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Methods for purification of single domain antigen-binding molecules

A combination and molecular technology, applied in the preparation method of peptides, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve ligand leakage, cumbersome size exclusion chromatography, and contamination of eluted products, etc. question

Inactive Publication Date: 2011-12-07
ABLYNX NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Size-exclusion chromatography tends to be cumbersome and results in severe dilution of the product, which is a hindrance in large-scale, efficiency-based manufacturing processes
Ligand leakage from the affinity chromatography column can also occur, which leads to unwanted contamination of the eluted product (Steindl, J. Immunol. Methods 235:61-69 (2000))

Method used

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  • Methods for purification of single domain antigen-binding molecules
  • Methods for purification of single domain antigen-binding molecules
  • Methods for purification of single domain antigen-binding molecules

Examples

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preparation example Construction

[0110] Preparation of SDAB molecules

[0111] The SDAB molecule may comprise one or more recombinant, CDR-grafted, humanized, camelized, deimmunized and / or in vitro generated (e.g., selected by phage display) single domain molecules ( For example, Nanobody molecules). Techniques for generating antibodies and SDAB molecules and modifying them recombinantly are known in the art and are described in detail below.

[0112] Various methods known to those skilled in the art can be used to obtain antibodies. For example, monoclonal antibodies can be produced by generating hybridomas according to known methods. Then using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE TM ) assay to screen the hybridomas formed in this manner to identify one or more hybridomas that produce a Nanobody that specifically binds to a given antigen. Any form of a given antigen can be used as an immunogen, for example, recombinant antigens, natura...

Embodiment 1

[0187] Example 1: Description of the ATN-103 coding sequence

[0188] ATN-103 is a trivalent nanobody molecule targeting TNFα and HSA. Nanobodies were isolated from a phage library derived from llama by selection for TNFα or HSA as described in WO 06 / 122786. The specific activity of the Nanobodies was tested and TNF1 was selected as a Nanobody inhibitor of human TNFα and ALB1 as a human anti-HSA Nanobody for half-life extension. TNF1 and ALB1 were humanized by CDR grafting onto the closest human framework (DP51 / DP53). During the humanization of TNF1, 2 camelid residues (P84 and R103) were retained and this version was named TNF30. During the humanization of ALB1, seven camelid residues (N16, N73, T76, P84, T93, I94 and S103) were retained and this version was named ALB8. Each of the two TNF30 Nanobodies was linked by a 9 amino acid glycine-serine linker (Gly 4 SerGly 3 Ser (SEQ ID NO:9)) was linked to the central ALB8 Nanobody resulting in a trivalent molecule referred to...

Embodiment 2

[0189] Embodiment 2: ATN-103 purification process

[0190] The ATN-103 purification process consists of two chromatography steps and three membrane filtration steps (see image 3 ). All steps were performed at room temperature unless otherwise indicated.

[0191] The rationale, purpose and description of each purification step are provided below.

[0192] MabSelect Protein A affinity chromatography and low pH virus inactivation

[0193] MabSelect TM The main purpose of the protein A chromatography step included product capture from the clarified cell-free conditioned media and separation of ATN-103 from process-derived impurities (e.g., host cell DNA and proteins, media components and adventitious agents).

[0194] MabSelect Protein A is an affinity resin composed of a highly cross-linked agarose matrix covalently derivatized by thioether linkage with recombinant Protein A produced from E. coli fermentation.

[0195] MabSelect Protein A columns were equilibrated with Tr...

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Abstract

Processes and methods of purifying or separating Single Domain Antigen Binding (SDAB) molecules that include one or more single binding domains (e.g., one or more nanobody molecules), substantially devoid of a complementary antibody domain and an immunoglobulin constant region, using Protein A-based affinity chromatography, are disclosed.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Serial No. 61 / 109,481, filed October 29, 2008, the entire contents of which are hereby incorporated by reference in their entirety. [0003] sequence listing [0004] This application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 28, 2009, is named w223738w.txt and is 12,853 bytes in size. Background technique [0005] Recombinant proteins such as antibodies often contain various impurities that need to be removed before the protein product becomes pharmaceutically acceptable. Some of these impurities may include host cell proteins (HCPs), DNA molecules, variant and / or misfolded forms of product proteins, and high molecular weight aggregates (HMWA). Aggregate formation is problematic during antibody production because it can negatively impact product safety by causing...

Claims

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Application Information

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IPC IPC(8): C07K16/06C07K16/24
CPCC07K1/18C07K2317/569C07K16/065C07K16/241C07K16/18C07K1/22A61P1/04A61P17/06A61P19/02A61P25/00A61P29/00A61P37/06A61P43/00C07K1/36C07K16/06C12N15/11
Inventor 保罗.R.布朗斯科特.A.托布勒安德鲁.M.伍德奥斯汀.W.伯施
Owner ABLYNX NV
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