No abstract

No 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.

Bacterial endotoxin is a Gram-negative bacterial cell wall component that is harmful to humans at threshold concentrations, and it is not expected to be in aseptically-produced pharmaceutical medicines. It has been suggested that endotoxin cannot be detected over time in certain biopharmaceutical drug product formulations containing citrate, phosphate, and polysorbate components via an unknown masking mechanism. We have generated and present data here that indicate that endotoxin can be recovered in a variety of matrices, and under various experimental conditions.

No abstract