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Title: Addressing lipid impurities in Pichia pastoris cells producing virus like particles : genetic and bioprocess studies
Author: Bandyopadhyay, Sushobhan Kalyankumar
ISNI:       0000 0004 8507 7516
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Production of hepatitis B surface antigen (HBsAg) virus like particles (VLPs) occurs intracellularly in Pichia pastoris (P. pastoris). During extraction by cell disruption, host cell DNA and lipid impurities are released into the feed stream fouling the downstream process membranes and matrices. Effect of commercial triacylglycerol Candida rugosa (C. rugosa) lipase (CRL) on P. pastoris homogenate is investigated. P. pastoris homogenate was treated with CRL (0.0001 to 0.1 mg/mL) resulting in removal of triacylglycerol and increase in throughput by 60%, during constant flux filtration using 0.45μm polyethylene sulfone single layer disc membrane at 800 litres per square meter per hour. A strategy was employed where C. rugosa lipase 3 (CRL lip3) gene was localized in three different cellular locations (cytosol, endoplasmic reticulum, and cell surface) and encoded them under the control of PENO1 promoter alongside of the HBsAg gene under PAOX1. Developed strains were tested for expression of HBsAg along with optimisation of potentially toxic intracellular CRL lip3 expression. The CRL lip3 production did not influence HBsAg expression. A cell growth and gene induction regimes were identified with CRL lip3 localised to cell cytosol which enabled maximal HBsAg expression with independent control and CRL lip3 expression to peak only at the end of cultivation. Strains from the strategies which produced both, HBsAg as well as CRL lip3, were selected and further scaled-up to 0.25L bioreactor. The maximal sequential expression of HBsAg protein up to 20mg/L and CRL lip3 up to 150mg/L was confirmed. The formation of HBsAg VLP produced from co-expression culture was also confirmed using transmission electron microscopy. Downstream benefits of introducing lipases to VLP-containing P. pastoris homogenate and feasibility of cell engineering to produce CRL lip3 in a manner that does not otherwise compromise host cell performance was confirmed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available