Purification method of human glucagon like peptide-1 (hGLP-1) analogue fusion proteins

A glucagon and fusion protein technology, applied in the preparation methods of glucagon and peptides, animal/human proteins, etc., can solve problems such as differences in physical and chemical properties, large differences in impurities, difficult antibody purification methods, etc., to achieve Improving the quality control standard and removing the effect of HCP content

Active Publication Date: 2019-12-03
CHENGDU JINKAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difference in the amino acid composition of the antibody, the physical and chemical properties of the antibody are significantly different, and the differences in the respective impuritie...

Method used

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  • Purification method of human glucagon like peptide-1 (hGLP-1) analogue fusion proteins
  • Purification method of human glucagon like peptide-1 (hGLP-1) analogue fusion proteins
  • Purification method of human glucagon like peptide-1 (hGLP-1) analogue fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1. A purification method of hGLP-1 analog fusion protein

[0084] 1. Clarify and filter cell culture harvest fluid

[0085] (1) For the 0-5L cell culture harvest liquid, centrifugation can be used to remove large particles of insoluble matter such as cells and debris, and then filtered with a 0.22 μm filter membrane to obtain a clarified filtrate containing hGLP-1 analogue fusion protein. The conditions for the centrifugation operation are: 3500g, 7-12min, 2-8°C.

[0086] (2) For the 50L and 250L cell culture harvest liquid, the DOHC and B1HC two-stage series connection method of Millipore Company can be used to perform depth filtration and filter with a 0.2 μm filter element to obtain a clarified filtrate.

[0087] 2. Crude purification of Protein A affinity chromatography

[0088] The clarification filtrate that above-mentioned step 1 obtains is carried out the rough purification of Protein A affinity chromatography, and the step of Protein A affinity chroma...

Embodiment 2

[0130] Embodiment 2 A kind of purification method of hGLP-1 analogue fusion protein

[0131] 1. Clarify and filter cell culture harvest fluid

[0132] With embodiment 1 step 1 (1);

[0133] 2. Crude purification of Protein A affinity chromatography

[0134]Step is with embodiment 1 step 2, and concrete step parameter sees Table 2 for details;

[0135] 3. Low pH incubation inactivated virus

[0136] With embodiment 1 step 3;

[0137] 4. Fine purification

[0138] Step is with embodiment 1 step 4, and concrete step parameter sees Table 2 for details;

[0139] 5. Prepare the target protein stock solution by filtration

[0140] Same as Step 5 of Example 1.

[0141] Table 2 crude purification and fine purification chromatography methods, steps and parameter conditions

[0142]

[0143] Using the purification method of this example, the HCP content in the target protein can also be reduced to below 100ppm, and charge variants and other trace impurities can be effectively ...

Embodiment 3

[0144] Embodiment 3 A kind of purification method of hGLP-1 analogue fusion protein

[0145] 1. Clarify and filter cell culture harvest fluid

[0146] With embodiment 1 step 1 (1);

[0147] 2. Crude purification of Protein A affinity chromatography

[0148] Step is the same as embodiment 1 step 2, and the specific step parameters are shown in Table 3;

[0149] 3. Low pH incubation inactivated virus

[0150] With embodiment 1 step 3;

[0151] 4. Fine purification

[0152] Step is with embodiment 1 step 4, and concrete step parameter sees Table 3 for details;

[0153] 5. Prepare the target protein stock solution by filtration

[0154] Same as Step 5 of Example 1.

[0155] Table 3 crude purification and fine purification chromatography methods, steps and parameter conditions

[0156]

[0157] Using the purification method of this example, the HCP content in the target protein can also be reduced to below 100ppm, and charge variants and other measured impurities can be ...

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Abstract

The invention provides a purification method of human glucagon like peptide-1 (hGLP-1) analogue fusion proteins (including dulaglutide). The purification method includes the step that a three-step chromatography method is adopted for purification, specifically, a sample is firstly subjected to a coarse purification step of Protein A affinity chromatography so as to effectively remove impurities such as HCP and endotoxin and maintain the high objective protein yield; and then anion exchange chromatography and hydrophobic chromatography are used for further fine purification so as to effectivelyremove charge isomers, residual HCP and other trace impurities in the sample, the content of all the impurities is controlled within the administration safety range, and the high objective protein yield and activity are maintained. According to the purification method, during separation and purification of an objective protein, good reproducibility and stability are achieved, and the purificationmethod is suitable for downstream purification process linear amplification of the hGLP-1 analogue fusion proteins so as to be used for production and preparation of drugs for treating type 2 diabetes or slimming drugs.

Description

technical field [0001] The invention belongs to the field of protein separation and purification, and in particular relates to a new purification process of human glucagon-like peptide-1 analog fusion protein. The process is suitable for large-scale separation and purification of GLP-1 analog fusion protein and its application in the preparation of protein medicine for treatment and / or prevention. Background technique [0002] Human glucagon-like peptide-1 (human glucogan like peptide-1, hGLP-1) is a polypeptide composed of 37 amino acids synthesized and secreted by intestinal L cells. hGLP-1 has the functions of stimulating insulin secretion and promoting glucose absorption. and blood sugar lowering; hGLP-1 can also reduce glucagon secretion, inhibit gastric emptying, control appetite, reduce body weight and increase glucose utilization; in addition, hGLP-1 can also promote the regeneration of islet β cells and prevent their apoptosis hGLP-1 is dead, and the effect of hGLP...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/36C07K1/34C07K1/18C07K1/20C07K1/22
CPCC07K14/605C07K2319/00
Inventor 谷类燃罗天学荣艳珍
Owner CHENGDU JINKAI BIOTECH CO LTD
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