高效体积排阻色谱法定量检测口蹄疫疫苗中146S的疫苗预处理方法

Vaccine pretreatment for quantification of 146S antigen in foot-and-mouth disease vaccines by high performance size exclusion chromatography

摘要:

旨在建立一种口蹄疫灭活病毒疫苗的处理方法，去除尺寸排阻色谱法（HPSEC）检测146S抗原过程中的杂质干扰，获得最佳的检测信号并实现146S抗原含量的准确测定。分别考察了超速离心法、PEG沉淀法、核酸酶消化法对两批疫苗样品中的HPSEC检测干扰杂质的去除效果。在优化条件下，超速离心法处理后146S检测结果分别为7.1、7.6 μg/mL，PEG沉淀法为9.7、10.4 μg/mL；酶消化法处理后的检测值最高，分别为10.5、10.4 μg/mL，且杂质去除完全、处理速度快、操作条件温和。通过响应面法确定最优酶消化处理条件如下：200 μL水相中添加终浓度421 U/mL的Benzonase，25.1 ℃下反应1.29 h。在该最优条件下，对4家企业各3种不同血清型共12批疫苗样品进行146S含量检测，结果表明建立的方法对不同疫苗均有良好适用性，且重复性好（RSD<5.3%，n=3）。通过该处理方法，解决了杂质对146S定量检测的干扰，扩大了HPSEC检测技术的应用范围，进一步确保了检测结果的准确可靠。

Abstract:

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.