流感疫苗血凝素含量测定替代方法在甲型H1N1流感疫苗研发中的应用

目的: 甲型H1N1流感爆发,使血凝素(haemagglutinin,HA)含量测定成为疫苗研发的关键制约环节。本研究采用替代方法来检测疫苗原液中HA含量,以解决这一制约瓶颈。 方法: 采用经验证的改良SDS-PAGE方法,测定HA在总蛋白中比例,结合总蛋白含量,计算出疫苗原液中HA含量,用以配制疫苗。以WHO提供的国际参考品对成品疫苗进行复测。 结果: 各企业采用该方法,检测疫苗原液,各原液中总蛋白在267~770 μg·mL^-1间,HA比例在23.1%~62.8%间,HA含量在104~303 μg·mL^-1间。各企业根据HA含量配制成品疫苗用于临床试验,复测结果证明临床剂量准确。 结论: 在甲型H1N1流感疫苗研发中,可采用HA含量测定替代方法。

Objective: After the outbreak of pandemic A (H1N1) influenza,vaccine development process was limited by the unavailability of reference materials from WHO.In this study,the alternative methods were used to resolve this problem,hence to boost the development of this vaccine. Methods: Manufacturers in China used the established SDS-PAGE method to measure the Haemagglutinin (HA) percentage in vaccine bulks,thus,the HA content can be calculated based on the total protein contents.The exact HA contents of final products were re-tested by SRID using the references materials from WHO. Results: For various vaccines bulks,the total protein content are 267-770 μg·mL^-1,the ratio of HA in total proteins are 23.1%-62.8%,the HA content are 104-303 μg·mL^-1,facilitating the formulation of final products for clinical trials.The re-test results proved that the HA content measured by SDS-PAGE were relative accurate. Conclusion: The alternative method was successfully applied during the development of pandemic A (H1N1) influenza vaccine.

[1] LI Chang-gui(李长贵),WANG Jun-zhi(王军志).Issues in developing the pandemic influenza vaccine and related regulatory activities(大流行流感疫苗研发和质量管理中存在的问题探讨). Chin Pharm Aff (中国药事),2007,21(8):563
[2] Changgui Li,Stella Chun,Gary Van Domselaar,et al.Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins.Vaccine.2008,26:6068
[3] WHO guidelines on the use of vaccines and antivirals during influenza pandemics.World Health Organization.WHO/CDS/CSRRMD/2004.8.Geneva,Swizerland.2004
[4] SHAO Ming(邵铭),LI Juan(李娟),SONG Ying-li(宋颖丽),et al. Development and validation of alternative method for determination of haemagglutinin content in influenza vaccine(流感疫苗血凝素含量测定替代方法的建立及验证).Chin J Biological(中国生物制品学杂志),2010,23 (7):770
[5] Li CG,Shao M,Cui XY,et al.Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of
[2009] pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China.Biologicals,2010,38(2):284
[6] Wood JM,Schild GC,Newman RW,et al.An improved single-radial-immunodiffusion technique for assay of influenza haemagglutinin antigen:application for potency determinations of inactivated whole virus and subunit vaccines.J Biol Stand,1977,5 (3):237