An automated microscale chromatographic purification of virus‐like particles as a strategy for process development

Abstract

The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.

Micropipette Tip-Based Immunoassay with Electrochemical Detection of Antitissue Transglutaminase to Diagnose Celiac Disease Using Staples and a Paper-Based Platform

“…They have also been used for facilitating sample preparation (packed with commercial resins , ) and for performing electrochemical measurements as containers of electrodes and electrolytes , or just as a part of the electrode body (commonly working or reference electrodes). The tip can also be used as a miniaturized column that can be filled with commercial resins for purification purposes . The ZipTip is a special micropipette tip with a bed of chromatography media (C18, C4, or strong cationic) fixed at its end, intended for purifying and concentrating protein, peptide, or oligonucleotide samples by solid-phase extraction prior to analysis (usually mass spectrometry).…”

“…The tip can also be used as a miniaturized column that can be filled with commercial resins for purification purposes. 24 The ZipTip is a special micropipette tip 25 with a bed of chromatography media (C18, C4, or strong cationic) fixed at its end, intended for purifying and concentrating protein, peptide, or oligonucleotide samples by solid-phase extraction prior to analysis (usually mass spectrometry). Specific purifications can be made by modifying the micropipette tip with molecularly imprinted monoliths as in the case of selective extraction of berberine in plasma and urine samples.…”

Scale‐down characterization of post‐centrifuge flocculation processes for high‐throughput process development

“…Such technologies are required to generate the process understanding early in process development so that time to market, risk of failure and associated costs are reduced (Titchener-Hooker et al, 2008 ). The USD approach has been successfully established for centrifugation (Tait et al, 2009 ), normal flow filtration (Jackson et al, 2006 ; Kong et al, 2010 ) and chromatography (Wenger et al, 2007 ; Wiendahl et al, 2008 ), but it is yet to be proved for other unit operations such as flocculation.…”

Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay

“…Therefore, the applicability of AMP clay to enhance cell disruption was investigated to assess its efficiency on cell lysis. The demonstration system involves the recombinant capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) which was expressed in P. pastoris GS115 and spontaneously assembled into icosahedral virus-like particles (VLPs) (Wenger et al 2007; Wu et al 2011). …”