Insights into virus inactivation by polysorbate 80 in the absence of solvent
Insights into virus inactivation by polysorbate 80 in the absence of solvent
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DOI：
10.1002/btpr.2953
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发表时间：
2019-12-27
影响因子：
2.9
通讯作者：
O'Donnell, Sean
中科院分区：
文献类型：
--
作者：
Chen, Dayue;Luo, Wen;O'Donnell, Sean
Triton X-100长期以来一直单独使用或与溶剂结合使用，以在生物制药生产中消除包膜病毒。然而，欧洲化学品管理局（ECHA）于2017年正式将Triton X-100列入附件XIV授权清单，因为Triton X-100的降解产物4-（1，1，3，3-四甲基丁基）苯酚具有有害的内分泌干扰活性。因此，在欧洲经济区使用Triton X-100将需要在2021年1月4日日落日期之后获得ECHA的授权。在寻找Triton X-100的可能替代品时，我们发现聚山梨酯80（PS 80）在不存在任何溶剂的情况下能够有效灭活包膜病毒，如异嗜性鼠白血病病毒和伪狂犬病病毒，其疗效相当，如通过对数减少因子测量的。有趣的是，PS 80在磷酸盐缓冲液（PBS）中未显示任何杀病毒活性，而在无细胞中国仓鼠卵巢（CHO）生物反应器收获物中实现了稳健的病毒灭活。这一有趣的观察结果使我们推测PS 80的病毒灭活涉及PBS中不存在的无细胞CHO生物反应器收获物中的组分。具体而言，我们假设无细胞生物反应器中的酯酶和/或脂肪酶收获水解的PS 80以产生油酸，一种已知的有效杀病毒剂，其进而灭活病毒。使用PBS中的纯化重组溶酶体磷脂酶A2异构体（rLPLA 2）证实了该理论。随后的表征工作表明，在与Triton X-100相当的温度和浓度范围内，PS 80的病毒灭活有效且耐用。与Triton X-100类似，PS 80的病毒灭活作用同时依赖于处理时间和温度。与Triton X-100不同，PS 80的失活与浓度没有简单的相关性。此外，我们已经证明PS20表现出与PS 80相似的病毒灭活活性。基于目前工作中描述的发现，我们认为PS 80可能是Triton X-100的可行替代品，可用于广谱生物药物的生产工艺，以实现理想的病毒清除。最后，在GMP生产的背景下，使用PS 80的病毒灭活的优点和缺点进行了讨论。
Triton X-100 has long been used either alone or in combination with solvent to inactivate enveloped viruses in biopharmaceutical manufacturing. However, European Chemicals Agency (ECHA) officially placed Triton X-100 on the Annex XIV authorization list in 2017 because 4-(1,1,3,3-tetramethylbutyl) phenol, a degradation product of Triton X-100, is of harmful endocrine disrupting activities. As a result, any use of Triton X-100 in the European Economic Area would require an ECHA issued authorization after the sunset date of January 4, 2021. In search of possible replacements for Triton X-100, we discovered that polysorbate 80 (PS80) in absence of any solvents was able to effectively inactive enveloped viruses such as xenotropic murine leukemia virus and pseudorabies virus with comparable efficacy as measured by log reduction factors. Interestingly, PS80 did not show any virucidal activities in phosphate buffered saline (PBS) while achieving robust virus inactivation in cell-free Chinese hamster ovary (CHO) bioreactor harvests. This intriguing observation led us to speculate that virus inactivation by PS80 involved components in the cell-free CHO bioreactor harvests that were absent in PBS. Specifically, we hypothesized that esterase and/or lipases in the cell-free bioreactor harvests hydrolyzed PS80 to yield oleic acid, a known potent virucidal agent, which in turn inactivated viruses. This theory was confirmed using purified recombinant lysosomal phospholipase A2 isomer (rLPLA2) in PBS. Subsequent characterization work has indicated that virus inactivation by PS80 is effective and robust within temperature and concentration ranges comparable to those of Triton X-100. Similar to Triton X-100, virus inactivation by PS80 is dually dependent on treatment time and temperature. Unlike Triton X-100, PS80 inactivation does not correlate with concentrations in a simple manner. Additionally, we have demonstrated that PS20 exhibits similar virus inactivation activities as PS80. Based on the findings described in the current work, we believe that PS80 is potentially a viable replacement for Triton X-100 and can be used in manufacturing processes for wide spectrum of biopharmaceuticals to achieve desirable virus clearance. Finally, the advantages and disadvantages of using PS80 for virus inactivation are discussed in the contexts of GMP manufacturing.