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Purification method of veterinary recombinant chorionic gonadotrophin

A purification method and eluent technology, which is applied in the field of protein purification and can solve problems such as environmental pollution

Pending Publication Date: 2022-04-15
BEIJING VJT BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the purification process, environmental pollution or harmful reagents such as isopropanol, dithiothreitol (DTT) and β-mercaptoethanol are used to varying degrees, and there is a risk of environmental pollution.

Method used

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  • Purification method of veterinary recombinant chorionic gonadotrophin
  • Purification method of veterinary recombinant chorionic gonadotrophin
  • Purification method of veterinary recombinant chorionic gonadotrophin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Deep filtration of recombinant hCG cell culture fluid

[0048]Equipment: Depth Filter Model: MD0HC05FS1 0.55m 2 and MXOHC01FS1 0.11m 2 Or equivalent product; peristaltic pump model: JMCD175TOC2 or equivalent product.

[0049] Materials: recombinant hCG cell culture fluid (initial weight: 30 g of rhCG target protein measured based on HPLC method (30L bioreactor)); sodium hydroxide; purified water.

[0050] Depth filtration process: All operations are carried out under cooling (4-10°C).

[0051] Depth filter recombinant hCG cell culture fluid (19L) filtered under the following conditions:

[0052] Permeation flow rate: 300mL / min;

[0053] Inlet pressure: 1.5-2.0ba;

[0054] Transmembrane pressure: 1.5-2.0ba;

[0055] Among them: transmembrane pressure (TMP) = (inlet pressure + outlet pressure) / 2;

[0056] The retentate part was washed with the equilibration buffer of the complex chromatography; the washing step was repeated three times until the eff...

Embodiment 2

[0058] Example 2 MMC Composite Chromatography of Recombinant hCG Protein on the Composite Mode Filler of Ion Exchange and Hydrophobic Interaction

[0059] Equipment: chromatography column GE 150 / 500; chromatography system AKTA pilot; pH meter; conductivity meter; balance.

[0060] Materials: rhCG supernatant obtained by deep filtration; initial amount: 30 g of rhCG measured by HPLC; sodium acetate, acetic acid, arginine, sodium hydroxide pellets; purified water; hydrochloric acid; sodium chloride; ethanol .

[0061] Buffer:

[0062] Equilibrium buffer: 50mM NaAC / HAC, 0.08M NaCl, pH5.5;

[0063] Wash buffer: 50mM NaAC / HAC, 0.28M NaCl, 0.06M Arg, pH5.5;

[0064] Eluent buffer: 50mM NaAC / HAC, 0.3M NaCl, 0.3M Arg, pH5.5;

[0065] Regeneration solution: 1M NaCl;

[0066] Cleaning solution: 0.5M NaOH;

[0067] Cleaning solution: H 2 O;

[0068] Storage solution: 20% ethanol.

[0069] Packing: Pack the column according to the manufacturer's instructions. The installed colum...

Embodiment 3

[0086] Example 3 Ion-exchange chromatography of recombinant hCG protein based on monodisperse polystyrene-divinylbenzene copolymer-based spheres

[0087] Equipment: chromatography column GE 100 / 500; chromatography system: AKTApilot; pH meter; conductivity meter; balance.

[0088] Materials: rhCG after composite chromatography; purified water; sodium hydroxide; Tris; hydrochloric acid; ethanol; sodium chloride.

[0089] Buffer:

[0090] Equilibration buffer: 10mM Tris-HCl, 0.12M NaCl, pH 5.0;

[0091] Elution buffer: 10mM Tris-HCl, 0.4M NaCl, pH 5.5;

[0092] Regeneration solution: 1M NaCl;

[0093] Cleaning solution: 0.5M NaOH;

[0094] Cleaning solution: H 2 O;

[0095] Storage solution: 20% ethanol.

[0096] Packing: Pack the column according to the manufacturer's instructions. The installed column dimensions are as follows:

[0097] Diameter: 100mm;

[0098] Column bed height: 12±2cm;

[0099] Column bed volume: 0.8-1.2L.

[0100] Purification process: all opera...

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Abstract

The invention relates to the technical field of protein purification, and particularly discloses a purification method of veterinary recombinant chorionic gonadotrophin. The purification method provided by the invention comprises MMC composite chromatography, ion exchange chromatography and hydrophobic chromatography. The recombinant hCG obtained through the purification method is high in purity (98% or above), high in specific activity (20900 IU / mg) and high in purification yield (65% or above). The whole production process does not involve organic solvents polluting the environment, is more environment-friendly and lower in cost, and can be suitable for large-scale purification of rhCG for veterinary use.

Description

technical field [0001] The invention relates to the field of protein purification, in particular to the purification of veterinary recombinant human chorionic gonadotropin (rhCG). Background technique [0002] Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by syncytiotrophoblast cells of human placenta, which can promote the maturation of ovarian follicles in female animals, induce ovulation, and promote the synthesis and secretion of estrogen. In human clinical practice, hCG is often used in assisted reproductive therapy, treatment of infertility and so on. In the animal health industry, hCG is often used in estrus synchronization and superovulation in female animals, including pigs, cattle, and sheep. In the aquaculture industry, hCG is often used for artificial ripening, ovulation and sperm production of fish, and is one of the preferred oxytocic agents for fish artificial breeding. [0003] Commercialized hCG was originally extracted and purified...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/59C07K1/16C07K1/18C07K1/20
Inventor 罗昊澍于金库裴慧玲师磊韩国熊剑胜
Owner BEIJING VJT BIO CO LTD
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