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Method of detecting protein charge variant and method for determining production process of bioproduct

A technology for biological products and variants, applied in the field of biomedicine, can solve the problems of uneven molecular size, charge, glycosylation modification, etc., and achieve the effect of sensitive detection

Inactive Publication Date: 2018-07-27
HUBEI BIO PHARMA IND TECHCAL INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] At present, human recombinant DNA protein products or human recombinant monoclonal antibody products are mostly produced using mammalian cell expression systems. During the production, storage, and transportation of products, product aggregation, degradation, and various Post-translational modification, which leads to the heterogeneity of the product's molecular size, charge, glycosylation modification, etc., and the corresponding variants

Method used

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  • Method of detecting protein charge variant and method for determining production process of bioproduct
  • Method of detecting protein charge variant and method for determining production process of bioproduct
  • Method of detecting protein charge variant and method for determining production process of bioproduct

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Experimental program
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Embodiment 1

[0056] The inventors explored a lot of test methods in the early stage (including mobile phase composition, mobile phase pH value, elution time and chromatographic column screening), and finally determined the key parameters of the test method, and successfully applied to the detection and analysis of protein charge variants. The determination of critical process parameters (CPPs) in the process sector provides a theoretical basis and reference to ensure the safety and effectiveness of clinical medication.

[0057] 1.1 Exploration of mobile phase components

[0058] Condition 1:

[0059] The composition of PIT mobile phase is shown in Table 4.

[0060] Table 4:

[0061]

[0062] The chromatographic conditions are shown in Table 5.

[0063] table 5:

[0064] conditional content

[0065] The elution program is shown in Table 6, and the elution time is 20 min.

[0066] Table 6:

[0067] time

[0068] The obtained chromatogram is as figure 1 shown. T...

Embodiment 2

[0189] Embodiment 2 charge variant peak identification

[0190] The inventor, on the basis of completely separating the variant peaks of the target protein (protein A, monoclonal antibody A), used sialidase (neuraminidase) digestion to identify the variant peaks. The identification results are as follows: Figure 12 and Figure 13 shown. in, Figure 12 The pH-HPLC chromatogram before protein A digestion is shown, Figure 13 The pH-HPLC chromatogram after protein A digestion is shown.

[0191] Depend on Figure 12 and Figure 13 The results showed that after the target protein A was digested with sialidase, all variant peaks disappeared before 21.2 minutes, indicating that the previous variant peaks were all acidic variant peaks, and it was speculated that the post-translational modification of the target protein A was mainly sialic acid modification .

[0192] According to the above test results, it is determined that the control range of the acidic charge variant of th...

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Abstract

A method of detecting a protein charge variant includes the steps of: analyzing to-be-detected protein via pH-IEC ion exchange chromatography to obtain a chromatogram map; on the basis of the chromatogram map, determining content of the to-be-detected protein charge variant. According to the embodiment, the method can detect the protein charge variant quickly, accurately and sensitively. The method especially is suitable for detection of the protein charge variant of recombinant DNA protein products, of which the pI value (isoelectric point) is 7.0-9.0, or a recombinant monoclonal antibody product.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular, the invention relates to a method for detecting protein charge variants and a method for determining the production process of biological products. Background technique [0002] At present, human recombinant DNA protein products or human recombinant monoclonal antibody products are mostly produced using mammalian cell expression systems. During the production, storage, and transportation of products, product aggregation, degradation, and various Post-translational modification, resulting in the heterogeneity of the product's molecular size, charge, glycosylation modification, and its corresponding variants. The formation of these variants may occur at any stage throughout the life cycle of the product, and data show that only post-translational modifications (such as N-terminal glutamate cyclization, C-terminal lysine truncation, N-terminal glutamate aminoamide / glutamic acid cyclization...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 周昌茂马铮黄璐董思聪
Owner HUBEI BIO PHARMA IND TECHCAL INST
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