低pH孵放法灭活生物制品中异嗜性小鼠白血病病毒的方法学研究
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摘要
生物制品包括血液制品、基因制品、病毒类疫苗等,为了提高生物制品临床使用的安全性,生产工艺要具有一定的去除或灭活部分病毒的能力,生产过程中应有特定的去除或灭活病毒的方法。重组融合蛋白属于基因制品的一种,目前上市销售的重组融合蛋白类生物制品包括TNF-α、IFN-γ、IL-18、IL-12等,此类蛋白主要以中国仓鼠卵巢细胞(Chinese Hamster Ovary, CHO)作为表达载体。目前,CHO细胞表达抗体药物已经产业化。然而,CHO细胞内本身存在内源性逆转录病毒,这一结论已经得到证实；但这种内源性逆转录病毒对人类不具有致病性。尽管这种内源性逆转录病毒对于人类是非致病性的,但采用CHO细胞作为药用蛋白质的表达载体,其分泌的蛋白质对于人用药的安全性仍然具有的一定潜在风险。因此,各国新药研发相关法规要求对于CHO细胞来源的药用蛋白质,其生产工艺中必须具备病毒去除或灭活工艺,而且,必须对该工艺在实验室进行病毒去除或灭活的验证检验。我国相关生物制品生产厂家及生产品种逐年增多,但实验室验证研究的方法国内目前尚未建立,需在美国进行试验,因而时间长、费用高成为制约国内相关新药研发的关键问题。本课题根据SFDA相关法规和技术要求,建立此类蛋白药物的病毒灭活验证方法,以填补国内空白。
此外,本课题中建立的异嗜性小鼠白血病病毒(X-MulV)致猫神经星形胶质细胞(PG-4)产生病变效应的体外模型,不仅可以用于生物制品中病毒灭活验证检验,亦可用于抗逆转录病毒新药的筛选及药效学评价。
美国FDA及我国SFDA要求：对于CHO细胞来源的重组蛋白在低pH生产工艺的验证性试验中,采用X-MulV作为CHO细胞内源性逆转录病毒的指示病毒(又称作模拟病毒、替代病毒),建立低pH孵放法病毒灭活验证方法。
本课题共分三个部分进行研究：
一、X-MuIV体外培养及活性评价体系的建立
目前国外研究者采用Mus. Dunni细胞对X-MulV进行传代,由于该细胞株在国内即为少见,同时考虑到X-MulV能够在非小鼠细胞系生长的特点,我们选择以CHO细胞作为X-MulV的传代细胞,通过SYBR Green荧光定量聚合酶连反应(SYBR Green real-time PCR assay),以gag基因作为靶基因,定量X-MulV在CHO细胞中生长2-7天的相对表达量,并通过病毒感染性试验(PG-4细胞空斑试验)确定病毒数量显著增加时间点的病毒滴度。
结果显示：分别以培养时间作为横坐标、以指示病毒gag基因RNA的相对表达量作为纵坐标,绘制gag基因RNA相对表达量随时间变化的曲线。曲线显示：gag基因在CHO细胞中生长2-7天的生长曲线符合指数形式,指数方程拟合相关系数R2=0.9125,生长第7天的数量比第2天显著增长(p<0.01),是第2天的13.89倍。
经CHO细胞传代培养7天的X-MulV悬液,其在指示细胞PG-4上的病毒滴度为8.78±0.25logioPFU/mL。病毒灭活方法指导原则中规定：作为指示病毒的病毒,其病毒滴度必须达到7.0log1o以上。
因此,以CHO细胞作为X-MulV的传代细胞,经培养7天得到的病毒悬液能够用于病毒灭活验证试验。
二、低pH法灭活生物制品中X-MulV方法的建立
1.缓冲体系的建立：在灭活验证试验中,模拟重组蛋白质的生产工艺,以Tris-碱-柠檬酸缓冲液体系作为样本缓冲液,将样本缓冲液与X-MulV储备液以9：1体积比混匀后,进行病毒灭活验证试验。
2.检测指标：以病毒滴度作为检测指标,当降低的病毒滴度大于4.0log10PFU视为有效灭活。
3.灭活参数考察：包括pH值、灭活温度、灭活时间以及蛋白质浓度对灭活效果的影响。灭活试验中设置不同因素、不同水平参数如下：灭活pH值：3.00、3.50、4.00以及5.00；灭活温度：4℃、10℃、20℃以及25℃；灭活时间：2h、4h、6h、8h以及10h；蛋白质浓度(以卵清白蛋白作为模拟蛋白,OVA):0mg/mL、1.0mg/mL、2.0mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL以及10.0mg/mL。将不同因素、不同水平按照排列组合的方式组合后,分别进行病毒灭活验证试验。
对所有得到的残余病毒滴度进行统计学分析,通过正交分析主成分分析得出：不同因素对病毒滴度的影响大小依次是：pH>温度>时间；pH值和温度对病毒滴度有显著的影响(p<0.01),时间不具有显著性影响(p=0.264)；通过正交分析交互作用分析得出：温度和时间之间的交互作用对病毒滴度具有显著性影响(p=0.016)；通过正交分析筛选出的最优灭活条件为：pH3.50、4℃、4h。多元线性回归分析显示：pH值能够显著影响病毒滴度(p<0.01),温度能够影响病毒滴度,但影响不具有显著性(p=0.081),时间对病毒滴度没有影响(p=0.941)。结合上述分析结果,同时考虑到实际灭活工艺在常温条件下进行；因此,在考察蛋白质浓度对病毒滴度的影响时,在pH3.50、4h一致的前提下,同时考察4℃和25℃的灭活效度。经灭活后,蛋白质浓度从0mg/mL～10.0mg/mL范围内,4℃组和25℃组病毒滴度降低分别大于4.1log10PFU和4.7log10PFU;且25℃组样本病毒降低滴度基本大于5.0log10PFU。
在此基础上,进行pH值、温度及时间因素允许范围的考察。通过病毒感染性试验,当灭活温度为25℃、孵育4h时,pH值在3.60-3.90范围内,X-MulV病毒滴度降低均大于5.0log10PFU;当pH为3.60、孵育4h时,温度在23℃、24℃、25℃、26℃以及27℃范围时,X-MulV病毒滴度降低均大于5.0log10PFU;当pH值为3.60、孵育温度为25℃时,灭活0～180min不能有效灭活X-MulV,灭活240min能够有效灭活X-MulV。通过参数允许变化范围的考察,结合相关文献报道：pH3.50时,生物制品会有少量损失。我们将最优灭活条件设为：pH3.60、25℃及4h。
4.逆转录酶活性检测：为了进一步评价灭活效度,采用产物增强的逆转录酶反应法(简称为PERT法)考察X-MulV灭活前后逆转录酶活性的变化。结果显示：以MMLV作为已知浓度的标准逆转录酶,通过逆转录酶浓度-CT值标准曲线,计算样本中逆转录酶的含量。经SYBR Green荧光定量PCR反应检测逆转录酶活性,经pH3.50、4h灭活后,逆转录酶含量比未灭活样本降低两个数量级,具有显著性差异(p<0.01),且4℃组与25℃组之间没有显著性差异(p>0.05)。
5.低pH对生物制品活性的影响：选择L929细胞株作为靶细胞,采用MTT比色法检测rhTNF-α的对L929细胞的杀伤活性。将rhTNF-α经pH3.60、25℃孵育4h后,其杀伤L929细胞活性与中性rhTNF-α相比,未发生变化。
6.低pH对病毒包膜基因的影响：利用传统聚合酶链式反应,考察X-MulV在有效灭活前后,包膜基因env3’末端1626～1930位点片段的扩增情况。X-MulV经pH3.60、25℃灭活4h后,env3’末端305bp片段未扩增出,而阳性对照中该片段条带清晰,且亮度高。
三、试验方法的实际应用
采用pH3.60、25℃、4h的灭活条件对百奥泰(广州)生物科技有限公司提供的注射用rhTNF-a融合蛋白进行X-MulV的灭活验证,结果显示病毒滴度降低4.0loglo以上,说明此条件能够有效灭活指示病毒X-MulV,符合国际规定的病毒滴度降低大于4.0log1o的要求。
通过以上研究,我们得出结论：
1.以CHO细胞作为X-MulV的传代载体,培养7天,获得的X-MulV病毒悬液,在PG-4细胞中的病毒滴度能够达到7.0log10PFU以上,符合生物制品病毒灭活指导原则关于指示病毒的要求；
2.采用pH3.60、25℃、4h灭活CHO细胞来源的人重组融合TNF-a蛋白药物中指示病毒X-MulV,能够实现有效灭活,其病毒滴度降低大于4.0log10PFU,说明通过本研究建立的验证性试验方法可行；
3.此灭活条件对所检测样品的生物学活性无影响；
4.低pH影响包膜基因env3’末端1626-1930位点,X-MulV能够被有效灭活可能与该位点有关。
CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in Chinese hamster ovary (CHO) cells. Inactivation process is required in manufacturing for the safety of human use. In this study, we established a low pH method for evaluation of murine xenotropic gamma retrovirus (X-MulV) clearance in manufacturing of recombinant human TNF-a receptor immunoglobin G fusion proteins (rhTNF-a) for injection. X-MulV is used as a model virus in evaluation of viral inactivation in biological products derived from CHO cells. Mus. dunni cells are used to support the replication of X-MulV particles at present. In consideration of the characters of X-MulV, we tried to propagate the viruses on CHO cells, a non-murine cell line. The viruses were harvested from CHO cells from day2to day7post-infection, respectively. Reverse transcription-SYBR green fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the X-MulV particles on different days. The development of X-MulV on CHO cells was a typical single-step growth curve. The titers of viral stock harvested on day7were8.78±0.25log10PFU per mL, which were assayed on PG-4cells. Based on these data, we conclude that CHO cells could be a host organism for X-MulV particles. X-MulV produced on CHO cells could be used in the evaluation at a lab-scale of viral clearance in pharmaceutical proteins derived from CHO cells.
Relevant parameters were analyzed and screened by orthogonal analysis and multiple linear regression analysis. Parameters including pH, temperatures, time and protein content were investigated for their impact on X-MulV inactivation. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-a were spiked with X-MulV and were inactivated by the sifted conditions. Reverse transcriptase (RT) activities in inactivated samples were detected by real-time SYBR green product-enhanced reverse transcriptase assay (PERT). The pH and temperatures were critical factors in X-MulV inactivation. Simultaneously, there was an interaction between temperature and time on inactivation. Protein concentration and time had a less impact on inactivation. Inactivation conditions of pH3.50,4℃or25℃and4hours were chosen to achieve greater than4.0log10inactivation of X-MulV. RT activities were decreased significantly in samples that were inactivated with low pH and there were no difference between4℃or25℃.
We performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of rhTNF-a. RhTNF-a were spiked with X-MulV and were inactivated at pH3.60-3.90,25±2℃, and0-240min, respectively. Samples incubated at the conditions for15～180min were not inactivated effectively. For4h incubation, log10reductions were achieved5.0log10. Biological activity of rhTNF-a incubated at pH3.60,25℃for4h, which was assayed on murine L929fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH3.60, and it may be the mechanism of low pH inactivation. This method was used for the evaluation for the viral clearance in CHO-derived recombinant TNF-a protein, which was provided by a manufacturer in Guangzhou.
The optimal conditions of inactivation were pH3.60,25℃and4hours. This method could be used at the lab scale for evaluation of viral clearance in pharmaceutical proteins derived from CHO cells in manufacturing process.
此外,本课题中建立的异嗜性小鼠白血病病毒(X-MulV)致猫神经星形胶质细胞(PG-4)产生病变效应的体外模型,不仅可以用于生物制品中病毒灭活验证检验,亦可用于抗逆转录病毒新药的筛选及药效学评价。
美国FDA及我国SFDA要求：对于CHO细胞来源的重组蛋白在低pH生产工艺的验证性试验中,采用X-MulV作为CHO细胞内源性逆转录病毒的指示病毒(又称作模拟病毒、替代病毒),建立低pH孵放法病毒灭活验证方法。
本课题共分三个部分进行研究：
一、X-MuIV体外培养及活性评价体系的建立
目前国外研究者采用Mus. Dunni细胞对X-MulV进行传代,由于该细胞株在国内即为少见,同时考虑到X-MulV能够在非小鼠细胞系生长的特点,我们选择以CHO细胞作为X-MulV的传代细胞,通过SYBR Green荧光定量聚合酶连反应(SYBR Green real-time PCR assay),以gag基因作为靶基因,定量X-MulV在CHO细胞中生长2-7天的相对表达量,并通过病毒感染性试验(PG-4细胞空斑试验)确定病毒数量显著增加时间点的病毒滴度。
结果显示：分别以培养时间作为横坐标、以指示病毒gag基因RNA的相对表达量作为纵坐标,绘制gag基因RNA相对表达量随时间变化的曲线。曲线显示：gag基因在CHO细胞中生长2-7天的生长曲线符合指数形式,指数方程拟合相关系数R2=0.9125,生长第7天的数量比第2天显著增长(p<0.01),是第2天的13.89倍。
经CHO细胞传代培养7天的X-MulV悬液,其在指示细胞PG-4上的病毒滴度为8.78±0.25logioPFU/mL。病毒灭活方法指导原则中规定：作为指示病毒的病毒,其病毒滴度必须达到7.0log1o以上。
因此,以CHO细胞作为X-MulV的传代细胞,经培养7天得到的病毒悬液能够用于病毒灭活验证试验。
二、低pH法灭活生物制品中X-MulV方法的建立
1.缓冲体系的建立：在灭活验证试验中,模拟重组蛋白质的生产工艺,以Tris-碱-柠檬酸缓冲液体系作为样本缓冲液,将样本缓冲液与X-MulV储备液以9：1体积比混匀后,进行病毒灭活验证试验。
2.检测指标：以病毒滴度作为检测指标,当降低的病毒滴度大于4.0log10PFU视为有效灭活。
3.灭活参数考察：包括pH值、灭活温度、灭活时间以及蛋白质浓度对灭活效果的影响。灭活试验中设置不同因素、不同水平参数如下：灭活pH值：3.00、3.50、4.00以及5.00；灭活温度：4℃、10℃、20℃以及25℃；灭活时间：2h、4h、6h、8h以及10h；蛋白质浓度(以卵清白蛋白作为模拟蛋白,OVA):0mg/mL、1.0mg/mL、2.0mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL以及10.0mg/mL。将不同因素、不同水平按照排列组合的方式组合后,分别进行病毒灭活验证试验。
对所有得到的残余病毒滴度进行统计学分析,通过正交分析主成分分析得出：不同因素对病毒滴度的影响大小依次是：pH>温度>时间；pH值和温度对病毒滴度有显著的影响(p<0.01),时间不具有显著性影响(p=0.264)；通过正交分析交互作用分析得出：温度和时间之间的交互作用对病毒滴度具有显著性影响(p=0.016)；通过正交分析筛选出的最优灭活条件为：pH3.50、4℃、4h。多元线性回归分析显示：pH值能够显著影响病毒滴度(p<0.01),温度能够影响病毒滴度,但影响不具有显著性(p=0.081),时间对病毒滴度没有影响(p=0.941)。结合上述分析结果,同时考虑到实际灭活工艺在常温条件下进行；因此,在考察蛋白质浓度对病毒滴度的影响时,在pH3.50、4h一致的前提下,同时考察4℃和25℃的灭活效度。经灭活后,蛋白质浓度从0mg/mL～10.0mg/mL范围内,4℃组和25℃组病毒滴度降低分别大于4.1log10PFU和4.7log10PFU;且25℃组样本病毒降低滴度基本大于5.0log10PFU。
在此基础上,进行pH值、温度及时间因素允许范围的考察。通过病毒感染性试验,当灭活温度为25℃、孵育4h时,pH值在3.60-3.90范围内,X-MulV病毒滴度降低均大于5.0log10PFU;当pH为3.60、孵育4h时,温度在23℃、24℃、25℃、26℃以及27℃范围时,X-MulV病毒滴度降低均大于5.0log10PFU;当pH值为3.60、孵育温度为25℃时,灭活0～180min不能有效灭活X-MulV,灭活240min能够有效灭活X-MulV。通过参数允许变化范围的考察,结合相关文献报道：pH3.50时,生物制品会有少量损失。我们将最优灭活条件设为：pH3.60、25℃及4h。
4.逆转录酶活性检测：为了进一步评价灭活效度,采用产物增强的逆转录酶反应法(简称为PERT法)考察X-MulV灭活前后逆转录酶活性的变化。结果显示：以MMLV作为已知浓度的标准逆转录酶,通过逆转录酶浓度-CT值标准曲线,计算样本中逆转录酶的含量。经SYBR Green荧光定量PCR反应检测逆转录酶活性,经pH3.50、4h灭活后,逆转录酶含量比未灭活样本降低两个数量级,具有显著性差异(p<0.01),且4℃组与25℃组之间没有显著性差异(p>0.05)。
5.低pH对生物制品活性的影响：选择L929细胞株作为靶细胞,采用MTT比色法检测rhTNF-α的对L929细胞的杀伤活性。将rhTNF-α经pH3.60、25℃孵育4h后,其杀伤L929细胞活性与中性rhTNF-α相比,未发生变化。
6.低pH对病毒包膜基因的影响：利用传统聚合酶链式反应,考察X-MulV在有效灭活前后,包膜基因env3’末端1626～1930位点片段的扩增情况。X-MulV经pH3.60、25℃灭活4h后,env3’末端305bp片段未扩增出,而阳性对照中该片段条带清晰,且亮度高。
三、试验方法的实际应用
采用pH3.60、25℃、4h的灭活条件对百奥泰(广州)生物科技有限公司提供的注射用rhTNF-a融合蛋白进行X-MulV的灭活验证,结果显示病毒滴度降低4.0loglo以上,说明此条件能够有效灭活指示病毒X-MulV,符合国际规定的病毒滴度降低大于4.0log1o的要求。
通过以上研究,我们得出结论：
1.以CHO细胞作为X-MulV的传代载体,培养7天,获得的X-MulV病毒悬液,在PG-4细胞中的病毒滴度能够达到7.0log10PFU以上,符合生物制品病毒灭活指导原则关于指示病毒的要求；
2.采用pH3.60、25℃、4h灭活CHO细胞来源的人重组融合TNF-a蛋白药物中指示病毒X-MulV,能够实现有效灭活,其病毒滴度降低大于4.0log10PFU,说明通过本研究建立的验证性试验方法可行；
3.此灭活条件对所检测样品的生物学活性无影响；
4.低pH影响包膜基因env3’末端1626-1930位点,X-MulV能够被有效灭活可能与该位点有关。
CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in Chinese hamster ovary (CHO) cells. Inactivation process is required in manufacturing for the safety of human use. In this study, we established a low pH method for evaluation of murine xenotropic gamma retrovirus (X-MulV) clearance in manufacturing of recombinant human TNF-a receptor immunoglobin G fusion proteins (rhTNF-a) for injection. X-MulV is used as a model virus in evaluation of viral inactivation in biological products derived from CHO cells. Mus. dunni cells are used to support the replication of X-MulV particles at present. In consideration of the characters of X-MulV, we tried to propagate the viruses on CHO cells, a non-murine cell line. The viruses were harvested from CHO cells from day2to day7post-infection, respectively. Reverse transcription-SYBR green fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the X-MulV particles on different days. The development of X-MulV on CHO cells was a typical single-step growth curve. The titers of viral stock harvested on day7were8.78±0.25log10PFU per mL, which were assayed on PG-4cells. Based on these data, we conclude that CHO cells could be a host organism for X-MulV particles. X-MulV produced on CHO cells could be used in the evaluation at a lab-scale of viral clearance in pharmaceutical proteins derived from CHO cells.
Relevant parameters were analyzed and screened by orthogonal analysis and multiple linear regression analysis. Parameters including pH, temperatures, time and protein content were investigated for their impact on X-MulV inactivation. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-a were spiked with X-MulV and were inactivated by the sifted conditions. Reverse transcriptase (RT) activities in inactivated samples were detected by real-time SYBR green product-enhanced reverse transcriptase assay (PERT). The pH and temperatures were critical factors in X-MulV inactivation. Simultaneously, there was an interaction between temperature and time on inactivation. Protein concentration and time had a less impact on inactivation. Inactivation conditions of pH3.50,4℃or25℃and4hours were chosen to achieve greater than4.0log10inactivation of X-MulV. RT activities were decreased significantly in samples that were inactivated with low pH and there were no difference between4℃or25℃.
We performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of rhTNF-a. RhTNF-a were spiked with X-MulV and were inactivated at pH3.60-3.90,25±2℃, and0-240min, respectively. Samples incubated at the conditions for15～180min were not inactivated effectively. For4h incubation, log10reductions were achieved5.0log10. Biological activity of rhTNF-a incubated at pH3.60,25℃for4h, which was assayed on murine L929fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH3.60, and it may be the mechanism of low pH inactivation. This method was used for the evaluation for the viral clearance in CHO-derived recombinant TNF-a protein, which was provided by a manufacturer in Guangzhou.
The optimal conditions of inactivation were pH3.60,25℃and4hours. This method could be used at the lab scale for evaluation of viral clearance in pharmaceutical proteins derived from CHO cells in manufacturing process.
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