PEG enhances viral clearance on ceramic hydroxyapatite

Abstract

PEG enhances viral clearance on ceramic hydroxyapatiteViral clearance across ceramic hydroxyapatite (CHT TM ) was examined in two elution systems: sodium chloride and sodium chloride plus poly(ethylene glycol) (PEG). In both cases clearance of xenotropic murine leukemia virus was significant (3-4 log) while that of minute virus of mice varied between 1.7 and 2.7 log; in addition, the addition of PEG to the elution buffer enhanced viral clearance. The data are in agreement with the previous results and demonstrate that additional clearance can be obtained by adding PEG to a ceramic hydroxyapatite buffer system. IntroductionViral clearance via hydroxyapatite chromatography has been studied for at least 45 years [1][2][3][4]. The mechanism of binding can be due to either synergistic effects of multi-site binding [5][6][7], strong interactions of clustered surface phosphates (in the case of lipid-enveloped viruses) with calcium [8,9] or both. The original eluant used almost exclusively for hydroxyapatite was phosphate. However, sodium chloride as an eluant has also been used for some 50 years [10], with improvements noted for antibody purity [11,12] and aggregate removal [9,13]. In more recent years, a variety of other additives, such as poly(ethylene glycol) (PEG) [14], have been employed as elution modifiers. PEG has been shown to improve aggregate separation by differentially enhancing retention of larger solutes [14]. This implies that retention of viral particles should also be enhanced. The size of minute virus of mice (MVM) particles is approximately 18-26 nm [15] while that of xenotropic murine leukemia virus (x-MuLV) is 80-110 nm.A study performed several years ago [16][17][18] demonstrated 43 and 2 log clearance of x-MuLV and MVM, respectively, in a sodium chloride gradient. This study expands on these data and confirms that PEG provides additional clearance of x-MuLV and MVM at an antibodyloading level consistent with current manufacturing processes. Materials and methods Antibody and viral solutionsA monoclonal antibody, purified over a protein A column, was generously supplied by Avid Bioservices (Tustin, CA, USA). The eluate was neutralized to pH 7.0 and 0.5 M sodium phosphate, pH 7.0, was added to a final concentration of 10 mM. Antibody was loaded onto ceramic hydroxyapatite (CHT) columns at $10 mg protein/mL bed volume.Stock solutions of either MVM or x-MuLV (Charles River Laboratories, Malvern, PA, USA) were used for this study. CHTCHT TM , type I, 40 m, was supplied by Bio-Rad Laboratories (Hercules, CA, USA). The CHT was packed in 11.3 Â 100 mm columns (Atoll GmbH, Weingarten, Germany). For all steps, the linear flow rate was 300 cm/h. All runs were performed at room temperature. Chromatography buffersThe following buffers were employed for this study. Each buffer was tested for cytotoxicity and interference in the infectivity assay systems used to quantitate each virus.(A) 10 mM sodium phosphate, pH 7.0 (B) 10 mM sodium phosphate, 10% PEG-1000 (SigmaAldrich, St. Louis, MO, USA), pH 7.0 (C) 10 m...

A tribute to Dr. Serge N. Timasheff, our mentor

“…This established the foundation for a method now known as Steric Exclusion Chromatography, which uses PEG to promote binding on hydroxylated chromatography surfaces [Lee et al 2012;Gagnon et al 2014]. The solute size dependency effects noted by Arakawa and Timasheff meanwhile inspired inclusion of PEG in ion exchange and hydroxyapatite buffers to promote separation of aggregates from non-aggregated antibodies [Milby et al 1989;Gagnon et al 1996;Yoshimoto et al 2015;Kluters et al 2015aKluters et al , 2015bKluters et al , 2015cZhou et al 2011;Gagnon 2008;Snyder et al 2009]. Hydroxyapatite, of course, was also a subject of interest for Professor Timasheff, who contributed to Marina Gorbunoff's still-classic publications on its retention mechanisms [Gorbunoff 1984a[Gorbunoff , 1984bGorbunoff and Timasheff 1984].…”

Protein–solvent interaction

“…A decreasing PEG concentration is used to dissociate and elute the bound macromolecules. As PEG is more effective for aggregates, it should enhance the binding of larger solutes more effectively (Snyder et al 2009). Thus, SXC has been successfully used to purify viruses and immunoglobulin M, which is a pentamer antibody (Lee et al 2012).…”

Next generation vaccines and vectors: Designing downstream processes for recombinant protein‐based virus‐like particles

“…A novel chromatography technique incorporating the selectivity of PEG‐induced precipitation was developed by the Gagnon group [92–95]. This technique is referred to as steric exclusion chromatography.…”