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.
Background: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K + current ( I KACh ), called I KH ; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. Methods: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. Results: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating I KACh by a mechanism involving frequency-dependent activation of PKC ε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA–treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of I KACh . Attenuated membrane translocation of PKC ε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Conclusions: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.
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