Separation and purification method for monoclonal antibody

A monoclonal antibody, separation and purification technology, applied in the preparation methods of peptides, chemical instruments and methods, anti-animal/human immunoglobulins, etc. The effect of high product yield

Active Publication Date: 2016-11-23
HUBEI BIO PHARMA IND TECHCAL INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the current isolation and purification methods for recombinant anti-human-EGFR monoclonal antibodies still need to be improved

Method used

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  • Separation and purification method for monoclonal antibody
  • Separation and purification method for monoclonal antibody
  • Separation and purification method for monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The cell culture supernatant harvested from the 150L fermenter was divided into three parts, and the Mabselect SuRe chromatography column was used for the first step of purification three times, and 40L of the first sample was loaded on the equilibrated Mabselect SuRe chromatography column (BPG200 / 50) In, after loading the sample, rinse the chromatography column with buffer A (containing 20mM PB, 150mM NaCl, pH 7.2), and then wash the column with buffer B (containing 20mM PB, 1M NaCl, pH 6.5) and buffer A ( Contains 20mM PB, 150mM NaCl, pH 7.2) to wash the chromatographic column, then elute with buffer C (ie 0.1M NaAC-HAc, pH 3.4), collect the eluted fraction; repeat this purification step twice, the purification process chromatogram picture see figure 1 ; Each collected elution fraction (pH 3.7, referred to as "elution fraction I") was left at room temperature for 2 hours, then diluted with pH 7.2 phosphate buffer to neutralize to pH 5.2; three dilutions were combined ...

Embodiment 2

[0046] Load the cell culture supernatant harvested from the 7.5L fermenter on the equilibrated Mabselect SuRe LX chromatographic column (XK50 / 30). ) to wash the chromatography column, then wash the chromatography column with buffer B (containing 20mM PB, 1M NaCl, pH 7.0) and buffer A (containing 20mM PB, 120mM NaCl, pH 7.5) in sequence, and then wash the chromatography column with buffer C ( That is, 0.1M glycine, pH 3.2) was eluted, and the eluted fraction was collected, called eluted fraction I; the eluted fraction I (pH 3.5) was left at room temperature for 1 hour, and then diluted with phosphate buffer solution of pH 7.2 Neutralize (to pH 5.5), load the sample on the equilibrated POROS XS chromatography column (XK50 / 20), after loading, wash the column with buffer D (20mM PB, pH 6.2), and then use buffer Solution E (20mM PB, 100mMNaCl, pH 6.2) was eluted, and the eluted fraction was collected, called eluted fraction II; the eluted fraction II was diluted 2 times with 20mM P...

Embodiment 3

[0048] Load the cell culture supernatant harvested from the 7.5L fermenter on the equilibrated ProSep Ultra Plus chromatography column. After loading, wash the chromatography with buffer A (including 20mM PB, 180mM NaCl, pH 7.0) Column, then wash the column with buffer B (containing 20mM PB, 1.5M NaCl, pH 6.0) and buffer A (containing 20mM PB, 180mM NaCl, pH 7.0) in sequence, and then wash the chromatography column with buffer C (that is, 0.1M glycine , pH 3.0) elution, collect the elution fraction, called elution fraction I; leave the elution fraction I (pH 3.2) at room temperature for 0.5 hours, then dilute and neutralize to pH 5 with pH 8.0 phosphate buffer .8. Load the sample on the equilibrated Eshmuno CPX chromatography column. After loading the sample, wash the column with buffer D (20mM PB, 50mM NaCl, pH 5.8), and then wash the column with buffer E (20mM PB, 200mM NaCl , pH 5.8) elution, collect the elution fraction, called elution fraction II; dilute the elution fract...

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Abstract

The invention discloses a separation and purification method for a monoclonal antibody. The method includes the steps that cell culture supernatant containing the monoclonal antibody is subjected to Protein A affinity chromatography so as to obtain a first elution component; the first elution component is placed for a specified time at the room temperature with a specified pH value kept so as to obtain the first elution component subjected to low-pH-value inactivated virus treatment; the first elution component subjected to low-pH-value inactivated virus treatment is neutralized and then subjected to cation exchange chromatography so as to obtain a second elution component; the second elution component is diluted and then subjected to anion exchange chromatography so as to obtain a through flow; the through flow is filtered with a virus removal filter so as to obtain filter liquor; the filter liquor is subjected to ultrafiltration concentration, filter wash and buffer solution replacement in sequence so as to obtain stoste of the monoclonal antibody. The method is simple and easy to implement, and the endotoxin content and other quality indexes of the obtained product meet the quality standard of monoclonal antibody drugs.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to the field of development and production of antibody drugs, in particular to a method for separating and purifying recombinant anti-human-EGFR monoclonal antibodies. Background technique [0002] Malignant tumor is a disease that seriously endangers human health in the world today, ranking second after heart disease among the deaths caused by various diseases. With the aging of the population and the continuous development of urbanization, the incidence of malignant tumors is on the rise. [0003] Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene C-erbB-1, and erbB-1 is widely distributed on the epithelial cell membrane except vascular tissue. Overexpression of EGFR has been found in various solid tumors such as colorectal cancer, head and neck cancer, and lung cancer. EGFR is a transmembrane glycoprotein with a molecular weight of ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/22C07K16/24C07K1/22C07K1/18
Inventor 王学海陈爱芳何昆许勇杨仲文李莉娥马梵辛张帆吕兴凯田吕明肖强黄璐田华于静
Owner HUBEI BIO PHARMA IND TECHCAL INST
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