A method for determining the glycosylation and terminal modification of immunoglobulin charge variants

An immunoglobulin and charge isomerization technology, applied in the field of kits for glycosylation and terminal modification, can solve the problems of complex sample processing, high cost, long time consumption, etc., and achieve the effect of simple sample processing and improved accuracy.

Active Publication Date: 2015-12-02
LIVZON MABPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method for determining the glycosylation and terminal modification of immunoglobulin charge variants
  • A method for determining the glycosylation and terminal modification of immunoglobulin charge variants
  • A method for determining the glycosylation and terminal modification of immunoglobulin charge variants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Condition Screening of Immunoglobulin Reduction Method

[0050] 1.1 Investigate the amount of reducing agent DTT

[0051] 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 0.1 MDTT solution, and 2 μL and 4 μL of 0.5 MDTT solution respectively, and finally add an appropriate amount of 6M guanidine hydrochloride solution to make the final concentration of DTT respectively 10 mM, 25mM, 50mM and 100mM, and react with the IgG1 protein at 65°C for 45min.

[0052] 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, ACQUITYUPLCcolumn, BEHC4, 1.7μm (particle size), (Aperture), 2.1×50mm. The chromatographic...

Embodiment 2

[0081] 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)

[0082] Using optimized reducing conditions (5 μg antibody A was added to 10 μL of 6M guanidine hydrochloride solution, then 2 μL of 0.5 MDTT solution was added, and finally an appropriate amount of 6M guanidine hydrochloride solution was added to make the final concentration of DTT 50 mM, and the reaction was carried out at 65°C for 45 minutes). 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 t...

Embodiment 3

[0085] Example 3 Using the UPLC-MS method of the present invention to determine the glycosylation and terminal modification of the charge variant of antibody A

[0086] Take 200 μg of antibody A, add carboxypeptidase B 1 μg, 4 μg, 10 μg and 20 μg respectively, and react at 37°C for about 3 hours. Then adopt cation exchange chromatography (CEX-HPLC) to analyze; Chromatographic column can adopt DionnexBioLCMAbPacSCX-104 * 250mm, use mobile phase E (20mMMES and 20mMNaCl) and mobile phase F (20mMMES and 200mMNaCl) to carry out 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 when the amount of carboxypeptidase B is 1 μg and 4 μg, the enzyme digestion is insufficient, and when the amount is ≥10 μg, the enzyme digestion is sufficient, so 10 μg carboxypeptidase B / 200 μg antibody is preferred. In addition, several parts of 200 μg of antibody A were added to 10 μg of carboxypeptidase B...

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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 present invention relates to the field of biotechnology. Specifically, the present invention provides a method for simultaneously determining the glycosylation and terminal modification of the charge isoform of immunoglobulin, and the present invention also relates to a method for determining the glycosylation and terminal modification of the charge isoform of immunoglobulin. Kit for end modification status. 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 modifica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
Inventor 朱保国彭育才杨嘉明
Owner LIVZON MABPHARM
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