Methods of Evaluating Peptide Mixtures

The presently disclosed subject matter provides methods for evaluating and characterizing peptides, peptide mixtures, and polypeptide mixtures. More particularly, the presently disclosed subject matter provides methods for evaluating or characterizing complex peptide or polypeptide mixtures comprising glutamic acid, alanine, tyrosine, and lysine, e.g., Copolymer-1 or glatiramer acetate, including, but not limited to, methods of identifying. isolating, quantifying, and purifying amino acids, peptides, polypeptides, and combinations thereof having a diethylamide group instead of a carboxyl group present on the C-terminus. The presently disclosed methods can be used to determine the mole percent of polypeptides having a diethylamide group at a C-terminus thereof and can be used to evaluate one or more properties of a sample of one polypeptide mixture as compared to one or more properties of a different sample of a polypeptide mixture. 1. A method for detecting a modification of at least one C-terminus of one or more amino acids , peptides , polypeptide chains , and combinations thereof in a sample , the method comprising:(a) providing a sample suspected of containing one or more amino acids, peptides, polypeptide chains, and combinations thereof, having at least one modified C-terminus; and(b) analyzing the sample by a method capable of detecting a modification of at least one C-terminus of an amino acid, peptide, polypeptide chains, and combinations thereof in the sample.2. The method of claim 1 , wherein the modification of at least one C-terminus includes at least one C-terminus of the one or more amino acids claim 1 , peptides claim 1 , polypeptide chains claim 1 , and combinations thereof in the sample having a diethylamide moiety bound thereto.3. The method of claim 1 , wherein the one or more amino acids is selected from the group consisting of alanine claim 1 , glutamic acid claim 1 , lysine claim 1 , and tyrosine.4. The method of claim 3 , wherein one or more ...

EVALUATION OF COPOLYMER DIETHYLAMIDE

Methods of analyzing glatiramer acetate (GA) or a polymeric precursor thereof are provided. The methods can include determining a level of one or more diethylamide-modified amino acids in a sample comprising GA or a polymeric precursor thereof, and selecting at least a portion of the sample based on the assessment of the one or more diethylamide-modified amino acids in the sample. 1. (canceled)2. A method of manufacturing glatiramer acetate drug product , the method comprising:preparing an amino acid copolymer of L-glutamic acid, L-alanine, L-Lysine, and L-tyrosine;measuring, in at least one sample of the copolymer, the levels of at least two, three, or four individual diethylamide-modified amino acids selected from the group consisting of: diethylamide-modified alanine, diethylamide-modified lysine, diethylamide-modified glutamic acid, and diethylamide-modified tyrosine; andprocessing the copolymer to produce glatiramer acetate drug product if the measured levels of at least one of the at least two, three, or four measured individual diethylamide-modified amino acids meets commercial release specification (i), (ii), (iii), or (iv):(i) the level of diethylamide-modified alanine in the sample is 59.5-76.1% of the total diethylamide-modified amino acids in the sample on a mol percent basis;(ii) the level of diethylamide-modified lysine detected in the sample is 11.3-17.3% of the total diethylamide-modified amino acids in the sample on a mol percent basis;(iii) the level of diethylamide-modified glutamic acid detected in the sample is 9.9-15.0% of the total diethylamide-modified amino acids in the sample on a mol percent basis; or(iv) the level of diethylamide-modified tyrosine detected in the sample is 4.8-7.2% of the total diethylamide-modified amino acids in the sample on a mol percent basis,thereby manufacturing glatiramer acetate drug product.3. The method of wherein:the step of preparing comprises co-polymerizing N-carboxy anhydrides of L-alanine, benzyl- ...

EVALUATION OF COPOLYMER DIETHYLAMIDE

Methods of analyzing glatiramer acetate (GA) or a polymeric precursor thereof are provided. The methods can include determining a level of one or more diethylamide-modified amino acids in a sample comprising GA or a polymeric precursor thereof, and selecting at least a portion of the sample based on the assessment of the one or more diethylamide-modified amino acids in the sample. 1. (canceled)2. A method of comprising(a) providing a batch of glatiramer acetate(b) obtaining one or more samples of the batch of glatiramer acetate; (i) cleaving the glatiramer acetate to generate peptide fragments;', '(ii) combining the peptide fragments with detectably labeled diethylamide-modified peptide standards;', '(iii) fractioning the mixture of peptide fragments and detectably labeled diethylamide-modified peptide standards to generate subpopulations; and', '(iv) detecting the peptide fragments and detectably labeled diethylamide-modified peptide standards in one or more subpopulations by a method selected from mass spectroscopy analysis, MS-MRM detection, tandem mass spectrophotometry (MS/MS), and NMR., '(c) measuring, in at least one of the one or more samples of the batch, the level of at least one individual diethylamide-modified amino acid selected from the group consisting of: diethylamide-modified alanine, diethylamide-modified lysine, diethylamide-modified glutamic acid, and diethylamide-modified tyrosine using a method that comprises3. The method of wherein the peptide fragments comprise peptides and amino acids.4. The method of wherein the average length of the peptide fragments is less than 4 amino acids.5. The method of wherein detectably labeled diethylamide-modified peptide standards comprise diethylamide-modified peptides and diethylamide-modified amino acids.6. The method of wherein the step of providing a batch of glatiramer acetate comprises: (i) polymerizing N-carboxy anhydrides of L-alanine claim 2 , benzyl-protected L-glutamic acid claim 2 , trifluoroacetic ...

METHODS OF EVALUATING PEPTIDE MIXTURES

The presently disclosed subject matter provides methods for evaluating and characterizing peptides, peptide mixtures, and polypeptide mixtures. More particularly, the presently disclosed subject matter provides methods for evaluating or characterizing complex peptide or polypeptide mixtures comprising glutamic acid, alanine, tyrosine, and lysine, e.g., Copolymer-1 or glatiramer acetate, including, but not limited to, methods of identifying, isolating, quantifying, and purifying amino acids, peptides, polypeptides, and combinations thereof having a diethylamide group instead of a carboxyl group present on the C-terminus. The presently disclosed methods can be used to determine the mole percent of polypeptides having a diethylamide group at a C-terminus thereof and can be used to evaluate one or more properties of a sample of one polypeptide mixture as compared to one or more properties of a different sample of a polypeptide mixture. 198-. (canceled)99. A method of evaluating diethylamide formed during the manufacture of glatiramer acetate , the method comprising:{'figref': {'@idref': 'DRAWINGS', 'FIG. 1'}, 'preparing a copolymer of glutamic acid, alanine, lysine, and tyrosine according to the process of ;'}measuring, in a sample of the copolymer, a level of diethylamide to determine an amount of polypeptides in the sample having a diethylamide-modified C-terminal amino acid;processing the copolymer to produce glatiramer acetate if the amount of polypeptides in the sample having a diethylamide-modified C-terminal amino acid is within 7 mol % to 20 mol % polypeptides having a diethylamide-modified C-terminal amino acid.100. The method of claim 99 , comprising processing the copolymer to produce glatiramer acetate if the amount of polypeptides in the sample having a diethylamide-modified C-terminal amino acid is within 8 mol % to 18 mol % polypeptides having a diethylamide-modified C-terminal amino acid101. The method of claim 99 , processing the copolymer to produce ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains. 1. (canceled)2. A method for treating an inflammatory or autoimmune or immune disease comprising administering to a subject in need thereof an Fc construct comprising: i) A comprises a first Fc domain monomer;', 'ii) L is a linker; and', 'iii) B comprises a second Fc domain monomer;, 'a) a first polypeptide having the formula A-L-B; wherein'} i) A′ comprises a third Fc domain monomer;', 'ii) L′ is a linker; and', 'iii) B′ comprises a fourth Fc domain monomer;, 'b) a second polypeptide having the formula A′-L′-B′; wherein'}c) a third polypeptide that comprises a fifth Fc domain monomer; andd) a fourth polypeptide that comprises a sixth Fc domain monomer;wherein A and A′ combine to form a first Fc domain, B and the fifth Fc domain monomer combine to form a second Fc domain, and B′ and the sixth Fc domain monomer combine to form a third Fc domain;the first polypeptide comprises the same amino acid sequence as the second polypeptide, and the third polypeptide comprises the same amino acid sequence as the fourth polypeptide;the second Fc domain monomer comprises a different amino acid sequence than the fifth Fc domain monomer, and the fourth Fc domain monomer comprises a different amino acid sequence than the sixth Fc domain monomer, andeach of the first, second, third, fourth, fifth, and sixth Fc domain monomers comprises a human IgG1 CH2 domain monomer and a human IgG1 CH3 domain monomer, wherein each CH3 domain monomer has no more than 10 single amino acid substitutions to promote complementary dimerization of Fc domain monomers.3. The method of claim 2 , whereineach of the CH3 domain monomers of the first and third Fc domain monomers comprises a complementary dimerization selectivity module that promote dimerization between the first Fc domain monomer and the third Fc domain monomer,each of the CH3 domain monomers of ...

Sialylated Glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described. 1. A method of producing a sialylated IgG antibody preparation wherein at least 60% of the branched glycans on the Fc domain of the IgG antibodies comprise a terminal sialic acid on both the alpha 1 ,3 arm and the alpha 1 ,6 arm , the method comprising:providing an IgG antibody preparation;combining the IgG antibody preparation with a beta 1,4 galactosyltransferase and uridine diphosphate galactose a galactose donor to provide a galactosylation reaction mixture;incubating the galactosylation reaction mixture to allow galactosylation of branched glycans; adding a ST6 beta-galactoside alpha-2,6-sialyltransferase 1 having at least 95% identity to amino acids 95-416 of SEQ ID NO:1 and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA) an ST6-Gal1 sialyltransferase and a sialic acid donor to the galactosylation reaction mixture to provide a sialylation reaction mixture; andincubating the sialylation reaction mixture for a sufficient period of time to allow least 60% of the branched glycans on the Fc domain of the IgG antibodies in the preparation to be sialylated on both the alpha 1,3 arm and on the alpha 1,6 arm, wherein additional CMP-NANA sialic acid donor is added to the sialylation reaction mixture during the incubation of the sialylation reaction mixture.2. The method of claim 1 , wherein the CMP-NANA sialic acid donor is added periodically.3. The method of or claim 1 , wherein the galactosylation reaction mixture and the sialylation reaction mixture comprise MnCl. This application is a continuation of U.S. application Ser. No. 16/589,634, filed on Oct. 1, 2019, which is a continuation of U.S. application Ser. No. 15/954,146, filed on Apr. 16, 2018, which is a continuation of U.S. application Ser. No. 14/787,403, filed on Oct. 27, 2015 (now abandoned), which is a U.S. national stage application under 35 USC § 371 of International ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present disclosure relates to engineered IgG Fc constructs and uses thereof. 112.-. (canceled)14. The method of claim 13 , wherein the subject has an autoimmune disease.17. The cell culture medium of claim 16 , wherein the substantially homogenous population of the Fc construct is at least 85% homogenous.19. The method of claim 18 , wherein the substantially homogenous population of the Fc construct is at least 85% homogenous. This application is a divisional application of Nonprovisional patent application Ser. No. 16/303,793 filed Nov. 21, 2018, which is a National Stage Application under U.S.C. § 371 of International Application No. PCT/US2017/034084, filed May 23, 2017, which claim the benefit of prior co-pending U.S. Provisional Application Ser. No. 62/340,322, filed May 23, 2016, and or prior co-pending U.S. Provisional Application Ser. No. 62/443,451, filed Jan. 6, 2017. The disclosures of the above applications are hereby incorporated by reference in their entirety.This document includes a sequence listing submitted to the United States Patent and Trademark Office via the electronic filing system as an ASCII text file. The sequence listing, which is incorporated-by-reference herein, is titled “12320197.txt”, was created on Sep. 9, 2021, and has a size of 71.6 kilobytes.Therapeutic proteins, e.g., therapeutic antibodies and Fc-fusion proteins, have rapidly become a clinically important drug class for patients with immunological and inflammatory diseases, cancers, and infections.The present disclosure features biologically active Fc domain-containing therapeutic constructs. Such constructs may have desirable serum half-life and/or binding affinity and/or avidity for Fc receptors.In general, the disclosure features Fc constructs having 2-10 Fc domains, e.g., Fc constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains. In some embodiments, the Fc construct includes 2-10 Fc domains, 2-5 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 3-5 Fc domains, 2-8 Fc ...

Sialylated glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

COMPOSITIONS AND METHODS RELATED TO ENGINEERED FC-ANTIGEN BINDING DOMAIN CONSTRUCTS

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain. 1. An Fc-antigen binding domain construct comprising enhanced effector function , wherein the Fc-antigen binding domain construct comprises an antigen binding domain and a first Fc domain joined to a second Fc domain by a linker , wherein the Fc-antigen binding domain construct has enhanced effector function in an antibody-dependent cytotoxicity (ADCC) assay , an antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) assay relative to a construct having a single Fc domain and the antigen binding domain.2. A polypeptide comprising an antigen binding domain; a linker; a first IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a C3 domain ,wherein at least one Fc domain monomer comprises mutations forming an engineered protuberance.3. The polypeptide of wherein the antigen binding domain comprises an antibody heavy chain variable domain.4. The polypeptide of wherein the antigen binding domain comprises an antibody light chain variable domain.5. The polypeptide of wherein the first IgG1 Fc domain monomer comprises two or four reverse charge mutations and the second IgG1 Fc domain monomer comprises mutations forming an engineered protuberance.6. The polypeptide of wherein the first IgG1 Fc domain monomer comprises mutations forming an engineered protuberance and the second IgG1 Fc domain monomer comprises two or four reverse charge mutations.7. The polypeptide of wherein both the first IgG1 Fc domain monomer and the second IgG constant domain monomer ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc-ANTIGEN BINDING DOMAIN CONSTRUCTS TARGETED TO CTLA-4

Fc-antigen binding constructs having a CTLA-4 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

SIALYLATED GLYCOPROTEINS

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described. 1. A method of producing a preparation of glycoproteins comprising Fc regions comprising branched glycans comprising an α1 ,3 arm and an α1 ,6 arm , the preparation comprising (i) a target level of branched glycans having a sialic acid on an α1 ,3 arm and/or (ii) a target level of branched glycans having a sialic acid on an α1 ,6 arm , the method comprising:providing a plurality of glycoproteins comprising Fc regions comprising branched glycans comprising an α1,3 arm and an α1,6 arm; andcontacting the glycoproteins with an ST6 sialyltransferase in the presence of a limited reaction condition, thereby producing a glycoprotein preparation having (i) the target level of branched glycans having a sialic acid on the α1,3 arm and/or (ii) the target level of branched glycans having a sialic acid on an α1,6 arm.2. The method of claim 1 , wherein the limited reaction condition is sufficient for the ST6 sialyltransferase substantially to add a sialic acid to an α1 claim 1 ,3 arm of a branched glycan and not sufficient for the ST6 sialyltransferase substantially to add a sialic acid to an α1 claim 1 ,6 arm of a branched glycan.3. The method of claim 1 , further comprising isolating the glycoprotein preparation.4. The method of claim 3 , further comprising measuring a level of branched glycans comprising a sialic acid on an α1 claim 3 ,3 arm and/or measuring a level of branched glycans having a sialic acid on an α1 claim 3 ,6 arm.5. The method of claim 1 , wherein the target level of branched glycans having a sialic acid on an α1 claim 1 ,3 arm is at least 20% claim 1 , 25% claim 1 , 30% claim 1 , 35% claim 1 , 40% claim 1 , 45% claim 1 , 50% claim 1 , 55% claim 1 , 60% claim 1 , 65% claim 1 , 70% claim 1 , 75% claim 1 , 80% claim 1 , 85% claim 1 , 90% claim 1 , 95% claim 1 , or 100% of sialylated branched glycans.6. The method of claim 1 , wherein the target ...

ENGINEERED Fc CONSTRUCTS

The present disclosure relates to engineered IgG Fc constructs and uses thereof.

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc-ANTIGEN BINDING DOMAIN CONSTRUCTS

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain. 1. A polypeptide comprising an antigen binding domain; a linker; a first IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain ,wherein at least one Fc domain monomer comprises mutations forming an engineered protuberance,and wherein at least one other Fc domain monomer comprises at least one, two or three reverse charge mutations.254.-. (canceled)55. A polypeptide complex comprising a polypeptide of joined to a second polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain claim 1 , a CH2 domain and a CH3 domain claim 1 , wherein the polypeptide and the second polypeptide are joined by disulfide bonds between cysteine residues within the hinge domain of the first claim 1 , second or third IgG1 Fc domain monomer of the polypeptide and the hinge domain of the second polypeptide.5678.-. (canceled)79. The polypeptide complex of claim 55 , wherein the polypeptide complex is further joined to a third polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain claim 55 , a CH2 domain and a CH3 domain claim 55 , wherein the polypeptide and the third polypeptide are joined by disulfide bonds between cysteine residues within the hinge domain of the first claim 55 , second or third IgG1 Fc domain monomer of the polypeptide and the hinge domain of the third polypeptide claim 55 , wherein the second and third polypeptides join to different IgG1 Fc domain monomers of the polypeptide.8082.-. (canceled)83. The polypeptide complex of claim 55 , wherein the ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present disclosure relates to compositions and methods of engineered IgG Fc constructs, wherein the Fc constructs include one or more Fc domains. 1. An Fc construct comprising: i) A comprises a first Fc domain monomer;', 'ii) L is a linker; and', 'iii). B comprises a second Fc domain monomer;, 'a) a first polypeptide having the formula A-L-B; wherein'}b) a second polypeptide comprising a third Fc domain monomer; andc) a third polypeptide comprising a fourth Fc domain monomer;wherein the first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain and the second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain; andwherein at least one of the Fc domains comprises at least one amino acid modification that alters: one or more of: (i) binding affinity to one or more Fc receptors, (ii) an effector function, (iii) the level of Fc domain sulfation, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and/or (vii) susceptibility to degradation.2. The Fc construct of claim 1 , wherein the first Fc domain monomer and the third Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the third Fc domain monomer.3. The Fc construct of claim 1 , wherein the second Fc domain monomer and the fourth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the second Fc domain monomer and the fourth Fc domain monomer.4. The Fc construct of claim 1 , wherein A consists of an Fc domain monomer.5. The Fc construct of claim 1 , wherein B consists of an Fc domain monomer.6. The Fc construct of claim 1 , wherein the second polypeptide consists of an Fc domain monomer.7. The Fc construct of claim 1 , wherein the third polypeptide consists of an Fc domain monomer.8. (canceled)9. The Fc construct of claim 1 , further comprising an IgG Cantibody constant domain and an IgG C1 ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc-ANTIGEN BINDING DOMAIN CONSTRUCTS

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain. 1. A polypeptide comprising an antigen binding domain; a linker; a first IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain , a CH2 domain and a CH3 domain ,wherein at least one Fc domain monomer comprises mutations forming an engineered protuberance,and wherein at least one other Fc domain monomer comprises at least one, two or three reverse charge mutations.259.-. (canceled)60. A polypeptide complex comprising a polypeptide of joined to a second polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain claim 1 , a CH2 domain and a CH3 domain claim 1 , wherein the polypeptide and the second polypeptide are joined by disulfide bonds between cysteine residues within the hinge domain of the first claim 1 , second or third IgG1 Fc domain monomer of the polypeptide and the hinge domain of the second polypeptide.6164.-. (canceled)65. The polypeptide complex of claim 60 , wherein the polypeptide complex is further joined to a third polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain claim 60 , a CH2 domain and a CH3 domain claim 60 , wherein the polypeptide and the third polypeptide are joined by disulfide bonds between cysteine residues within the hinge domain of the first claim 60 , second or third IgG1 Fc domain monomer of the polypeptide and the hinge domain of the third polypeptide claim 60 , wherein the second and third polypeptides join to different IgG1 Fc domain monomers of the polypeptide.6668.-. (canceled)69. The polypeptide complex of wherein the second ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains. 1. An Fc construct comprising: i). A comprises a first Fc domain monomer;', 'ii). L is a linker; and', 'iii). B comprises a second Fc domain monomer;, 'a). a first polypeptide having the formula A-L-B; wherein'} i). A′ comprises a third Fc domain monomer;', 'ii). L′ is a linker; and', 'iii). B′ comprises a fourth Fc domain monomer;, 'b). a second polypeptide having the formula A′-L′-B′; wherein'}c). a third polypeptide that comprises a fifth Fc domain monomer; andd). a fourth polypeptide that comprises a sixth Fc domain monomer;wherein A and A′ combine to form a first Fc domain, B and said fifth Fc domain monomer combine to form a second Fc domain, and B′ and said sixth Fc domain monomer combine to form a third Fc domain.2. The Fc construct of claim 1 , wherein each of said first and third Fc domain monomers comprises a complementary dimerization selectivity module that promote dimerization between said first Fc domain monomer and said third Fc domain monomer claim 1 ,each of said second and fifth Fc domain monomers comprises a complementary dimerization selectivity module that promote dimerization between said second Fc domain monomer and said fifth Fc domain monomer, and/oreach of said fourth and sixth Fc domain monomers comprises a complementary dimerization selectivity module that promote dimerization between said fourth Fc domain monomer and said sixth Fc domain monomer.3. The Fc construct of claim 2 , wherein the complementary dimerization selectivity modules of said first and third Fc domain monomers each comprise reverse charge mutations in at least two positions within a ring of charged residues at the interface between C3 domains.4. The Fc construct of claim 3 , wherein the reverse charge mutations in at least two positions are K409D/D339K claim 3 , K392D/D399K claim 3 , E357K/K370E claim 3 , D356K/K439D ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains. 1. A method for treating inflammation comprising administering to a subject in need thereof an Fc construct comprising: i). A comprises a first Fc domain monomer;', 'ii). L is a linker; and', 'iii). B comprises a second Fc domain monomer;, 'a). a first polypeptide having the formula A-L-B; wherein'} i). A′ comprises a third Fc domain monomer;', 'ii). L′ is a linker; and', 'iii). B′ comprises a fourth Fc domain monomer;, 'b). a second polypeptide having the formula A′-L′-B′; wherein'}c). a third polypeptide that comprises a fifth Fc domain monomer; andd). a fourth polypeptide that comprises a sixth Fc domain monomer;wherein A and A′ combine to form a first Fc domain, B and the fifth Fc domain monomer combine to form a second Fc domain, and B′ and the sixth Fc domain monomer combine to form a third Fc domain;the first polypeptide comprises the same amino acid sequence as the second polypeptide, and the third polypeptide comprises the same amino acid sequence as the fourth polypeptide;the second Fc domain monomer comprises a different amino acid sequence than the fifth Fc domain monomer, and the fourth Fc domain monomer comprises a different amino acid sequence than the sixth Fc domain monomer, and{'sub': H', 'H', 'H, 'each of the first, second, third, fourth, fifth, and sixth Fc domain monomers comprises a human IgG1 C2 domain monomer and a human IgG1 C3 domain monomer, wherein each C3 domain monomer has no more than 10 single amino acid substitutions to promote complementary dimerization of Fc domain monomers.'}2. The method of claim 1 , wherein{'sub': 'H', 'each of the C3 domain monomers of the first and third Fc domain monomers comprises a complementary dimerization selectivity module that promote dimerization between the first Fc domain monomer and the third Fc domain monomer,'}{'sub': 'H', 'each of the C3 domain ...

GLYCOPROTEIN PREPARATIONS

Preparations of glycoproteins, e.g., therapeutic preparations of glycoproteins, having altered levels of affinity for Fcγ receptors relative to reference glycoprotein preparations, and methods of making and methods of using such preparations, are described. 1. A glycoprotein composition comprising a glycoform comprising an Fc region comprising a target level of glycans selected from the group consisting of a high mannose glycan , a sialylated glycan , a galactosylated glycan , a glycan comprising a terminal N-acetylglucosamine , a fucosylated glycan , a sulfated glycan , and combinations thereof ,which composition is characterized in that, when it is contacted with an FcγIIIa receptor having a terminal glycan selected from the group consisting of mannose, N-acetylglucosamine, and beta-1,4 galactose, the composition shows an altered affinity for the FcγIIIa receptor as compared with a reference glycoprotein composition lacking the glycoform.2. The glycoprotein composition of claim 1 , wherein the target level of glycans is selected from one or more of:(a) about 10% to 100% high mannose glycan,(b) about 10% to 100% sialylated glycan,(c) about 10% to 100% galactosylated glycan,(d) about 10% to 100% terminal N-acetylglucosamine,(e) about 10% to 100% fucosylated glycan, and(f) about 10% to 100% sulfated glycan.3. A therapeutic preparation comprising the glycoprotein composition of or claim 1 , wherein the therapeutic preparation comprises about 10% to about 100% of said glycoform.4. The composition of or claim 1 , comprising a second glycoform comprising a second target level of (a)-(f).5. The preparation of claim 4 , wherein the composition is characterized in that claim 4 , when it is contacted with an FcγIIIa receptor having a terminal glycan selected from the group consisting of mannose claim 4 , N-acetylglucosamine claim 4 , and beta-1 claim 4 ,4 galactose claim 4 , the composition shows an altered affinity for the FcγIIIa receptor as compared with a reference ...

Sialylated Glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described. 1. A method of producing a sialylated IgG antibody preparation wherein at least 60% of the branched glycans on the Fc domain of the IgG antibodies comprise a terminal sialic acid on both the alpha 1 ,3 arm and the alpha 1 ,6 arm , the method comprising:providing an IgG antibody preparation;combining the IgG antibody preparation with a beta 1,4 galactosyltransferase and uridine diphosphate galactose a galactose donor to provide a galactosylation reaction mixture;incubating the galactosylation reaction mixture to allow galactosylation of branched glycans; adding a ST6 beta-galactoside alpha-2,6-sialyltransferase 1 having at least 95% identity to amino acids 95-416 of SEQ ID NO:1 and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA) to the galactosylation reaction mixture to provide a sialylation reaction mixture; andincubating the sialylation reaction mixture for a sufficient period of time to allow least 60% of the branched glycans on the Fc domain of the IgG antibodies in the preparation to be sialylated on both the alpha 1,3 arm and on the alpha 1,6 arm, wherein additional CMP-NANA sialic acid donor is added to the sialylation reaction mixture during the incubation of the sialylation reaction mixture.2. The method of claim 1 , wherein the CMP-NANA sialic acid donor is added periodically.3. The method of or claim 1 , wherein the galactosylation reaction mixture and the sialylation reaction mixture comprise MnCl. This application is a continuation of U.S. application Ser. No. 15/954,146, filed on Apr. 16, 2018, which is a continuation of U.S. application Ser. No. 14/787,403, filed on Oct. 27, 2015 (now abandoned), which is a U.S. national stage application under 35 USC § 371 of International Application Number PCT/US2014/036413, filed on May 1, 2014, which claims benefit to U.S. Provisional Application No. 61/818,563, filed May 2, 2013, ...

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present invention relates to engineered IgG Fc constructs and uses thereof. 3. The Fc construct of claim 1 , wherein the first polypeptide and the second polypeptide have the same amino acid sequence and wherein the third polypeptide and the fourth polypeptide have the same amino acid sequence.8. (canceled)9. The Fc construct of claim 1 , wherein one or more of the Fc domain monomers is an IgG Fc domain monomer.10. The Fc construct of claim 1 , wherein one or more of the Fc domain monomers is an IgG Fc domain monomer having up to ten amino acid modifications.11. The Fc construct of claim 1 , wherein each Fc domain monomer has no more than ten amino acid modifications.12. A pharmaceutical composition comprising the Fc construct of .13. A method of reducing immune cell activation of the immune response in a subject claim 1 , the method comprising administering to the subject an Fc construct of .14. The method of claim 13 , wherein the subject has an autoimmune disease.15. A method of treating inflammation in a subject claim 1 , the method comprising administering to the subject an Fc construct of .17. The cell culture medium of claim 16 , wherein the substantially homogenous population of the Fc construct is at least 85% homogenous.18. A method of preparing a cell culture medium comprising a substantially homogenous population of an Fc construct of claim 1 , the method comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'a) providing host cells in a cell culture medium, wherein the cells comprise polynucleotides encoding each of the first, second, third, and fourth polypeptides of ;'}b) expressing the polypeptides in the host cells under conditions that allow for the formation of the Fc construct and secretion into the cell culture medium; andc) collecting the cell culture medium.19. The method of claim 18 , wherein the substantially homogenous population of the Fc construct is at least 85% homogenous. This application claims the benefit of prior co- ...

Sialylated Glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described. 151-. (canceled)52. A method for treating inflammation , comprising administering a pharmaceutical composition comprising administering , to a patient in need thereof , a composition comprising modified IVG wherein greater than 60% of the branched glycans are sialylated on both the α1 ,3 arm and the α1 ,6 arm.53. The method of wherein greater than 70% of the branched glycans are sialylated on both the α1 claim 52 ,3 arm and the α1 claim 52 ,6 arm.54. The method of claim 52 , wherein the sialylated branched glycans have a NeuAc-α2 claim 52 ,6-Gal terminal linkage. This application claims benefit to U.S. Provisional Application No. 61/818,563, filed May 2, 2013, which is hereby incorporated by reference.The invention relates generally to glycobiology and glycoproteins.Therapeutic glycoproteins are an important class of therapeutic biotechnology products, and therapeutic Fc containing glycoproteins, such as IVIG, Fc-receptor fusions, and antibodies (including murine, chimeric, humanized and human antibodies and fragments thereof) account for the majority of therapeutic biologic products.The invention encompasses the discovery of a novel mechanism of sialylation by a sialyltransferase (ST6 Gal-I), which sialylates a substrate (e.g., an Fc-containing glycoprotein comprising branched glycans comprising an α1,3 arm and an α1,6 arm) in an ordered fashion. Specifically, under certain conditions, ST6 sialyltransferase catalyzes addition of a sialic acid on an α1,3 arm, followed by addition of a second sialic acid on an α1,6 arm, followed by removal of sialic acid from an α1,3 arm. Accordingly, activity of ST6 sialyltransferase can be controlled using methods described herein to produce glycoproteins having particular branch sialylation patterns.In one aspect, the invention features a method of producing a preparation of glycoproteins comprising Fc regions ...

Compositions and Methods Related to Engineered Fc Constructs

The present disclosure relates to engineered IgG Fc constructs and uses thereof. 36.-. (canceled)7. The Fc construct of claim 2 , wherein each amino acid substitution at position I253 is I253A.8. The Fc construct of claim 2 , wherein at least one Fc domain monomer comprises an amino acid substitution at position 8292.916.-. (canceled)1862.-. (canceled)83. The Fc construct of claim 2 , wherein each of the first and second polypeptides comprises the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGG GGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 78) claim 2 , andeach of the third and fourth polypeptides comprises the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73).84. The Fc construct of claim 2 , wherein each of the first and second polypeptides comprises the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGG GGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG (SEQ ID NO: 49) claim 2 , andeach of the third and fourth polypeptides comprises the sequence ...

compositions and methods related to manipulated fc constructs

a presente invenção refere-se a construtos de fc de igg manipulados e seus usos. The present invention relates to manipulated IgG Fc constructs and their uses.

Compositions and methods related to engineered fc constructs

The present disclosure relates to engineered IgG Fc constructs and uses thereof.

Sialylated glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

Compositions and methods related to engineered Fc constructs

The present invention relates to engineered IgG Fc constructs and uses thereof.

Engineered Fc constructs

The present disclosure relates to engineered IgG Fc constructs and uses thereof.

Compositions and methods related to engineered Fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Compositions and methods related to engineered Fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Compositions and methods related to engineered fc constructs

The present invention relates to engineered IgG Fc constructs and uses thereof.

Compositions and methods related to engineered fc-antigen binding domain constructs

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain.

COMPOSITIONS AND METHODS RELATED TO HANDLED FC-ANTIGEN CONNECTION CONSTRUCTIONS

composições e métodos relacionados a construtos do domínio de ligação fc-antígeno manipulados. a presente divulgação se refere a composições e métodos de construtos do domínio de ligação fc-antígeno manipulados, em que os construtos do domínio de ligação fc-antígeno incluem pelo menos dois domínios fc e pelo menos um domínio de ligação ao antígeno. compositions and methods related to manipulated fc-antigen binding domain constructs. the present disclosure relates to compositions and methods of constructs of the manipulated fc-antigen binding domain, wherein the constructs of the fc-antigen binding domain include at least two fc domains and at least one antigen binding domain.

Glycoprotein preparations

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to ctla-4

Fc-antigen binding constructs having a CTLA-4 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to ctla-4

Fc-antigen binding constructs having a CTLA-4 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization

Compositions and methods related to engineered fc-antigen binding domain constructs

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain.

Compositions and methods related to engineered Fc-antigen binding domain constructs

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain.

Compositions and methods related to engineered fc constructs

Evaluation of copolymer diethylamide

Methods of analyzing glatiramer acetate (GA) or a polymeric precursor thereof are provided. The methods can include determining a level of one or more diethylamide-modified amino acids in a sample comprising GA or a polymeric precursor thereof, and selecting at least a portion of the sample based on the assessment of the one or more diethylamide-modified amino acids in the sample.

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to ccr4

Fc-antigen binding constructs having a CCR4 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization.

Compositions and methods related to engineered fc constructs

The present disclosure relates to compositions and methods of engineered IgG Fc constructs, wherein the Fc constructs include one or more Fc domains.

Compositions and methods related to engineered fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Sialylated glycoproteins

compositions and methods related to manipulated fc constructs

a presente invenção refere-se a composições e métodos de construtos de fc manipulados, em que os construtos de fc incluem um ou mais domínios fc. The present invention relates to manipulated fc constructs compositions and methods, wherein the fc constructs include one or more fc domains.

Evaluation of copolymer diethylamide

Methods of analyzing glatiramer acetate (GA) or a polymeric precursor thereof are provided. The methods can include determining a level of one or more diethylamide-modified amino acids in a sample comprising GA or a polymeric precursor thereof, and selecting at least a portion of the sample based on the assessment of the one or more diethylamide-modified amino acids in the sample.

Compositions and methods related to engineered fc constructs

Compositions and methods related to engineered fc-antigen binding domain constructs

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain.

Compositions and methods related to engineered fc constructs

The present invention relates to engineered IgG Fc constructs and uses thereof.

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc-ANTIGEN BINDING DOMAIN CONSTRUCTS

The present disclosure relates to compositions and methods of engineered Fc-antigen binding domain constructs, where the Fc-antigen binding domain constructs include at least two Fc domains and at least one antigen binding domain.

Compositions and methods related to engineered fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Compositions and methods related to engineered fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Kompozycje i sposoby powiązane z uzyskanymi metodami inżynierii konstruktami fc

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS

The present disclosure relates to compositions and methods of engineered IgG Fc constructs, wherein the Fc constructs include one or more Fc domains.

Compositions and methods related to engineered fc constructs

Compositions and methods related to engineered fc constructs

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to cd38

Fc-antigen binding constructs having a CD38 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization.

Compositions and methods related to engineered fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Methods related to engineered fc constructs

Engineered Fc constructs

The present disclosure relates to engineered IgG Fc constructs and uses thereof.

Sammensætninger og fremgangsmåder relateret til genmanipulerede fc-konstrukter

Sialylated Glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to cd38

Fc-antigen binding constructs having a CD38 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization.

Kompozycje i sposoby związane z projektowanymi konstruktami Fc

Compositions and methods related to engineered Fc constructs

The present disclosure relates to compositions and methods of engineered IgG Fc constructs, wherein the Fc constructs include one or more Fc domains.

Methods related to engineered fc constructs

The present invention relates to methods of using biologically active Fc domain-containing therapeutic constructs (Fc constructs) to induce immune cell activation of the immune response in a subject, and for example, to treat diseases such as cancers and infections in a subject. Such Fc constructs may include 5 or more Fc domains.

ENGINEERED Fc CONSTRUCTS

The present disclosure relates to engineered IgG Fc constructs and uses thereof.

Composiciones y procedimientos relacionados con construcciones de Fc modificadas genéticamente

La presente invención se refiere a construcciones de IgG Fc diseñadas y a sus usos. (Traducción automática con Google Translate, sin valor legal)

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to pd-l1

Fc-antigen binding constructs having a PD-L1 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization.

Compositions and methods related to engineered fc-antigen binding domain constructs targeted to pd-l1

Fc-antigen binding constructs having a PD-L1 binding domain and two or more Fc domains are described as are methods for using such constructs. Also described are polypeptides making up such constructs. Fc domain monomers that are included in the constructs can include amino acid substitutions that promote homodimerization or heterodimerization.

Sialylated Glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

Compositions and methods related to engineered Fc constructs

The present invention relates to engineered IgG Fc constructs and uses thereof.

Compositions and methods related to engineered Fc constructs

The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Sialylated Glycoproteins

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

CONSTRUTO DE Fc, MÉTODO DE PREPARAÇÃO DO MESMO, COMPOSIÇÃO FARMACÊUTICA, USOS DA MESMA E CÉLULA HOSPEDEIRA

COMPOSIÇÕES E MÉTODOS RELACIONADOS COM CONSTRUTOS DE Fc MANIPULADOS. A presente invenção refere-se a composições e métodos de construtos de Fc de IgG manipulados, em que os referidos construtos de Fc incluem um ou mais domínios Fc.

Compositions and methods related to engineered Fc constructs

COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS ABSTRACT The present invention relates to compositions and methods of engineered IgG Fc constructs, wherein said Fc constructs include one or more Fc domains.

Sammensætninger og fremgangsmåder relateret til genmanipulerede fc-konstrukter