Chromatofocusing

Lukačin, Richard; Deppert, Wolfgang R.

doi:10.1016/s0301-4770(08)60535-7

Cited by 1 publication

(2 citation statements)

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“…The protein commonly elutes at a pH value ±0.5 of the pI measured by IEF, which is referred to as the pI app (Sluyterman & Kooistra, 1989). Due to different contribution of topological residues to the net surface charge, the adsorption‐based retention by chromatofocusing can separate proteins that have the same pI but different surface charge distributions (Hearn et al, 1988; Huchens, 1998; Likacin & Deppert, 2000). The determination of the pI app by chromatofocusing may somewhat differ from the pI measured by electrophoretic mobility or IEF.…”

Section: Resultsmentioning

confidence: 99%

“…The specifically designed Mono P resin and polyampholytes added in the mobile phase allow the establishment of an internal linear pH gradient, which focuses the analyzed macromolecule close to its pI. The protein commonly elutes at a pH value ±0.5 of the pI measured by IEF, which is referred to as the pI app (Sluyterman & Kooistra, 1989 (Hearn et al, 1988;Huchens, 1998;Likacin & Deppert, 2000). The determination of the pI app by chromatofocusing may somewhat differ from the pI measured by electrophoretic mobility or IEF.…”

Section: Determination Of the Pi Of Viruses By Chromatofocusingmentioning

confidence: 99%

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Impact of the isoelectric point of model parvoviruses on viral retention in anion‐exchange chromatography

Leisi

Wolfisberg

Nowak

et al. 2020

Biotech & Bioengineering

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Anion-exchange chromatography (AEX) is used in the downstream purification of monoclonal antibodies to remove impurities and potential viral contamination based on electrostatic interactions. Although the isoelectric point (pI) of viruses is considered a key factor predicting the virus adsorption to the resin, the precise molecular mechanisms involved remain unclear. To address this question, we compared structurally homologous parvoviruses that only differ in their surface charge distribution. A single charged amino acid substitution on the capsid surface of minute virus of mice (MVM) provoked an increased apparent pI (pI app) 6.2 compared to wild-type MVM (pI app = 4.5), as determined by chromatofocusing. Despite their radically different pI app , both viruses displayed the same interaction profile in Mono Q AEX at different pH conditions. In contrast, the closely related canine parvovirus (pI app = 5.3) displayed a significantly different interaction at pH 5. The detailed structural analysis of the intricate three-dimensional structure of the capsids suggests that the charge distribution is critical, and more relevant than the pI, in controlling the interaction of a virus with the chromatographic resin. This study contributes to a better understanding of the molecular mechanisms governing virus clearance by AEX, which is crucial to enable robust process design and maximize safety. K E Y W O R D S anion-exchange chromatography, isoelectric point, parvovirus, virus clearance 1 | INTRODUCTION Recombinant proteins, such as therapeutic monoclonal antibodies (mAbs), are commonly produced in mammalian cell culture systems, which carry an inherent risk of being contaminated with viral agents. Some mammalian cell lines (e.g., Chinese hamster ovarian cells) are known to host endogenous retroviruses and to express retroviruslike particles (Dinowitz et al., 1992; Lieber, Benveniste, Livingston, & Todaro, 1973). In addition, mammalian cells can be infected by adventitious viruses, such as the minute virus of mice (MVM), which has, at very low frequency, repetitively been introduced into bioreactors through contaminated raw materials (Berting, Farcet, &

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Section: Resultsmentioning

confidence: 99%