周庆玲;于晶;谢英林;李春艳;韩云飞;李杨;董焕;岳振奇;汪一名;胡晓明;管洪超;于洪涛;
ZHOU Qing-ling;YU Jing;XIE Ying-lin;LI Chun-yan;HAN Yun-fei;LI Yang;DONG Huan;YUE Zhen-qi;WANG Yi-ming;HU Xiao-ming;GUAN Hong-chao;YU Hong-tao;Center of Development of Drugs, Jilin Yatai(Group)Co., Ltd.;

• 1:吉林亚泰(集团)股份有限公司药物与研发中心
• 2:吉林省职业病医院监测科

摘要(Abstract)：

目的筛选大流行流感疫苗的稳定剂。方法将流感病毒株Influenza A/Vietnam/1194/2004(H5N1)接种Vero细胞制备5批疫苗原液,加入Al(OH)3吸附制备成品疫苗。取同一批疫苗原液,分成5份,分别加入吐温-80(20μg/ml)及0.5%、1%和2%人血白蛋白稳定剂,以不加入稳定剂的疫苗原液作为空白对照,于37℃放置4周,检测流感病毒的血凝滴度,筛选疫苗的稳定剂;将加入筛选出的稳定剂的5批疫苗原液及3批成品疫苗于37℃放置不同时间,检测血凝素(hemagglutinin,HA)含量。结果不加入稳定剂的疫苗原液37℃放置1周后,血凝滴度明显下降;加入20μg/ml吐温-80和0.5%人血白蛋白的疫苗原液37℃放置1周后,血凝滴度亦明显下降;而加入1%和2%人血白蛋白的疫苗原液于37℃放置4周,血凝滴度仍未下降,后续试验选择1%人血白蛋白作为疫苗稳定剂。加入1%人血白蛋白的5批疫苗原液于37℃放置1⁴周与放置前比较,HA含量差异均无统计学意义(P>0.05);加入1%人血白蛋白的3批成品疫苗37℃放置6周,疫苗HA含量有下降趋势,37℃放置14周与放置前比较,HA含量差异均无统计学意义(P>0.05);加入1%人血白蛋白的3批成品疫苗37℃放置6周,疫苗HA含量有下降趋势,37℃放置1⁵周,相邻两周间HA含量差异无统计学意义(P>0.05),放置5周,HA含量下降幅度≤1μg/ml。结论 1%人血白蛋白对流感病毒具有良好的稳定作用。
Objective To screen the stabilize for pandemic influenza vaccine. Methods Five batches of bulks of pandemic influenza vaccine were prepared by inoculation of strains Influenza A / Vietnam / 1194 / 2004(H5N1)into Vero cells, to which aluminium hydroxide was added to prepare final product. The same batch of bulk was divided into five groups. The bulks in four test groups were added with Tween-80(20 μg / ml)as well as 0. 5%, 1% and 2% human serum albumin respectively, using that containing no stabilizer as blank control. After storage at 37 ℃ for 4 weeks, the bulks were determined for haemagglutination titer, based on which the stabilizer was screened. Five batches of bulks and three batches of final products, containing the screened stabilizer, were stored at 37 ℃ for various weeks, and determined for haemagglutinin(HA)content. Results The haemagglutination titers of bulks containing no stabilizer, containing 20 μg / ml Tween-80 and containing 0. 5% human serum albumin decreased significantly after storage at 37 ℃ for one week. However, the haemagglutination titers of bulks containing 1% and 2% human serum albumin showed no decrease after storage at 37 ℃ for 4 weeks. The 1% human serum albumin was screened as stabilizer for further study. The HA contents of five batches of bulks containing 1% human serum albumin after storage at 37 ℃ for 1 5周,相邻两周间HA含量差异无统计学意义(P>0.05),放置5周,HA含量下降幅度≤1μg/ml。结论 1%人血白蛋白对流感病毒具有良好的稳定作用。
Objective To screen the stabilize for pandemic influenza vaccine. Methods Five batches of bulks of pandemic influenza vaccine were prepared by inoculation of strains Influenza A / Vietnam / 1194 / 2004(H5N1)into Vero cells, to which aluminium hydroxide was added to prepare final product. The same batch of bulk was divided into five groups. The bulks in four test groups were added with Tween-80(20 μg / ml)as well as 0. 5%, 1% and 2% human serum albumin respectively, using that containing no stabilizer as blank control. After storage at 37 ℃ for 4 weeks, the bulks were determined for haemagglutination titer, based on which the stabilizer was screened. Five batches of bulks and three batches of final products, containing the screened stabilizer, were stored at 37 ℃ for various weeks, and determined for haemagglutinin(HA)content. Results The haemagglutination titers of bulks containing no stabilizer, containing 20 μg / ml Tween-80 and containing 0. 5% human serum albumin decreased significantly after storage at 37 ℃ for one week. However, the haemagglutination titers of bulks containing 1% and 2% human serum albumin showed no decrease after storage at 37 ℃ for 4 weeks. The 1% human serum albumin was screened as stabilizer for further study. The HA contents of five batches of bulks containing 1% human serum albumin after storage at 37 ℃ for 1 4 weeks showed no significant difference with those before storage(P > 0. 05). The HA contents of three batches of final products containing the stabilizer showed decreasing tendencies after storage at 37 ℃ for 6 weeks, while showed no significant difference between various weeks within 1 4 weeks showed no significant difference with those before storage(P > 0. 05). The HA contents of three batches of final products containing the stabilizer showed decreasing tendencies after storage at 37 ℃ for 6 weeks, while showed no significant difference between various weeks within 1 5 weeks after storage(P > 0. 05), of which the decrease was not more than 1 μg / ml after storage for 5 weeks. Conclusion The 1% human serum albumin is a stabilize suitable for pandemic influenza vaccine.

关键词(KeyWords)： 稳定剂;血凝滴度;血凝素含量
Stabilizer;Haemagglutination titer;Haemagglutinin content

Abstract:

Keywords:

基金项目(Foundation): 吉林省2012年重大科技攻关“双十项目”(2012ZDGG011)

作者(Authors): 周庆玲;于晶;谢英林;李春艳;韩云飞;李杨;董焕;岳振奇;汪一名;胡晓明;管洪超;于洪涛;
ZHOU Qing-ling;YU Jing;XIE Ying-lin;LI Chun-yan;HAN Yun-fei;LI Yang;DONG Huan;YUE Zhen-qi;WANG Yi-ming;HU Xiao-ming;GUAN Hong-chao;YU Hong-tao;Center of Development of Drugs, Jilin Yatai(Group)Co., Ltd.;

DOI: 10.13200/j.cnki.cjb.000847

参考文献(References)：

• [1]Centers for Disease Control and Prevention.Update:isolation of avian influenza A(H5N1)viruses from humans-Hong Kong,1997-1998[J].JAMA,1998,279(5):347-348.
• [2]Luo DY.Design and development of pandeminc influenza vaccine using reverse genetics[D].Chongqing:Third Military Medical University of Chinese P.L.A,2008.罗德炎.大流行流感裂解疫苗的研制及关键技术的研究[D].重庆:第三军医大学,2008.
• [3]Leroux-Roels I,Borkowski A,Vanwolleghem T,et al.Antigen sparing and cross-reactive immunity with an adjuvanted r H5N1prototype pandemic influenza vaccine:a randomised controlled trial[J].Lancet,2007,370(9587):580-589.
• [4]Xu CL,Yang L,Wen LY,et al.The epidemiological profile of the 1918 influenza pandemic[J].Chin J Virol,2009(Suppl):23-26.(in Chinese)徐翠玲,杨磊,温乐英,等.1918年流感大流行的流行病学概述[J].病毒学报,2009,25(增刊):23-26.
• [5]Miyuki C,Quintilio W,Miyaji EN,et al.Production of H5N1(NIBRG-14)inactivated whole virus and split virion influenza vaccines and analysis of immunogenicity in mice using different adjuvant for mutations[J].Vaccine,2010,28(13):2505-2509.
• [6]Li YF,Xie B,Hui MZ.Development of influenza virus and its cell-culture-derived vaccine production[J].J Biology,2012,29(2):59-64.(in Chinese)李一锋,谢波,惠觅宙.流感病毒及其疫苗传代细胞制备研究进展[J].生物学杂志,2012,29(2):59-64.
• [7]Lv HL,Fu XH,Zheng M.Advance in cell derived influenza virus vaccine process development[J].Chin J Vet Med,2009,43(6):43-48.(in Chinese)吕宏亮,付秀花,郑明.传代细胞制备流感病毒疫苗工艺研究进展[J].中国兽药杂志,2009,43(6):43-48.
• [8]Ehrlich HJ,Müller M,Oh HM,et al.A clinical trial of a whole-virus H5N1 vaccine derived from cell culture[J].N Engl J Med,2008,358(24):2573-2584.