Impact of clarification strategy on chromatographic separations: Pre‐processing of cell homogenates

Abstract

This research focused on how the extent and type of primary solid-liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original "soluble" state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration.

Evaluation of the impact of lipid fouling during the chromatographic purification of virus‐like particles from Saccharomyces cerevisiae

“…Alternatively and of potentially greater utility could be the introduction of feed pre-treatment steps to reduce the lipid level prior to chromatographic purification. Possible options include reduced pressure homogenization followed by an additional centrifugation step to remove cytosolic materials, 28 selective precipitation with ammonium sulphate, 5 the use of an adsorptive step such as a depth filter, e.g. Zeta Plus Delipid filters (3M, Bracknell, UK), or a particulate sorbent, e.g.…”

“…13 Conversely, Bracewell et al reported that lipid fouling can have a major impact on the performance of chromatography. 5 Owing to the hydrophobic nature of lipids, which are co-released with HBsAg VLP in the upstream process, this subsequent HIC step will thus be particularly susceptible to lipid fouling. In the extreme case, it can be envisaged that lipid fouling might be so acute as to render a whole process economically unfeasible.…”

“…This can be particularly problematic in terms of the deleterious effect on the performance of the downstream processing steps that follow, such as chromatography. 5 As with all biopharmaceuticals, chromatography is the core operation about which the downstream processing of VLPs is centred due to the unparalleled resolution it is capable of achieving. 6 Nevertheless, it is also prone to fouling by components in process feed streams under repeated or prolonged exposure, 7 and although standard clean-in-place (CIP) conditions will help to restore most column types toward their original states, the fouling caused by certain types of material such as lipids is irreversible to some extent.…”

A monolith purification process for virus-like particles from yeast homogenate

“…Using a homogenised and clarified crude S. cerevisiae feed, containing the HBsAg VLP, Jin et al [24] saw fouling effects from the lipid released during the detergent VLP liberation step during purification. Pretreatment of the feed before loading on to the column can greatly reduce the effect of fouling [26,29] which can be visualised using confocal microscopy [24,30] to confirm the position and degree of fouling.…”

Yeast cell disruption strategies for recovery of intracellular bio-active compounds — A review

“…Attempts to maximize product recovery through increases in the intensity of cell disruption usually result in micronization of cell debris and a reduction in flux during membrane filtration, which is caused by the release of bio-active components and the co-release of contaminants. In addition, the contaminant suspensions also reduce the ease of sanitization and the lifetime of column during chromatography separation performance (Lyddiatt, 2002;Bracewell, 2008). Thus, cell disruption should be optimized by considering the process economics of all unit operations in the upstream and downstream processing.…”