fc-binding affinity chromatography resin;b) loading a sample comprising at least two Fc-comprising immunoglobulin variants onto said column;c) optionally washing said column with a washing liquid; andd) conveying an eluent through said column to elute at least a target immunoglobulin variant from said column and recovering one or more eluate fractions comprising said target immunoglobulin variant in enriched form.2. The method of claim 1 , wherein in step d) claim 1 , said one or more eluate fractions comprise said target immunoglobulin variant in enriched form.3. The method of claim 1 , wherein in step d) claim 1 , the ratio of target Fc-comprising immunoglobulin variant concentration to total Fc-comprising immunoglobulin variant concentration is at least 50% higher than in said sample.4. The method of claim 1 , wherein one of said Fc-comprising immunoglobulin variants is an intact immunoglobulin claim 1 , while another one of said Fc-comprising immunoglobulin variants is an Fc-comprising fragment of an immunoglobulin.5. The method of claim 1 , wherein one of said Fc-comprising immunoglobulin variants is an intact immunoglobulin with an Fc region and two Fab regions claim 1 , while another one of said Fc-comprising immunoglobulin variants is a half-antibody with an Fc region and only one Fab region.6. The method ...

Antibody Purification

Methods for the purification of antibodies are provided. Purification methods provided involve the use of hydroxyapatite resin (HA) to separate an antibody of interest from one or more impurities. The impurity may be a clipped antibody that comprises a cleaved peptide bind in the VH domain. 1. A method of purifying an antibody comprising: 'the antibody preparation comprises: I) an intact antibody of interest and II) a clipped version of the antibody of interest, wherein the clipped version of the antibody of interest is a degradation production from the intact antibody of interest, and has a mass that is less than 10% different than the mass of the intact antibody of interest; and', 'A) loading an antibody preparation in a load buffer onto a hydroxyapatite (HA) resin, whereinB) eluting the intact antibody of interest from the HA resin with an elution buffer comprising an ion, wherein the concentration of the ion in the elution buffer is increased during the elution.2. A method of purifying a bispecific antibody comprising: 'the antibody preparation comprises: I) an intact bispecific antibody of interest; and II) at least one impurity species, wherein the impurity species are selected from the group consisting of: a) a clipped version of the bispecific antibody of interest, wherein the clipped version of the bispecific antibody of interest is a degradation production from the intact bispecific antibody of interest, and has a mass that is less than 10% different than the mass of the intact bispecific antibody of interest; b) a first parent antibody, wherein the first parent antibody is a monospecific antibody having the same antigen specificity as a first arm of the intact bispecific antibody; c) a second parent antibody, wherein the second parent antibody is a monospecific antibody having the same antigen specificity as a second arm of the intact bispecific antibody; and d) high molecular mass species (HMMS); and', 'A) loading an antibody preparation in a load buffer ...

ANTIBODY FOR DETECTING EARLY DAMAGE OF BLOOD-BRAIN BARRIER IN ISCHEMIC STROKE AND APPLICATION THEREOF

A specific antibody for detecting the blood brain barrier early injury of cerebral ischemic stroke is characterized by specifically identifying KTRRKMDRYDKSNIL in degradation fragments of an occludin protein, but not identifying the full-length occludin protein. Therefore, the antibody can be used for specifically detecting the blood brain barrier early injury of the cerebral ischemic stroke, and can eliminate the influence of the full-length occludin protein in serum on a detection result, so that the specificity and accuracy of detecting the blood brain barrier early injury of the cerebral ischemic stroke are obviously improved. 1. An antibody for specifically detecting blood brain barrier early injury of cerebral ischemic stroke , wherein the antibody only specifically identifies KTRRKMDRYDKSNIL in degradation fragments of an occludin protein , but does not identify the occludin protein at full length.2. The antibody according to claim 1 , wherein the antibody is prepared with a method comprising:(1) synthesizing a polypeptide KTRRKMDRYDKSNIL;(2) synthesizing an antigen: coupling the polypeptide synthesized in the step (1) with KLH to obtain KLH-polypeptide as an immune antigen;(3) immunizing animals with the immune antigen to obtain antiserum; and(4) performing antibody purification on the antiserum with an antigen affinity column to obtain a specific antibody.3. The antibody according to claim 2 , wherein the animals are New Zealand white rabbits.4. The antibody according to claim 3 , wherein the animals are immunized with the immune antigen claim 3 , and subjected to booster immunization once every 14 days claim 3 , for a total of 4 times claim 3 , and then carotid artery blood of the animals is taken to obtain the antiserum.5. The antibody according to claim 2 , wherein the animals are immunized with the immune antigen claim 2 , and subjected to booster immunization once every 14 days claim 2 , for a total of 4 times claim 2 , and then carotid artery blood of ...

REMOVAL OF LEAKED AFFINITY PURIFICATION LIGAND

The invention provides for the removal of a large fraction of contaminants from protein preparations while maintaining a high level of recovery using tentacle anion exchange matrix chromatography medium. Using the methods of the invention, leached affinity chromatography contaminants can be removed from recombinant protein preparations. 1. A method for purifying a recombinant protein from a sample containing the recombinant protein and a second protein that binds to the protein , comprising subjecting the sample to a tentacle anion exchange matrix chromatography medium under conditions whereby the recombinant protein binds to the tentacle anion exchange matrix chromatography medium , followed by eluting the recombinant protein bound to the chromatography medium in an eluant , whereby at least 85% of the recombinant protein is recovered in the eluant and at least 75% of the second protein is removed from the eluant.2. The method of claim 1 , wherein the tentacle anion exchange matrix chromatography medium contains a strong anion functional group.3. The method of claim 2 , wherein the strong anion functional group is trimethyl-ammoniumethyl (TMAE).4. The method of any one of to claim 2 , wherein the resin substrate of the tentacle anion exchange matrix chromatography medium is a methacrylate polymeric resin or a polyvinylstyrene polymeric resin.5. The method of any one of to claim 2 , wherein the chromatography medium is Fractogel® EMD TMAE HiCap.6. The method of any one of to claim 2 , wherein the recombinant protein contains a C2/C3 region of an antibody.7. The method of claim 6 , wherein the second protein is Protein A or Protein G.8. The method of any one of to claim 6 , wherein the sample is obtained from affinity purification of the protein over a Protein A chromatography medium.9. The method of any one of to claim 6 , wherein the recombinant protein is an antibody or an Fc fusion protein.10. The method of claim 9 , wherein the recombinant protein is a tumor ...

METHOD FOR PURIFYING IMMUNOGLOBULIN

The present invention relates to a method for purifying an immunoglobulin, and more particularly, to a method for purifying an immunoglobulin, which comprises: dialyzing and concentrating an immunoglobulin-containing plasma protein fraction II paste; removing thrombotic substances from the dialyzed and concentrated fraction by a purification process using ceramic cation exchange resin; and performing elution while maintaining salt concentration at a constant level to maintain the polymer content of the immunoglobulin at a low level. When the immunoglobulin purification method according to the present invention is used, the efficiency with which impurities and thrombotic substances are removed can be increased and the polymer content of the immunoglobulin can be maintained, and thus a stable immunoglobulin with improved quality can be produced. 1. A method for purifying an immunoglobulin , comprising the steps of:(a) dissolving an immunoglobulin-containing plasma protein fraction II paste, followed by filtration to obtain a fraction II solution;(b) dialyzing and/or concentrating the obtained fraction II solution, subjecting the dialyzed and/or concentrated solution to anion exchange chromatography, and recovering a fraction not attached to a column of the anion exchange chromatography;(c) treating the recovered fraction with a solvent and/or a detergent to inactivate viruses, followed by subjecting the fraction to cation exchange chromatography to remove the solvent and/or the detergent, and thrombotic substances;(d) dialyzing and/or concentrating an eluate obtained from the cation exchange chromatography; and(e) filtering the dialyzed and/or concentrated solution, thereby obtaining a purified immunoglobulin.2. The method of claim 1 , wherein the dissolving of the plasma protein fraction II paste in step (a) is performed by adding a sodium chloride solution in an amount equivalent to 2-10 times the volume of the plasma protein fraction.3. The method of claim 1 , ...

METHOD FOR PURIFYING DARBEPOETIN ALFA

The present invention relates to a method for purifying darbepoetin alfa by selectively separating only a structural isoform having a high content of sialic acid from a mixture of structural isoforms of darbepoetin alfa having various contents of sialic acid. Since the method of the present invention is a novel method for purifying darbepoetin alfa which can be conveniently and simply produced, it is possible to remarkably increase productivity due to process efficiency improvement, as well as to yield high purity darbepoetin alfa when mass-producing darbepoetin alfa according to the present invention. 1. A method for purifying darbepoetin alfa , comprising:(a) binding darbepoetin alfa to an anion exchange chromatography column by loading a mixture comprising darbepoetin alfa having various contents of sialic acid into the anion exchange chromatography column;(b) washing the chromatography column with a washing buffer solution containing arginine; and(c) eluting the darbepoetin alfa, which remains bound to the chromatography column, from the column.2. The method of claim 1 , wherein the darbepoetin alfa eluted in step (c) has an isoelectric point ranging from pI 2.0 to 4.0.3. The method of claim 1 , wherein the washing buffer solution containing arginine has a pH ranging from 3.0 to 5.0.4. The method of claim 1 , wherein the washing buffer solution containing arginine further comprises at least one selected from the group consisting of NaCl and urea.5. The method of claim 4 , wherein the washing buffer solution comprises NaCl at a concentration ranging from 5 mM to 90 mM.6. The method of claim 4 , wherein the washing buffer solution comprises urea at a concentration ranging from 3 M to 8 M.7. The method of claim 1 , wherein step (b) is washing structural isoforms of darbepoetin alfa having higher isoelectric points than a desired isoelectric point claim 1 , to obtain a structural isoform of darbepoetin alfa having the desired isoelectric point.8. The method of claim ...

System and Method for Producing Interleukin Receptor Antagonist (IRA)

The present invention relates to a system and method for producing high levels of autologous IL-1RA cytokine, comprising: a blood collection vessel (), a cover (), a portion of separation gel (), an anticoagulant portion (), a plasma collection syringe comprising a sharp needle (), a buffy coat collection syringe () and an incubation tube with cover (). The vessel is adapted such that, when containing the whole blood (), and centrifuged after treatment, yields separation fractions comprising, a first fraction of RBCs sediment (), a second fraction of said gel (), a third fraction comprising WBCs, platelets and), and fourth fraction of plasma solution (). 1122-. (canceled)12415. The system according to claim 123 , wherein said incubation tube () is adapted for incubation for sufficient time and temperature yielding high levels of said autologous IL-1RA cytokine; said incubation tube is selected from the group consisting of: a plastic tube claim 123 , a glass tube and any combination thereof.125. The system according to claim 123 , wherein said system further comprises at least one of the following:a. a filter unit having pores size of about of 0.2-50 μm;b. a stopcock adapted as a turning plug which controls the flow of fluid from said plasma collection syringe to said blood collection tube;c. a stopcock adapted as a turning plug which controls the flow of fluid from said buffy coat collection syringe to said blood collection tube;d. said an anticoagulant selected from the group consisting of: a citrate based anticoagulant, EDTA salt, heparin salt based anticoagulant, oxalate based anticoagulant and any combination thereof; and{'sup': '3', 'e. said separation gel at density of about 1.06-1.09 gr/cm.'}126. The system according to claim 123 , wherein said centrifugation is being performed for about 10 to about 30 minutes.127. The system according to claim 123 , wherein at least one of the following holds true:a. said plasma is discarded up to 80% of plasma from said ...

PURIFICATION METHOD FOR RECOMBINANT PROTEINS AND NANOPARTICLES

The invention is directed to methods for purifying recombinant proteins, e.g. HIV-1 envelope trimers and/or nanoparticles, wherein the methods do not use an affinity step. 1. A method of purifying a recombinant viral envelope protein , the method comprising:a. step (a) contacting an anion exchange (AEX) chromatography resin with a fraction (1) comprising recombinant viral envelope protein,b. step (b) eluting a fraction (2) from the resin of step (a),c. step (c) contacting a mixed-mode chromatography resin with the fraction (2) from step (b), and 'i. wherein fraction (3) has fewer product-related impurities compared to fraction (1) or fraction (2).', 'd. step (d) eluting a fraction (3) from the resin of step (c),'}2. The method of claim 1 , wherein the method further comprises:e. step (e) contacting a hydrophobic interaction chromatography (HIC) resin with fraction (3) from step (d), and 'i. wherein fraction (4) has fewer product-related impurities compared to fraction (1), fraction (2), or fraction (3).', 'f. step (f) collecting unbound flow through as fraction (4),'}3. A method of purifying a recombinant viral envelope protein claim 1 , the method comprising:a. step (a) contacting an anion exchange (AEX) chromatography resin with a fraction (1) comprising a recombinant viral envelope protein,b. step (b) eluting a fraction (2) from the resin of step (a),c. step (c) contacting a HIC resin with fraction (2) from step (b), and 'i. wherein fraction (3) has fewer product-related impurities compared to fraction (1) or fraction (2).', 'd. step (d) collecting flow through from step (c) as fraction (3),'}4. A method of purifying a recombinant viral envelope protein claim 1 , the method comprising:a. step (a) contacting a mixed-mode chromatography resin with a fraction (1) comprising a recombinant viral envelope protein,b. step (b) eluting a fraction (2) from the resin of step (a),c. step (c) contacting a HIC resin with fraction (2) from step (b), and 'i. wherein fraction (3) ...

ENHANCED LIQUID FORMULATION STABILITY OF ERYTHROPOIETIN ALPHA THROUGH PURIFICATION PROCESSING

A method for purifying rHuEPO through the use of a multi-step filtration process which provides for a rHuEPO product having superior stability and shelf-life. 1. A recombinant human erythropoietin alpha product having a shelf life of at least four years manufactured by the following process wherein the process comprises the following steps:a. manufacturing a recombinant human erythropoietin alpha product and collecting the supernatant,b. clarifying the supernatant and subjecting the supernatant to a multi-step purification process comprising the following procedures in order: ultradiafiltration, anion exchange chromatography, ammonium sulfate precipitation, hydrophobic interaction chromatography, acetonitrile addition, reversed phase chromatography, a second anion exchange chromatography step, gelfiltration, and nano filtration for virus removal; andc. producing a recombinant human erythropoietin alpha product that is substantially free of non-O-glycosylated recombinant human erythropoietin alpha isoforms.2. The product made by the process of claim 1 , wherein said purified human erythropoietin alpha product is filled into a Type 1 glass container.3. The product made by the process of wherein step b wherein the urea in a wash buffer in the first anion exchange chromatography step is at a concentration of less than 3M.4. The product made by the process of wherein step b wherein the urea in a wash buffer in the first anion exchange chromatography step is at a concentration of less than 0.1M. This application is a divisional of U.S. application Ser. No. 14/775,248, filed Sep. 11, 2015, which is a National Stage Entry of International Application No. PCT/CA2014/000252 filed on Mar. 14, 2014, which claims priority to U.S. Provisional Application No. 61/792,991 filed Mar. 15, 2013.The present application is generally related to methods and products resulting from said methods resulting in a highly stable erythropoietin alpha product having a substantially increased shelf ...

OVERLOAD AND ELUTE CHROMATOGRAPHY

The present invention provides methods for purifying a polypeptide from a composition comprising the polypeptide and at least one contaminant by overloading a chromatography material and eluting the product. 1. A method for purifying a polypeptide from a composition comprising the polypeptide and one or more contaminants , said method comprisinga) loading the composition onto a chromatography material in an amount in excess of the dynamic binding capacity of the chromatography material for the polypeptide,b) eluting the polypeptide from the chromatography material under conditions wherein the one or more contaminants remain bound to the chromatography material, andc) pooling fractions comprising the polypeptide in the chromatography effluent from steps a) and b).2. (canceled)3. The method of claim 1 , wherein the polypeptide is an immunoadhesin.4. The method of claim 1 , wherein the polypeptide is an antibody.5. The method of claim 4 , wherein the antibody is a monoclonal antibody.6. The method of claim 5 , wherein the monoclonal antibody is a chimeric antibody claim 5 , humanized antibody claim 5 , or human antibody.7. The method of claim 5 , wherein the monoclonal antibody is an IgG monoclonal antibody.8. (canceled)9. The method of claim 4 , wherein the antibody is an antigen binding fragment claim 4 , wherein the antigen binding fragment is a Fab fragment claim 4 , a Fab′ fragment claim 4 , a F(ab′)2 fragment claim 4 , a scFv claim 4 , a di-scFv claim 4 , a bi-scFv claim 4 , a tandem (di claim 4 , tri)-scFv claim 4 , a Fv claim 4 , a sdAb claim 4 , a tri-functional antibody claim 4 , a BiTE claim 4 , a diabody or a triabody.10. The method of claim 1 , wherein the polypeptide is an enzyme claim 1 , a hormone claim 1 , a fusion protein claim 1 , an Fc-containing protein claim 1 , an immunoconjugate claim 1 , a cytokine or an interleukin.11. The method of claim 1 , wherein the at least one contaminant is any one or more of Chinese Hamster Ovary Protein (CHOP) claim ...

CEX CHROMATOGRAPHY MEDIA AND LOW SALT ELUTION OF TARGET PROTEINS FROM BIOPHARMACEUTICAL FEEDS

A bind/elute chromatography method and compositions for low salt/low solution conductivity separation of target proteins from a mixture of aggregates and other impurities. 1. A method of separating a monomeric protein of interest from a mixture comprising aggregates of the protein of interest in a sample , the method comprising contacting the sample with a solid support , the solid support comprising a polyvinyl ether resin functionalized with a 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and N ,N-dimethylacrylamide (DMMA) , wherein the molar ratio of DMMA to AMPS is greater than 2.0 , and eluting the monomeric protein of interest from the solid support with a buffer having a solution conductivity between about 10 mS/cm and 20 mS/cm.2. The method of claim 1 , wherein the monomeric protein of interest is a monoclonal antibody.3. The method of claim 1 , wherein the protein of interest is a recombinant protein.4. The method of claim 1 , wherein the mixture comprises at least 1% aggregates of the protein of interest.5. The method of claim 1 , wherein the solid support is a bead.6. The method of claim 1 , wherein the solid support is a membrane. The present application claims the benefit of priority of U.S. Patent Application No. 62/651,878, filed Apr. 3, 2018, which is incorporated by reference herein in its entirety.Described herein are methods for purifying target proteins, such as therapeutic proteins and antibody molecules antibodies, from a biopharmaceutical feed using bind/elute cation exchange chromatography.Biopharmaceutical products of interest are produced by cells grown in culture. The product of interest is harvested and purified to remove impurities using a cascade of separation technologies. Examples of impurities include aggregates, host cell protein (HCP), and nucleic acids, endotoxins, viruses, etc. (see, e.g., State-of-the-Art in Downstream Processing of Monoclonal Antibodies: Process Trends in Design and Validation Biotechnol. Prog., 2012, 899- ...

Method for Purifying Antibodies from Raw Milk

The present application relates to a method for preparing an antibody or antibody fragment composition from raw milk from a non-human mammal expressing said antibody or antibody fragment in its milk, comprising steps a) precipitation of the raw milk with caprylic acid, b) separation, consisting of centrifugation or filtration through a depth filter, and optionally c) filtration through an active carbon depth filter. 1. Method for preparing a composition comprising a monoclonal antibody or a monoclonal antibody fragment from raw milk of a non-human mammal expressing said monoclonal antibody or monoclonal antibody fragment in its milk , comprising:a) a step of precipitation of the raw milk with caprylic acid,b) a step of separation consisting of a centrifugation or filtration through a depth filter, and optionallyc) a step of filtration through an activated carbon depth filter.2. Method according to claim 1 , characterized in that step a) makes it possible to both clarify the milk and to render it biologically safe and to purify the monoclonal antibody or monoclonal antibody fragment.3. Method according to or claim 1 , characterized in that step a) precipitates the β-lactoglobulins.4. Method according to any one of to claim 1 , characterized in that the raw milk has not undergone any prior step of clarification and/or skimming and/or acidification.5. Method according to any one of to claim 1 , characterized in that the final percentage (w/w) of caprylic acid used in step a) is comprised between 0.5 and 3.0% claim 1 , preferably comprised between 1.0 and 2.5% claim 1 , more preferably between 1.3% and 2.0% claim 1 , and is preferably 1.7%.6. Method according to any one of to claim 1 , characterized in that in step a) after addition of caprylic acid the pH of the mixture is adjusted to a value less than 4.8 claim 1 , preferably comprised between 4.0 and 4.8 claim 1 , preferably at a value of 4.3.7. Method according to any one of to claim 1 , characterized in that claim ...

PROCESS FOR OBTAINING AN IgG COMPOSITION THROUGH HEAT TREATMENT

A process for obtaining an IgG composition involves heat treatment. This process obtains an IgG composition from an IgG solution partly purified from human plasma, in which by applying intermediate heat treatment and without using reagents to precipitate high molecular weight aggregates/polymers and/or proteins virtually total elimination of the IgG polymers generated during the process is achieved. Furthermore this process offers high productivity, lower production costs and is easy to implement in comparison with the processes of the know art. In addition to this, by using this process stability is imparted to the final product in liquid. 1. A process for obtaining an IgG composition from an IgG solution partly purified from human plasma comprising the steps of:a) diafiltering the partly purified IgG solution;b) stabilising the solution obtained in step a);c) heat treating the solution obtained in step b);d) selectively adsorbing high molecular weight aggregates and/or polymers from the solution heat treated in step c) through cation chromatography; ande) diafiltering and formulating the solution obtained in step d).2. A process according to claim 1 , wherein the said process is carried out starting with an IgG solution purified from human plasma having an IgG content of more than 95% in relation to total proteins.3. A process according to claim 1 , wherein said process is carried out starting with an IgG solution purified from human plasma having an IgG content of more than 97% in relation to total proteins.4. A process according to claim 1 , wherein the diafiltration step (a) is carried out until the ethanol concentration is less than 0.5% (weight/volume) claim 1 , preferably less than 0.1% (weight/volume).5. A process according to claim 1 , wherein the diafiltration step (a) is carried out until the concentration of non-denatured precipitation reagents such as PEG claim 1 , octanoic acid claim 1 , compatible non-ionic detergents or any mixture thereof is less ...

IMMUNOGLOBULIN A ANTIBODIES AND METHODS OF PRODUCTION AND USE

The presently disclosed subject matter provides antibodies, e.g., IgA antibodies and IgG-IgA fusion molecules, and compositions comprising such antibodies, as well as methods of making and using such antibodies and compositions. 1. An isolated IgA antibody , or a fragment thereof , wherein the IgA antibody comprises one or more of the following:(a) a substitution at amino acid V458, N459 and/or S461;(b) a substitution at amino acid 1458; and(c) a substitution at an amino acid selected from the group consisting of N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, S461 and a combination thereof.2. The isolated IgA antibody of claim 1 , wherein:(a) amino acid V458 is substituted with an isoleucine (V4581), amino acid N459 is substituted with a glutamine (N459Q), a glycine (N459G) or an alanine (N459A), and/or amino acid S461 is substituted with an alanine (S461A);(b) amino acid I458 is substituted with a valine (I458V); and/or(c) the substitutions at amino acids N166, S212, N263, N337, I338, T339 and N459 are N166A, S212P, N263Q, N337T, I338L, T339S and N459Q.3. The isolated IgA antibody of claim 1 , wherein the IgA antibody is an IgA1 claim 1 , IgA2m1 claim 1 , IgA2m2 or IgA2mn antibody.4. The isolated IgA antibody of claim 1 , wherein the IgA antibody comprises substitutions at amino acids N337 claim 1 , 1338 and T339 and one or more substitutions at T168 claim 1 , N211 claim 1 , S212 claim 1 , S213 claim 1 , N263 claim 1 , T265 claim 1 , N459 claim 1 , S461 and a combination thereof.5. An isolated nucleic acid encoding the IgA antibody of .6. A host cell comprising the nucleic acid of .7. A method of producing an IgA antibody culturing the host cell of so that the IgA antibody is produced.8. A pharmaceutical composition comprising one or more IgA antibodies of and a pharmaceutically acceptable carrier.9. A method of treating an individual having a disease claim 1 , wherein the method comprises administering to the individual an effective amount of ...

Methods of Engineering Surface Charge for Bispecific Antibody Production

The present disclosure relates to methods of modifying the isoelectric point of an antibody. The method includes providing an antibody comprising a first polypeptide comprising a heavy chain variable region and a second polypeptide comprising heavy chain variable region and substituting, in at least one of the first and second polypeptides of the antibody, one or more amino acid residues of the heavy chain variable region (V) at positions 7, 9, 11, 14, 41, 70, 74, 82a, 84, and 113, according to the Kabat numbering system, wherein the substituting increases or decreases the isoelectric point of the antibody. 1. A method of modifying the isoelectric point of an antibody , said method comprising: a first polypeptide comprising a heavy chain variable region and', 'a second polypeptide comprising heavy chain variable region; and, 'providing an antibody, said antibody comprising{'sub': 'H', 'substituting, in at least one of the first and second polypeptides of the antibody, one or more amino acid residues of the heavy chain variable region (V) at positions 7, 9, 11, 14, 41, 70, 74, 82a, 84, and 113, according to the Kabat numbering system, wherein said substituting increases or decreases the isoelectric point of the antibody.'}2. The method of claim 1 , wherein when the one or more amino acid residues of the Vat positions 7 claim 1 , 9 claim 1 , 11 claim 1 , 14 claim 1 , 41 claim 1 , 74 claim 1 , 84 claim 1 , and 113 (Kabat numbering) are neutrally charged amino acid residues claim 1 , said substituting comprises:exchanging said one or more neutrally charged amino acid residues with one or more positively charged amino acid residues to increase the isoelectric point of the antibody.3. The method of claim 1 , wherein when the one or more amino acid residues of the Vat positions 7 claim 1 , 9 claim 1 , 11 claim 1 , 14 claim 1 , 41 claim 1 , 74 claim 1 , 84 claim 1 , and 113 (Kabat numbering) are negatively charged amino acid residues claim 1 , said substituting comprises: ...

POLYPEPTIDE OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

The present invention relates to a field of biotechnology. It discloses a novel amino acid sequence of recombinant human bone morphogenetic protein-2 (rhBMP-2) and encoded nucleotide sequences thereof, and a method for preparing rhBMP-2. In the present invention, by further optimizing the nucleotide sequence of rhBMP-2, novel amino acid sequence of rhBMP-2 that can express good ectopic induced osteogenic activity and has good renaturation and purification effect is screened out. The engineered bacteria constructed by the present invention can induce the production of recombinant rhBMP-2 protein with an expression level of about 55%, and the produced target protein is more easily denatured and purified than the existing recombinant rhBMP-2, with better denaturation and purification effect and higher osteoinductive activity. 1. A polypeptide of a recombinant human bone morphogenetic protein-2 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 1.2. The polypeptide according to claim 1 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 2.3. The polypeptide according to claim 2 , wherein the polynucleotide encoding the amino acid sequence SEQ ID NO: 2 is SEQ ID NO: 3.4. The polypeptide according to claim 2 , wherein the recombinant human bone morphogenetic protein-2 is prepared according to the following method and the method comprises the following steps:1) designing DNA sequences encoding recombinant human bone morphogenetic protein-2;{'i': 'E. coli', '2) constructing an expression vector to transform host cells;'}3) screening positive clones for culture and inducing the expression of the target protein;4) renaturing and purifying the expression product to obtain the recombinant human bone morphogenetic protein-2;wherein the DNA sequence is shown in SEQ ID NO: 3.5. The polypeptide according to claim 4 , wherein a dilution method is used for renaturation in the step (4) and the final concentration of the protein in the ...

PURIFICATION METHOD FOR VITAMIN K DEPENDENT PROTEINS BY ANION EXCHANGE CHROMATOGRAPHY

The present invention relates to a method for the purification of Vitamin K dependent proteins with high yield and high purity, particularly enriched in active protein, on anion exchange resin materials, to Vitamin K dependent proteins obtainable by said method, and to a kit comprising means for carrying out said method. 1. A method for the purification of a Vitamin K dependent protein comprising the steps of:(a) loading an anion exchange resin material (in the following also “the first anion exchange resin material”) with the Vitamin K dependent protein in a loading buffer in the absence or low concentration of divalent cations, optionally followed by one to three wash steps; 1-3 mM calcium is comprised in the eluant', 'the eluant has a conductivity of 14-23 mS/cm (25° C.), and', 'the pH of the eluant is between 7.0 and 9.0., '(b) eluting the Vitamin K dependent protein with an eluant comprising calcium and a counter-anion to form an eluate containing the Vitamin K dependent protein, wherein2. The method for purification of further comprising the following steps:(c) diluting the obtained eluate pool, ((1) to optionally lower the conductivity), and increasing the concentration of the calcium,(d) loading a second anion exchange resin material with the eluate as obtained after step (c); and(e) collecting the flow-through containing the Vitamin K dependent protein.3. The method according to or claim 1 , wherein in step (b):{'sup': '++', 'the conductivity is between 18 and 20 mS/cm (25° C.) at a pH of 7.0-7.4 and 1 mM Ca,'}{'sup': '++', 'the conductivity is between 18.5 and 21 mS/cm (25° C.) at a pH of 7.4-7.6 and 1 mM Ca,'}{'sup': '++', 'the conductivity is between 19 and 22.5 mS/cm (25° C.) at a pH of 7.6-8.0 and 1 mM Ca,'}{'sup': '++', 'the conductivity is between 20 and 23 mS/cm (25° C.) at a pH of 8.0-9.0 and 1 mM Ca,'}{'sup': '++', 'the conductivity is between 15 and 17.0 mS/cm (25° C.) at a pH of 7.0-7.4 and 2 mM Ca,'}{'sup': '++', 'the conductivity is between 16 ...

Capture Purification Processes for Proteins Expressed in a Non-Mammalian System

Methods of purifying proteins expressed in non-mammalian expression systems in a non-native soluble form directly from cell lysate are disclosed. Methods of purifying proteins expressed in non-mammalian expression systems in a non-native limited solubility form directly from a refold solution are also disclosed. Resin regeneration methods are also provided.

PROCESS FOR PRODUCTION OF FIBRINOGEN AND FIBRINOGEN PRODUCED THEREBY

A process for purifying fibrinogen from a fibrinogen containing source by precipitation of fibrinogen by a precipitating agent from a fibrinogen containing solution in the presence of one or more chelating agent(s) and removal of the supernatant from the fibrinogen paste, characterised in that fibrinogen is extracted from the paste forming a liquid fraction containing fibrinogen, and an undissolved residue, which is separated from the liquid. 1. A process for purifying fibrinogen from a fibrinogen containing source comprising precipitating fibrinogen with a precipitating agent from a fibrinogen containing solution in the presence of one or more chelating agent(s) and removing the supernatant from the fibrinogen paste , wherein the fibrinogen is extracted from the paste forming a liquid fraction containing fibrinogen and an undissolved residue which is separated from the liquid , wherein addition of one or more protease inhibitor(s) is omitted.2. The process of claim 1 , wherein the one or more protease inhibitor(s) is selected from the group consisting of C1-protease inhibitors claim 1 , trypsin inhibitors claim 1 , thrombin inhibitors claim 1 , antithrombin-III (AT-III) claim 1 , heparin-cofactor-II claim 1 , aprotinin claim 1 , pepstatin claim 1 , leupeptin and epsilon-aminocaproic acid.3. The process of claim 1 , wherein the one or more chelating agent(s) is a Ca-chelating agent selected from the group consisting of 1 claim 1 ,2-bis(o-amino)ethane-N claim 1 ,N claim 1 ,N′ claim 1 ,N′-tetraacetic acid (BAPTA) claim 1 , diethylene-triamine-pentaacetic acid (DTPA) claim 1 , ethylenediamine-tetraacetic acid (EDTA) claim 1 , ethylene-glycol-tetraacetic acid (EGTA) and nitrilo-triacetic acid (NTA).4. The process of claim 1 , wherein the concentration of the chelating agent is in a range of 3 mM to 100 mM.5. The process of claim 1 , wherein the concentration of the chelating agent is in a range of 5 mM to 50 mM.6. The process of claim 1 , wherein the concentration of ...

HUMAN ANTIBODIES THAT BIND HUMAN TNF-ALPHA AND METHODS OF PREPARING THE SAME

Methylglyoxal (MGO)-modified recombinant TNF-alpha antibodies (e.g., Adalimumab) are identified. MGO modification decreases binding between Adalimumab and TNF-alpha. Methods are disclosed for reducing the presence of MGO-modified antibodies in the production of Adalimumab TNF-alpha antibodies. 1. A method for purifying a composition comprising a target protein , said method comprising:(a) loading the composition to a cation exchange adsorbent using a loading buffer, wherein the pH of the loading buffer is lower than the pI of the target protein;(b) washing the cation exchange adsorbent with a washing buffer, wherein the pH of the washing buffer is lower than the pI of the target protein;(c) eluting the cation exchange adsorbent with an elution buffer, said elution buffer being capable of reducing the binding between the target protein and the cation exchange adsorbent; and(d) collecting the eluate, wherein the percentage of the target protein is higher in the eluate than the percentage of the target protein in the composition.2. The method of claim 1 , wherein the conductivity of the elution buffer is higher than the conductivity of the washer buffer.3. The method of claim 2 , wherein the conductivity of the elution buffer is raised by increasing the salt concentration of the elution buffer.4. The method of claim 2 , wherein the pH of the elution buffer is between 5.5 and 9.0.5. The method of claim 3 , wherein the salt concentration of the elution buffer is between 20 mM NaCl and 200 mM NaCl.6. The method of claim 1 , wherein the target protein is a human antibody or an antigen-binding portion thereof claim 1 , wherein the target protein dissociates from human TNF-alpha with a Kof 1×10M or less and a Krate constant of 1×10sor less claim 1 , both determined by surface plasmon resonance claim 1 , and wherein the target protein neutralizes human TNF-alpha cytotoxicity in a standard in vitro L929 assay with an ICof 1×10M or less.7. A method for purifying a composition ...

PURIFICATION OF IMMUNOGLOBULINS FROM PLASMA

The present invention relates to the purification of target molecules like immunoglobulins from plasma. The use of a certain type of ion exchanger based on a crosslinked polyvinylether results in especially high yields of the target molecule. 1. A method for purifying a target molecule from a plasma sample bya) Providing a plasma sample comprising the target moleculeb) subjecting said plasma sample to an ion exchange chromatography on a polyvinylether matrix carrying between 600 and 1200 μmol/g anionic groups whereby purified target molecule is eluted from the matrix.3. Method according to claim 1 , characterized in that the matrix is obtainable by copolymerisation of a hydrophilically substituted alkyl vinyl ether selected from the group of 1 claim 1 ,4-butanediol monovinyl ether claim 1 , 1 claim 1 ,5-pentanediol monovinyl ether claim 1 , diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether and divinylethyleneurea (1 claim 1 ,3-divinylimidazolin-2-one) as crosslinking agent.4. Method according to claim 1 , characterized in that the ionic groups have been attached to the matrix by subjecting the polyvinylether matrix to cerium catalyzed graft polymerization.5. Method according to claim 1 , characterized in that claim 1 , the polyvinylether matrix carries graft polymers with 700 to 1100 μmol/g positively charged anion exchange groups.6. Method according to claim 1 , characterized in that the anion exchange group is trimethylammoniumalkyl.7. Method according to claim 1 , characterized in that the ion exchange chromatography is performed in the flow-through mode.8. Method according to claim 1 , characterized in that the target molecule is an immunoglobulin.9. Method according to claim 1 , characterized in that the target molecule is separated from IgA claim 1 , IgM claim 1 , albumin claim 1 , and factor XIa.10. Method according to claim 1 , characterized in that the matrix in step b) is eluted with a buffer having a pH between 4 and 7.4.11. ...

METHOD FOR PRODUCING INJECTABLE FORMULATIONS OF BLOOD-DERIVED PROTEIN MATERIALS, AND MATERIALS OBTAINED USING SAID METHOD

A method for producing injectable pharmaceutical formulations of blood-derived protein materials includes the steps of fractioning the source material in a polymer/salt aqueous two-phase system in the presence of phenol, purifying the top phase of the system by means of precipitation with caprylic acid and purifying the bottom phase by means of thermocoagulation, increasing the purity of the materials in both phases through chromatography, removing viral particles by means of the nanofiltration of both preparations, and formulating, stabilizing, and packaging the resulting materials. 1. A method for the production of injectable formulations from plasma derived protein products with reduced viral load , wherein said method comprises the steps of:a. fractionation of the starting material in a system of two aqueous phases by adding a polymer and at least one salt;b. addition of phenol to the two phase system as a first step of viral inactivation;c. separation of upper and lower phases of the system into two aqueous phases;d. purification of the products in the system's upper phase through precipitation with a fatty acid;e. purification of the products in the system's lower phase through thermo coagulation;f. removal of denaturalized protein precipitates formed during the purification steps of the system's upper and lower phases in two steps;g. increasing the purity of the products obtained from the upper and lower phases through chromatography;h. nano-filtration of the products obtained in the previous step to remove viral particles; andi. formulation, stabilization and sterilization of the products thus obtained.2. The method according to claim 1 , wherein the stalling material is selected front the group consisting of blood plasma claim 1 , serum claim 1 , a fraction obtained through the Cohn method or any other material containing plasma derived protein products.3. The method according to claim 1 , wherein the polymer used in the fractionation of the starting ...

METHODS OF PURIFYING POLYPEPTIDES

The present invention provides methods for purifying a polypeptide from a composition comprising the polypeptide and at least one contaminant and formulations comprising the polypeptide purified by the methods. The methods for purifying include cation exchange material and/or mixed mode material.

INSULIN PRECURSOR PURIFYING METHOD USING ANION EXCHANGE CHROMATOGRAPHY

The present invention relates to an insulin precursor purifying method for improving the production yield of an insulin precursor by adjusting the pHs of a first buffer solution for equilibrating an ion exchange resin and a second buffer solution for eluting an insulin precursor bound to the ion exchange resins. The insulin precursor purifying method according to the present invention is a high-purity and high-yield insulin precursor purifying method for reinforcing by mean of suitable pH combination of buffer solutions, the binding force of an insulin precursor to ion exchange resins and enabling the insulin precursor to be effectively eluted thereafter, and is very useful in high-yield insulin production through enzymatic conversion after a purification process. 1. A method for purifying an insulin precursor comprising:(a) equilibrating an anion exchange resin with a first buffer solution;(b) introducing a solution comprising an insulin precursor into the equilibrated anion exchange resin; and(c) eluting the insulin precursor bound to the anion exchange resin using a second buffer solution having a pH higher than a pH of the first buffer solution.2. The method according to claim 1 , wherein the anion exchange resin is a diethylaminoethyl cellulose-based resin.3. The method according to claim 2 , wherein the anion exchange resin is Fractogel EMD DEAE or Capto DEAE.4. The method according to claim 1 , wherein the first buffer solution is 20 mM Tris-HCl at a pH of 7.0 to 8.0.5. The method according to claim 1 , wherein the second buffer solution is 10-100 mM Tris-HCl or borate at a pH of 8.0 to 10.0 containing 0-200 mM sodium chloride.6. The method according to claim 5 , wherein the second buffer solution is selected from the group consisting of:(i) 50 mM Tris-HCl at a pH of 8.0 to 9.0 containing 0-100 mM sodium chloride (NaCl);(ii) 50 mM borate at a pH of 9.2 containing 10 mM sodium chloride (NaCl);(iii) 50 mM borate at a pH of 9.4 containing 30 mM sodium chloride ( ...

Production of Immunotoxin D2C7 - (scdsFv) -PE38KDEL

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a recombinant exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a Phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in BLR (λ DE3). D2C7-IT was produced by a 10 L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein. 1. A method of producing clinical grade D2C7(scdsFv)-PE38KDEL immunotoxin , comprising the steps of:{'i': 'E. coli', 'a. culturing an lambda lysogen comprising a plasmid encoding said immunotoxin in a fermentor to produce a bacterial cell paste;'}{'sub': '4', 'b. lysing bacteria of the bacterial cell paste in a buffer comprising MgSO, DNaseI, and lysozyme;'}c. collecting inclusion bodies from the lysed bacteria;d. solubilizing the inclusion bodies and reducing proteins of the solubilized inclusion bodies to form reduced proteins of the inclusion bodies;e. refolding the reduced proteins of the inclusion bodies in the presence of a protease inhibitor to form single-chain disulfide stabilized immunotoxin;f. purifying the single-chain disulfide stabilized immunotoxin to remove the protease inhibitor and endotoxin; to form purified, single-chain disulfide stabilized immunotoxin;g. storing the purified single-chain disulfide stabilized immunotoxin at a temperature of −70° to −90° C.2E. coliE. coli. The method of wherein the lambda lysogen is made by transforming competent cells of an lambda lysogen with a plasmid encoding said immunotoxin claim 1 , and recovering a transformant.3. The method of wherein expression of the immunotoxin is inducible.4. The method of wherein expression of the immunotoxin is induced with ...

HUMAN ANTIBODIES THAT BIND HUMAN TNF-ALPHA AND METHODS OF PREPARING THE SAME

Methylglyoxal (MGO)-modified recombinant TNF-alpha antibodies (e.g., Adalimumab) are identified. MGO modification decreases binding between Adalimumab and TNF-alpha. Methods are disclosed for reducing the presence of MGO-modified antibodies in the production of Adalimumab TNF-alpha antibodies. 1. A composition comprising a binding protein capable of binding TNF-alpha , wherein said binding protein comprises at least one methylglyoxal (MGO)-susceptible amino acid , and wherein at least a portion of said binding protein comprises one or more MGO-modified amino acids.2. The composition of claim 1 , wherein the portion of the binding protein that comprises at least one MGO-modified amino acid is less than 12%.3. The composition of claim 1 , wherein the portion of the binding protein that comprises at least one MGO-modified amino acid is less than 6%.4. The composition of claim 1 , wherein the MGO-susceptible amino acid is an arginine.5. The composition of claim 1 , wherein the binding protein is a human antibody or an antigen-binding portion thereof claim 1 , wherein the binding protein dissociates from human TNF-alpha with a Kof 1×10M or less and a Krate constant of 1×10sor less claim 1 , both as determined by surface plasmon resonance claim 1 , and wherein the binding protein neutralizes human TNF-alpha cytotoxicity in a standard in vitro L929 assay with an ICof 1×10M or less.6. A composition comprising a binding protein capable of binding TNF-alpha claim 1 , said binding protein comprising a methylglyoxal (MGO)-susceptible amino acid claim 1 , wherein said composition is prepared by substantially removing molecules of said binding protein that comprise at least one MGO-modified amino acid.7. The composition of claim 6 , wherein more than 70% of said molecules that comprise at least one MGO-modified amino acid is removed.8. The composition of claim 6 , wherein more than 90% of said molecules that comprise at least one MGO-modified amino acid is removed.9. The ...

USE OF CAPRYLIC ACID PRECIPITATION FOR PROTEIN PURIFICATION

In certain embodiments, the invention provides a method of purifying a protein of interest from a mixture which comprises the protein of interest and one or more contaminants, comprising: a) subjecting the mixture to a first chromatography step; b) recovering the protein of interest in an elution solution; c) adding caprylic acid to the elution solution to form a contaminant precipitate; d) removing the contaminant precipitate from the elution solution; and e) subjecting the post-precipitated elution solution to a second chromatography column, thereby purifying the protein of interest. 1. A method of purifying a protein of interest from a mixture which comprises the protein of interest and one or more contaminants , comprising:a) subjecting the mixture to a first chromatography step;b) recovering the protein of interest in an elution solution;c) adding caprylic acid to the elution solution to form a contaminant precipitate;d) removing the contaminant precipitate from the elution solution; ande) subjecting the post-precipitated elution solution to a second chromatography column, thereby purifying the protein of interest.2. The method of claim 1 , wherein the contaminants are selected from host cell proteins claim 1 , host cell metabolites claim 1 , host cell constitutive proteins claim 1 , nucleic acids claim 1 , endotoxins claim 1 , viruses claim 1 , product related contaminants claim 1 , lipids claim 1 , media additives and media derivatives.3. The method of claim 1 , wherein the first chromatography is selected from an affinity chromatography claim 1 , an ion exchange chromatography claim 1 , a hydrophobic interaction chromatography claim 1 , and a mix-mode chromatography.4. The method of claim 3 , wherein the first chromatography is a protein A affinity chromatography.5. The method of claim 3 , wherein the first chromatography is an anion exchange chromatography.6. The method of claim 3 , wherein the first chromatography is a cation exchange chromatography.7. The ...

VARIANT DOMAINS FOR MULTIMERIZING PROTEINS AND SEPARATION THEREOF

The current invention relates to means and methods for producing and isolating immunoglobulin proteins comprising a first and a second immunoglobulin polypeptide, in particular to means and methods for producing, and separating proteins comprising said first and second immunoglobulin polypeptide. By including variations to amino acids, and variant separation domains from a cell producing the desired immunoglobulin protein, a desired immunoglobulin protein as produced can be separated from mixtures of immunoglobulin proteins. 1. An immunoglobulin CH1 , CH2 , CH3 region comprising a variation of an amino acid that is non-surface exposed in an immunoglobulin or a combination of said regions , wherein the variation is selected fromneutral amino acid to a negatively charged amino acid;a positively charged amino acid to a neutral amino acid;a positively charged amino acid to a negatively charged amino acid;a neutral amino acid to a positively charged amino acid;a negatively charged amino acid to a neutral amino acid; anda negatively charged amino acid to a positively charged amino acid.2. (canceled)3. The immunoglobulin region of claim 1 , wherein said variation of an amino acid is not in the CH1/CL claim 1 , CH2/CH2 domain or CH3/CH3 domain interface.4. The immunoglobulin region of claim 1 , which is an IgG region.5. (canceled)6. An immunoglobulin CH1 region comprising a variation of an amino acid selected from N159 claim 1 , N201 claim 1 , T120 claim 1 , K147 claim 1 , D148 claim 1 , Y149 claim 1 , V154 claim 1 , A172 claim 1 , Q175 claim 1 , S190 claim 1 , and K213 (EU-numbering);an immunoglobulin CH2 region comprising a variation of amino acid V303; oran immunoglobulin CH3 region comprising a variation of an amino acid selected from K370, E382 and E388.79-. (canceled)1113-. (canceled)14. The immunoglobulin region of claim 6 , wherein the variation is selected froma neutral amino acid to a negatively charged amino acid;a positively charged amino acid to a neutral amino ...

METHODS FOR PURIFYING RECOMBINANT POLYPEPTIDES

A method of purifying a recombinant polypeptide from a Host Cell Protein (HCP), wherein the amino acid sequence of the recombinant polypeptide comprises a cathepsin L cleavage site is provided. The method includes: (a) applying a solution containing the recombinant polypeptide and HCP to a superantigen chromatography solid support, (b) washing the superantigen chromatography solid support with a wash buffer containing caprylate and arginine; and (c) eluting the recombinant polypeptide from the superantigen chromatography solid support. A method of purifying an anti-OX40 antigen binding polypeptide from a Host Cell Protein (HCP) is also provided. 1. A method of purifying a recombinant polypeptide from a Host Cell Protein (HCP) , wherein the amino acid sequence of the recombinant polypeptide comprises a cathepsin L cleavage site; the method comprising: (a) applying a solution comprising the recombinant polypeptide and HCP to a superantigen chromatography solid support , (b) washing the superantigen chromatography solid support with a wash buffer comprising greater than about 50 mM caprylate and greater than about 0.5 M arginine; and (c) eluting the recombinant polypeptide from the superantigen chromatography solid support.2. The method according to claim 1 , wherein the caprylate is sodium caprylate.3. The method according to claim 1 , wherein the wash buffer comprises about 1.1 M arginine.4. The method according to claim 1 , wherein the wash buffer comprises about 150 mM caprylate.5. The method according to claim 1 , wherein the wash buffer comprises about 1.1 M arginine and about 150 mM caprylate.6. The method according to claim 1 , wherein the HCP is cathepsin L.7. The method according to claim 1 , wherein the recombinant polypeptide is an IgG1.8. The method according to claim 1 , wherein the recombinant polypeptide is an anti-OX40 antigen binding polypeptide.9. The method according to claim 1 , wherein step (b) comprises: (b1) washing the superantigen ...

ANTIBODY PURIFICATION BY CATION EXCHANGE CHROMATOGRAPHY

A method for purifying an antibody by cation exchange chromatography is described in which a high pH wash step is used to remove of contaminants prior to eluting the desired antibody using an elution buffer with increased conductivity. 1. A method for purifying an antibody from a composition comprising the antibody and at least one contaminant , which method comprises the sequential steps of:(a) loading the composition onto a cation exchange material wherein the composition is at a first pH;(b) washing the cation exchange material with a first wash buffer at a pH which is greater than that of the composition in (a), wherein the pH of the first wash buffer is from about 6.8 to about 9.0;(c) washing the cation exchange material with a second wash buffer at a pH which is less than that of the first wash buffer; and(d) eluting the antibody from the cation exchange material with an elution buffer at a conductivity which is substantially greater than that of the second wash buffer.2. The method of wherein the pH of the second wash buffer and the pH of the elution buffer are approximately the same.3. The method of wherein the antibody binds to human CD20.4. The method of wherein the antibody binds to human vascular endothelial growth factor (VEGF).5. The method of wherein the pH of the composition in (a) is from about 4.0 to about 6.0 claim 1 , the pH of the first wash buffer is from about 6.8 to about 8.0 claim 1 , the pH of the second wash buffer is from about 5.0 to about 6.0 claim 1 , and the pH of the elution buffer is from about 5.0 to about 6.0.6. The method of wherein the conductivity of the elution buffer is from about 10 mS/cm to about 100 mS/cm.7. The method of wherein the elution buffer comprises about 100 to about 300 mM NaCl.8. The method of wherein the cation exchange material comprises cross-linked poly(styrene-divinylbenzene) flow-through particles coated with a polyhydroxylated polymer functionalized with sulfopropyl groups.9. The method of wherein the ...

METHOD FOR INCREASING PYRO-GLUTAMIC ACID FORMATION OF A PROTEIN

A method for conversion of an N-terminal glutamine and/or glutamic acid residue of a protein to pyro-glutamic acid within a purification process. 1. A method for purification of a protein containing an N-terminal glutamine and/or an N-terminal glutamic acid , the method comprising a step of incubating said protein under conditions to promote cyclization of the N-terminal glutamine and/or an N-terminal glutamic acid of said protein to N-terminal pyro-glutamic acid.2. The method according to wherein the protein is an antibody.3. The method according to claim 1 , wherein the protein is an anti-CD20 antibody.4. The method of claim 3 , wherein the anti-CD20 antibody comprises a light chain sequence identical to SEQ ID NO:1 and a heavy chain sequence identical to SEQ ID NO:2.5. The method of claim 1 , wherein the conversion takes place at a temperature of 20-45° C.6. The method of wherein the conversion is carried out in the cell media claim 1 , or in a buffer.7. The method of wherein the concentration of the buffer is selected from the range of from 20 mM to 150 mM.8. The method of wherein the pH is in the range of from 3.5-9.0.9. The method of wherein the conversion from N-terminal glutamine and/or glutamic acid to pyro-glutamic acid reaches at least 50%.10. The method of wherein the conversion step comprises non-enzymatic conversion of the N-terminal glutamine and/or the glutamic acid to pyro-glutamic acid.11. The method of wherein the protein has from 1 to 4 N-terminal glutamine residues and/or from 1 to 4 N-terminal glutamic acid residues before conversion.12. The method of further comprising the steps of a protein A chromatography with a suitable resin and an ion exchange chromatography with a suitable resin.13. The method of wherein the method comprises the steps:(a) protein A capture with a suitable resin,(b) a viral inactivation at a pH in the range of from 3.0-4.0,(c) an anion exchange chromatography,(d) the conversion of the N-terminal glutamine and/or glutamic ...

Method of Isolating and Purifying Fusion Protein Comprising Factor VII

The present invention provides a method of isolating and purifying a fusion protein comprising factor VII, and more specifically relates to a method of isolating and purifying a fusion protein comprising factor VII and transferrin, to a high degree of purity. Because the present invention provides a method whereby a recombinant fusion protein comprising factor VII can be isolated and purified to a high degree of purity, the invention is useful in producing a pharmaceutical preparation comprising factor VII that can be used in situations in which copious bleeding occurs such as surgery. 2. The method of claim 1 , wherein the animal cells are selected from the group consisting of CHO cells claim 1 , BHK21 cells claim 1 , HEK293 cells claim 1 , and Hep G2 cells.3. The method of claim 1 , wherein the fusion protein comprising the factor VII is a protein in which the factor VII is fused to transferrin.4. The method of claim 3 , wherein the fusion protein comprises a linker between the factor VII and the transferrin.5. The method of claim 4 , wherein the linker is a peptide having an amino acid sequence set forth in one of SEQ ID NOs: 3 to 12.6. The method of claim 1 , wherein the affinity chromatography in step 1 uses an aqueous buffer (pH 5.0 to 8.0) containing sodium thiocyanate as an elution buffer.7. The method of claim 1 , wherein the mixed-mode chromatography in step 1 uses an aqueous buffer (pH 5.0 to 8.0) comprising sodium phosphate and sodium chloride.8. The method of claim 1 , wherein the anion exchange chromatography in step 2 uses an aqueous buffer (pH 5.0 to 8.0) claim 1 , which comprises 10 mM to 50 mM Tris claim 1 , 1 mM to 10 mM calcium chloride claim 1 , and 50 mM to 150 mM sodium chloride claim 1 , as an elution buffer. The present invention relates to a method of isolating and purifying a fusion protein containing factor VII (FVII), and more particularly, to a method of isolating and purifying a fusion protein including FVII and transferrin to a high ...

FRACTIONATION METHOD

Provided herein is a novel method of purifying an IgG antibody from a preparation by use of an electropositive membrane having a defined porosity. 1. A method of purifying an IgG antibody from a preparation containing the antibody and contaminants comprising:(a) contacting the preparation with an electropositive membrane having a porosity that retains at least 50% of non-adsorbed solutes with a hydrodynamic diameter greater than a selected size but permits passage of non-adsorbed solutes with a hydrodynamic diameter less than the selected size, wherein the selected size is about 10 nm, further wherein during at least a portion of the contacting step the preparation comprises (i) a salt at a concentration less than about 50 mM and a pH in a range from about 3 to within about 0.5 pH units of-an isoelectric point of a most alkaline glycoform of the IgG antibody in the preparation; or (ii) a salt at concentration greater than about 50 mM and a pH from about 3 to about 9; and(b) a final contacting step defined by either (i) an absence of excess salt in the preparation or a non-zero salt concentration in the preparation of not greater than 20 mM, and (ii) a pH value in a range from about 5 to within about 0.5 pH units of the isoelectric point of the most alkaline glycoform of the IgG antibody.2. The method of claim 1 , wherein the selected size is approximately 11 nm claim 1 , approximately 12 nm claim 1 , approximately 13 nm claim 1 , or approximately 14 nm.35-. (canceled)6. The method of claim 1 , wherein the electropositive membrane has a porosity that retains at least 60% to at least 99% of the non-adsorbed solutes.711-. (canceled)12. The method of claim 1 , wherein during a portion of the contacting step of (a)(i) claim 1 , the preparation comprises an absence of salt or during a portion of the contacting step of (a)(ii) claim 1 , the preparation comprises the presence of a salt at 50 mM up to saturation.13. The method of claim 1 , wherein the salt is selected from ...

Antibody Preparation

The present invention relates to the field of biotechnology, and in particular to the field of antibodies. Provided herein are novel methods for removing glycosylated antibody variants from an antibody preparation, an antibody preparation obtained by said method, and a pharmaceutical composition comprising the same. 1. A method for reducing the amount of a CDR-H1 glycosylated antibody variant that competes for binding to human VEGF-A with the antibody bevacizumab in a preparation comprising said antibody variant , the method comprising the following steps: (i) subjecting said preparation to cation exchange chromatography (CEX) , size exclusion chromatography (SEC) or lectin affinity purification (LAP) , (ii) analyzing fractions obtained in step (i) for the presence of said CDR-H1 glycosylated antibody variant by HPLC-MS(/MS); and (iii) removing fractions comprising said CDR-H1 glycosylated antibody variant.2. The method of claim 1 , further comprising changing a purification process for bevacizumab claim 1 , by adjusting splitting criteria for a CEX-step and/or introducing an SEC purification step.3. The method of claim 1 , wherein said competition is measured by ELISA claim 1 , flow cytometry or surface plasmon resonance (SPR) assay.4. The method of claim 1 , wherein said antibody comprises a CDR-H1 having the amino acid sequence shown in SEQ ID NO: 1 (SGYTFTNYGMN) claim 1 , wherein the first N is glycosylated.5. The method of claim 1 , wherein said N-glycosylation is a complex type glycosylation.6. The method of claim 1 , wherein said CDR-H1 glycosylated variant (i) has a reduced binding affinity to VEGF in comparison to a non-CDR-H1 glycosylated variant and/or (ii) shows a reduced inhibition on the VEGF-dependent proliferation of HUVEC cells in comparison to a non-CDR-H1 glycosylated variant.7. The method of claim 1 , wherein said antibody comprises a CDR-H1 shown in SEQ ID NO: 1 claim 1 , CDR-H2 shown in SEQ ID NO: 2 claim 1 , CDR-H3 shown in SEQ ID NO: 3 claim ...

RECOMBINANT PICHIA PASTORIS STRAIN AND METHOD FOR PRODUCING ACTIVE PEDIOCIN PA-1

This invention provides a recombinant strain which has been genetically modified for highly expressing pediocin PA-1 and secreting it into the medium so that the pediocin PA-1 can be easily collected and purified from the culture medium. In this invention, we also demonstrated that the obtained pediocin PA-1 had specific antimicrobial activities against ATCC 13932 and several other bacteria strains. Therefore, pediocin PA-1 produced from this recombinant strain has a great potential for the application in food preservation and pharmaceutical industry. 1. A pediocin PA-1 substance produced from a recombinant yeast strain which is transformed with a DNA sequence coding for pediocin PA-1.2Pachia pastoris. The pediocin PA-1 substance according to wherein said yeast strain is a strain which is deposited as NRRL Y-67635.3. The pediocin PA-1 substance according to wherein said DNA sequence encoded as SEQ ID No. 1.4. The pediocin PA-1 substance according to wherein said pediocin PA-1 substance expresses antimicrobial activity against a plurality of target bacterial strains.5Listeria monocytogenesPediococcus acidilacticiListeria innocua, Listeria monocytogenesVibrio parahaemolyticus, Enterococcus faecalis, Streptococcus thermophllusLactobacillus plantarum.. The pediocin PA-1 substance according to wherein said target bacterial strains further comprises ATCC 1393 claim 4 , PAC1.0. VSVTCC claim 4 , claim 4 , and6. The pediocin PA-1 substance according to wherein said antimicrobial activity of said pediocin PA-1 is fully maintained for at least one hour in 100° C. and at least 20 minutes in 121° C.7. The pediocin PA-1 substance according to wherein said antimicrobial activity is stable in a pH levels ranging from 1 to 12.8. A method for producing an active pediocin PA-1 from a yeast strain claim 5 , comprising:(i) transforming a DNA sequence coding for pediocin PA-1 into a yeast strain under a preset condition to generate a recombinant yeast strain capable of producing said ...

APPARATUS AND PROCESS FOR PURIFICATION OF PROTEINS

The invention is directed to an apparatus and method for purifying a protein. The apparatus involves the use of a capture chromatography resin, a depth filter arranged after the capture chromatography resin, and a mixed-mode chromatography resin arranged after the depth filter. The method involves providing a sample containing the protein, processing the sample through a capture chromatography resin, a depth filter, and a mixed-mode chromatography resin. A membrane adsorber or monolith may be substituted for the mixed-mode chromatography column. 120-. (canceled)21. A method for purifying a protein comprising:a. providing a sample containing the protein;b. processing the sample through a capture chromatography resin to provide a first eluate comprising the protein;c. after the sample is processed through the capture chromatography resin, processing the first eluate through a depth filter to provide a filtered eluate comprising the protein; andd. after the first eluate is processed through the depth filter, processing the filtered eluate through a mixed-mode chromatography resin to provide a second eluate comprising the protein.22. The method of wherein the capture chromatography resin is selected from the group consisting of an affinity resin claim 21 , an ion exchange resin claim 21 , and a hydrophobic interaction resin.23. The method of wherein the capture chromatography resin is selected from the group consisting of a protein A resin claim 21 , a protein G resin claim 21 , a protein A/G resin claim 21 , and a protein L resin.24. The method of wherein the protein is selected from the group consisting of a protein fragment claim 21 , an antibody claim 21 , a monoclonal antibody claim 21 , an immunoglobulin claim 21 , and a fusion protein.25. The method of wherein the sample is a cell culture.26. The method of wherein the sample is clarified prior to processing through the capture chromatography resin.27. The method of wherein the sample is clarified by a ...

METHOD FOR PREPARING A CONCENTRATE OF POLYVALENT IMMUNOGLOBULIN

The present invention relates to a method for preparing a concentrate of polyvalent immunoglobulins with view to therapeutic use, from an initial solution of blood plasma or a plasma fraction enriched with immunoglobulins, comprising the steps for removing the protein contaminants by precipitation with caprylic acid in order to obtain a solution free of proteases, and for separating by chromatography on a fluidized bed the solution free of proteases, said method allowing a concentrate of human polyvalent immunoglobulins with a yield of more than 4.5 g of immunoglobulins per liter of blood plasma applied to be obtained. 1. A method for preparing a concentrate of human polyvalent immunoglobulins from an initial solution of blood plasma or a fraction of plasma enriched with immunoglobulins , comprising:(a) removing protein contaminants by contacting the initial solution of blood plasma or a fraction of plasma enriched with immunoglobulins with caprylic acid in order to obtain a solution free of proteases,(b) fluidized bed chromatography of the solution free of proteases, to obtain a concentrate of human polyvalent immunoglobulins with a yield of more than 4.5 g of immunoglobulins per liter of blood plasma.2. The method according to claim 1 , wherein the concentration of caprylic acid ranges from 0.5 to 1.5.3. The method according to claim 1 , wherein the step for removing protein contaminants with caprylic acid (a) is carried out at a pH between 4.3 and 4.9.4. The method according to claim 1 , further comprising between the step for removing protein contaminants with caprylic acid (a) and the fluidized bed chromatography step (b) claim 1 , a clarification step at an acid pH.5. The method according to claim 1 , wherein the fluidized bed chromatography step for the solution free of proteases (b) is carried out with a chromatographic support of the ion exchange type claim 1 , of the affinity type of the pseudo-affinity type or of the mixed-mode type.6. The method ...

Method for separation of monomeric polypeptides from aggregated polypeptides

The present invention relates to methods for obtaining a polypeptide in a monomeric form, the method comprising a) providing a solution containing the polypeptide in monomeric form and in aggregated form, wherein the ratio of monomeric to aggregated form is 4:1 or less, as determined by size exclusion chromatography, b) performing mixed-mode chromatography in bind-and-elute mode, or hydrophobic interaction chromatography in flow-through mode, or a size-exclusion chromatography, and c) performing a weak cation exchange chromatography in bind-and-elute mode or flow-through mode, and thereby obtaining the polypeptide in monomeric form.

PROTEIN PURIFICATION METHODS TO REDUCE ACIDIC SPECIES

The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for controlling the amount of charge variants, aggregates, and fragments of a protein of interest, as well as host cell proteins, present in purified preparations by applying particular chromatography conditions during such protein purification. 1. A method of making a pharmaceutical composition , comprising mixing(a) a composition comprising adalimumab, wherein the composition comprises less than 10% total acidic species of adalimumab, wherein the acidic species of adalimumab correspond to the peaks that elute earlier than the main peak in a WCX-10 HPLC chromatogram of adalimumab, wherein the WCX-10 HPLC chromatogram is generated using a first mobile phase of 10 mM Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and wherein the WCX-10 HPLC chromatogram is generated using detection at 280 nm; and(b) a pharmaceutically acceptable carrier, thereby making a pharmaceutical composition.2. The method of claim 1 , wherein the acidic species of adalimumab comprise a first acidic region (AR1) and a second acidic region (AR2).3. The method of claim 1 , wherein the adalimumab is produced in a mammalian host cell grown in cell culture.4. The method of claim 3 , wherein the mammalian host cell is selected from the group consisting of a CHO cell claim 3 , an NS0 cell claim 3 , a COS cell claim 3 , and an SP2 cell.5. The method of claim 1 , wherein the low acidic species composition comprises less than 3.8% total acidic species of adalimumab.6. The method of claim 2 , wherein the low acidic species composition comprises 0.8% AR1 and 3.0% AR2.7. The method of claim 1 , wherein the low acidic species composition comprises less than 2.4% total acidic species of adalimumab.8. The method of claim 2 , wherein the low acidic species composition comprises 0.2% AR1 and 2.2% AR2.9. ...

BIVALENT BISPECIFIC ANTIBODY HYBRID PROTEIN EXPRESSION AND PREPARATION METHODS

The present disclosure relates to the field of biological technologies, and discloses a method for expressing and preparing a bivalent bispecific antibody. In the present disclosure, each portion of a bivalent bispecific antibody and an immune hybrid protein thereof is respectively expressed in a suitable prokaryotic or eukaryotic cell system, separated and purified by high-performance affinity chromatography, and then spliced in vitro by trans-splicing reaction mediated by an intein, to prepare the bivalent specific antibody and an immune hybrid protein thereof. The method has high production efficiency and wide scope of application, and facilitates the separation and purification of the products. The product prepared through the present method has bivalent bispecific immune affinity and cytotoxic bioactivity. Therefore, the present disclosure provides a novel method for preparing biological drugs for directionally attacking cancers or other diseases. 1. A method for expressing and preparing a bivalent bispecific antibody , the bivalent bispecific antibody comprising a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen , and a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen , the method comprising the following steps:S1: splitting an expressed sequence of the bivalent bispecific antibody, to obtain a portion A antibody and a portion B antibody, where the portion A antibody comprises the first light chain, the first heavy chain, and an Fc chain of the second heavy chain having Ic fused to the N terminus; and the portion B antibody comprises the second light chain and a VH+CH1 chain of the second heavy chain having In fused to the C terminus;S2: constructing a mammalian cell expression vector by whole-gene synthesis, to obtain a vector or vectors expressing the portion A antibody and a vector or vectors expressing the portion B antibody;S3: expressing mammalian ...

METHOD FOR PREPARING GLY-TB4

The present invention relates to a method for preparing glycine-thymosin β4 (Gly-Tβ4). The present invention has an excellent effect in that, according to the present invention, a large amount of high-purity Gly-Tβ4 can be obtained with high productivity through chromatography, enzyme treatment, and filtration of a sample containing GST fusion thymosin β4 (GST-Tβ4). 1. A method of preparing glycine-thymosin β4 (Gly-thymosin β4 , Gly-Tβ4) , comprising:(S1) loading a sample in a primary anion exchange chromatography column equilibrated with an equilibration buffer, washing the column with a wash buffer, and eluting a fraction attached to the column with the elution buffer;(S2) performing affinity chromatography on an eluate;(S3) enzymatically cleaving the eluate by thrombin treatment;(S4) loading the cleaved product in a secondary anion exchange chromatography column equilibrated with an equilibration buffer, washing the column with a washing buffer, and eluting a fraction attached to the column with an elution buffer at a dynamic binding capacity (DBC) of 3 mg/mL or less; '(S6) filtrating the eluate.', '(S5) loading the eluate in a cation exchange chromatography column equilibrated with an equilibration buffer, washing the column with a wash buffer, and eluting a fraction attached to the column with an elution buffer at a DBC of 3 to 8 mg/mL; and'}2. The method of claim 1 , wherein claim 1 , in Step (S4) claim 1 , the cleaved product is loaded in the column at pH 8 to 9 and a conductivity of 4 mS/cm or less.3. The method of claim 1 , wherein the fraction attached to the column in the anion exchange chromatography in Step (S4) is eluted with a buffer of pH 7 to 9 claim 1 , which contains 10 to 30 mM Tris-HCl and 30 to 130 mM NaCl claim 1 , at a flow rate of 100 to 300 cm/hr.4. The method of claim 1 , wherein claim 1 , in Step (S5) claim 1 , the eluted product is loaded in the column at pH 4.5±0.1 and a conductivity of 2±0.2 mS/cm or less.5. The method of claim 1 , ...

Method Of Maintaining Narrow Residence Time Distributions In Continuous Flow Systems

Methods of maintaining narrow residence time distributions in continuous flow systems, particularly applicable to virus inactivation such as during a protein purification process. Fluid sample is introduced into an axial flow channel and caused to flow therein in discrete packets or zones to minimize residence time distribution and axial dispersion. Embodiments described herein obviate or minimize the need for using large tanks or reservoirs for performing virus inactivation during a protein purification process; reduce the overall time required for virus inactivation, and/or reduce the overall physical space required to perform the virus inactivation operation during a protein purification process, which in turn reduces the overall footprint for the purification process. 1. A method for maintaining a narrow residence time distribution of a fluid sample flowing in a fluid channel having an axial length , comprising causing said fluid sample to flow in discrete packets along said axial length within said fluid channel by introducing an immiscible fluid into said fluid channel to create an interface that separates the fluid into said discrete packets.2. The method of claim 1 , wherein said fluid sample has a nominal residence time in said fluid channel of one to two minutes.3. The method of claim 1 , wherein said fluid sample has a nominal residence time in said fluid channel of two to four minutes.4. The method of claim 1 , wherein said fluid sample has a nominal residence time in said fluid channel of four to six minutes.5. The method of claim 1 , wherein said fluid sample has a nominal residence time in said fluid channel of six to eight minutes.6. The method of claim 1 , wherein said fluid sample has a nominal residence time in said fluid channel of eight to ten minutes.7. The method of claim 1 , wherein said fluid sample has a nominal residence time in said fluid channel of ten to fifteen minutes.8. The method of claim 1 , wherein said fluid sample has a nominal ...