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Method for measuring sialic acid content of protein

A technology of sialic acid and protein, which is applied in the field of determination of sialic acid content of protein, can solve problems that need to be further developed, and achieve the effects of improving denaturation and precipitation efficiency, good ionization degree, and improving accuracy

Active Publication Date: 2017-08-25
HUBEI BIO PHARMA IND TECHCAL INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, methods for the determination of sialic acid content in monoclonal antibodies and recombinant protein biomacromolecular drugs still need to be further developed

Method used

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  • Method for measuring sialic acid content of protein
  • Method for measuring sialic acid content of protein
  • Method for measuring sialic acid content of protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 measures the method for the sialic acid content of protein and corresponding condition screening

[0042] 1) Prepare the sample

[0043] In the process of sialidase digestion and hydrolysis, the hydrolysis time plays a decisive factor for the hydrolysis efficiency (when the sialidase is excessive). The inventors compared three different hydrolysis times, namely 37°C for 1h (sample No. 01), 37°C for 4h (sample No. 02) and overnight hydrolysis at 37°C (12h) (sample No. 03). In order to facilitate the subsequent protein precipitation process, the hydrolysis volume was adjusted to 25 microliters. Simultaneously, the inventors carried out in parallel only the protein sample, the control without sialidase (No. 04 sample) and the sialidase control without protein sample (No. 05 sample). The specific sample processing scheme is shown in Table 1.

[0044] Table 1

[0045] sample 01 02 03 04 05 0.25 mg / ml VEGF-Trap (µl) 16 16 16 16 0 ...

Embodiment 2

[0052] Embodiment 2 establishes quantitative curve

[0053] Before mass spectrometry quantitative analysis of the protein samples to be tested, the international standard of sialic acid was used to establish a quantitative curve. Sialic acid standard product: product number A187000, lot number: 4-SCC-101-1, molecular weight 309.27, purchased from TRC (Canada). The sialic acid standard was dissolved in MilliQ ultrapure water to make a stock solution of 1 mg / ml, and then frozen at -80°C. Serially dilute the 1 mg / ml sialic acid standard solution with ultrapure water to 0.1 mg / ml, 0.01 mg / ml, 1 μg / ml, 0.1 μg / ml, 0.08 μg / ml, 0.04 μg / ml, 0.02 μg / ml Milliliter, 0.01 μg / ml, 0.005 μg / ml, blank zero point is ultrapure water.

[0054] The quantitative curve established is as image 3 as shown, image 3 The results show that: the method has good specificity, and the blank has no interference (such as being used to detect samples 04 and 05); the linear relationship of sialic acid is go...

Embodiment 3

[0055] The mensuration of embodiment 3 detection limit and quantitative limit

[0056] Depend on Figure 4 As a result, it can be seen that the signal-to-noise ratio (S / N) recorded by the standard solution of 0.04 micrograms / ml is 104.6991 (injection volume is 1 microliter), thus drawing the detection limit (LOD) of the method =S / N=3) was about 1.0 pg; the limit of quantitation (LQD=S / N=10) was about 3.5 pg. Therefore, this method meets the quantitative detection requirements of total sialic acid in protein biomacromolecular drugs.

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Abstract

The invention provides a method for measuring sialic acid content of protein. The method comprises the steps of 1, using sialidase to conduct digestive treatment on a protein sample, so that digestive juice containing sialic acid is obtained; 2, conducting sedimentation treatment on the digestive juice, and separating supernatant containing sialic acid; 3, using an ultra-high performance liquid chromatography-mass spectrometry method to analyze the supernatant, so that a mass spectrogram is obtained; 4, based on the mass spectrogram, determining the sialic acid content of protein. In the method for measuring the sialic acid content of protein, on the condition that there is no need to conduct derivatization on sialic acid, the total sialic acid content in the protein sample is subjected to rapid quantitative detection, the method is easy to operate, the sensitivity is high, the accuracy is high, the elapsed time is short, and the method can be used for intermediate quality control over biomacromolecule production or quality inspection of final products.

Description

technical field [0001] The invention relates to the technical field of detection, in particular, the invention relates to a method for determining the sialic acid content of proteins. Background technique [0002] Sialic acid is a kind of monosaccharide derivative of neuraminic acid with a skeleton of 9 carbon atoms. The most common sialic acid is N-acetylneuraminic acid (Neu5Ac), which is widely distributed in animal tissues, mainly in the form of glycoproteins and gangliosides. Sialic acid performs multiple functions in living organisms, including promoting brain development, enhancing memory, and promoting intestinal absorption of vitamins and minerals, etc. At the same time, because sialic acid is negatively charged, a relatively concentrated negatively charged area is formed on the cell, which is also a binding receptor for viruses such as influenza. [0003] In recent years, biomacromolecules represented by monoclonal antibodies and recombinant proteins have develope...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 董思聪马梵辛王学海许勇李莉娥黄璐
Owner HUBEI BIO PHARMA IND TECHCAL INST
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