High throughput screening method for monoclonal antibody product cell strains through two-dimensional liquid phase

A cell line, high-throughput technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of lengthy sample processing and storage steps, reduce the accuracy of analysis results, increase the probability of sample degradation, etc., to save manpower , improve efficiency and avoid degradation

Inactive Publication Date: 2019-03-15
张骐
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, in the research and development and production of antibody products, in order to obtain the polymer content data of monoclonal antibodies secreted by a certain cell line, two steps of experiments must be completed manually in sequence: first, collect and capture the monoclonal antibodies in the cell culture medium , and then use the captured monoclonal antibody as a sample, and use other chromatographic analysis to obtain the analysis results reflecting the physical and chemical properties of the monoclonal antibody. The whole process takes several days, and is accompanied by a large amount of manpower and coordination within the organization. Laborious, and due to lengthy sample handling and storage steps, the probability of sample degradation is increased, reducing the accuracy of analytical results

Method used

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  • High throughput screening method for monoclonal antibody product cell strains through two-dimensional liquid phase
  • High throughput screening method for monoclonal antibody product cell strains through two-dimensional liquid phase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The cell line of bevacizumab was placed in SFM Ⅰ Ⅰ medium supplemented with 4 mmol / L glutamic acid, and the medium was filtered with a 0.2 μm filter membrane to remove cells and other larger particles to prepare the sample to be tested.

[0024] The detection of bevacizumab by two-dimensional liquid phase is used for high-throughput screening of bevacizumab cell lines. The conditions for separation by first-dimensional liquid chromatography are as follows: an affinity column is used to add 40 mmol The Tris-HCl buffer solution with a pH value of 6.3 as the mobile phase A, the buffer solution with a pH value of 2.5 as the mobile phase B, the flow rate is 0.3mL / min, the column temperature is 25°C, and the detection wavelength 280nm, gradient elution conditions: 0~3min, 0%B; 3~3.1min, 0%~100%B; 3.1~6min, 100%B; 6~6.1min, 100%~0%B; 6.1 ~10min, 0% B; the condition that the second-dimension liquid chromatography carries out detection is: adopt size-exclusion chromatographic co...

Embodiment 2

[0031] The cell line of bevacizumab was placed in SFM Ⅰ Ⅰ medium supplemented with 4 mmol / L glutamic acid, and the medium was filtered with a 0.2 μm filter membrane to remove cells and other larger particles to prepare the sample to be tested.

[0032] Bevacizumab is detected by two-dimensional liquid phase to perform high-throughput screening of bevacizumab cell lines. The conditions for separation by first-dimensional liquid chromatography are: use an affinity chromatographic column to add 60 mmol The Tris-HCl buffer solution whose pH value is 7.3 of the NaCl eluent is mobile phase A, and the buffer solution with a pH value of 3.5 is mobile phase B. The flow rate is 0.5mL / min, the detection wavelength is 280nm, and the column temperature is 25°C, gradient elution conditions: 0~3min, 0%B; 3~3.1min, 0%~100%B; 3.1~6min, 100%B; 6~6.1min, 100%~0%B; 6.1 ~10min, 0% B; the condition that the second-dimension liquid chromatography carries out detection is: adopt size-exclusion chroma...

Embodiment 3

[0039] The cell line of bevacizumab was placed in SFM Ⅰ Ⅰ medium supplemented with 4 mmol / L glutamic acid, and the medium was filtered with a 0.2 μm filter membrane to remove cells and other larger particles to prepare the sample to be tested.

[0040]The detection of bevacizumab by two-dimensional liquid phase is used for high-throughput screening of bevacizumab cell lines. The conditions for separation by first-dimensional liquid chromatography are as follows: an affinity column is used to add 50 mmol The Tris-HCl buffer solution with a pH value of 6.8 as the mobile phase A, the buffer solution with a pH value of 3.0 as the mobile phase B, the flow rate is 0.5mL / min, the detection wavelength is 280nm, and the column temperature is 25°C, gradient elution conditions: 0~3min, 0%B; 3~3.1min, 0%~100%B; 3.1~6min, 100%B; 6~6.1min, 100%~0%B; 6.1 ~10min, 0% B; the condition that the second-dimension liquid chromatography carries out detection is: adopt size-exclusion chromatographic ...

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Abstract

The invention relates to a high throughput screening method for monoclonal antibody product cell strains through a two-dimensional liquid phase. The high throughput screening method comprises detection for a monoclonal antibody product through the two-dimensional liquid phase, wherein the monoclonal antibody product is bevacizumab. The method specifically comprises the following steps: (1) separating a first-dimensional liquid chromatogram; and (2) detecting a second-dimensional liquid chromatogram: draining fractions obtained by cutting the first-dimensional liquid chromatogram according to retention time to the second-dimensional liquid chromatogram for detection. According to the method, the screening efficiency is improved, the manpower is saved, and the possibility that a sample is degraded is avoided due to the fact that tedious sample treatment and storage steps are avoided, so that the accuracy of an analysis result is improved.

Description

technical field [0001] The invention relates to a method for high-throughput screening of cell lines of monoclonal antibody products by two-dimensional liquid, and belongs to the technical field of analysis of antibody products including monoclonal antibodies and fusion proteins. Background technique [0002] Nowadays, monoclonal antibodies ("monoclonal antibodies" for short) have been produced on a large scale and widely used, especially therapeutic monoclonal antibodies, and how to screen out monoclonal antibodies that can secrete stable enough physical and chemical properties before the development of antibody technology The cell line has become a key starting point for process development. At this stage, in the research and development and production of antibody products, in order to obtain the polymer content data of monoclonal antibodies secreted by a certain cell line, two steps of experiments must be completed manually in sequence: first, collect and capture the mono...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 张骐
Owner 张骐
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