Abstract
AbstractBACKGROUNDThe yeast Pichia pastoris is a popular host organism for production of a range of biological products, several of which are intracellular. The disruption of yeast cells by homogenization also releases large quantities of lipids, which can foul the downstream membranes and chromatography matrices used for purification. This work examines lipid removal from yeast cells following homogenization by enzymatic degradation and its impact on the performance of the subsequent centrifugation and filtration.RESULTSLipase treatment of cell homogenate at 37 °C for 2 h, followed by clarification using a scaled‐down mimic of disc stack centrifugation, resulted in a 6.5‐fold improvement in solids removal when compared to untreated feed material. The lipase‐treated and untreated materials that had undergone initial centrifugation were then tested for filtration performance by passing the material through a 0.45 μm polyethylene sulfone membrane under constant flux. A 50% increase in throughput was observed in comparison to the untreated material.CONCLUSIONThese proof‐of‐concept data suggest enzymatic digestion of lipids, analogous to the widely performed DNA reduction using nucleases, could be a valuable process improvement strategy. © 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).
Highlights
Yeast expression systems are increasingly used to produce proteins of commercial importance, many of which are produced as an intracellular product
In this study we investigate whether an analogous approach of enzymatic digestion can be utilized to reduce membrane fouling and increase filtration efficiency
Lipid was extracted from the P. pastoris homogenate using the method described by Ejsing et al.[5] before and after treatment; we studied the reduction of TAG content using high-performance liquid chromatography (HPLC)-ELSD techniques, showing the impact on filter performance
Summary
Yeast expression systems are increasingly used to produce proteins of commercial importance, many of which are produced as an intracellular product. The first step in purification of these products is cell lysis, often by means of high-pressure homogenization. This releases intracellular product along with other cellular material and debris. The first step in the downstream processing of this lysate is the removal of this debris by clarification At scale this is usually accomplished by filtration. Large molecular components can cause membrane fouling and limit the efficiency of this process The disruption of yeast cells by homogenization releases large quantities of lipids, which can foul the downstream membranes and chromatography matrices used for purification. This work examines lipid removal from yeast cells following homogenization by enzymatic degradation and its impact on the performance of the subsequent centrifugation and filtration
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