Identification and characterization of a Triton X-100 replacement for virus inactivation.

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.

Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry.

Osteopontin (OPN) is a highly modified protein that is found in many tissues and has been associated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor of hydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well as others, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) to perform a comprehensive analysis of the post-translational modification of OPN purified from rat bone. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) MS showed masses of 37.6 and 36.8 kDa before and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately 10.4 phosphate groups. Using proteolytic digestion and tandem MS, we localized 29 sites of phosphorylation: S10, S11, S46, S47, T50, S60, S62, S65, S146, T154, S160, S164, S167, S193, S196, S203, S220, S223, S232, S241, S245, S257, S262, S267, S278, S290, S295, S296, and S297. In addition, Y150 was shown to be sulfated and T107, T110, T116, and T121 are O-glycosylated. No glycan was detected at the potential N-glycosylation site. Other modifications, including deamidation, oxidation, and carbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzed in detail, even after sialidase treatment, presumably because of the presence of a large number of acidic residues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated and has an additional site of glycosylation, many different sites of phosphorylation, and a lower overall phosphate content.