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Method for determining immunoglobulin glycosylation and terminal modification states during fermentation process

A technology of immunoglobulin and terminal modification, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex sample processing, complicated processing process, unsuitable sample batch detection, etc., and achieve simple sample processing and high resolution. Effect

Active Publication Date: 2013-07-24
LIVZON MABPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic fluorescent labeling method is a classic quantitative method for the determination of IgG1 glycosylation, but the sample processing process is quite complicated, time-consuming, and requires a large amount of sample; there are also mass spectrometry used to detect IgG enzymatic fragments for glycosylation analysis, such as papain enzyme and IdeS enzyme, etc., but these methods have some shortcomings, such as the selectivity of the enzyme cutting site is not strong, or the cost is too high, or the sample processing is complicated, etc., and it is not suitable for the batch detection of routine or process development samples.
Using LC-MS for peptide map analysis can theoretically detect the glycosylation and terminal modification of antibodies at the same time. However, there are many technical difficulties in the separation, measurement and data analysis of glycopeptides, which are not suitable for quantitative analysis of glycosylation. The process is complex and time-consuming, and the enzyme digestion process may also affect the original terminal modification of the antibody
During the protein fermentation process, the purity of the protein in the fermentation broth needs to be tested at any time to better control the fermentation process. However, the detection requires a large amount of samples and the time-consuming detection is still the biggest problem at present.
Therefore, there is still no report on simultaneous rapid determination of IgG glycosylation and terminal modification suitable for antibody development

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  • Method for determining immunoglobulin glycosylation and terminal modification states during fermentation process
  • Method for determining immunoglobulin glycosylation and terminal modification states during fermentation process
  • Method for determining immunoglobulin glycosylation and terminal modification states during fermentation process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Condition Screening of Immunoglobulin Reduction Method

[0051] 1.1 Investigate the amount of reducing agent DTT

[0052] The effects of four different amounts of DTT on the separation of light and heavy chains were investigated. Take 4 parts of 5 μg antibody protein A, add them to 10 μL 6M guanidine hydrochloride solution respectively, then add 2 μL and 5 μL of 0.1M DTT solution, and 2 μL and 4 μL of 0.5M DTT solution, and finally add an appropriate amount of 6M guanidine hydrochloride solution to make the final concentration of DTT respectively 10mM, 25mM, 50mM and 100mM, and react with the IgG1 protein at 65°C for 45min.

[0053] The light chain and heavy chain obtained in the reaction were separated by C4 reverse ultra-high pressure liquid chromatography, and the liquid phase system used was UPLC (Waters, ACQUITY). Chromatographic column: Waters, ACQUITY UPLC column, BEH C4, 1.7μm (particle size), (Aperture), 2.1×50mm. The chromatographic conditions...

Embodiment 2

[0082] Example 2 Using the UPLC-MS method of the present invention to measure the glycosylation and terminal modification of antibody A and antibody B (IgG1)

[0083] Using optimized reducing conditions (5 μg antibody A was added to 10 μL 6M guanidine hydrochloride solution, then 2 μL 0.5M DTT solution was added, and finally an appropriate amount of 6M guanidine hydrochloride solution was added to make the final concentration of DTT 50 mM, 65 ° C for 45 minutes), UPLC separation ( Consistent with Example 1.1), ESI-MS detection (consistent with Example 1.1) and normalized data processing (consistent with Example 1.2) to analyze the glycosylation and terminal modification of Antibody A and Antibody B. The first amino acid at the N-terminal of the light chain and heavy chain of antibody A is glutamine (Gln), which is prone to pyroglutamic acid cyclization; the first amino acid at the N-terminal of the light chain of antibody B is glutamic acid (Glu), which is less likely to occu...

Embodiment 3

[0086] Example 3 Using the UPLC-MS method of the present invention to measure the glycosylation and terminal modification of antibody A in the reactor cell culture medium

[0087] In the Antibody A cell culture process, collect the cell culture fluid on days 9, 11 and 13; use conventional small-scale Protein A affinity chromatography to obtain preliminary purified IgG1, and then use an ultrafiltration centrifuge tube (molecular weight cut-off 10K ) to concentrate the sample to 1-5 mg / mL. Then adopt the optimized reducing conditions in the present invention (5 μg of antibody A is added to 10 μL of 6M guanidine hydrochloride solution, then 2 μL of 0.5M DTT solution is added, and finally an appropriate amount of 6M guanidine hydrochloride solution is added to make the final concentration of DTT 50 mM, react at 65°C for 45 minutes) , UPLC separation (consistent with Example 1.1), ESI-MS detection (consistent with Example 1.1) and normalized data processing (consistent with Examp...

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Abstract

The invention provides a method for determining immunoglobulin fermentation broth sample glycosylation and terminal modification states. With the method, immunoglobulin glycosylation, N-terminal pyroglutamic acidification, and C-terminal de-lysine can be simultaneously determined rapidly. The method comprises the steps that: (1) immunoglobulin is preliminarily purified with an affinity chromatography (Protein A), and the sample is concentrated by using an ultrafiltration centrifugation device; (2) the immunoglobulin concentrated in the step (1) is denatured by using a denaturant, and is reduced by using a reducing agent, such that light chain and heavy chain are split; (3) the immunoglobulin light chain and heavy chain in the step (2) are separated with reversed-phase ultrahigh-pressure liquid chromatography; (4) molecular weights of the light chain and heavy chain obtained in the step (3) are determined by using mass spectrometry; and (5) the chromatographic data in the step (3) and the mass spectral data in the step (4) are analyzed, such that the glycosylation and terminal modification states of the immunoglobulin are determined.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention provides a method for measuring the glycosylation and terminal modification of immunoglobulins during cell culture. At the same time, the invention also provides a kit for measuring the glycosylation and terminal modification of the immunoglobulin fermentation liquid sample. Background technique [0002] In the past ten years, monoclonal antibodies have achieved great success and great development in the biomedical field and even the entire pharmaceutical industry. Compared with traditional small molecule drugs, monoclonal antibodies have the advantages of strong specificity, significant curative effect, less side effects, and less dosage. Antibodies have greater heterogeneity in terms of drug molecular identity. This characteristic of antibodies is caused by many factors, among which post-translational modification is the most important intrinsic factor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 朱保国彭育才杨嘉明
Owner LIVZON MABPHARM