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An improved method for measuring the isoelectric point of recombinant protein by capillary isoelectric focusing

An isoelectric focusing and recombinant protein technology, applied in the field of electric point, can solve the problems of inaccurate detection results, poor separation effect, and inability to produce peaks for samples, and achieve the effect of accurate detection results and good separation effect.

Active Publication Date: 2019-04-05
CHENGDU JINKAI BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although cIEF has been widely used in the quality analysis of recombinant protein drugs, when separating and analyzing recombinant protein samples, the anode is often used as the inlet port of the capillary electrophoresis instrument to detect the sample, and the cathode is the outlet port (such as Alejandro Cifuentes et al., Journal of Chromatography A, 830 (1999) 453–463; Scott Mack et al., Electrophoresis 2009, 30, 4049–4058, etc.), so that the peak order of the charge isomers of the sample is basic and then acidic, so for acidic samples, the separation peaks are usually in the detection spectrum The separation effect is poor, especially for charge isoforms and isoelectric point markers with pI<4.1, even if the separation time is prolonged, the peak of the sample cannot be released, resulting in inaccurate detection results

Method used

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  • An improved method for measuring the isoelectric point of recombinant protein by capillary isoelectric focusing
  • An improved method for measuring the isoelectric point of recombinant protein by capillary isoelectric focusing
  • An improved method for measuring the isoelectric point of recombinant protein by capillary isoelectric focusing

Examples

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Effect test

Embodiment 1

[0056] Determination of the isoelectric point of recombinant human follicle-stimulating hormone by capillary isoelectric focusing

[0057] 1) Concentration and desalting of recombinant human follicle-stimulating hormone (rhFSH) samples

[0058] Take 400 μg of rhFSH solution, add it to a 10kDa ultrafiltration spin column (Merck Millipore Company), centrifuge at 12000×g for 15 minutes, discard the waste liquid; add eCAP Tris buffer, pH8.0 (Beckman Company), centrifuge at 12000×g for 15 minutes, take out Concentrate the column tube, invert the recombinant human follicle-stimulating hormone sample into a new clean centrifuge tube, centrifuge at 3000×g for 3 minutes, collect the pretreated sample with a concentration of about 10 mg / ml, and obtain a concentrated protein solution.

[0059] 2), preparation of rhFSH sample mixture

[0060] The rhFSH concentrated protein solution prepared by desalination in step 1) was mixed with the components in Table 1 according to the ratio to prep...

Embodiment 2

[0074] Example 2. Determination of the isoelectric point of recombinant human follicle-stimulating hormone by capillary isoelectric focusing

[0075] 1), using the same method as in Example 1 to concentrate and desalt the rhFSH sample to obtain a rhFSH concentrated solution;

[0076] 2) Mix the concentrated rhFSH solution prepared by desalination in step 1 with the components in Table 2-3 according to the proportioning ratio to prepare the rhFSH sample mixture to be tested;

[0077] 3), using the same method as in Example 1, the capillary is activated with different solutions;

[0078] 4), using the same method as in Example 1, the rhFSH sample mixture is loaded and tested;

[0079] 5) Using the same method as in Example 1, record the isoelectric focusing spectrum of the rhFSH sample.

[0080] Test results such as figure 1 As shown, when the concentration of urea gel is 3M or 4.5M, the isoelectric focusing spectrum of rhFSH sample has good separation effect, high peak capac...

Embodiment 3

[0086] Example 3. Determination of the isoelectric point of recombinant human follicle-stimulating hormone by capillary isoelectric focusing

[0087] 1), using the same method as in Example 1 to concentrate and desalt the rhFSH sample to obtain a rhFSH concentrated solution;

[0088] 2) Mix the concentrated rhFSH solution prepared by desalination in step 1 with the components in Table 4-7 according to the proportions to prepare the rhFSH sample mixture to be tested;

[0089] 3), using the same method as in Example 1, the capillary is activated with different solutions;

[0090] 4), using the same method as in Example 1, the rhFSH sample mixture is loaded and tested;

[0091] 5) Using the same method as in Example 1, record the isoelectric focusing spectrum of the rhFSH sample.

[0092] Test results such as figure 2 As shown, when the volume ratio of Pharmalyte (2.5-5) and Pharmalyte (3-10) is 10-90% respectively, the separation effect of charge variants of rhFSH sample is ...

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Abstract

The invention provides an improving method for measuring recombinant protein isoelectric points through cIEF (capillary isoelectric focusing). The method comprises the following steps of (1) taking recombination protein; performing concentration desalting to obtain a concentrated protein solution; (2) mixing carrier ampholyte, gel, cosolvents, place holding agents and isoelectric point markers with the concentrated protein solution in the step (1); preparing a sample mixing solution for cIEF analysis; (3) using different solutions for activating capillary tubes; (4) performing sample loading on a sample mixed solution in the step (2) into the capillary tubes; performing focusing separation at 20 DEG C; using a migration solution for migration; recording a cIEF atlas through an ultraviolet absorption detector; (5) determining the isoelectric point of each isomer of the recombinant protein in a sample mixture to be analyzed according to the cIEF atlas in the step (4). The used method has the advantages of good separation effect, high peak volume, high repeatability, high reproducibility and the like, and is applicable to the quality control of acid recombinant protein.

Description

technical field [0001] The invention belongs to the field of separation and analysis of biological products, in particular to a method for measuring the isoelectric point of acidic recombinant protein by capillary isoelectric focusing. Background technique [0002] In recent years, with the rapid development of recombinant DNA technology, the use of recombinant protein drugs to treat various diseases has become one of the hot spots in the research and development of the biomedical industry. At present, most recombinant proteins are glycoproteins, and the glycosylation and sugar structure of recombinant protein drugs are closely related to in vivo stability and biological activity. During the production process, host animal cells, medium composition, culture conditions, culture environment and protein structure, as well as separation and purification processes will all affect the glycosylation level of recombinant proteins. Glycoprotein biotechnology drugs produce various is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
CPCG01N27/44795
Inventor 李楠罗荣罗双儒黎耘
Owner CHENGDU JINKAI BIOTECH CO LTD
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