Research On A Production Technology For Influenza A(H1N1) Virus Vaccine (SUB-UNIT)

Influenza A(H1N1) is defined as an acute respiratory infectious disease.Distinctive from the previous or current seasonal flu virus, the strains of the virusgene fragments contains three kinds of influenza virus including swine flu, avian fluand human influenza. The crowd in general are highly susceptible to InfluenzaA(H1N1) virus and the virus is able to spread through human-to-human transmission.The world has had a pandemic of influenza A(H1N1)since2009, and it brought greatpanic to people all over the globe. The purpose of this study is to seek a more safe,economic and feasible production technology for Influenza A(H1N1) virus vaccine（SUB-UNIT） through a systematic experimental design and solutionimplementation.In this study, we use300mesh filter cloth to obtain allantoic fluid of viral chickembryo culture from the coarse filter.Continuous low speed centrifugation method isused afterwards for clarification. The100-300K Dultrafiltration membraneconcentration method is used for30to50times after the clarification phase of theviral allantoic fluid,followed by sucrose density gradient centrifugation of theinfluenza virus purification. The purpose is to remove the vast majority ofovalbumin and other impurities in the virus concentrate.The virus fluid was purifiedwith the addition of formaldehyde by ratio1:2000. Virus inactivation is carried outof2-8degree centigrade for4days. The100K Dultrafiltration membrane is used tofiltrate，inactivate and purify virus liquid in order to remove formaldehyde andsucrose. The purified virus liquid after inactivation is removed of sugar by100KDmembrane ultrafiltration. According to the final concentration of1:4000,we addTween-80at4degree centigrade and stir for1hour. Then join the sixteen alkyl three methyl ammonium bromide(CTAB) by0.2%, split virus for2hours and purified byIRC-50ion exchange column;We use the IRC-50ion exchange resin column topurify again, and eluted with PBS. Finally the H1N1influenza viral stock isacquired.A finished product of the vaccine is produced eventually after a preparationof follow-up semi-finished products.Single radial immunodiffusion method is used in the research to detect antigencontent of vaccines and specific assay.SDS-polyacrylamide gel electrophoresis isused to detect molecular weight and purity of the vaccine. The 《Pharmacopoeia ofChina (2010) III》 appendix VIL has been taken into account to test freeformaldehyde content, and we use appendix VIH to test content of polysorbate80.Colorimetry is used to detect-3-methyl sixteen alkyl ammonium bromide(CTAB)content. The use of method two in appendix VIB is to detect the total proteincontent and enzyme-linked immunosorbent assay to detect ovalbumincontent.Appendix XIIF is used for testing abnormal toxicity and Appendix XIIE isused for the content of bacterial endotoxin. We have checked the safety andimmunogenicity of the vaccine through certain animal experiments, and to place thevaccine at37degree centigrade,20~25degree centigrade,2~8degree centigrade toinvestigate the stability of the vaccine.Experiments results show that by using300mesh filter cloth first and then byusing the8000rpm continuous flow low speed centrifuge to remove macromolecularsubstances, the clarifying effect is quite significant. In addition, the operation isrelatively simple and fast. It can ensure the continuity of the production and reducethe chance of contamination. At the same time, the finished products’ pyrogen canbe effectively controlled. The results of total protein removal rate and the virusharvest rate are ideal after each batch of allantoic virus was concentrated and throughsucrose density gradient centrifugation. The virus can achieve inactivation effectwithin three days. By using100KD ultrafiltration diafiltration, test results of the virus showed formaldehyde content is less than25μg/ml and sugar content is lessthan1%. The removal effect is quite ideal. The electron microscope shows obviousviral lytic subunit structure after cracking and no complete virus particles. Thecracking effect is quite good. The hemagglutinin content of the concentratemonovalent influenza virus is higher after cracking and purification. Total proteinand egg albumin levels are relatively lower.Tween-80and CTAB have mostly beenremoved. And the purity of each batch of the influenza viral stock electrophoreticbands is higher.Test results show that the vaccine antigens and specificity are error free. Thecontent of HA is in full compliance with the national standards. The non-activeingredients including formaldehyde,total protein,ovalbumin,Tween-80and CTABwere consistent with the requirements of Pharmacopoeia of China.The content ofbacterial endotoxin is also in accordance with the requirements of Pharmacopoeia ofChina. All three batches of vaccine have good immune effect and each batch canachieve higher neutralizing antibody titers. Animal experimental results showthat the vaccine is safe. Vaccine stability results show that the influenza vaccinesubunit developed in this study has a good stability. Eventually, the product’s qualitystandards were determined through statistical analysis methods.In summary,this study established a H1N1influenza vaccine subunit productionpreparation process and determined the product’s quality standards through a seriesof quality researches. Testing results are all in line with national standards. The thirdgeneration of qualified influenza vaccine products are prepared and produced.Finished products of the vaccine only retain HA NA antigen that help the humanbody create protective antibodies. It has greatly reduced the side effects on humanbody and guaranteed the safety of the vaccine. To conclude, the vaccine can be usedas a first choice of H1N1influenza prevention.