聚乙二醇修饰的脂质体四价流感疫苗稳定性

Study on stability of lyophilized preparation of PEG-modified influenza vaccine liposomes

目的:通过影响因素实验、加速实验考察聚乙二醇(PEG)修饰的脂质体流感疫苗冻干粉的物理和生物稳定性,并预测其有效期。方法:(40±2)℃下进行高温影响因素实验,4℃,(25±2)℃下进行加速实验。以外观性状、粒径、包封率、稳定系数作为物理稳定性评价指标;采用脾淋巴细胞增殖实验和血凝抑制实验以刺激指数(SI)和免后与免前抗体滴度比(HI)作为指标考察生物稳定性。采用经典恒温法,利用Arrhenius公式计算有效期。结果:影响因素实验:PEG2000(3000/6000)修饰的脂质体流感疫苗冻干粉于(40±2)℃下放置20 d,粒径和稳定系数逐渐增大,包封率逐渐下降;放置10 d时,各PEG组SI值比原液组大,与原液组比较差异有统计学意义(P<0.05);各PEG组免后与免前抗体滴度比均>4;放置20 d时,各PEG组SI值比原液组小,与原液组比较有显著性差异(P<0.05);且各PEG组免后与免前抗体滴度比均<4。加速实验:3批PEG6000修饰的脂质体流感疫苗冻干粉在4℃和(25±2)℃下放置3个月,4℃下粒径、包封率、稳定系数基本不变,(25±2)℃下粒径和稳定系数增大,包封率下降;4℃下PEG6000组SI值与原液组比较有显著性差异(P<0.05);(25±2)℃下PEG6000组SI值与原液组比较均无显著性差异;4℃和(25±2)℃PEG6000组免后与免前抗体滴度比均≥4。有效期预测:25℃,30℃,35℃,40℃的降解速率常数(K)分别为1.039×10^(-3),2.649×10-3,3.574×10^(-3),4.868×10^(-3)d^(-1),lnK对1/T进行线性回归,回归方程为:lnK=20.195-8148.5/T(r=0.9179)。结论:PEG修饰的脂质体流感疫苗冻干粉在(40±2)℃下放置10 d,PEG6000修饰的脂质体流感疫苗冻干粉在4℃和(25±2)℃下放置3个月都具有良好稳定性;通过推算PEG6000修饰的脂质体流感疫苗在4℃,25℃,37℃下分别大约能储存2.2年、3.3月、24 d。

Objective:To investigate the physical and biological stability of PEG-modified liposome influenza vaccine lyophilized powder by influencing factor experiment and accelerated experiment and predict the validity period.Methods:High temperature influencing factor test was carried out at(40±2)℃,and accelerated test was carried out at 4℃ and(25±2)℃.Physical stability was evaluated by appearance,particle size,encapsulation rate and stability coefficient.Spleen lymphocyte proliferation test and hemagglutination inhibition test were used to evaluate the biological stability,in which the stimulation index(SI)and antibody titer ratio(HI)were used as indexes.The validity period was calculated by using the classical constant temperature method and Arrhenius formula.Results:In the influencing factor experiment,the particle size and stability coefficient increased and the encapsulation rate decreased gradually after placing PEG2000(3000/6000)-modified liposome influenza vaccine freeze-dried powder at(40±2)℃ for 20 days.On the 10th day,the SI value of each PEG group was significantly bigger than that of the original solution group(P<0.05).The titer ratio of antibody before and after immunization was >4 for all PEG groups;on the 20th day,the SI value of each PEG group was significantly smaller than that of the original solution group(P<0.05);the antibody titer ratio of each PEG group after and before immunization was less than 4.In the accelerated experiment,three batches of PEG6000-modified liposome influenza vaccine lyophilized powder were placed at 4℃ and(25±2)℃ for 3 months,the sample was stable at 4℃ with minor changing in particle size,encapsulation rate and stability coefficient,while at(25±2)℃ the particle size and stability coefficient gradually increased,the encapsulation rate gradually decreased.There was significant difference in SI value between PEG6000 group and stock solution group under 4℃ storage condition(P<0.05),and no significant difference under(25±2)℃ storage condition.For PEG6000 group,the ratio of antibody titer after and before immunization were≥4 under both storage condition of 4℃ and(25±2)℃.For the validity period prediction,the values of decomposition velocity constants K at 25℃,30℃,35℃ and 40℃ were 1.039×10^(-3),2.649×10^(-3),3.574×10^(-3),and 4.868×10^(-3)d^(-1),respectively.The regression equation of lnK to 1/T was as following:lnK=20.195-8148.5/T(r=0.9179).Conclusion:PEG-modified liposome influenza vaccine freeze-dried powder has good stability at(40±2)℃ for 10 days,PEG6000-modified liposome influenza vaccine freeze-dried powder has good stability at 4℃ and(25±2)℃ for 3 months.It is estimated that PEG6000-modified liposome influenza vaccine can be stored for 2.2 years,3.3 months and 24 days at 4℃,25℃ and 37℃ respectively.