Use of high-throughput tools to optimise polishing-chromatography sequences for complex feed mixtures

• Main Author: Jurlewicz, Kosma Michal
• Published: University College London (University of London) 2018
• Subjects: 660.6
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.763285

Polishing chromatography is a critical element of a bioprocess, because it is currently the only scalable separation technique that can remove process-related impurities, thereby achieving the high purity required of a biotherapeutic. Optimising the polishing chromatography of complex feeds has not been systematically addressed in the literature. This thesis identified a novel, academically affordable ternary protein mixture and systematically developed an optimal two-column polishing train for it. The ternary protein feed mixture was selected using many criteria, but had no special feature to aid identification, such as a chromophore, making it more difficult to characterise. The resulting analytical chromatogram could not be fully resolved, which is typical of industrially relevant products, such as glycoproteins. The selected HPLC column produced fast separations, resulting in a comparatively rapid quantification of preparative chromatograms. Many chromatographic resins and operating conditions were screened, resulting in the non-obvious sequence a hydrophobic interaction (HIC) followed by an anion-exchange (AX) adsorbent. Systematic experimental studies optimised the sequence with respect to yield, purity and amount recovered. Although the loading exceeded the binding capacity of the HIC column, runs at extremely high loadings (60 - 150 g/L) gave very efficient separation in an unusual combination of flow-through and bind-and-elute modes. It was found to achieve >200 mg of acceptably pure product from a single run. A variety of problems were encountered during the development of this polishing train, to which solutions were developed. While these problems are not uncommon, the literature does not contain systematic solutions to them. Examples include decisions about sequence design, protein solubility issues, and the detailed characterisation of samples from preparative runs (not achieved by analytical HPLC). In particular, a system-specific deconvolution methodology was developed that allowed complete characterisation of the mixture; the approach is likely to be widely applicable to industrially relevant biological feed mixtures.