A method for regulating antibody galactosylation level using ala-gln

A galactose and glycoprotein technology, applied in the biological field, can solve the problems of reducing the production of cell culture protein, reducing the proportion of antibody galactose modification, etc.

Active Publication Date: 2021-09-28
兴盟生物医药(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, reducing the proportion of antibody galactose modification by cooling method will also reduce the production of cell culture protein
[0007] Ala-Gln is mainly used in cell culture, and has the effect of increasing cell culture density, cell viability and expression. The method of Ala-Gln regulating the ratio of antibody galactose modification has not been reported yet

Method used

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  • A method for regulating antibody galactosylation level using ala-gln
  • A method for regulating antibody galactosylation level using ala-gln
  • A method for regulating antibody galactosylation level using ala-gln

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 A method for regulating antibody galactosylation level using Ala-Gln

[0063] In this embodiment, the method of adding Ala-Gln on the day of inoculation is adopted, and the specific steps are as follows:

[0064] (1) FortiCHO+1g / L P188+0.1g / L DEX was used as the basal medium, and Efficient FeedA, Efficient Feed B, Efficient Feed C, Cell boost 1, HYPEP 1511, and GlycosylationAdjust were used as supplementary materials;

[0065] (2) Press 0.35±0.15×10 6 Cell / mL inoculation density CHO cells (expressing Denosumab) were inoculated in a 250mL shake flask, and the volume of the shake flask was 60mL;

[0066] (3) Cell culture temperature is 36.5±0.5°C, shaker speed is 120-130rpm, CO 2 The concentration is 5%-8%;

[0067] (4) Add 4mM Ala-Gln on the day of inoculation;

[0068] (5) Harvest the cells after culturing for 21 days, and perform antibody protein purification after centrifugation.

[0069] (6) Poros-HPLC was used to detect antibody expression and galacto...

Embodiment 2

[0074] Example 2 A method for regulating antibody galactosylation level using Ala-Gln

[0075] Referring to Example 1, wherein step (4) is to add 8mM Ala-Gln on the day of inoculation.

[0076] The results are as follows:

[0077] Glycoform 8mM Ala-Gln *G0F-GN 0.95 *G0 0 G0F 60.69 Man5 2.56 G1F[6] 13.15 G1F[3] 15.13 G2F 4.68 Galactose % 32.96 Fucose% 94.6

[0078] Note: Galactose%=G1F[6]+G1F[3]+G2F; Fucose%=G0F-GN+G0F+G1F[6]+G1F[3]+G2F.

[0079] The CHO cell antibody expression level after 21 days of culture was 2.10g / L ( image 3 ), the galactose modification ratio was 32.96%. During the culture period, the viable cell density changes as figure 1 , the cell viability changes as figure 2 .

Embodiment 3

[0080] Example 3 A method for regulating antibody galactosylation level using Ala-Gln

[0081] Referring to Example 1, wherein step (4) is to add 8mM Ala-Gln on the day of inoculation, and add 2mM Ala-Gln on the 6th day (D6) and the 8th day (D8) after inoculation respectively.

[0082] A control group is set with reference to Example 2.

[0083] The results are analyzed in the following table:

[0084] Glycoform control Add 2mM Ala-Gln to D6 / D8 *G0F-GN 0.95 1.11 *G0 0 0 G0F 60.69 66.1 Man5 2.56 2.96 G1F[6] 13.15 11 G1F[3] 15.13 12.76 G2F 4.68 3.5 Galactose % 32.96 27.26 Fucose% 94.6 94.47

[0085] Note: Galactose%=G1F[6]+G1F[3]+G2F; Fucose%=G0F-GN+G0F+G1F[6]+G1F[3]+G2F.

[0086] The CHO cell antibody expression level after 21 days of culture was 1.91g / L ( Figure 6 ), the galactose modification ratio was 27.26%. During the culture period, the viable cell density changes as Figure 4 , the cell...

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Abstract

The invention provides a method for regulating antibody galactosylation level by using Ala-Gln. By adding Ala-Gln to the culture process of protein-expressing cells to adjust the ratio of galactose modification, while ensuring the protein yield, it solves the problem that the commonly used cooling method reduces the ratio of antibody galactose modification and also reduces the protein yield of cell culture. The method provided by the invention has simple operation, controllable program and is suitable for quantitative production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for regulating antibody galactosylation level by using Ala-Gln. Background technique [0002] Glycosylation of proteins is the most common post-translational modification of proteins, which is the transfer of sugar molecules to specific positions in proteins under the action of glycosyltransferases. The structure and composition of sugar chains on proteins can significantly affect the stability, safety and efficacy of proteins. Therefore, understanding the efficacy of protein glycoforms and controlling glycoform synthesis is important. [0003] The glycosylation types of proteins in mammals can be divided into three types: N-glycosylation, O-glycosylation and GPI glycosylphosphatidylinositol anchor. The synthesis of N-linked sugar chains starts in the endoplasmic reticulum and completes in the Golgi apparatus. The first step in the synthesis of N-sugar chains ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/107
CPCC07K1/1077
Inventor 靳志刚王鑫
Owner 兴盟生物医药(苏州)有限公司
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