Quantifying variants of a proteinaceous product is a challenging problem in bioprocess engineering. However, such variants as glycoforms can substantially affect the behaviour of the drug in the body, and therefore represents valuable product information. Here, the phosphorylated and glycosylated variants of ovalbumin that are found in typical commercial samples (e.g., from Sigma) are completely characterised by analytical chromatography and subsequent deconvolution. Several typical analytical columns were tried, and a monolithic ion exchanger was found to provide better resolution than the other resins in substantially shorter times. Results from this monolithic column were then deconvolved by hand, so that each physical component within the feed mixture was assigned a single peak. The results are therefore quite different from, and appreciably more robust than, those produced by a typical computational deconvolution package. It was found that this deconvolution allowed ready analysis of fractions from preparative chromatographic runs of the commercial ovalbumin sample on a standard commercial resin (Capto Q). It is suggested that this kind of hand-tuned deconvolution offers significant promise for the detailed analysis of complex product variants that have very similar chromatographic retention behaviour.

Chromatographic quantification of a complex protein mixture using problem-specific deconvolution

251st National Meeting (2016)

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