Igf-i poly (ethylene glycol) conjugates

Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5' to 3' direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with an amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.

Purification of non-glycosylated polypeptides

The current invention reports a method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein the method comprises three chromatography steps of which the first chromatography step selected from i) hydrophobic charge induction chromatography, or ii) hydrophobic interaction chromatography, or iii) affinity chromatography, or iv) ion exchange chromatography, the second chromatography step is selected from i) anion exchange chromatography, or ii) cation exchange chromatography, or iii) hydroxylapatite chromatography, or iv) hydrophobic interaction chromatography, and the a third chromatography step is selected from i) hydrophobic charge induction chromatography, or ii) anion exchange chromatography, or iii) cation exchange chromatography, or iv) hydrophobic interaction chromatography, whereby the first chromatography step is an affinity chromatography in case of polypeptides capable of interacting with metal ligands, the second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, and the third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point.

Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

A conjugate consisting of an insulin-like growth factor-1 (IGF-I) variant and one or two poly(ethylene glycol) group(s), characterized in that said IGF-I variant has an amino acid alteration at up to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-I amino acid sequence so that one or two of said amino acids is/are lysine and amino acid 27 is a polar amino acid but not lysine, is conjugated via the primary amino group(s) of said lysine(s) and said poly(ethylene glycol) group(s) have an overall molecular weight of from 20 to 100 kDa is disclosed. This conjugate is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease.

IGF-I poly (ethylene glycol) conjugates

Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5′ to 3′ direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with an amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.

Method for reduction of 1->2 reading frame shifts

Herein is reported a method for the recombinant production of a polypeptide, which comprises the dipeptide AR, characterized in that the method comprises the recovering of the polypeptide from the cells or the cultivation medium of a cultivation of a cell comprising a nucleic acid encoding the polypeptide and thereby producing the polypeptide, whereby the dipeptide AR comprised in the polypeptide is encoded by the oligonucleotide gca cgt, or the oligonucleotide gcg cgt, or the oligonucleotide gcc cgt.

Prokaryotic expression construct

Herein is reported a pro-polypeptide which is useful for the expression of a polypeptide of interest in a prokaryotic cell. Therefore the pro-polypeptide is fused to the N-terminus of the polypeptide of interest. The pro-polypeptide as reported herein provides for improved expression yields and improves the handling of the fusion polypeptide (downstream processing, purification). For example, efficient endotoxin removal is effected while the protein of interest comprising the pro-polypeptide is bound e.g. to an affinity chromatography material. Thereafter the pro-polypeptide can efficiently be cleaved from the polypeptide of interest by the incorporated protease cleavage site with the cognate protease.

METHOD FOR THE PRODUCTION OF CONJUGATES OF INSULIN-LIKE GROWTH FACTOR-1 AND POLY(ETHYLENE GLYCOL)

The present invention relates to a fusion protein comprising IGF-I or an IGF-I variant N-terminally linked to the C-terminus of a propeptide. The invention relates also to a method involving the use of the aforementioned fusion protein in the production of a lysine-PEGylated IGF-I or IGF-I variant. The method comprises the steps of cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding the fusion protein and causing the cell to express the fusion protein, recovering and PEGylating said fusion protein, cleaving said PEGylated fusion protein with IgA protease, and recovering lysine-PEGylated IGF-I or IGF-I variant. The invention relates also to a lysine-PEGylated IGF-I or IGF-I variant produced using the above method. In addition, the invention relates to a method for treating a neurodegenerative disorders like Alzheimer's Disease using the lysine-PEGylated IGF-I or IGF-I variant and a composition comprising the lysine-PEGylated IGF-I or IGF-I variant. 1. A method for treating a patient suffering from Alzheimer's disease comprising administering to said patient a pharmaceutically-effective amount of a lysine-PEGylated insulin-like growth factor I (IGF-I) (SEQ ID NO: 1) or a lysine-PEGylated IGF-I variant , said variant being a polypeptide which differs from SEQ ID NO: 1 in that one or two of the lysine residues at positions 27 , 65 , or 68 thereof is independently substituted by a polar amino acid selected from the group consisting of: cysteine; aspartic acid; glutamic acid; histidine; asparagine; glutamine; arginine; serine; and threonine; and the remainder of the amino acid sequence of said variant is the same as that of SEQ ID NO: 1.2. The method of wherein the lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant is administered in an amount of from about 0.1 to about 100 mg/ml.3. The method of claim 1 , wherein the lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant is prepared by a process comprising(A) ...

PURIFICATION OF NOT-GLYCOSYLATED POLYPEPTIDES

The current invention reports a method for the purification of a non-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein the method comprises three chromatography steps of which the first chromatography step selected from i) hydrophobic charge induction chromatography, or ii) hydrophobic interaction chromatography, or iii) affinity chromatography, or iv) ion exchange chromatography, the second chromatography step is selected from i) anion exchange chromatography, or ii) cation exchange chromatography, or iii) hydroxylapatite chromatography, or iv) hydrophobic interaction chromatography, and the a third chromatography step is selected from i) hydrophobic charge induction chromatography, or ii) anion exchange chromatography, or iii) cation exchange chromatography, or iv) hydrophobic interaction chromatography, whereby the first chromatography step is an affinity chromatography in case of polypeptides capable of interacting with metal ligands, the second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, and the third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point. 2. The method of claim 1 , characterized in that said method comprises an additional step after step d) which ise) PEGylating said polypeptide.3. The method of claim 2 , characterized in that said steps b) and c) are cation exchange chromatography steps.4. A method for the recombinant production of a non-glycosylated heterologous polypeptide in a prokaryotic cell claim 2 , characterized in that said method comprises the following steps:{'i': 'E. coli', 'a) cultivating a prokaryotic cell comprising a nucleic acid encoding said heterologous polypeptide under conditions suitable for the expression of said heterologous polypeptide, wherein the prokaryotic cell is an cell,'}b) recovering said heterologous polypeptide ...

CONJUGATES OF INSULIN-LIKE GROWTH FACTOR-1 AND POLY(ETHYLENE GLYCOL)

A conjugate consisting of an insulin-like growth factor-1 (IGF-I) variant and one or two poly(ethylene glycol) group(s), characterized in that said IGF-I variant has an amino acid alteration at up to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-I amino acid sequence so that one or two of said amino acids is/are lysine and amino acid 27 is a polar amino acid but not lysine, is conjugated via the primary amino group(s) of said lysine(s) and said poly(ethylene glycol) group(s) have an overall molecular weight of from 20 to 100 kDa is disclosed. This conjugate is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease. 2. A conjugate according to wherein said poly(ethylene glycol) has an overall molecular weight of from 20 to 100 kDa.3. A conjugate according to wherein said IGF-I variant is additionally conjugated to poly(ethylene glycol) at the N-terminal amino acid.4. A conjugate according to wherein said IGF-I variant is conjugated to poly(ethylene glycol) at a member selected from the group consisting of lysine 65 claim 1 , lysine 68 claim 1 , and lysine 37 or is conjugated to poly(ethylene glycol) at both K65 and K68.5. A conjugate according to wherein up to three amino acids at the N-terminus are truncated.6. A conjugate according to wherein the poly(ethylene glycol) group(s) is/are branched poly(ethylene glycol) group(s).7. A conjugate according to wherein the poly(ethylene glycol) group(s) have an overall molecular weight of 20 kDa to 100 kDa.8. An IGF-I variant selected from the group consisting of:(A) a variant which differs from wild-type IGF-I in that the amino acid sequence of the variant has an alteration at residue position 27 such that lysine is replaced with arginine;(B) a variant which differs from wild-type IGF-I in that the amino acid sequence of the variant has alterations at residue positions 27 and 65 such that both lysines are replaced with arginine;(C) a variant which differs from wild-type IGF-I in ...

ON-COLUMN ENZYMATIC CLEAVAGE

Herein is reported a method for obtaining a polypeptide by an immobilized metal ion affinity chromatography from a pro-polypeptide that comprise at its N- or C-terminus an metal ion affinity chromatography tag and a protease cleavage site comprising the step of recovering the polypeptide from the immobilized metal ion affinity chromatography column by incubating the bound pro-polypeptide with a protease, whereby the immobilized metal ion affinity chromatography material has been washed at least once with an urea solution. 1. A method for producing a polypeptide from a pro-polypeptide , whereby the pro-polypeptide comprises at its N- or C-terminus a metal ion affinity chromatography tag and a protease cleavage site located between the tag and the polypeptide , by an on-column enzymatic cleavage of the protease cleavage site on an immobilized metal ion affinity chromatography column comprising the following steps:denaturing the pro-polypeptide bound to the metal ion affinity chromatography material,renaturing the pro-polypeptide bound to the metal ion affinity chromatography material, andincubating the bound pro-polypeptide with a protease and thereby producing the polypeptide.2. A method for producing a polypeptide from a pro-polypeptide , whereby the pro-polypeptide comprises at its N- or C-terminus a metal ion affinity chromatography tag and a protease cleavage site located between the tag and the polypeptide , by an on-column enzymatic cleavage of the protease cleavage site on an immobilized metal ion affinity chromatography column comprising the following steps:contacting the bound pro-polypeptide with a solution comprising a denaturing agent,optionally contacting the bound pro-polypeptide with a solution comprising urea or a urea derivatives if the solution comprising a denaturing agent employed in the previous step was free of urea or a urea derivative or contacting the bound pro-polypeptide with a solution comprising urea or a urea derivatives if the solution ...

METHOD FOR REDUCTION OF 1->2 READING FRAME SHIFTS

Herein is reported a method for the recombinant production of a polypeptide, which comprises the dipeptide AR, characterized in that the method comprises the recovering of the polypeptide from the cells or the cultivation medium of a cultivation of a cell comprising a nucleic acid encoding the polypeptide and thereby producing the polypeptide, whereby the dipeptide AR comprised in the polypeptide is encoded by the oligonucleotide gca cgt, or the oligonucleotide gcg cgt, or the oligonucleotide gcc cgt. 3. The method according to claim 1 , characterized in that the dipeptide AR comprised in the polypeptide is encoded by the oligonucleotide gca cgt claim 1 , or the oligonucleotide gcg cgt claim 1 , or the oligonucleotide gcc cgt.4. The method according to any one of to claim 1 , characterized in that the cell is a prokaryotic cell.5E. coli. The method according to any one of to claim 1 , characterized in that the prokaryotic cell is an cell.6. The method according to any one of to claim 1 , characterized in that the polypeptide is an apolipoprotein A-I claim 1 , or a variant thereof having apolipoprotein A-I activity claim 1 , or a fusion polypeptide thereof having apolipoprotein A-I activity.7. The method according to claim 6 , characterized in that the polypeptide has an amino acid sequence selected from the group comprising SEQ ID NO: 09 to SEQ ID NO: 14.8. The method according to any one of to claim 6 , characterized in that the polypeptide has the amino acid sequence of SEQ ID NO: 09 or SEQ ID NO: 11. This application is a continuation of International Application No. PCT/EP2013/053547 having an international filing date of Feb. 22, 2013, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119 to European Patent Application Nos. 12157512.0 filed Feb. 29, 2012 and 12162810.1 filed Apr. 2, 2012.The instant application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its ...

METHOD FOR REDUCTION OF 1->3 READING FRAME SHIFTS

Herein is reported a method for the recombinant production of a polypeptide, which comprises the tripeptide QKK, characterized in that the method comprises the step of recovering the polypeptide from the cells or the cultivation medium of a cultivation of a cell comprising a nucleic acid encoding the polypeptide and thereby producing the polypeptide, whereby the tripeptide QKK comprised in the polypeptide is encoded by the oligonucleotide cag aaa aaa or the oligonucleotide caa aag aaa. 2E. coli. A method for reducing the by-product formation during the recombinant production of a full length polypeptide in , which comprises the tripeptide QKK , comprising the step of:substituting in the polypeptide encoding nucleic acid one to three nucleotides in the tripeptide QKK encoding oligonucleotide caa aaa aag (SEQ ID NO. 01), or the oligonucleotide caa aag aag (SEQ ID NO: 02), or the oligonucleotide cag aag aag (SEQ ID NO: 03) to obtain the oligonucleotide caa aag aaa (SEQ ID NO: 04), or the oligonucleotide cag aaa aaa (SEQ ID NO: 05), thereby producing a substituted polypeptide encoding nucleic acid, andrecovering the polypeptide from the cells or the cultivation medium of a cultivation of a cell comprising the substituted nucleic acid encoding the polypeptide and thereby reducing the by-product formation during the recombinant production of a polypeptide, which comprises the tripeptide QKK.3. The method according to any one of or , characterized in that the method comprises one or more of the following further steps:providing the amino acid sequence or the encoding nucleic acid of a polypeptide comprising the tripeptide QKK, and/ortransfecting a cell with the substituted nucleic acid encoding the polypeptide, and/orcultivating the cell transfected with the substituted nucleic acid (under conditions which are suitable for the expression of the polypeptide), and/orrecovering the polypeptide from the cell or the cultivation medium, and/oroptionally purifying the produced ...

CONJUGATES OF INSULIN-LIKE GROWTH FACTOR-1 AND POLY(ETHYLENE GLYCOL)

A conjugate consisting of an insulin-like growth factor-1 (IGF-I) variant and one or two poly(ethylene glycol) group(s), characterized in that said IGF-I variant has an amino acid alteration at up to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-I amino acid sequence so that one or two of said amino acids is/are lysine and amino acid 27 is a polar amino acid but not lysine, is conjugated via the primary amino group(s) of said lysine(s) and said poly(ethylene glycol) group(s) have an overall molecular weight of from 20 to 100 kDa is disclosed. This conjugate is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease. 1. A conjugate comprising an IGF-I (insulin-like growth factor I) variant and poly(ethylene glycol) , wherein: 'a variant which differs from wild-type IGF-I in that the amino acid sequence of the variant has alterations at residue positions 27 and 65 such that both lysines are replaced with arginine; and', 'said IGF-I variant issaid poly(ethylene glycol) is conjugated to said IGF-I variant via one or more primary amino groups.2. The conjugate according to wherein said poly(ethylene glycol) has an overall molecular weight of from 20 to 100 kDa.3. The conjugate according to wherein said IGF-I variant is additionally conjugated to poly(ethylene glycol) at the N-terminal amino acid.4. The conjugate according to wherein said IGF-I variant is conjugated to poly(ethylene glycol) at a member selected from the group consisting of lysine 65 claim 1 , or is conjugated to poly(ethylene glycol) at K65.5. The conjugate according to wherein up to three amino acids at the N-terminus are truncated.6. The conjugate according to wherein the poly(ethylene glycol) group(s) is/are branched poly(ethylene glycol) group(s).7. The conjugate according to wherein the poly(ethylene glycol) group(s) have an overall molecular weight of 20 kDa to 100 kDa.8. An IGF-I variant wherein the:variant differs from wild-type IGF-I in that the amino acid ...

PURIFICATION OF NOT-GLYCOSYLATED POLYPEPTIDES

The current invention reports a method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein the method comprises three chromatography steps of which the first chromatography step selected from i) hydrophobic charge induction chromatography, or ii) hydrophobic interaction chromatography, or iii) affinity chromatography, or iv) ion exchange chromatography, the second chromatography step is selected from i) anion exchange chromatography, or ii) cation exchange chromatography, or iii) hydroxylapatite chromatography, or iv) hydrophobic interaction chromatography, and the a third chromatography step is selected from i) hydrophobic charge induction chromatography, or ii) anion exchange chromatography, or iii) cation exchange chromatography, or iv) hydrophobic interaction chromatography, whereby the first chromatography step is an affinity chromatography in case of polypeptides capable of interacting with metal ligands, the second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, and the third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point. 1. A method for purifying a non-glycosylated polypeptide , the method comprising a sequence of three successive chromatography steps comprising i) hydrophobic charge induction chromatography,', 'ii) hydrophobic interaction chromatography,', 'iii) affinity chromatography, and', 'iv) ion exchange chromatography;, 'a) a first chromatography step selected from the group consisting of i) anion exchange chromatography,', 'ii) cation exchange chromatography,', 'iii) hydroxylapatite chromatography,', 'iv) hydrophobic interaction chromatography, and', 'v) hydrophobic charge induction chromatography; and, 'b) a second chromatography step selected from the group consisting of i) hydrophobic charge induction chromatography ...

BISPECIFIC 2+1 CONTORSBODIES

The invention relates to novel bispecific antibodies consisting of two fusion polypeptides comprising two antigen binding domains capable of specific binding to a first target and one antigen binding domain capable of specific binding to a second target, and to methods of producing these molecules and to methods of using the same. 1. A bispecific antibody consisting of two fusion polypeptides and comprising two antigen binding domains capable of specific binding to a first target and one antigen binding domain capable of specific binding to a second target comprising: the spacer domain is a polypeptide and comprises at least 25 amino acid residues,', 'the first part of the first antigen binding domain capable of specific binding to the first target is fused either directly to, or via a first peptide linker to, the N-terminus of the spacer domain,', 'the second part of the first antigen binding domain capable of specific binding to the first target is fused either directly to, or via a second peptide linker to, the C-terminus of the spacer domain, and', 'the first part of an antigen binding domain capable of specific binding to a second target is fused either directly to, or via a third peptide linker to, the C-terminus of the second part of the first antigen binding domain capable of specific binding to the first target or is fused either directly to, or via a third peptide linker to, the N-terminus of the first part of the first antigen binding domain capable of specific binding to the first target, and, '(a) a first fusion polypeptide comprising a first part of a first antigen binding domain capable of specific binding to the first target, a spacer domain, a second part of a first antigen binding domain capable of specific binding to the first target and a first part of an antigen binding domain capable of specific binding to the second target, wherein'} the spacer domain is a polypeptide and comprises at least 25 amino acid residues,', 'the first part of the ...

IGF-I POLY (ETHYLENE GLYCOL) CONJUGATES

Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5′ to 3′ direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with man amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.

NOVEL TNF FAMILY LIGAND TRIMER-CONTAINING ANTIGEN BINDING MOLECULES

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising two different fusion polypeptides that comprise a spacer domain, an antigen binding domain and three ectodomains of a TNF ligand member or fragments thereof, wherein two of said ectodomains are separated from each other by a spacer domain comprising at least 25 amino acids and wherein the two fusion polypeptides are covalently associated to each other in the spacer domain 1. A TNF family ligand trimer-containing antigen binding molecule comprising the spacer domain is a polypeptide and comprises at least 25 amino acid residues,', 'the first ectodomain of a TNF ligand family member or a fragment thereof is fused either directly or via a first peptide linker to the N-terminus of the spacer domain and', 'the second ectodomain of said TNF ligand family member or a fragment thereof is fused either directly or via a second peptide linker to the C-terminus of the spacer domain,, '(a) a first fusion polypeptide comprising a first ectodomain of a TNF ligand family member or a fragment thereof, a spacer domain and a second ectodomain of said TNF ligand family member or a fragment thereof, wherein'} the spacer domain is a polypeptide and comprises at least 25 amino acid residues, and', 'wherein the second part of the antigen binding domain is fused either directly or via a third peptide linker to the C-terminus of the spacer domain or is present in form of a light chain, and, '(b) a second fusion polypeptide comprising a first part of an antigen binding domain and a spacer domain, wherein'} either the C-terminus of the second ectodomain of said TNF ligand family member in the first fusion polypeptide or to the C-terminus of the spacer domain in the second fusion polypeptide, or', 'in case the second part of the antigen binding domain is fused to the C-terminus of the spacer domain of the second fusion protein, to the C-terminus of the second ectodomain of said TNF ligand ...

THE CONTORSBODY - A SINGLE CHAIN TARGET BINDER

Herein is reported a circular fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain and a spacer domain, wherein the spacer domain is a polypeptide and comprises at least 25 amino acid residues, the first part of the binding domain is a polypeptide and is fused via a first linker to the N-terminus of the spacer domain, the second part of the binding domain is a polypeptide and is fused via a second linker to the C-terminus of the spacer domain, the first part of the binding domain and the second part of the binding domain are associated with each other and form a binding site that specifically binds to a target. 117-. (canceled)18: A dimeric fusion polypeptide comprising: the spacer domain of the first fusion polypeptide is a polypeptide and comprises at least 25 amino acid residues,', 'the first part of the binding domain of the first fusion polypeptide is a polypeptide that is fused either directly or via a first linker to the N-terminus of the spacer domain of the first fusion polypeptide,', 'the second part of the binding domain of the first fusion polypeptide is a polypeptide that is fused either directly or via a second linker to the C-terminus of the spacer domain of the first fusion polypeptide, and', 'the first part of the binding domain and the second part of the binding domain of the single chain first fusion polypeptide associate and form a functional binding site that specifically binds to the first target, and, 'wherein, 'a first fusion polypeptide specifically binding to a first target, the first fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain, and a spacer domain,'} the spacer domain of the second fusion polypeptide is a polypeptide and comprises at least 25 amino acid residues,', 'the first part of the binding domain of the second fusion polypeptide is a polypeptide that is fused either directly or via a first linker to the N-terminus of the spacer domain ...

The contorsbody - a single chain target binder

Herein is reported a circular fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain and a spacer domain, wherein the spacer domain is a polypeptide and comprises at least 25 amino acid residues, the first part of the binding domain is a polypeptide and is fused via a first linker to the N- terminus of the spacer domain, the second part of the binding domain is a polypeptide and is fused via a second linker to the C-terminus of the spacer domain, the first part of the binding domain and the second part of the binding domain are associated with each other and form a binding site that specifically binds to a target.

Novel tnf family ligand trimer-containing antigen binding molecules

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising two different fusion polypeptides that comprise a spacer domain, an antigen binding domain and three ectodomains of a TNF ligand member or fragments thereof, wherein two of said ectodomains are separated from each other by a spacer domain comprising at least 25 amino acids and wherein the two fusion polypeptides are covalently associated to each other in the spacer domain.

Bispecific 2+1 contorsbodies

The invention relates to novel bispecific antibodies consisting of two fusion polypeptides comprising two antigen binding domains capable of specific binding to a first target and one antigen binding domain capable of specific binding to a second target, and to methods of producing these molecules and to methods of using the same.

The contorsbody - a single chain target binder

Herein is reported a circular fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain and a spacer domain, wherein the spacer domain is a polypeptide and comprises at least 25 amino acid residues, the first part of the binding domain is a polypeptide and is fused via a first linker to the N-terminus of the spacer domain, the second part of the binding domain is a polypeptide and is fused via a second linker to the C-terminus of the spacer domain, the first part of the binding domain and the second part of the binding domain are associated with each other and form a binding site that specifically binds to a target.

CONTORSBODY - A BIND OF DIANA MONOCATENARY

En la presente memoria se informa de un polipéptido de fusión circular que comprende una primera parte de un dominio de unión, una segunda parte de un dominio de unión y un dominio espaciador, en el que el dominio espaciador es un polipéptido y comprende por lo menos 25 residuos aminoácidos, la primera parte del dominio de unión es un polipéptido y se fusiona mediante un primer conector con el extremo N-terminal del dominio espaciador, la segunda parte del dominio de unión es un polipéptido y se fusiona mediante un segundo conector con el extremo C-terminal del dominio espaciador, la primera parte del dominio de unión y la segunda parte del dominio de unión se asocian entre sí y forman un sitio de unión que se une específicamente a una diana.

2 + 1 BISPECIFIC ANTIBODIES (CONTORSBODIES)

Purification of not-glycosylated polypeptides

The current invention reports a method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein the method comprises three chromatography steps of which the first chromatography step selected from i) hydrophobic charge induction chromatography, or ii) hydrophobic interaction chromatography, or iii) affinity chromatography, or iv) ion exchange chromatography, the second chromatography step is selected from i) anion exchange chromatography, or ii) cation exchange chromatography, or iii) hydroxylapatite chromatography, or iv) hydrophobic interaction chromatography, and the a third chromatography step is selected from i) hydrophobic charge induction chromatography, or ii) anion exchange chromatography, or iii) cation exchange chromatography, or iv) hydrophobic interaction chromatography, whereby the first chromatography step is an affinity chromatography in case of polypeptides capable of interacting with metal ligands, the second chromatography step is not a hydroxylapatite chromatography step in case of polypeptides with an isoelectric point below 6.0, and the third chromatography step can be performed in flow-through mode with polypeptides having a low or high isoelectric point.

target-specific fusion polypeptide, dimeric fusion polypeptide, isolated nucleic acid, isolated nucleic acid pair, host cell, method for producing a fusion polypeptide, immunoconjugate, pharmaceutical formulation, fusion polypeptide and use of the fusion polypeptide

aqui é descrito um polipeptídio de fusão circular que compreende uma primeira parte de um domínio de ligação, uma segunda parte de um domínio de ligação e um domínio de espaçamento, em que o domínio de espaçamento é um polipeptídio e compreende pelo menos 25 resíduos de aminoácidos, a primeira parte do domínio de ligação é um polipeptídio e é fundido através de um primeiro ligante ao terminal n do domínio de espaçamento, a segunda parte do domínio de ligação é um polipeptídio e é fundido através de um segundo ligante ao terminal c do domínio de espaçamento, a primeira parte do domínio de ligação e a segunda parte do domínio de ligação estão associadas uma com a outra e formam um sítio de ligação que se liga de forma específica a um alvo. described herein is a circular fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain and a spacing domain, wherein the spacing domain is a polypeptide and comprises at least 25 amino acid residues. , the first binding domain part is a polypeptide and is fused through a first n-terminal linker to the spacing domain, the second binding domain part is a polypeptide and is fused through a second c-terminal linker to the spacing domain spacing, the first binding domain part and the second binding domain part are associated with each other and form a binding site that specifically binds to a target.

On-column enzymatic cleavage

Herein is reported a method for obtaining a polypeptide by an immobilized metal ion affinity chromatography from a pro-polypeptide that comprise at its N- or C-terminus an metal ion affinity chromatography tag and a protease cleavage site comprising the step of recovering the polypeptide from the immobilized metal ion affinity chromatography column by incubating the bound pro-polypeptide with a protease, whereby the immobilized metal ion affinity chromatography material has been washed at least once with an urea solution.

Bispecific 2+1 contorsbodies

Prokaryotic expression construct

Herein is reported a pro-polypeptide which is useful for the expression of a polypeptide of interest in a prokaryotic cell. Therefore the pro-polypeptide is fused to the N-terminus of the polypeptide of interest. The pro-polypeptide as reported herein provides for improved expression yields and improves the handling of the fusion polypeptide (downstream processing, purification). For example, efficient endotoxin removal is effected while the protein of interest comprising the pro-polypeptide is bound e.g. to an affinity chromatography material. Thereafter the pro-polypeptide can efficiently be cleaved from the polypeptide of interest by the incorporated protease cleavage site with the cognate protease.

Igf-i poly (ethylene glycol) conjugates

Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5' to 3' direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with an amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.

Process for Preparation of Conjugates of Insulin-Like Growth Factor-1 and Poly (Ethylene Glucole)

Metode for fremstilling av en lysinPEGylert IGF-I eller IGF-I variant. Nevnte variant omfatter at én eller to aminosyrer valgt fra gruppen bestående av lysin 27, 65 og/eller 68 er substituert uavhengig av en annen polar aminosyre,dyrking av en prokaryot vertscelle som omfatter en ekspresjonsvektor som inneholder en nukleinsyre som koder for et fusjonsprotein som omfatter nevnte IGF-I eller IGF-I variant N-terminalt bundet til C-terminus av et propeptid, hvor nevnte propeptid ender C-terminalt med aminosyrene -Y-Pro, hvor Y er valgt fra gruppen bestående av Pro, Pro-ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr-Pro, Arg-Pro eller Pro-Arg-Pro, gjenvinning og PEGylering av nevnte fusjonsprotein, spaltning av nevnte PEGylerte fusjonsproteiner med IgA protease og gjenvinning av nevnte PEGylerte IGF-I eller IGF-I variant. PEGylert IGF-I eller IGF-I variant er anvendelig for behandling av neurodegenerative lidelser som Alzheimers sykdom

Recombinant proteinase from clostridium hystolyticum and the use thereof for isolating cells and cell groups

A process is proposed for breaking down cellular tissue and releasing the constituent cells or cell groups by incubating the cellular tissue with a recombinant neutral protease from Clostridium hystolyticum. The protease in question is coded by (a) a DNA of the nucleotide 1027-1965 from SEQ ID NO:3 or by a DNA complementary to it; (b) nucleic acids which hybridise with a DNA of the nucleotide 1027-1965 from SEQ ID NO:3; (c) nucleic acids which would hybridise with one of the nucleic acids mentioned in (a) or (b) without degeneration of the genetic code. The protease is a product of a prokaryotic or eukaryotic expression of an exogenic nucleic acid. Incubation continues until the release of cells or cell groups in the desired quantity and separation of the cells or cell groups from the cellular tissue fractions.

Interferon-alpha polypeptides and conjugates

The present invention provides interferon-alpha polypeptides and conjugates, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.

Method for the production of conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

Method for the production of a lysine-PEGylated IGF-I or IGF-I variant, said variant comprising one or two amino acid(s) selected from the group consisting of lysine 27, 65 and/or 68 substituted independently by another polar amino acid, characterized in cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding a fusion protein comprising said IGF-I or IGF-I variant N-terminally linked to the C-terminus of a propeptide, that said propeptide ends C-terminally with amino acids -Y-Pro, wherein Y is selected from the group consisting of Pro, Pro-Ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr-Pro, Arg-Pro, or Pro-Arg-Pro, recovering and PEGylating said fusion protein, cleaving said PEGylated fusion protein with IgA protease, and recovering said PEGylated IGF-I or IGF-I variant. The PEGylated IGF-I or IGF-I variant is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease.

Method for reduction of 1->3 reading frame shifts

Herein is reported a method for the recombinant production of a polypeptide, which comprises the tripeptide QKK, characterized in that the method comprises the step of recovering the polypeptide from the cells or the cultivation medium of a cultivation of a cell comprising a nucleic acid encoding the polypeptide and thereby producing the polypeptide, whereby the tripeptide QKK comprised in the polypeptide is encoded by the oligonucleotide cag aaa aaa or the oligonucleotide caa aag aaa.

Bispecific 2+1 contorsbodies

The invention relates to novel bispecific antibodies consisting of two fusion polypeptides comprising two antigen binding domains capable of specific binding to a first target and one antigen binding domain capable of specific binding to a second target, and to methods of producing these molecules and to methods of using the same.

On-column enzymatic cleavage.

En la presente se da a conocer un método para obtener un polipéptido por una cromatografía de afinidad de iones metálicos inmovilizados a partir de un pro-polipéptido que comprende en su N- o C-término una marca de cromatografía de afinidad de iones metálicos y un sitio de escisión de proteasa, el cual comprende el paso de recuperar el polipéptido a partir de la columna de cromatografía de afinidad de iones metálicos inmovilizados al incubar el pro-polipéptido unido con una proteasa, por lo que el material de cromatografía de afinidad de iones metálicos inmovilizados se ha lavado al menos una vez con una solución de urea.

Recombinant collagenase type i from clostridium histolyticum and its use for isolating cells and groups of cells

A polypeptide having the properties of a Class I Clostridium histolyticum collagenase (CHC I) and the amino acid sequence SEQ ID NO:2, the N terminal of which has been optionally extended in one or more amino acids of the sequence SEQ ID NO:3, is advantageously adequate for isolating cells in tissues from mammals and human tissues.

Method for the purification of an n-terminal fragment of hepatocyte growth factor

A method for the production of the N-terminal four kringle-containing fragment of hepatocyte growth factor (NK4) by expression of a nucleic acid encoding said NK4 in a microbial host cell, isolation of inclusion bodies containing said NK4 in denatured form, solubilization of the inclusion bodies and naturation of the denatured NK4, characterized in that solubilization and naturation are performed at pH 7-9 in phosphate buffered solution, provides NK4 in high purity and high yield.

Recombinant proteinase from Clostridium histolyticum and its use for the isolation of cells and cell assemblies

A process is proposed for breaking down cellular tissue and releasing the constituent cells or cell groups by incubating the cellular tissue with a recombinant neutral protease from Clostridium hystolyticum. The protease in question is coded by (a) a DNA of the nucleotide 1027-1965 from SEQ ID NO:3 or by a DNA complementary to it; (b) nucleic acids which hybridise with a DNA of the nucleotide 1027-1965 from SEQ ID NO:3; (c) nucleic acids which would hybridise with one of the nucleic acids mentioned in (a) or (b) without degeneration of the genetic code. The protease is a product of a prokaryotic or eukaryotic expression of an exogenic nucleic acid. Incubation continues until the release of cells or cell groups in the desired quantity and separation of the cells or cell groups from the cellular tissue fractions.

Recombinant type ii collagenase from clostridium histolyticum and its use for isolating cells and groups of cells

Process for disintegrating cell tissue and releasing cells or groups of cells contained therein by incubation of the cell tissue with a class II collagenase from Clostridium histolyticum, which still has a considerable degree of collagenase activity after 12 freeze/thaw cycles and contains SEQ ID NO:2 or SEQ ID NO:10 in a characteristic partial region or it is coded in this region by SEQ ID NO:1, SEQ ID NO:9 or a sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code and is the product of a prokaryotic or eukaryotic expression of an exogenous DNA, until the cells or groups of cells are released to the desired extent and separating the cells or the groups of cells from the cell tissue fractions.

Prokaryotic expression construct

Herein is reported a pro-polypeptide which is useful for the expression of a polypeptide of interest in a prokaryotic cell. Therefore the pro-polypeptide is fused to the N-terminus of the polypeptide of interest. The pro-polypeptide as reported herein provides for improved expression yields and improves the handling of the fusion polypeptide (downstream processing, purification). For example, efficient endotoxin removal is effected while the protein of interest comprising the pro-polypeptide is bound e.g. to an affinity chromatography material. Thereafter the pro-polypeptide can efficiently be cleaved from the polypeptide of interest by the incorporated protease cleavage site with the cognate protease.

Recombinant type ii collagenase from clostridium histolyticum and its use for isolating cells and groups of cells

Process for disintegrating cell tissue and releasing cells or groups of cells contained therein by incubation of the cell tissue with a class II collagenase from Clostridium histolyticum, which still has a considerable degree of collagenase activity after 12 freeze/thaw cycles and contains SEQ ID NO:2 or SEQ ID NO:10 in a characteristic partial region or it is coded in this region by SEQ ID NO:1, SEQ ID NO:9 or a sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code and is the product of a prokaryotic or eukaryotic expression of an exogenous DNA, until the cells or groups of cells are released to the desired extent and separating the cells or the groups of cells from the cell tissue fractions.

Method for the production of insulin-like growth factor-i

Method for the production of IGF-I, characterized by cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding a fusion protein comprising said IGF-I N-terminally linked to the C-terminus of a propeptide, whereby said propeptide ends C-terminally with amino acids -Y-Pro, wherein Y is selected from the group consisting of Pro, Pro-Ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr-Pro, Arg-Pro, or Pro-Arg-Pro, recovering and cleaving said fusion protein with IgA protease, and recovering said IGF-I. IGF-I is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease.

METHOD FOR PURIFYING AN N-TERMINAL FRAGMENT OF THE HEPATOCYTE GROWTH FACTOR

On-column enzymatic cleavage

Herein is reported a method for obtaining a polypeptide by an immobilized metal ion affinity chromatography from a pro-polypeptide that comprise at its N- or C-terminus an metal ion affinity chromatography tag and a protease cleavage site comprising the step of recovering the polypeptide from the immobilized metal ion affinity chromatography column by incubating the bound pro-polypeptide with a protease, whereby the immobilized metal ion affinity chromatography material has been washed at least once with an urea solution.

Method for the production of conjugates of insulin-like growth factor-I and poly(ethylene glycol)

Method for the production of a lysine-PEGylated IGF-I or IGF-I variant, said variant comprising one or two amino acid(s) selected from the group consisting of lysine 27, 65 and/or 68 substituted independently by another polar amino acid, characterized in cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding a fusion protein comprising said IGF-I or IGF-I variant N-terminally linked to the C-terminus of a propeptide, that said propeptide ends C-terminally with amino acids -Y-Pro, wherein Y is selected from the group consisting of Pro, Pro-Ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr-Pro, Arg-Pro, or Pro-Arg-Pro, recovering and PEGylating said fusion protein, cleaving said PEGylated fusion protein with IgA protease, and recovering said PEGylated IGF-I or IGF-I variant. The PEGylated IGF-I or IGF-I variant is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease.

Bispecific 2+1 contorsbodies

The invention relates to novel bispecific antibodies consisting of two fusion polypeptides comprising two antigen binding domains capable of specific binding to a first target and one antigen binding domain capable of specific binding to a second target, and to methods of producing these molecules and to methods of using the same.

method for reducing 1-> 2 reading frame offsets

1/1 resumo método para a redução de 1->2 deslocamentos de estruturas de leitura descreve-se neste contexto um método para a produção recombinante de um polipeptídeo, que compreende o dipeptídeo ar, caracterizado por o método compreender a recuperação do polipeptídeo a partir das células ou do meio de cultivo da cultura de uma célula que compreende um ácido nucléico que codifica a polipeptídeo e produzir desse modo o polipeptídeo, pelo que o dipeptídeo ar compreendido no polipeptídeo é codificado pelo oligonucleotídeo gca cgt, ou pelo oligonucleotídeo gcg cgt, ou pelo oligonucleotídeo gcc cgt. In this context, a method for recombinant production of a polypeptide comprising the dipeptide ar is characterized in that the method comprises recovering the polypeptide from of the cells or the culture medium of a cell comprising a polypeptide-encoding nucleic acid and thereby producing the polypeptide, whereby the dipeptide ar comprised in the polypeptide is encoded by the gca cgt oligonucleotide, or the gcg cgt oligonucleotide, or by the gcc cgt oligonucleotide.

Prokaryotic expression construct

Herein is reported a pro-polypeptide which is useful for the expression of a polypeptide of interest in a prokaryotic cell. Therefore the pro-polypeptide is fused to the N-terminus of the polypeptide of interest. The pro-polypeptide as reported herein provides for improved expression yields and improves the handling of the fusion polypeptide (downstream processing, purification). For example, efficient endotoxin removal is effected while the protein of interest comprising the pro-polypeptide is bound e.g. to an affinity chromatography material. Thereafter the pro-polypeptide can efficiently be cleaved from the polypeptide of interest by the incorporated protease cleavage site with the cognate protease.

Method for the purification of an n-terminal fragment of hepatocyte growth factor

A method for the production of the N-terminal four kringle-containing fragment of hepatocyte growth factor (NK4) by expression of a nucleic acid encoding said NK4 in a microbial host cell, isolation of inclusion bodies containing said NK4 in denatured form, solubilization of the inclusion bodies and naturation of the denatured NK4, characterized in that solubilization and naturation are performed at pH 7-9 in phosphate buffered solution, provides NK4 in high purity and high yield.

Interferon-alpha polypeptides and conjugates

The present invention provides interferon-alpha polypeptides and conjugates, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.

Recombinant proteinase from clostridium hystolyticum and the use thereof for isolating cells and cell groups

Verfahren zur Auflösung von Zellgewebe und Freisetzung von darin enthaltenen Zellen oder Zellverbänden durch Inkubation des Zellgewebes mit einer rekombinanten neutralen Protease aus Clostridium histolyticum, welche codiert wird von a) einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 oder einer hierzu komplementären DNA, b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 hybridisieren, c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden, und das Produkt einer prokaryontischen oder eukaryontischen Expression einer exogenen Nukleinsäure ist, bis zur Freisetzung der Zellen oder Zellverbände im gewünschten Umfang und Abtrennung der Zellen oder der Zellverbände von den Zellgewebeanteilen.

Method for the production of insulin-1-like growth factor conjugates and poly (ethylene glycol)

Patente de Invenção:"MÉTODO PARA A PRODUÇçO DE CONJUGADOS DE FATOR DE CRESCIMENTO SEMELHANTE À INSULINA-1 E POLI(ETILENO GLICOL)". Método para a produção de um IGF-I PEGuilado com lisina ou variante de IGF-I PEGuilado com lisina, a referida variante compreendendo um ou dois aminoácidos selecionados do grupo que consiste em lisina 27, 65 e/ou 68 independentemente substituídos por um outro aminoácido polar, caracterizado pelo fato de cultivar uma célula hospedeira procariótica que compreende um vetor de expressão contendo um ácido nucleico que codifica uma proteína de fusão compreendendo o referido IGF-I ou variante de IGF-I ligada pelo teminal N ao terminal C de um propeptídio, pelo fato de o referido propeptídio ter seu terminal C terminado com aminoácidos -Y-Pro, onde Y é selecionado do grupo que consiste em Pro, Pro-Ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr-Pro, Arg-Pro, ou Pro-Arg-Pro, recuperar e PEGuilar a referida proteína de fusão, clivar a referida proteína de fusão PEGuilada com protease de IgA, e recuperar o referido IGF-I PEGuilado ou variante de IGF-I PEGuilado. O IGF-I PEGuilado ou variante de IGF-I é útil para o tratamento de distúrbios neurodegenerativos como o mal de Alzheimer Invention Patent: "METHOD FOR PRODUCTION OF INSULIN-1 AND POLY (ETHYLENE GLYCOL) GROWTH FACTOR CONJUGATES". A method for producing a lysine-pegylated IGF-I or lysine-pegylated IGF-I variant, said variant comprising one or two amino acids selected from the group consisting of lysine 27, 65 and / or 68 independently substituted by another amino acid polar, characterized in that it cultivates a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding a fusion protein comprising said IGF-I or N-terminally linked IGF-I variant to a C-terminal of a propeptide, said propeptide has its C-terminus terminated with amino acids -Y-Pro, where Y is selected from the group consisting of Pro, Pro-Ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr - ...

Method for the production of conjugates of insulin-like growth factor-i and poly(ethylene glycol).

Se describe un método para la producción de IGF-I o variante de IGF-I PEGilado en la lisina, y la variante comprende uno o dos aminoácido(s) seleccionados del grupo que consiste en lisina 27, 65 y/o 68 sustituidos independientemente por otro aminoácido polar, que se caracteriza por cultivar una célula huésped procariota que comprende un vector de expresión que contiene un ácido nucleico que codifica una proteína de fusión que comprende el IGF-I o variante de IGF-I unido en el extremo N-terminal al extremo C-terminal de un propéptido, en el que el propétido finaliza en el C-terminal con los aminoácidos -Y-Pro, en el que Y se selecciona del grupo que consiste en Pro, Pro-Ala, Pro-Gly, Pro-Thr, Ala-Pro, Gly-Pro, Thr-Pro, Arg-Pro, o Pro-Arg-Pro, recuperando y PEGilando la proteína de fusión, escindiendo la proteína de fusión PEGilada con la proteasa IgA, y recuperando el IGF-I o variante de IGF-I PEGilado. El IGF-I o variante de IGF-I PEGilado es útil para el tratamiento de trastornos neurodegenerativos como la enfermedad de Alzheimer.

METHOD FOR PURIFYING AN N-TERMINAL FRAGMENT OF THE HEPATOCYTE GROWTH FACTOR

igf-i poly (ethylene glycol) conjugates

POLIPEPTÍDEO, SEU MÉTODO DE PRODUÇÃO CONJUGADO A UM RESÍDUO DE POLI(ETILENO GLICOL), SUA COMPOSIÇÃO FARMACÊUTICA E SEU USO. A presente invenção se refere a um método para produção de um polipeptídeo conjugado a um poli(etileno glicol) compreendendo a) prover um ácido nucleico codificando em construto de expressão compreendendo na direção 5' a 3' um ácido nucleico codificando um polipeptídeo, e um ácido nucleico codificando um local de tripsina de SEQ ID NO: 01, b) expressar o ácido nucleico de a) em uma célula e recuperar ao construto de expressão da célula e/ou o meio de cultivo, c) prover um peptídeo-alvo com uma sequência de aminoácido da SEQ ID NO: 02 covalentemente conjugada a um poli(etileno glicol) no resíduo de lisina C-terminal d) incubar o construto de expressão e o peptídeo-alvo com a tripsina mutante D189K K60E, N143H, E151H, e e) recuperar e, dessa forma, produzir o polipeptídeo conjugado a um poli(etileno glicol) da mistura de incubação. POLIPEPTIDE, ITS PRODUCTION METHOD CONJUGATED TO A POLY (ETHYLENE GLYCOL) WASTE, ITS PHARMACEUTICAL COMPOSITION AND ITS USE. The present invention relates to a method for producing a polypeptide conjugated to a poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in the 5 'to 3' direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) express the nucleic acid of a) in a cell and recover to the cell's expression construct and / or the culture medium, c) provide a target peptide with an amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue d) incubate the expression construct and the target peptide with the mutant trypsin D189K K60E, N143H, E151H, ee ) recover and thereby produce the polypeptide conjugated to a poly (ethylene glycol) from the incubation mixture.

Igf-i poly (ethylene glycol) conjugates

Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5' to 3' direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with an amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.

The contorsbody - a single chain target binder

Herein is reported a circular fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain and a spacer domain, wherein the spacer domain is a polypeptide and comprises at least 25 amino acid residues, the first part of the binding domain is a polypeptide and is fused via a first linker to the N- terminus of the spacer domain, the second part of the binding domain is a polypeptide and is fused via a second linker to the C-terminus of the spacer domain, the first part of the binding domain and the second part of the binding domain are associated with each other and form a binding site that specifically binds to a target.

Rekombinante collagenase typ i aus clostridium histolyticum und ihre verwendung zur isolierung von zellen und zellverbänden

Verfahren zur aufreinigung eines n-terminalen fragments des hepatozytenwachstumsfaktors

Antigen binding molecules

The present invention relates to a pair of binding molecules comprising complementary parts of an effector domain, such binding molecules being capable of forming a functional effector domain when bound to their target antigens on the surface of a cell. Specifically, the invention relates to a pair of binding molecules wherein the complementary parts of the effector domain are complemented with inert complementing domains while the functional effector domain is not formed, providing advantageous properties, such as produceability, stability and/or biological functionality, to the binding molecules.

Contorsbody - a single chain target binder

Herein is reported a circular fusion polypeptide comprising a first part of a binding domain, a second part of a binding domain and a spacer domain, wherein the spacer domain is a polypeptide and comprises at least 25 amino acid residues, the first part of the binding domain is a polypeptide and is fused via a first linker to the N-terminus of the spacer domain, the second part of the binding domain is a polypeptide and is fused via a second linker to the C-terminus of the spacer domain, the first part of the binding domain and the second part of the binding domain are associated with each other and form a binding site that specifically binds to a target.