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Single domain antigen combined molecule purifying method

A binding and molecular technology, applied in the preparation method of peptides, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve problems such as obstruction, elution product contamination, product dilution, etc.

Pending Publication Date: 2016-06-08
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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  • Single domain antigen combined molecule purifying method
  • Single domain antigen combined molecule purifying method
  • Single domain antigen combined molecule purifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0190] Example 1: Description of the ATN-103 coding sequence

[0191] ATN-103 is a trivalent nanobody molecule targeting TNFα and HSA. Nanobodies were isolated from llama-derived phage libraries by selection for TNFα or HSA as described in WO06 / 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 herei...

Embodiment 2

[0192] Embodiment 2: ATN-103 purification process

[0193] 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 noted.

[0194] The rationale, purpose and description of each purification step are provided below.

[0195] MabSelect Protein A affinity chromatography and low pH virus inactivation

[0196] The main purpose of the MabSelect™ Protein A chromatography step includes product capture from the clarified cell-free conditioned media and separation of ATN-103 from process-derived impurities (eg, host cell DNA and proteins, media components and foreign agents).

[0197] 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.

[0198] MabSelect Protein A columns were equilibrated with Tris-buffered so...

Embodiment 3

[0212] Example 3: Protein A capture comparison

[0213] The ability of the following Protein A-based matrices to capture ATN-103 was assessed: MabSelect TM (GE Healthcare), MabSelect Xtra TM (GE Healthcare), VaUltraPlus (Millipore) and MabSelectSuRe TM(GE Healthcare). MabSelect TM A protein A ligand containing a Z domain is used, and the resin backbone is more hydrophobic due to cross-linking. MabSelectXtra TM Uses the same ligands as MabSelect with a 30% increase in density, and has smaller beads and larger pore sizes. VaUltraPlus has a glass-based backbone and native protein A ligands. It is designed for higher capacity at higher flow rates. MabSelectSuRe TM Binding of Fc-containing molecules (ATN-103 has no Fc region) and its novel ligand allows for greater caustic stability.

[0214] When using MabSelect TM When the purified protein A peak pool was used as loading material (pH=7.0, diluted to 1g / L (expected condition medium concentration)), VaUltraPlus showe...

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Abstract

The invention discloses a method of purifying or separating a single domain antigen combined molecule (SDAB) which includes one or more single combined domains (e.g., one or more nano antibody molecules) and basically does not include complementary antibody domain and immonoglubolin constant domain on the basis of affinity chromatography of protein A.

Description

[0001] This application is a divisional case of a Chinese invention patent application (application date: October 29, 2009; application number: 200980153069.5 (international application number: PCT / US2009 / 062651); invention name: purification method for single-domain antigen-binding molecules) Apply. [0002] Cross References to Related Applications [0003] 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. [0004] sequence listing [0005] 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 [0006] Recombinant proteins such as antibodies often contain various impurities that need to be removed before the protein product becomes pharmaceutically accep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22C07K1/18C07K16/06C07K16/18C07K16/24
CPCC07K1/18C07K1/22C07K16/065C07K16/18C07K16/241C07K2317/569A61P1/04A61P17/06A61P19/02A61P25/00A61P29/00A61P37/06A61P43/00C07K1/36C07K16/06C12N15/11
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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