High performance liquid chromatography for analyzing sialic acid content in sample

A liquid chromatography column and sialic acid technology, which is applied in the direction of analyzing materials, measuring devices, and material separation, can solve the problems of poor repeatability, low specificity of analysis technology, high cost, etc., and achieve good accuracy, good linear relationship, Solve the phenomenon of insufficient acid hydrolysis or affect the effect of derivatization reaction

Pending Publication Date: 2022-05-10
鼎康(武汉)生物医药有限公司
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is the problems of low specificity, poor repeatability and high cost in the existing analysis technology, thereby providing a high performance liquid chromatography method for analyzing the content of sialic acid in samples

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  • High performance liquid chromatography for analyzing sialic acid content in sample
  • High performance liquid chromatography for analyzing sialic acid content in sample
  • High performance liquid chromatography for analyzing sialic acid content in sample

Examples

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Embodiment 1

[0032] In this example, the content of sialic acid in the biological analogues of sumetrumab is analyzed by HPLC. Firstly, the sialic acid is released by acetic acid treatment, and then labeled by derivatization reagent. The specific operation steps are as follows:

[0033] Step 1), take 400 μ G. put the sample into the centrifuge tube, and then add PBS to make up to 400 μ L。 After mixing, transfer to the ultrafiltration tube and centrifuge at 13000 rpm at 8 ± 2 ℃ for 10 min. After centrifugation, supplement the volume with PBS to the scale of 400 μ 50. Mix well, centrifuge at 13000rpm at 8 ± 2 ℃ for 10min, repeat this step for 4 times, and add PBS to make up the volume to the scale of 100 μ 50. Transfer the intercepted liquid in the ultrafiltration tube to a new centrifuge tube. After mixing, the protein concentration was measured by nanodrop.

[0034] Step 2), 25 μ L 4m acetic acid was added to 25 μ L 1mg / ml standard (Neu5Ac and Neu5Gc) / protein sample (xiumeile), mix well an...

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Abstract

The invention discloses a high performance liquid chromatography method for analyzing the content of sialic acid in a sample, and the method comprises the following steps: mixing acetic acid with a protein sample to obtain a mixed solution, so that the sialic acid in the protein sample is released. The invention solves the problems of low specificity of spectrophotometry, poor repeatability of thin-layer method, high cost of mass spectrometry and the like in the existing analysis technology, thereby providing a simple high performance liquid chromatography with high specificity and high accuracy for analyzing the content of sialic acid in different types of antibodies.

Description

technical field [0001] The invention relates to the field of chemical substance detection, in particular to an HPLC method for analyzing the content of sialic acid in a sample. Background technology [0002] New theories and technologies such as bispecific antibodies, antibody drug conjugates, glycosyl engineering antibodies and nano antibodies are emerging. Antibody drugs are mostly expressed in mammalian cells. The correct folding, spatial conformation and modification after gene translation determine the normal function of antibody drugs. Therefore, controlling the type and number of post-translational modified variants is one of the great challenges in the process of antibody drug development. The mass of sugar chain in antibody drugs accounts for 2% of the whole antibody molecule. For monoclonal antibodies for the treatment of autoimmune diseases, the amount of sialic acid modification at the end of sugar chain may have a strong positive correlation with its anti-inflammator...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 邓辉郑子荣李程亮吴俊明
Owner 鼎康(武汉)生物医药有限公司
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