Insights into virus inactivation by polysorbate 80 in the absence of solvent

Abstract

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.

Supported Lipid Bilayer Platform for Characterizing the Membrane-Disruptive Behaviors of Triton X-100 and Potential Detergent Replacements

“…This issue has led biopharmaceutical manufacturers to search for more environmentally friendly detergents that can replace TX-100 while displaying functionally equivalent virus inactivation properties [ 9 , 10 , 11 ]. Accordingly, to test detergent candidates, the major focus has been on treating selected membrane-enveloped viruses with different detergent concentrations under varying environmental conditions and exposure times and measuring the resulting effects on viral infectivity.…”

In vitro activity evaluation ofLippia albaessential oil against Zika virus

“…No assays above 668 μg/mL were performed because the high concentrations of the Tween 80 and ethanol mixture, which were shown to generate a total cytotoxicity in Vero 76. Moreover, essential forming protein-lipid complexes, avoiding the entry of the viral genome inside the host by adsorption (28,29). Additionally, high temperatures generate faster virus inactivation due to accelerated hydrolysis of PS80 and subsequent interactions between oleic acid and the viral envelope (29).…”

“…Moreover, essential forming protein-lipid complexes, avoiding the entry of the viral genome inside the host by adsorption (28,29). Additionally, high temperatures generate faster virus inactivation due to accelerated hydrolysis of PS80 and subsequent interactions between oleic acid and the viral envelope (29). With respect to the solvent, ethanol induces the activity of the enzyme 2",5" oligoadenylate synthetase whose principal role is to promote cellular resistance against viral infection (developing an antiviral state).…”

“…Tween 80 inactivates viruses, and has a low micellar concentration. Other emulsifiers are similar but more toxic, like Triton X-100 (29).…”

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

“…In an attempt to identify the components leading to oleic acid production from PS-80 for virus inactivation, Chen et al. evaluated CHO cell lines that had one to four lipase/esterase genes knocked out through genetic disruption [ 57 ]. It was observed that PS-80 was still able to inactivate virus, albeit to somewhat reduced efficiency, in the cell-free culture harvests from these genetically engineered CHO cell lines, including the quadruplet knockout CHO.…”