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Method of removing recombinant expression antibody aggregates and degradation products

A technology for degradation products and antibodies, applied in peptide preparation methods, chemical instruments and methods, anti-animal/human immunoglobulins, etc., can solve problems such as difficult removal of antibody aggregates and degradation fragments, increase of antibody protein retention time, etc.

Active Publication Date: 2020-12-01
MABWELL (SHANGHAI) BIOSCIENCE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Method of removing recombinant expression antibody aggregates and degradation products
  • Method of removing recombinant expression antibody aggregates and degradation products
  • Method of removing recombinant expression antibody aggregates and degradation products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Recombinant human anti-TNF-α monoclonal antibody protein sequence.

[0080] A human antibody for treating TNF-α-related diseases is recombinant human anti-TNF-α monoclonal antibody is expressed in CHO cells, and is purified by a series of standard chromatography steps. In this embodiment, the Ada-wood single-sequence of the Adta has a heavy chain variable region of SEQ ID NO: 1; and has the light chain variable region of SEQ ID NO: 2. Its molecular weight is about 148 kDa, consisting of 2 IgG1 heavy chains and 2 kappa light chains. Each heavy chain contains 451 amino acids, the molecular weight is about 49 kDa, and its sequence is shown in SEQ ID NO: 5; each light chain contains 214 amino acids, the molecular weight is about 24 kDa, and the sequence is shown in SEQ ID NO: 6.

Embodiment 2

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

[0082] The method of specific binding of TNF-α monoclonal antibody anti-TNF-α antibody is expressed in CHO cells. The expression vector containing the antibody gene was constructed by conventional molecularcloning, a derivative cell line of CHO-K1 cells (ATCCCCCL61) as a host cell expression. The construction process of high-yield stable cell line is briefly described as follows: Host cell suspension growth in CD-CHO medium (GIBCO, CA), takes place in a long-term host cell centrifugation, resuspended in fresh CD-CHO medium, count And regulate the cell density to 1.43 × 10 7 A / ml, take 600 ul of the cell suspension to add an electric shock cup, then add a linear plasmid 40 ug, and mix the cells and plasmids uniform with a pipette. Use the BIO-RAD radiograph to convert, instrument parameter setting To: Capacitor: 960ufd, voltage: 300V. Usually the electric shock time is 15-20 milliseconds. T...

Embodiment 3

[0083] Example 3: The effect of recombinant Ada Habitan common proximity and chromatography pre-washing conditions.

[0084] By adding pre-washing steps in affinity chromatography, it is mainly to add different pre-wash additives to examine the removal effect of pre-washing conditions for the polymer. If the pre-wash condition is inappropriate, the focus is focused on the optimization of the eluting step.

[0085] Affinity chromatography (different pre-wash):

[0086] Filler: Ge MabSelect 5ml, Chromatography: Bo Gron BXP10mm / 20cm, column height: 6.4cm;

[0087] Buffer:

[0088] Balance buffer buffer A1 (20 mM Pb + 0.15 M NaCl, pH 7.0);

[0089] Rebalance buffer Buffer A2 (20 mM Pb, pH 7.0);

[0090] 3 pre-wash dense Buffer B1-1 (20mm Pb + 1m urea, pH 7.0);

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

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

[0093] Elite buffer B2 (20mm citrate-Na 2 HPO 4 , PH3.4).

[0094] experiment procedure:

[0095] First use buffer A...

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Abstract

The invention provides an affinity chromatography method for removing antibody aggregates and degradation fragments and improving the yield and purity of antibody single bodies. The purity of the antibody single body is improved by optimizing parameter conditions such as pH value, conductivity and buffer solution type in the affinity chromatography process of the recombinant adalimumab, and then the yield of the antibody singer body is improved by adding urea or a PEG additive into an elution buffer solution. In a preferred embodiment, the yield of the recombinant adalimumab single body can reach 90%, and the purity of the single body in the product exceeds 95%.

Description

Technical field [0001] The present invention belongs to the field of biopharmaceutical, and more particularly to a separation purification method of genetic engineering recombinant expression antibody, more particularly to remove recombinant expression antibody polymers and degradation products from a culture solution containing recombinant antibody, and improve recombinant expression antibody orders. Method of content and purity of body. Background technique [0002] Tumor necrosis factor α (TNFα) is a cytokine produced by many cell types (including monocytes and macrophages), which is based on its ability to induce tumor necrosis of certain mice (see, for example, OLD, L. (1985) Science230: 630-632). Subsequently, a factor associated with the morbidity related to the malignanism is that the same molecule is the same as TNFα. It is believed that TNFα is involved in mediated shock (see, for example, Beutler, B. and Cerami, a. (1988) Annu.Rev. Biochem. 57: 505-518; Beutler, B. and...

Claims

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Application Information

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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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