A method for removing recombinantly expressed antibody aggregates and degradation products

An antibody and monoclonal antibody technology, applied in the preparation methods of peptides, chemical instruments and methods, anti-animal/human immunoglobulins, etc., can solve the problem of difficult to remove antibody aggregates and degraded fragments, increase antibody protein retention time, etc. question

Active Publication Date: 2021-01-12
MABWELL (SHANGHAI) BIOSCIENCE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the technical problem that affinity chromatography is difficult to remove antibody aggregates and degraded fragments, the present invention is based on the research on the removal of protein aggregates in the prior art and found that polyethylene glycol (PEG) increases the retention time of antibody proteins and removes aggregates The function mainly depends on PEG improving the hydration capacity of the stationary phase surface in the chromatography process. It is speculated that the effect of PEG on improving the hydration capacity of the matrix may also be applicable to the affinity matrix. Try to use PEG in the affinity chromatography to improve the hydration of the affinity chromatography. resolution, to remove aggregates and degraded fragments

Method used

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  • A method for removing recombinantly expressed antibody aggregates and degradation products
  • A method for removing recombinantly expressed antibody aggregates and degradation products
  • A method for removing recombinantly expressed antibody aggregates and degradation products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Protein sequence of recombinant human anti-TNF-α monoclonal antibody.

[0080] A human antibody for the treatment of TNF-α-related diseases is a recombinant human anti-TNF-α monoclonal antibody, which is expressed in CHO cells by means of genetic engineering, and purified by a series of standard chromatography steps. The adalimumab sequence specifically used in this example has the heavy chain variable region described in SEQ ID NO:1; and has the light chain variable region described in SEQ ID NO:2. Its molecular weight is about 148kDa and consists of 2 IgG1 heavy chains and 2 kappa light chains. Each heavy chain contains 451 amino acids with a molecular weight of about 49kDa, and its sequence is shown in SEQ ID NO:5; each light chain contains 214 amino acids with a molecular weight of about 24kDa, and its sequence is shown in SEQ ID NO:6.

Embodiment 2

[0081] Example 2: Recombinant expression and purification of recombinant human adalimumab.

[0082] Referring to the method of Wood et al., J Immunol. 145:3011 (1990), etc., the monoclonal antibody anti-TNF-α antibody that specifically binds to TNF-α was expressed in CHO cells. The expression vector containing the antibody gene was constructed by conventional molecular biology methods (Molecular Cloning), and a derived cell line of CHO-k1 cell (ATCCCCL61) was used as the host cell for expression. The construction process of high-yielding stable cell lines is briefly described as follows: host cells are grown in suspension in CD-CHO medium (Gibco, CA), centrifuged host cells in logarithmic growth phase, resuspended in fresh CD-CHO medium, and counted. and adjust the cell density to 1.43 × 10 7 Cells / ml, take 600ul of the above cell suspension and add it to the electric shock cup, then add 40ug of the linearized plasmid, (use a pipette to mix the cells and the plasmid evenly. U...

Embodiment 3

[0083] Example 3: Effects of recombinant adalimumab general affinity chromatography pre-washing conditions on yield and monomer purity.

[0084] By adding pre-wash steps in affinity chromatography, mainly adding different pre-wash additives, the effect of pre-wash conditions on the removal of aggregates was investigated. If prewash conditions are not suitable, focus on optimization studies of the elution step.

[0085] Affinity chromatography (different prewash):

[0086] Filler: GE Mabselect 5ml, column: Borgron BXP10mm / 20cm, column height: 6.4cm;

[0087] Buffer:

[0088] Equilibration buffer Buffer A1 (20mM PB+ 0.15M NaCl, pH 7.0);

[0089] Re-equilibration buffer Buffer A2 (20mM PB, pH 7.0);

[0090] 3 kinds of pre-elution buffer B1-1 (20mM PB + 1M urea, pH 7.0);

[0091] Buffer B1-2 (0.2 M Arg-HCl + 1 M NaCl, pH 7.0);

[0092]Buffer B1-3 (10 mM EDTA + 1 M NaCl, pH 7.0);

[0093] Eluent Buffer B2 (20mM citric acid-Na 2 HPO 4 , pH 3.4).

[0094] experiment procedu...

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Abstract

The present disclosure provides an affinity chromatography method for removing antibody aggregates and degraded fragments, and improving the yield and purity of antibody monomers. The purity of the antibody monomer was improved by optimizing the pH value, conductivity, buffer type and other parameters in the affinity chromatography process of recombinant adalimumab, and the purity of the antibody monomer was improved by adding urea or PEG additives to the elution buffer. Antibody monomer yield. In a preferred embodiment, the yield of recombinant adalimumab monomer can reach 90%, and the purity of the monomer in the product exceeds 95%.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a method for separating and purifying recombinantly expressed antibodies from genetically engineered bacteria, and more particularly to a method for removing recombinantly expressed antibody aggregates and degradation products from a culture solution containing recombinantly expressed antibodies, and improving the production of recombinantly expressed antibody monomers. method for the content and purity of the body. Background technique [0002] Tumor necrosis factor alpha (TNFα) is a cytokine produced by many cell types, including monocytes and macrophages, and its original identification was based on its ability to induce necrosis in certain mouse tumors (see, e.g., Old, L. (1985). ) Science 230: 630-632). Subsequently, a factor associated with cachexia called cachexia was shown to be the same molecule as TNFα. TNFα is believed to be involved in mediating shock (see, eg, Beu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C07K1/22
CPCC07K16/241C07K2317/56
Inventor 梅菲任杰方鹏季荣钰陈坤孙小伟曹雨霞
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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