Method for determining immunoglobulin charge isomer glycosylation and terminal modification states

An immunoglobulin and charge isomerization technology, applied in the field of glycosylation and terminal modification kits, can solve the problems of high cost, complicated sample processing, and the influence of the original terminal modification of antibodies, and achieves improved accuracy, sample Handling simple effects

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
Therefore, there is still no report on simultaneous rapid determination of IgG glycosylation and terminal modification suitable for antibody development, and there is no method that can quickly and accurately characterize and identify the structure of a small number of antibody charge variants

Method used

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  • Method for determining immunoglobulin charge isomer glycosylation and terminal modification states
  • Method for determining immunoglobulin charge isomer glycosylation and terminal modification states
  • Method for determining immunoglobulin charge isomer glycosylation and terminal modification states

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Condition screening of immunoglobulin reduction methods

[0050] 1.1 Investigate the dosage of DTT

[0051] The effects of 4 different amounts of DTT on the separation of light and heavy chains were investigated. Take 4 parts of 5μg antibody protein A and add them to 10μL 6M guanidine hydrochloride solution respectively, then add 2μL and 5μL of 0.1M DTT solution, and 2μL and 4μL of 0.5M DTT solution, and finally add an appropriate amount of 6M guanidine hydrochloride solution to make the final concentration of DTT respectively It is 10 mM, 25 mM, 50 mM and 100 mM to react with the IgG1 protein at 65° C. for 45 min.

[0052] The light chain and the heavy chain obtained by the reaction were separated by C4 reverse ultrahigh pressure liquid chromatography, and the liquid system used was UPLC (Waters, ACQUITY). Column: Waters, ACQUITY UPLC column, BEH C4, 1.7μm (particle size), (Aperture), 2.1×50mm. The chromatographic conditions were set as follows: the column temp...

Embodiment 2

[0081] Example 2 Using the UPLC-MS method of the present invention to determine the glycosylation and terminal modification of antibody A and antibody B (IgG1)

[0082] Using optimized reducing conditions (5μg antibody A was added to 10μL 6M guanidine hydrochloride solution, then 0.5M DTT solution 2μL was added, and finally an appropriate amount of 6M guanidine hydrochloride solution was added to make the final concentration of DTT 50mM, and the reaction at 65°C for 45min), UPLC separation (with Consistent with Example 1.1), ESI-MS detection (consistent with Example 1.1) and normalized data processing (consistent with Example 1.2) analyze the glycosylation and terminal modification of antibody A and antibody B. The first amino acid at the N-terminus of antibody A light chain and heavy chain are both glutamine (Gln), which is prone to pyroglutamate cyclization; the first amino acid at the N-terminus of antibody B light chain is glutamate (Glu), which is not prone to occur Pyroglu...

Embodiment 3

[0085] Example 3 The UPLC-MS method of the present invention is used to determine the glycosylation and terminal modification of the charge isomer of antibody A

[0086] Take 200μg of antibody A, add 1μg, 4μg, 10μg and 20μg of carboxypeptidase B respectively, and react at 37°C for about 3 hours. Then use cation exchange chromatography (CEX-HPLC) for analysis; the column can be Dionnex BioLC MAbPac SCX-104×250mm, using mobile phase E (20mM MES and 20mM NaCl) and mobile phase F (20mM MES and 200mM NaCl) for gradient Elution (0-3min, 10-20%F, 3-25min, 20-50%F, 25-38min, 50-70%F, 38-40min, 70%F, 40-42min, 70-10%F , 42-45min, 10% F). The results show that the digestion is insufficient when the amount of carboxypeptidase B is 1 μg and 4 μg, and the digestion is sufficient when the amount of carboxypeptidase B is ≥10 μg, so 10 μg carboxypeptidase B / 200 μg antibody is preferred. In addition, take several portions of 200μg of antibody A, add 10μg of carboxypeptidase B, and react at 37°...

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Abstract

The invention provides a method for determining immunoglobulin charge isomer 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 before and after carboxypeptidase B digestion are analyzed by using cation exchange chromatography (CEX-HPLC), and different immunoglobulin charge isomers are collected according to retention times after the column; (2) the immunoglobulin component 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 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 invention relates to the field of biotechnology. Specifically, the present invention provides a method for simultaneously measuring the glycosylation and terminal modification of immunoglobulin charge isomers. At the same time, the present invention also relates to a method for determining the glycosylation and terminal modification of immunoglobulin charge isomers. A kit for terminal modification. Background technique [0002] In the past ten years, monoclonal antibodies have achieved great success and tremendous development in the biomedical industry and 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. In terms of drug molecular characteristics, antibodies have greater heterogeneity. This characteristic of antibodies is caused by many factors, among which post-translational modification...

Claims

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Application Information

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IPC IPC(8): G01N30/88
Inventor 朱保国彭育才杨嘉明
Owner LIVZON MABPHARM
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