基于3种原理分离技术的多肽类药物比伐芦定杂质分析

Analysis of Impurities in Peptide Drug Bivalirudin Based on Three Kinds of Separation Principle Technology

关键词:  比伐芦定  有关物质  反相色谱法  亲水色谱法  分子排阻色谱

Key words:  bivalirudin  related substances  reversed-phase chromatography  hydrophilic chromatography  size exclusion chromatography

摘要:

目的 建立不同分离原理的高效液相色谱法，对7家企业的比伐芦定原料药中相关杂质进行分析，为全面控制比伐芦定有关物质提供依据。方法 采用反相高效液相色谱法(RP-HPLC)对11个杂质进行分离分析；采用亲水色谱法(HILIC-HPLC)对4个工艺杂质进行控制；采用分子排阻色谱法(SEC-HPLC)对聚合物进行测定。结果 建立的RP-HPLC方法可将主成分及11个杂质有效分离，11个杂质的校正因子均在0.8~1.2，比伐芦定检出浓度为0.1 μg·mL^-1，检测限为0.004%；建立的HILIC-HPLC方法可将主成分及4个工艺杂质有效分离，比伐芦定检出浓度为0.3 μg·mL^-1，检测限为0.01%；在SEC-HPLC条件下聚合物和比伐芦定依次出峰，两者分离度为2.9，比伐芦定检出浓度为6 ng·mL^-1，检测限为0.000 6%。采用主成分自身对照法计算7家企业15批样品中15种已知杂质及聚合物，不同企业的产品杂质含量与其生产工艺存在一定的相关性。结论 3种不同原理的方法均专属性良好，灵敏度高，耐用性好，结果可靠，可用于比伐芦定有关物质的质量控制。

Abstract:

OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises, to provide a basis for the comprehensive control of related substances of bivalirudin. METHODS Reversed-phase high performance liquid chromatography(RP-HPLC) was used to separate and analyze 11 kinds of impurities. Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities. Polymers were determined by size exclusion chromatography(SEC)-HPLC. RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities, the correction factors of 11 impurities were between 0.8-1.2, the detection concentration of bivalirudin was 0.1 μg·mL^-1, and the detection limit was 0.004%. The established HILIC-HPLC could effectively separate the principal components and four process impurities, and the detection concentration of bivalirudin was 0.3 μg·mL^-1, and the detection limit was 0.01%. Under SEC-HPLC conditions, the polymer and bivalirudin peaked sequentially, the resolution of the two was 2.9, the detection concentration of bivalirudin was 6 ng·mL^-1, and the detection limit was 0.000 6%. Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components, and the impurity contents from different enterprises had a certain correlation with their production processes. CONCLUSION The three different principles of the method have good specificity, high sensitivity, good durability, and reliable results, and can be used for quality control of substances related to bivalirudin.