Method for purifying antibody and buffer solution used therein

A buffer and antibody technology, applied in the field of biomedicine, can solve problems such as side effects and unsatisfactory effects, and achieve the effect of improving purification yield

Active Publication Date: 2017-12-08
越海百奥药业(绍兴)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Basic amino acids such as arginine and histidine and sucrose have been reported to prevent the aggregation of antibodies during the purification process. However, the research of our unit found that the effect of basic amino acids and sucrose on preventing the aggregation of IgG4 subtype antibodies is not ideal, and even There are also side effects

Method used

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  • Method for purifying antibody and buffer solution used therein
  • Method for purifying antibody and buffer solution used therein
  • Method for purifying antibody and buffer solution used therein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 、 Nivolumab （ IgG4 Subtype antibody representative) Preparation of antibody stock solution

[0038] The light chain and heavy chain genes of Nivolumab were cloned, inserted into the pcDNA3.1 expression vector, transfected into CHO-K1 expression host cells, and screened for high-expression clones. Carry out mass culture and induce expression under CO2 conditions. After 2 weeks of culture, collect the cell culture medium, and use a deep filter system (Pall Company) for clarification and filtration. First, use a deep filter with a pore size of 0.6-9 μm to remove cells, cell debris and insoluble substances. , and then use a depth filter with a pore size below 0.1 μm to remove fine particles, and collect the filtrate as the antibody stock solution for studying the Nivolumab purification process. The protein content in the antibody stock solution was determined to be 1.6 mg / ml by OD280 ultraviolet absorption method, and the pH value was determined to be 7...

Embodiment 2

[0039] Example 2 , using conventional techniques for Nivolumab antibody stock solution Protein A affinity chromatography and low pH Elution

[0040] ① ProteinA affinity chromatography and low pH Elution

[0041] Using GE’s rProteinA Sepharose 4 Fast Flow purification medium and Avant 150 protein purification system, the Nivolumab antibody stock solution was loaded at a dose of 30 mg protein / ml medium, and the binding buffer (Binding Buffer) was 20 mM phosphate buffer at pH 7.0 , the elution buffer (Elution Buffer) was 25mM citrate buffer with pH 3.5, the flow rate was 5ml / min, and the eluate containing protein was collected.

[0042] ②Ultra-high performance liquid chromatography ( UPLC ) combined with size exclusion chromatography ( SEC ) to analyze the eluate

[0043] The pH value of the eluate was determined to be 3.9, and the eluate containing the target protein (i.e. Nivolumab antibody) was sampled, using UPLC H-Class Bio ultra-high per...

Embodiment 3

[0044] Example 3 、Use ion exchange chromatography to separate and identify target proteins and aggregates in affinity chromatography eluates

[0045] Fractogel® EMD SO3 (S) strong ion exchange chromatography (Merck KGaA company) was used to separate the aggregates and target proteins in the affinity chromatography eluate in Example 2, and the equilibrium buffer was 20 mM vinegar at pH 5.0 Salt buffer, the elution buffer is 20 mM acetate buffer at pH 5.0 containing 1M sodium chloride, the UV monitoring of the protein purification instrument shows that there are 2 protein elution peaks, and the 2 elution peaks are collected The liquid was analyzed by ultra-high performance liquid chromatography combined with molecular size exclusion chromatography in Example 2, and the first elution peak was the elution peak of Nivolumab monomer (Monomer), containing only 1% of aggregates, and the second The elution peak is the elution peak of aggregates, containing 70% aggregates and 30% N...

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Abstract

The invention provides a method for purifying an antibody and a buffer solution used therein, belonging to the field of biomedicine. The method and the buffer solution are used for reducing aggregate formed in the low-pH value treatment process of antibody molecules. According to a technical scheme in the invention, the method comprises a step of adding mannitol with a certain concentration into the low-pH value buffer solution. The method can reduce the proportion of aggregate formed by IgG4 subtype antibodies (including a fusion protein containing an IgG4 subtype Fc segment) at a lower pH value to 2% from original 30%, so the purification yield of the antibody is improved, and the activity and safety of antibody drugs can be enhanced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for purifying antibodies and a used buffer. Background technique [0002] Antibody drugs are a type of drug that has grown rapidly in recent years. The variety and market share of new drugs have continued to increase, and they are playing an increasingly important role in the treatment of malignant tumors and autoimmune diseases. In the early stage of antibody drugs, antibodies of the IgG1 subtype dominate, and almost all early antibody drugs are of the IgG1 subtype. In recent years, antibody drugs of the IgG4 subtype have begun to enter the market. IgG4 subtype antibodies have weak ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) effects, and are the first choice for the development of antibody drugs with signal blocking, regulation or neutralization as the main mechanism Ideal for less side effects than ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C07K1/14
CPCC07K1/14C07K16/2818C07K2319/30
Inventor 郭亚军寇庚钱卫珠郭怀祖徐进
Owner 越海百奥药业(绍兴)有限公司
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