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Method for detecting sialic acid by using reversed phase chromatography

A reversed-phase chromatography, sialic acid technology, applied in the field of chromatographic analysis, can solve the problems of low accuracy and low sensitivity, and achieve the effect of prolonging the service life, high sensitivity and high accuracy

Active Publication Date: 2021-04-13
苏州药明生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the deficiencies in the prior art, the present invention provides a method for detecting sialic acid using reversed-phase chromatography in order to solve the problems of low accuracy and low sensitivity in the existing methods for detecting sialic acid content in protein drugs. The mobile phase formula in the liquid phase system reduces the proportion of the organic phase in the mobile phase, and adjusts the gradient of the mobile phase to improve the detection sensitivity and accuracy of the method; by increasing the steps of protein removal, the service life of the chromatographic column is extended

Method used

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  • Method for detecting sialic acid by using reversed phase chromatography
  • Method for detecting sialic acid by using reversed phase chromatography
  • Method for detecting sialic acid by using reversed phase chromatography

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Embodiment 1

[0039] The optimization of embodiment 1 mobile phase and mobile phase gradient

[0040] According to the commercialized LudgerTag DMB sialic acid release and labeling kit, the present invention improves its mobile phase and mobile phase gradient, as follows.

[0041] The traditional mobile phase (Ludger kit) gradient elution program is shown in Table 1:

[0042] Table 1 Traditional mobile phase gradient elution program

[0043]

[0044] Mobile phase gradient elution program of the present invention is as shown in table 2:

[0045] Table 2 Mobile phase gradient elution program of the present invention

[0046]

[0047]

[0048] Use the traditional mobile phase and the patented mobile phase to detect the same DMB-labeled blank control and sialic acid standard on the same column in the same instrument:

[0049] 1. DMB marking conditions

[0050] (1) Blank: Add 5 μL ultrapure water and 20 μL DMB labeling reagent to a 0.5 mL centrifuge tube, mix well and centrifuge, heat...

Embodiment 2

[0057] A method utilizing reverse phase chromatography to detect sialic acid, comprising the following specific steps:

[0058] (1) The protein sample to be tested is desalted and dried in a vacuum concentrator for 1-2 hours.

[0059] The specific steps of desalination treatment are as follows: take a certain amount of protein sample to be tested and add it to a 10kD ultrafiltration concentration tube, add ultrapure water to 400μL, centrifuge at 13000rpm to change the liquid, and repeat the operation twice. After centrifugation, reverse the 10kD ultrafiltration tube containing the sample into a new centrifuge tube, centrifuge at 1000rpm for 1min, and transfer the collected sample to a 1.5mL centrifuge tube.

[0060] (2) Perform acid hydrolysis on the dried sample to obtain an acid hydrolyzate.

[0061] In this comparative example, 25 μL of 2mol / L glacial acetic acid solution was used for acid hydrolysis.

[0062] (3) DMB label the acid hydrolyzate obtained in step (2). Take ...

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Abstract

The invention belongs to the technical field of chromatographic analysis, and particularly relates to a method for detecting sialic acid by utilizing reversed phase chromatography. According to the method, a to-be-detected protein sample is subjected to desalination, vacuum concentration and drying, acid hydrolysis, DMB labeling, protein removal and reversed-phase ultra-high performance liquid chromatography detection, and finally a detection result of the sialic acid content of the protein sample is obtained. By improving a mobile phase formula and a mobile phase gradient in a traditional liquid phase system, retention of N-acetylneuraminic acid and N-hydroxyacetylneuraminic acid on a chromatographic column is enhanced, the separation degree of the N-acetylneuraminic acid and the N-hydroxyacetylneuraminic acid from interference peaks of byproducts is good, quantification of the N-acetylneuraminic acid and the N-hydroxyacetylneuraminic acid is not influenced. In addition, due to the absence of the influence of interference peaks, the lowest point of the standard curve can be diluted to a lower concentration, the detection sensitivity of the method is improved, accurate quantification can still be realized when the content of sialic acid in the protein sample is low, and the method has the advantages of high accuracy and high sensitivity. The protein removal step is additionally arranged, so that the service life of the chromatographic column is prolonged.

Description

technical field [0001] The invention relates to the technical field of chromatographic analysis, in particular to a method for detecting sialic acid by reverse phase chromatography. Background technique [0002] Sialic acid is a kind of derivative of N-acetylneuraminic acid or its ester and its alcoholic hydroxyl group, located at the end of the side chain of glycoprotein. In therapeutic protein drugs, because sialic acid has a strong negative charge, the content of sialic acid directly affects the half-life of the drug. Proteins with no sialic acid or low sialic acid content are more likely to be degraded in the human body, while sialic acid The content may also affect its biological activity, and the ADCC (antibody-dependent cell-mediated cytotoxicity) activity of mutant antibodies with high sialic acid levels is decreased. The type of sialic acid also has an important impact on the immunogenicity of the drug. The type of humanized sialic acid in protein drugs is N-acetyl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/36G01N30/54
CPCG01N30/02G01N30/06G01N30/36G01N30/54
Inventor 崔爱艳李杰陶佳林王永忠
Owner 苏州药明生物技术有限公司
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