Affinity purification process capable of efficiently improving resolution of polymer separation
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A polymer and resolution technology, applied in the field of affinity purification technology, can solve problems such as protein instability, lower purification pressure, low pH elution conditions, etc., and achieve a wide range of applications and remarkable effects
Active Publication Date: 2019-07-30
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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As a key process step in the preparation of antibody drugs (including monoclonal antibodies, FC fusion proteins and double antibody protein drugs), affinity chromatography is currently considered to be the direct capture of products from the fermentation broth. However, due to Too low pH elution conditions may lead to protein instability, resulting in protein formation of polymers
However, for antibody purification, if the affinity capture step can remove part of the polymer, it will reduce the purification pressure in the subsequent purification step
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Embodiment 1
[0080] Example 1 The effect of adding salt ion additives to the elution solution in the Fc fusion protein affinity chromatography process
[0081] Use 50mM Tris-HAc, 150mM NaCl, pH 7.4 as the equilibration buffer for step 1a, step 1c, step 2a, step 5a, and step 5c; acetic acid-sodium acetate system, 50mM NaAc-HAc, 0.5M Imidazole, pH 5.5 (elution condition with salt) as the rinse 2 buffer of step 2b; 50mM NaAc-HAc, pH 5.5, as the rinse 3 buffer of step 2c; 0.1M NaOH solution as the step 1b, step 5b Disinfectant; 20% ethanol as the preservation solution for step 5d; acetic acid-sodium acetate system (50mM NaAc-HAc), with a linear gradient from pH 5.7 to 2.8, and from 0.05M NaCl, 0.05M histidine or 0.05M arginino Select the most suitable salt as an additive, and then evaluate the influence of 0.025M, 0.05M and 0.1M sodium chloride, histidine or arginine in the elution buffer on the resolution, and screen the most suitable elution salt Concentration, and finally the most suitable el...
Embodiment 2
[0092] Example 2 The effect of increasing the pH value of the affinity eluate in the process of antibody protein affinity chromatography
[0093] Use 50mM Tris-HAc, 150mM NaCl, pH 7.4 as the equilibration buffer for step 1a, step 1c, step 2a, step 5a, step 5c; acetic acid-sodium acetate system, 50mM NaAc-HAc, 1M NaCl, pH 5.5 (For elution conditions without salt, use 0.5M His-HCL for 15L) as the wash 2 buffer of step 2b; 50mM NaAc-HAc, pH 5.5, as the wash 3 buffer of step 2c; 0.1M NaOH solution is used as the disinfectant of step 1b and step 5b; 20% ethanol is used as the preservation solution of step 5d; acetic acid-sodium acetate system (50mM NaAc-HAc), with a linear gradient from pH 5.7 to 2.8, and then screened by one step elution The most suitable elution conditions, and the development results are verified through 15L production. The protein A column (GEMabselect SuRe LX) was used for loading, and the loading capacity was 20-45mg / mL.
[0094] Rinse the column with 2-5 column...
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Abstract
The invention provides a novel affinity purification process capable of efficiently improving the resolution of polymer separation. The process specifically includes the following steps that 1, affinity packing is treated, and sampling is performed; 2, drip washing is conducted, wherein a drip washing 2 buffer solution and a drip washing 3 buffer solution are used for drip washing a chromatographic column; 3, elution is conducted, wherein an eluting buffer solution with the pH of 2.8-5.7 or an eluting buffer solution including an additive is used for eluting products, and then the products arecollected; 4, in addition to the elution pH condition and an additive, the development of an elution method is optimized from linear gradient elution to one-step elution. The process breaks through atraditional thinking that an affinity step serves as a coarse purification step. According to the process, polymers can be effectively removed through the affinity step, the effect of fine purifyinga target protein is achieved, the process is amplified by a 15-200 L process and verified by a virus removal process, has a wide application range and can be widely used for antibody proteins, and thus strong technical support is provided for effective control over the content of the polymers in antibody drugs.
Description
Technical field [0001] The present invention relates to the field of biotechnology, in particular to an affinity purification process that effectively improves the separation resolution of multimers and the removal efficiency of multimers. Background technique [0002] The polymer (High Molecular Weight) in antibody drugs (including monoclonal antibodies, FC fusion proteins and double antibody protein drugs) is an antibody drug (including monoclonal antibodies, FC fusion proteins and double antibody protein drugs) that needs to be monitored during the process And the removed impurities, because it may cause the immunogenic reaction of the receptor, it is considered by the relevant regulatory agencies and the entire antibody drug (including monoclonal antibodies, FC fusion proteins and double antibody protein drugs) as an important quality of the product. Index (CQA). [0003] Multimer refers to the aggregate of antibody protein in the form of dimer or multimer. Polymers are mainl...
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