禽流感灭活疫苗主要生产工艺与原辅材料质量标准控制研究
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摘要
高致病性禽流感(High pathogenic avain influenza, HPAI)在全球范围的爆发和蔓延引起了世界各国的高度关注。为了有效防控HPAI,国际上主要采取隔离封锁疫点,扑杀销毁感染动物和受威胁动物,以及其他生物安全措施,许多发达国家和地区采用上述措施,扑灭了爆发的HPAI疫情。但当疫情面临大面积扩散,以上措施难以奏效、巨大经济损失难以承担时,免疫接种就成了控制疫情的关键措施之一。我们国家采取以禽流感疫苗免疫接种为主的综合防制措施。高致病性禽流感疫苗被纳入国家强制免疫品种,由农业部指定8家禽流感定点企业生产,执行政府招标采购制度。国家对于禽流感疫苗的研发、生产、销售、使用实施严格的监管措施,禽流感疫苗的质量受到了政府、生产企业、养殖户的高度关注。
本研究正是在上述背景下,在执行第四届兽药典委员会下达的“注射用白油质量标准及检测规范”、“黏度计替代吸管法检测黏度标准及方法研究”两项项目基础上,以影响禽流感疫苗质量的三个关键因素种蛋、注射用白油、黏度标准的研究和制定为主线,针对禽流感疫苗原辅材料质量、生产工艺、质量检验、市场应用等关键环节中的质量控制点进行系统研究,以解决当前禽流感疫苗在安全、高效、稳定、均一方面存在的问题,同时制定与国际接轨的行业及国家标准,以全面提升我国禽流感疫苗的质量水平,满足国际、国内市场的需求。
本研究内容分为三个主要部分：1禽流感病毒(AIV)增殖方法及生产用种蛋质量控制
在不同种蛋母源抗体水平下,采用矩阵方法对AIV最佳接种稀释度、最佳接种胚龄进行研究,结果显示采用母源抗体≤21og2的禽流感非免疫种蛋,病毒最佳稀释度为1:10000(104EID50/0.1mL),最佳接种胚龄为10日龄,每胚收获量达到12.5mL,平均HA效价为11log2,病毒EIDs0为108.5EID50/0.1mL;采用母源抗体≤6log2的禽流感低免疫种蛋,病毒最佳稀释度为1:10000(104EID50/0.1mL),最佳接种胚龄为10日龄,每胚收获量达到12.8mL,但平均HA效价为10log2,病毒EID50为107.3EID50/0.1ml;采用母源抗体≤8log2的禽流感高免疫种蛋,病毒最佳稀释度为1:1000(105EID50/0.1mL),最佳接种胚龄为10日龄,每胚收获量达到12mL,但平均HA效价为9log2,病毒EID50为107.6EID50/0.1mL,同期比较的SPF鸡胚每胚收获量达到13mL,平均HA效价为12log2,病毒EID50为108.8EID50/0.1mL,为保证实验的准确性,所有鸡胚的接种和收获均采用自动接种收获机进行。综合分析以上试验数据,我们选择将AIV1:10000(104EID50/0.1mL)倍稀释,接种10日龄非免疫鸡胚(母源抗体≤2log2)为最佳病毒增殖条件。
按照以上病毒增殖最佳条件,我们选择了国内14家主要禽流感灭活疫苗种蛋供应商提供的非免疫种蛋230万枚,生产重组AIV灭活疫苗14批共计5445万毫升。通过14批非免疫种蛋外观、重量、破损率、受精率、死胚率、利用率、每胚收获量统计,建立生产用种蛋内控质量标准。14批种蛋外观清洁、通过选蛋机保证蛋大小均匀、蛋重在50-60g之间,14批种蛋中,破损率在2%以下的有10批,受精率均在91%以上,接种前死胚率在4%以下的有11批,接种前种蛋利用率在85%以上的有8批。综合评价病毒接种后死胚率、每胚收获量、病毒效价指标,9批为接种后死胚率在10%以下,病毒每胚收获量在10mL以上、病毒效价在10log2以上,按照以上种蛋质量指标生产的禽流感疫苗14批中有11批HI平均抗体效价达到了8log2,高于禽流感疫苗成品效力检验6log2的指标,说明种蛋各项指标较为合理,能够有效保证产品质量。2注射用白油质量标准及操作规范研究
选择国内禽流感定点企业普遍使用的一种进口白油MC及两种不同黏度国产白油LH、HZ,采用《中华人民共和国兽药典》(2005版)“注射用白油质量标准”及“食品级白油质量标准”(GB48532008)进行全项检测,三种来源白油在运动黏度、稠环芳烃含量、碳数分布方面存在差异,其中4组LH来源白油运动黏度、稠环芳烃含量最低,碳数分布最窄,各组分清晰,三种指标分别为4.37-5.7mm2/s、0.005-0.015、15-19; MC来源白油三种指标为7.26mm2/s、0.162、15-24; HZ来源白油三种指标为11.16mm2/S、0.037、15-24。用这三类白油制备成油乳疫苗后在SPF鸡体做了比较试验。从安全性试验结果看,疫苗吸收状况存在一定的个体差异。采用胸部肌肉途径接种后,吸收最好的运动黏度为5.7mm2/S的LH白油组,在免疫后48天基本吸收,其次为HZ组,48天后有1只鸡局部有少量未吸收干酪物。稠环芳烃含量超标的MC组,局部炎性反应严重,吸收稍差。从效力检验指标看,LH来源4组白油制备的AI疫苗诱生的HI抗体效价全部高于进口的MC及国产HZ来源白油,疫苗免疫2周后HI抗体效价即能达到7.6log2,三周达到9.5log2。从免疫保存期及持续期数据看,用LH白油批量生产的ZSLHAI疫苗免疫后产生的HI抗体显著高于ZSMC、ZSHZ疫苗,免疫130天后,HI抗体仍然维持在9.4log2,保存12个月,ZSLH疫苗抗体效价维持在8.51og2,而保存9个月时,ZSMC、ZSHZ疫苗已达不到成品效力检验6log2的标准。从不同品种鸡鸭免疫保护效力试验数据看,免疫7日龄商品肉鸡,免疫后28天,ZSLH疫苗抗体效价为7.81og2, ZSMC疫苗为6.71og2, ZSHZ疫苗为6.1log2；免疫18日龄蛋雏鸡,免疫后38天达到抗体高峰,ZSLH疫苗为8.21og2, ZSMC疫苗为7.0log2,ZSHZ疫苗为6.9log2；免疫18周龄蛋种鸡,免疫后1个月达到抗体峰值,ZSLH疫苗为10.8log2,ZSMC疫苗为9.91og2, ZSHZ疫苗为8.2log2；免疫10日龄商品肉鸭,一次免疫4周达到抗体峰值,ZSLH疫苗为7.11og2, ZSMC疫苗为5.41og2, ZSHZ疫苗为5.2log2；3周龄加强免疫后,在第5周达到抗体峰值,ZSLH疫苗为9.31og2, ZSMC疫苗为8.31og2, ZSHZ疫苗为7.9log2；免疫14日龄易感蛋鸭,一次免疫ZSLH疫苗5周达到抗体峰值,为8.51og2, ZSMC. ZSHZ疫苗免疫4周达到峰值,分别为5.31og2,5.11og2;3周龄加强免疫后,在第6周达到抗体峰值,ZSLH疫苗为10.81og2, ZSMC疫苗为9.41og2, ZSHZ疫苗为9.1log2。综合分析,低黏度、低稠环芳烃含量、高纯度的国产LH白油安全性及免疫保护效力均优于进口和国产同类产品。3黏度计替代吸管法检测禽流感灭活疫苗黏度研究
研究发现,佐剂黏度是影响疫苗黏度的重要因素,在不改变生产工艺、配方的情况下,黏度范围为4.3-5.0mm2/s的LH来源白油制备的疫苗黏度最低,在40mPa.s以下,黏度范围为7.31-7.42mm2/s的MC来源白油制备的疫苗黏度在109mPa.s以内,黏度范围为8.48-11.29mm2/s的HZ来源白油制备的疫苗黏度最高为166mPa.s以内。通过改变佐剂黏度可显著降低疫苗黏度。
工艺配方也是影响禽流感灭活疫苗黏度的重要因素,在佐剂不变的情况下,采用水相：油相1：3的配方,加硬脂酸铝,疫苗黏度不高于60mPa.s;采用水相：油相1：3的配方,不加硬脂酸铝,疫苗黏度不高于40mPa.s;采用水相：油相1：1.5的配方加硬脂酸铝,疫苗黏度最高,达到165mPa.s,采用水相：油相1：1.5的配方不加硬脂酸铝,疫苗黏度达到119mPa.s。
不同禽流感疫苗生产企业生产相同品种的禽流感疫苗黏度存在差异,最低的为18.7mPa.s,最高的为91mPa.s,差异极显著,8个来源的相同品种疫苗中,有6个黏度在60mPa.s以下。综合统计所有品种禽流感疫苗黏度,60%产品黏度在60mPa.s以下,因此将禽流感灭活疫苗黏度企业内控标准定为不高于60mPa.s。
以44对刻度吸管及黏度计检测数据建立油乳剂疫苗黏度检测刻度吸管法和黏度计法的回归模型,通过回归关系方差分析表证明刻度吸管法与黏度计法检测疫苗黏度有极显著的回归关系。
本研究得出以下结论：(1)原辅材料质量是影响禽流感疫苗质量的关键因素,通过建立生产用种蛋内控质量标准及注射用白油质量标准,可保证禽流感疫苗的安全、稳定、高效、均一；(2)采用低黏度、低稠环芳烃、高纯度白油制备禽流感疫苗可在确保安全性的前提下显著提高疫苗免疫效价、延长免疫持续期,减少免疫次数并保证疫苗有效期内的抗原稳定性；(3)采用黏度计替代刻度吸管法检测疫苗黏度更加精确,并能够与国际接轨。在禽流感疫苗工艺研究中,佐剂黏度及工艺配方是影响疫苗黏度的重要因素。
It has been attracted high attention to the high pathogenic avian influenza (AI) broken out and spread in the world. The effective prevention and control methods for the disease generally include the quarantine blockade, killing the infected and threatened animals and other biological safety measures. At present, some countries and regions have eradicated m the epidemic high pathogenic AI by using those methods. However, when the epidemic disease spread wildly in some areas, it could not be prevented and controlled effectively by using the above measures, and it could cause great economic loss. And the vaccination management must become the one of the key measures to control the epidemic disease. In China, vaccination has been used as the one main measure of the comprehensive prevention system for avian influenza. The vaccine was produced by8bio-products factories and the marking of the products was arranged by Chinese government under the invited public bidding and stock system. The preparation, sale, administration of the vaccines and the development of the new vaccines against high pathogenic AI were administrated and inspected strictly by Chinese government. It has caused great attention to the quality of the vaccines from the state, the manufacturing enterprises and the animal breeders.
In order to improve the problems in safety, protective efficacy, stability and homogeneity of the current vaccine against AI, it was firstly studied on the quality criterion of seed eggs, paraffin injected and viscosity in this study by followed the two items administrated by the4th Veterinary Medicine Pharmacopoeia Committee, the quality criterion and inspection criterion for the injected paraffin and the criterion and the method for a substitute viscometer for straw. And the criterion of the vaccine production in the biological industry and state was formulated. It should be helpful for increasing the quality of the AI vaccine and satisfying the need of the internal and international markets. The content of the study are composed of three parts as following:
1. Studying on the method for propagation of avian influenza virus and the quality control of the seed eggs for production
Based on the levers of the maternal antibodies against AI of the seed egg from the chickens unvaccinated with AI vaccine, the optical concentration of the virus and the optical egg-embryo age for inoculation were titrated by the matrix method. The result showed that:(1) when the titer of the maternal antibody against AI was less than2Iog2, the optical diluted concentration of virus was1:1000(10EID50), the optical egg-embryo age for inoculation was10days old, the quantity of the harvested allanoic liquid for each egg was up to12.5ml, the average HA titer was111og2, and the virus titre was108.5EID50/0.1ml;(2)when the titer of the maternal antibody against AI was less than6log2, the optical diluted concentration of the virus was1:10000(104EID50), the optical egg-embryo age for inoculation was10days old, the volume of the harvested allanoic liquid for each egg is up to12ml, the average HA titer was121og2, and the virus content was108.8EID50/0.1ml. To make sure that the accuracy of the experiment, all the inoculation and harvest of the egg-embryos allanoic liquid were conducted by the automatic inoculation and harvest machine system. Analyzing the above data comprehensively, the best method for the propagation of the virus was selected as:the10days old egg embryos was inoculate by using virus dilution of1:10000(104EID50).
According to the above optical condition for virus propagation,14batches of the inactivated vaccine was produced in total5.445billion milliliter, by using2300thousand eggs provided by14main seed egg supplier for making AI inactivated vaccine. By the statistic analysis of the appearance,weight, dilapidation rate, the fertilized rate, dead embryo rate, utilization ratio and harvesting quantity14seed eggs unvaccinated, the internal quality control criterion of the seed eggs for production was established. The14batches of seed eggs appeared clean, homogenized in size chosen by chosen machine, weight of50-60grams, the dilapidation rate being lower than2%for10of14batches, the fertilized rate of all the eggs being over91%, dead embryo rate before inoculation being lower than4%for11of14, utilization ratio being over85%for8of14batches. Making a comprehensive evaluation of the dead embryo rate post inoculation, harvesting quantity per embryo, and the virus titer index, it showed that the dead embryo rate was below10%, harvesting quantity per embryo was above10ml, the virus titer was over101og2. Among the14batches of vaccine against avian influenza, llbatches had the HA titer of81og2, which being higher than the criterion of61og2. It indicated that the various indexes of the seed eggs were reasonable, which being available to guarantee the quality of the vaccine.
2. The study of the quality criterion and the Standard Operation Procedure of the paraffin injected
One type of imported paraffin oil MC, used popularly in the8fixed manufacturing enterprises of vaccine against AI, and two types of paraffin, LH and HZ, made in China with different viscosity are chosen and the quality was detected as the method for the quality criterion of the paraffin injected and the paraffin oil for food in China Veterinary Pharmacopoeia (2005edition), GB48532008. The results showed that the3different sources of paraffin had different movable viscosity, the content of condensed aromatics, and the distribution of the carbon numbers. The4LH with different origination had the lowest movable viscosity and the content of condensed aromatics, the least narrow of distribution of the carbon numbers and the distinct of each component, which being4.37-5.7mm2/s、0.005-0.015and15-19, respectively. The3indexes of the paraffin in MC were7.26mm2/s、0.162and15-24, respectively. The3indexes of the paraffin in HZ were11.16mm2/s、0.037and15-24, respectively. The result of the safety experiment demonstrated that the absorbing of the vaccines with different origin paraffin were different. When inoculation via muscle route, the vaccine with LH which originated with5.7mm2/s of movable viscosity could be best absorbed completely48days post inoculation. Meanwhile, the vaccine with HZ was less absorbed. And one bird had a few unabsorbed dry cheeses like materials in the injection area. In the group MC with over the standard of the content of the condensed aromatics, the vaccine with HZ was also less absorbed and there were severe local inflammation. The result of the efficacy test indicated that the levels of HI antibodies to AI in LH groups were higher than those in the MC and HZ groups, which being7.61og2and9.51og2at2and3weeks post inoculation. The HI antibody titer was dramatically higher from ZSLHAI vaccine than that from ZSMC、ZSHZ vaccines, which remaining9.41og2at130days post immunization. After the vaccines was stored for12months and inoculated with chicken, the HI antibody titer from ZSLH vaccine was8.5log2. But when ZSMC、 ZSHZ vaccines were preserved for9months, they only could provide61og2HI antibody to AI.
The results of immunological protection efficiency for different strains of chickens and ducks indicated as following:(1) when the commercial7days old broilers were vaccinated with ZSLH, ZSMC, ZSHZ, respectively, the antibodies were detected at28days post inoculation, which being7.81og2for the ZSLH group,6.71og2for the ZSMC group, and6.11og2for the6.11og2group.(2) When the18days old egg-laying chickens were vaccinated, the antibody reached the peak at4weeks post inoculation (wpi), which values for ZSLH, ZSMC, ZSHZ being7.11og2,5.41og2and5.21og2, respectively.(3)When the18weeks old breed layers were vaccinated, the antibody reached the peak at1month post inoculation, which values for ZSLH, ZSMC, ZSHZ being10.81og2,9.91og2and8.21og2, respectively.(4)When the commercial10days old meat-type ducks were firstly vaccination, the antibody reached the peak at4wpi, which values for ZSLH, ZSMC, ZSHZ being7.11og2,5.41og2and5.21og2, respectively. As the second enhancement at3weeks old, the antibody reached the peak5wpi, the values being9.31og2,8.31og2and7.91og2for ZSLH, ZSMC, ZSHZ, respectively.(5)When the susceptible14days old egg-laying ducks were vaccinated, the antibody reached the peak at5wpi, the value being8.51og2for the ZSLH group, but in ZSMC and ZSHZ groups, the antibody reached the peak at4wpi, which value being5.31og2and5.11og2, respectively. After the second enhanced vaccination at3weeks old, the antibody reached the peak at6wpi, which values being10.81og2,9.41og2and9.11og2for ZSLH, ZSMC, ZSHZ, respectively. It indicated that the low viscosity, low content of condensed aromatics and the purity of paraffin LH made in China were superior to those of the imported and the similar product made in China. The vaccine made with paraffin LH had higher safety and immunological protective efficiency than those with other oil adjuvant.
3. Studies on the method of a substitute viscometer for straw for detecting the viscosity of inactivated avian influenza vaccine
The viscosity of the adjuvant was the key factor affecting the viscosity of the vaccine. In this study, the viscosity of the vaccines made with3different paraffins was detected by using a method of a substitute viscometer for straw. The results showed that when no change on the manufacturing procedure and the prescription, the viscosity of the vaccine made with paraffin LH, which viscosity being4.3-5.0mm2/s, was the lowest (≤40mPa.s). Meanwhile, the viscosity of the vaccine made with paraffin MC, which viscosity being7.31-7.42mm2/s mm2/s, was less109mPa.s. And the viscosity of the vaccine made with paraffin HZ, which viscosity being8.48-11.29mm2/s, was lower than166mPa.s. It indicated that viscosity of the vaccine could be decreased dramatically by changing the viscosity of the oil adjuvant.
The prescription was also the important factor for the viscosity of the inactivated avian influenza vaccine. By using the same oil adjuvant, when the volume rate of the water phase to the oil phase was1:3, the viscosity of the vaccine was not higher than60mPa.s by using aluminium stearate, but not higher than40mPa.s without aluminium stearate. When the volume rate of the water phase to the oil phase was1:1.5, the viscosity of the vaccine was the highest,165mPa.s, by using aluminium stearate, but119mPa.s without aluminium stearate. After detection of8commercial vaccines, the results showed that the viscosity of the vaccines produced by different manufacturers were different. The lowest of the viscosity was18.7mPa.s and the highest was91mPa.s. Moreover, among the8commercial vaccines,6types of vaccines had low viscosity of less60mPa.s. It indicated that no more than60mPa.s could be used as the internal viscosity criterion for the inactivated avian influenza vaccine in industry. Finally, after the viscosity of64oil-emulsified vaccines samples were detected with the method of viscometer and the straw, the regression model was established. And it was found that there were significant regression relation between the method of viscometer and the straw.
In summary, because the quality of the raw materials was the key factor affecting the quality of the avian influenza vaccines, it was necessary to establish the internal control quality criterion of the seed eggs and the quality criterion of the paraffin injected for making sure the vaccine safe, stable, highly efficient and homogeneous. Secondly, the low viscosity, content of the condensed aromatics and high purity of the paraffin were helpful for producing the influenza vaccines with high safety and immune efficiency, antigen stability and the period of immunity. Third, the method viscometer could substitute for the method straw for detecting accurately the viscosity of the vaccine, which being in line with the international standards. The viscosity of the adjuvant and the prescription were the important factors in affecting the viscosity of the inactivated avian influenza vaccine.
本研究正是在上述背景下,在执行第四届兽药典委员会下达的“注射用白油质量标准及检测规范”、“黏度计替代吸管法检测黏度标准及方法研究”两项项目基础上,以影响禽流感疫苗质量的三个关键因素种蛋、注射用白油、黏度标准的研究和制定为主线,针对禽流感疫苗原辅材料质量、生产工艺、质量检验、市场应用等关键环节中的质量控制点进行系统研究,以解决当前禽流感疫苗在安全、高效、稳定、均一方面存在的问题,同时制定与国际接轨的行业及国家标准,以全面提升我国禽流感疫苗的质量水平,满足国际、国内市场的需求。
本研究内容分为三个主要部分：1禽流感病毒(AIV)增殖方法及生产用种蛋质量控制
在不同种蛋母源抗体水平下,采用矩阵方法对AIV最佳接种稀释度、最佳接种胚龄进行研究,结果显示采用母源抗体≤21og2的禽流感非免疫种蛋,病毒最佳稀释度为1:10000(104EID50/0.1mL),最佳接种胚龄为10日龄,每胚收获量达到12.5mL,平均HA效价为11log2,病毒EIDs0为108.5EID50/0.1mL;采用母源抗体≤6log2的禽流感低免疫种蛋,病毒最佳稀释度为1:10000(104EID50/0.1mL),最佳接种胚龄为10日龄,每胚收获量达到12.8mL,但平均HA效价为10log2,病毒EID50为107.3EID50/0.1ml;采用母源抗体≤8log2的禽流感高免疫种蛋,病毒最佳稀释度为1:1000(105EID50/0.1mL),最佳接种胚龄为10日龄,每胚收获量达到12mL,但平均HA效价为9log2,病毒EID50为107.6EID50/0.1mL,同期比较的SPF鸡胚每胚收获量达到13mL,平均HA效价为12log2,病毒EID50为108.8EID50/0.1mL,为保证实验的准确性,所有鸡胚的接种和收获均采用自动接种收获机进行。综合分析以上试验数据,我们选择将AIV1:10000(104EID50/0.1mL)倍稀释,接种10日龄非免疫鸡胚(母源抗体≤2log2)为最佳病毒增殖条件。
按照以上病毒增殖最佳条件,我们选择了国内14家主要禽流感灭活疫苗种蛋供应商提供的非免疫种蛋230万枚,生产重组AIV灭活疫苗14批共计5445万毫升。通过14批非免疫种蛋外观、重量、破损率、受精率、死胚率、利用率、每胚收获量统计,建立生产用种蛋内控质量标准。14批种蛋外观清洁、通过选蛋机保证蛋大小均匀、蛋重在50-60g之间,14批种蛋中,破损率在2%以下的有10批,受精率均在91%以上,接种前死胚率在4%以下的有11批,接种前种蛋利用率在85%以上的有8批。综合评价病毒接种后死胚率、每胚收获量、病毒效价指标,9批为接种后死胚率在10%以下,病毒每胚收获量在10mL以上、病毒效价在10log2以上,按照以上种蛋质量指标生产的禽流感疫苗14批中有11批HI平均抗体效价达到了8log2,高于禽流感疫苗成品效力检验6log2的指标,说明种蛋各项指标较为合理,能够有效保证产品质量。2注射用白油质量标准及操作规范研究
选择国内禽流感定点企业普遍使用的一种进口白油MC及两种不同黏度国产白油LH、HZ,采用《中华人民共和国兽药典》(2005版)“注射用白油质量标准”及“食品级白油质量标准”(GB48532008)进行全项检测,三种来源白油在运动黏度、稠环芳烃含量、碳数分布方面存在差异,其中4组LH来源白油运动黏度、稠环芳烃含量最低,碳数分布最窄,各组分清晰,三种指标分别为4.37-5.7mm2/s、0.005-0.015、15-19; MC来源白油三种指标为7.26mm2/s、0.162、15-24; HZ来源白油三种指标为11.16mm2/S、0.037、15-24。用这三类白油制备成油乳疫苗后在SPF鸡体做了比较试验。从安全性试验结果看,疫苗吸收状况存在一定的个体差异。采用胸部肌肉途径接种后,吸收最好的运动黏度为5.7mm2/S的LH白油组,在免疫后48天基本吸收,其次为HZ组,48天后有1只鸡局部有少量未吸收干酪物。稠环芳烃含量超标的MC组,局部炎性反应严重,吸收稍差。从效力检验指标看,LH来源4组白油制备的AI疫苗诱生的HI抗体效价全部高于进口的MC及国产HZ来源白油,疫苗免疫2周后HI抗体效价即能达到7.6log2,三周达到9.5log2。从免疫保存期及持续期数据看,用LH白油批量生产的ZSLHAI疫苗免疫后产生的HI抗体显著高于ZSMC、ZSHZ疫苗,免疫130天后,HI抗体仍然维持在9.4log2,保存12个月,ZSLH疫苗抗体效价维持在8.51og2,而保存9个月时,ZSMC、ZSHZ疫苗已达不到成品效力检验6log2的标准。从不同品种鸡鸭免疫保护效力试验数据看,免疫7日龄商品肉鸡,免疫后28天,ZSLH疫苗抗体效价为7.81og2, ZSMC疫苗为6.71og2, ZSHZ疫苗为6.1log2；免疫18日龄蛋雏鸡,免疫后38天达到抗体高峰,ZSLH疫苗为8.21og2, ZSMC疫苗为7.0log2,ZSHZ疫苗为6.9log2；免疫18周龄蛋种鸡,免疫后1个月达到抗体峰值,ZSLH疫苗为10.8log2,ZSMC疫苗为9.91og2, ZSHZ疫苗为8.2log2；免疫10日龄商品肉鸭,一次免疫4周达到抗体峰值,ZSLH疫苗为7.11og2, ZSMC疫苗为5.41og2, ZSHZ疫苗为5.2log2；3周龄加强免疫后,在第5周达到抗体峰值,ZSLH疫苗为9.31og2, ZSMC疫苗为8.31og2, ZSHZ疫苗为7.9log2；免疫14日龄易感蛋鸭,一次免疫ZSLH疫苗5周达到抗体峰值,为8.51og2, ZSMC. ZSHZ疫苗免疫4周达到峰值,分别为5.31og2,5.11og2;3周龄加强免疫后,在第6周达到抗体峰值,ZSLH疫苗为10.81og2, ZSMC疫苗为9.41og2, ZSHZ疫苗为9.1log2。综合分析,低黏度、低稠环芳烃含量、高纯度的国产LH白油安全性及免疫保护效力均优于进口和国产同类产品。3黏度计替代吸管法检测禽流感灭活疫苗黏度研究
研究发现,佐剂黏度是影响疫苗黏度的重要因素,在不改变生产工艺、配方的情况下,黏度范围为4.3-5.0mm2/s的LH来源白油制备的疫苗黏度最低,在40mPa.s以下,黏度范围为7.31-7.42mm2/s的MC来源白油制备的疫苗黏度在109mPa.s以内,黏度范围为8.48-11.29mm2/s的HZ来源白油制备的疫苗黏度最高为166mPa.s以内。通过改变佐剂黏度可显著降低疫苗黏度。
工艺配方也是影响禽流感灭活疫苗黏度的重要因素,在佐剂不变的情况下,采用水相：油相1：3的配方,加硬脂酸铝,疫苗黏度不高于60mPa.s;采用水相：油相1：3的配方,不加硬脂酸铝,疫苗黏度不高于40mPa.s;采用水相：油相1：1.5的配方加硬脂酸铝,疫苗黏度最高,达到165mPa.s,采用水相：油相1：1.5的配方不加硬脂酸铝,疫苗黏度达到119mPa.s。
不同禽流感疫苗生产企业生产相同品种的禽流感疫苗黏度存在差异,最低的为18.7mPa.s,最高的为91mPa.s,差异极显著,8个来源的相同品种疫苗中,有6个黏度在60mPa.s以下。综合统计所有品种禽流感疫苗黏度,60%产品黏度在60mPa.s以下,因此将禽流感灭活疫苗黏度企业内控标准定为不高于60mPa.s。
以44对刻度吸管及黏度计检测数据建立油乳剂疫苗黏度检测刻度吸管法和黏度计法的回归模型,通过回归关系方差分析表证明刻度吸管法与黏度计法检测疫苗黏度有极显著的回归关系。
本研究得出以下结论：(1)原辅材料质量是影响禽流感疫苗质量的关键因素,通过建立生产用种蛋内控质量标准及注射用白油质量标准,可保证禽流感疫苗的安全、稳定、高效、均一；(2)采用低黏度、低稠环芳烃、高纯度白油制备禽流感疫苗可在确保安全性的前提下显著提高疫苗免疫效价、延长免疫持续期,减少免疫次数并保证疫苗有效期内的抗原稳定性；(3)采用黏度计替代刻度吸管法检测疫苗黏度更加精确,并能够与国际接轨。在禽流感疫苗工艺研究中,佐剂黏度及工艺配方是影响疫苗黏度的重要因素。
It has been attracted high attention to the high pathogenic avian influenza (AI) broken out and spread in the world. The effective prevention and control methods for the disease generally include the quarantine blockade, killing the infected and threatened animals and other biological safety measures. At present, some countries and regions have eradicated m the epidemic high pathogenic AI by using those methods. However, when the epidemic disease spread wildly in some areas, it could not be prevented and controlled effectively by using the above measures, and it could cause great economic loss. And the vaccination management must become the one of the key measures to control the epidemic disease. In China, vaccination has been used as the one main measure of the comprehensive prevention system for avian influenza. The vaccine was produced by8bio-products factories and the marking of the products was arranged by Chinese government under the invited public bidding and stock system. The preparation, sale, administration of the vaccines and the development of the new vaccines against high pathogenic AI were administrated and inspected strictly by Chinese government. It has caused great attention to the quality of the vaccines from the state, the manufacturing enterprises and the animal breeders.
In order to improve the problems in safety, protective efficacy, stability and homogeneity of the current vaccine against AI, it was firstly studied on the quality criterion of seed eggs, paraffin injected and viscosity in this study by followed the two items administrated by the4th Veterinary Medicine Pharmacopoeia Committee, the quality criterion and inspection criterion for the injected paraffin and the criterion and the method for a substitute viscometer for straw. And the criterion of the vaccine production in the biological industry and state was formulated. It should be helpful for increasing the quality of the AI vaccine and satisfying the need of the internal and international markets. The content of the study are composed of three parts as following:
1. Studying on the method for propagation of avian influenza virus and the quality control of the seed eggs for production
Based on the levers of the maternal antibodies against AI of the seed egg from the chickens unvaccinated with AI vaccine, the optical concentration of the virus and the optical egg-embryo age for inoculation were titrated by the matrix method. The result showed that:(1) when the titer of the maternal antibody against AI was less than2Iog2, the optical diluted concentration of virus was1:1000(10EID50), the optical egg-embryo age for inoculation was10days old, the quantity of the harvested allanoic liquid for each egg was up to12.5ml, the average HA titer was111og2, and the virus titre was108.5EID50/0.1ml;(2)when the titer of the maternal antibody against AI was less than6log2, the optical diluted concentration of the virus was1:10000(104EID50), the optical egg-embryo age for inoculation was10days old, the volume of the harvested allanoic liquid for each egg is up to12ml, the average HA titer was121og2, and the virus content was108.8EID50/0.1ml. To make sure that the accuracy of the experiment, all the inoculation and harvest of the egg-embryos allanoic liquid were conducted by the automatic inoculation and harvest machine system. Analyzing the above data comprehensively, the best method for the propagation of the virus was selected as:the10days old egg embryos was inoculate by using virus dilution of1:10000(104EID50).
According to the above optical condition for virus propagation,14batches of the inactivated vaccine was produced in total5.445billion milliliter, by using2300thousand eggs provided by14main seed egg supplier for making AI inactivated vaccine. By the statistic analysis of the appearance,weight, dilapidation rate, the fertilized rate, dead embryo rate, utilization ratio and harvesting quantity14seed eggs unvaccinated, the internal quality control criterion of the seed eggs for production was established. The14batches of seed eggs appeared clean, homogenized in size chosen by chosen machine, weight of50-60grams, the dilapidation rate being lower than2%for10of14batches, the fertilized rate of all the eggs being over91%, dead embryo rate before inoculation being lower than4%for11of14, utilization ratio being over85%for8of14batches. Making a comprehensive evaluation of the dead embryo rate post inoculation, harvesting quantity per embryo, and the virus titer index, it showed that the dead embryo rate was below10%, harvesting quantity per embryo was above10ml, the virus titer was over101og2. Among the14batches of vaccine against avian influenza, llbatches had the HA titer of81og2, which being higher than the criterion of61og2. It indicated that the various indexes of the seed eggs were reasonable, which being available to guarantee the quality of the vaccine.
2. The study of the quality criterion and the Standard Operation Procedure of the paraffin injected
One type of imported paraffin oil MC, used popularly in the8fixed manufacturing enterprises of vaccine against AI, and two types of paraffin, LH and HZ, made in China with different viscosity are chosen and the quality was detected as the method for the quality criterion of the paraffin injected and the paraffin oil for food in China Veterinary Pharmacopoeia (2005edition), GB48532008. The results showed that the3different sources of paraffin had different movable viscosity, the content of condensed aromatics, and the distribution of the carbon numbers. The4LH with different origination had the lowest movable viscosity and the content of condensed aromatics, the least narrow of distribution of the carbon numbers and the distinct of each component, which being4.37-5.7mm2/s、0.005-0.015and15-19, respectively. The3indexes of the paraffin in MC were7.26mm2/s、0.162and15-24, respectively. The3indexes of the paraffin in HZ were11.16mm2/s、0.037and15-24, respectively. The result of the safety experiment demonstrated that the absorbing of the vaccines with different origin paraffin were different. When inoculation via muscle route, the vaccine with LH which originated with5.7mm2/s of movable viscosity could be best absorbed completely48days post inoculation. Meanwhile, the vaccine with HZ was less absorbed. And one bird had a few unabsorbed dry cheeses like materials in the injection area. In the group MC with over the standard of the content of the condensed aromatics, the vaccine with HZ was also less absorbed and there were severe local inflammation. The result of the efficacy test indicated that the levels of HI antibodies to AI in LH groups were higher than those in the MC and HZ groups, which being7.61og2and9.51og2at2and3weeks post inoculation. The HI antibody titer was dramatically higher from ZSLHAI vaccine than that from ZSMC、ZSHZ vaccines, which remaining9.41og2at130days post immunization. After the vaccines was stored for12months and inoculated with chicken, the HI antibody titer from ZSLH vaccine was8.5log2. But when ZSMC、 ZSHZ vaccines were preserved for9months, they only could provide61og2HI antibody to AI.
The results of immunological protection efficiency for different strains of chickens and ducks indicated as following:(1) when the commercial7days old broilers were vaccinated with ZSLH, ZSMC, ZSHZ, respectively, the antibodies were detected at28days post inoculation, which being7.81og2for the ZSLH group,6.71og2for the ZSMC group, and6.11og2for the6.11og2group.(2) When the18days old egg-laying chickens were vaccinated, the antibody reached the peak at4weeks post inoculation (wpi), which values for ZSLH, ZSMC, ZSHZ being7.11og2,5.41og2and5.21og2, respectively.(3)When the18weeks old breed layers were vaccinated, the antibody reached the peak at1month post inoculation, which values for ZSLH, ZSMC, ZSHZ being10.81og2,9.91og2and8.21og2, respectively.(4)When the commercial10days old meat-type ducks were firstly vaccination, the antibody reached the peak at4wpi, which values for ZSLH, ZSMC, ZSHZ being7.11og2,5.41og2and5.21og2, respectively. As the second enhancement at3weeks old, the antibody reached the peak5wpi, the values being9.31og2,8.31og2and7.91og2for ZSLH, ZSMC, ZSHZ, respectively.(5)When the susceptible14days old egg-laying ducks were vaccinated, the antibody reached the peak at5wpi, the value being8.51og2for the ZSLH group, but in ZSMC and ZSHZ groups, the antibody reached the peak at4wpi, which value being5.31og2and5.11og2, respectively. After the second enhanced vaccination at3weeks old, the antibody reached the peak at6wpi, which values being10.81og2,9.41og2and9.11og2for ZSLH, ZSMC, ZSHZ, respectively. It indicated that the low viscosity, low content of condensed aromatics and the purity of paraffin LH made in China were superior to those of the imported and the similar product made in China. The vaccine made with paraffin LH had higher safety and immunological protective efficiency than those with other oil adjuvant.
3. Studies on the method of a substitute viscometer for straw for detecting the viscosity of inactivated avian influenza vaccine
The viscosity of the adjuvant was the key factor affecting the viscosity of the vaccine. In this study, the viscosity of the vaccines made with3different paraffins was detected by using a method of a substitute viscometer for straw. The results showed that when no change on the manufacturing procedure and the prescription, the viscosity of the vaccine made with paraffin LH, which viscosity being4.3-5.0mm2/s, was the lowest (≤40mPa.s). Meanwhile, the viscosity of the vaccine made with paraffin MC, which viscosity being7.31-7.42mm2/s mm2/s, was less109mPa.s. And the viscosity of the vaccine made with paraffin HZ, which viscosity being8.48-11.29mm2/s, was lower than166mPa.s. It indicated that viscosity of the vaccine could be decreased dramatically by changing the viscosity of the oil adjuvant.
The prescription was also the important factor for the viscosity of the inactivated avian influenza vaccine. By using the same oil adjuvant, when the volume rate of the water phase to the oil phase was1:3, the viscosity of the vaccine was not higher than60mPa.s by using aluminium stearate, but not higher than40mPa.s without aluminium stearate. When the volume rate of the water phase to the oil phase was1:1.5, the viscosity of the vaccine was the highest,165mPa.s, by using aluminium stearate, but119mPa.s without aluminium stearate. After detection of8commercial vaccines, the results showed that the viscosity of the vaccines produced by different manufacturers were different. The lowest of the viscosity was18.7mPa.s and the highest was91mPa.s. Moreover, among the8commercial vaccines,6types of vaccines had low viscosity of less60mPa.s. It indicated that no more than60mPa.s could be used as the internal viscosity criterion for the inactivated avian influenza vaccine in industry. Finally, after the viscosity of64oil-emulsified vaccines samples were detected with the method of viscometer and the straw, the regression model was established. And it was found that there were significant regression relation between the method of viscometer and the straw.
In summary, because the quality of the raw materials was the key factor affecting the quality of the avian influenza vaccines, it was necessary to establish the internal control quality criterion of the seed eggs and the quality criterion of the paraffin injected for making sure the vaccine safe, stable, highly efficient and homogeneous. Secondly, the low viscosity, content of the condensed aromatics and high purity of the paraffin were helpful for producing the influenza vaccines with high safety and immune efficiency, antigen stability and the period of immunity. Third, the method viscometer could substitute for the method straw for detecting accurately the viscosity of the vaccine, which being in line with the international standards. The viscosity of the adjuvant and the prescription were the important factors in affecting the viscosity of the inactivated avian influenza vaccine.
引文
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