Method for detecting thymosin beta 4 content based on high performance liquid chromatograph

Abstract

The invention belongs to the technical field of biological chemistry and provides a high performance liquid chromatograph analysis method for thymosin beta 4 content. In the method, a high performance liquid chromatograph is used, and a gradient elution method is used for content detection. A conventional protein content detecting method for thymosin beta 4 has the defects of being complex in operation, likely to be affected by various factors and the like. Operation is simple, the peak type is good, the separation degree of a main peak and an impurity peak is good, and a simple and reliable analysis method is provided for detecting the thymosin beta 4 content.

Description

(1) Technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a method for determining the content of thymosin β4 based on high-performance liquid chromatography. (2) Background technology [0002] Thymosin β4 (hereinafter referred to as Tβ4) was isolated from the thymus in the 1980s, and later found to be a small peptide substance widely present in many tissues, and it is an important actin-binding protein. Tβ4 has various biological functions. Studies have shown that Tβ4 acts as a chemical inducer in the wound healing process of myocardium, cornea and skin, regulating the expression of various growth factors and the migration of chemotactic newborn cells. Due to patent factors, currently there are two main companies developing thymosin β4 as a drug, namely RegeneRX Biopharmaceutical Company of the United States, which is in the clinical research stage, and Beijing Northland Biotechnology Co., Ltd., which has applied for clinic...

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Problems solved by technology

The advantage of the Kjeldahl method is that it is not easily affected by other components in the liquid, and the measurement result is accurate, but it is time-consuming; the advantage of the biuret method is that it has good precision and less interfering substances, but its accuracy and sensitivity are low; The advantages of the Lowry method are rapidity and high sensitivity, but it is affected by the specificity of peptides, that is, the color intensity of different peptide drugs is slightly different due to the different contents of tyrosine and tryptophan (Dou Xiaorui et al. (Potency) Determination Method and Its Application. Advances in Drug Efficacy, 2011, 35(12): 536-541)

[0005] The conventional content determination of Tβ4 is not suitable for the above three methods for the following reasons: first, Tβ4 belongs to polypeptide drugs, and based on the particularity of the structure of polypeptide drugs, it is not suitable for detection by the Lowry method; second, the administration of polypeptide drugs The dose is small, and a detection method with high sensitivity is required, which is not suitable for detection by the biuret method; thirdly, from the perspective of drug development, the Kjeldahl method is too time-consuming, which is not conducive to production.

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