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Evaluation of Primary Capture Procedures for Recombinant Antibodies Manufactured in Pichia pastoris: Method, Viability and Economics

Bhavsar, Gaurav; (2023) Evaluation of Primary Capture Procedures for Recombinant Antibodies Manufactured in Pichia pastoris: Method, Viability and Economics. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The eukaryotic yeast Pichia pastoris is an increasingly applied platform for heterologous protein production for therapeutic and diagnostic purposes. Recent advances in high production titre have increased pressure on downstream processes to reduce the overall cost of production. The work in this thesis investigates a means of reducing the cost of P. pastoris production by combining three steps (centrifugation or filtration, concentration and affinity purification) in one step and comparing the primary capture of recombinant antibodies between radial and expanded bed adsorption chromatography systems, using Immobilized Metal Affinity Chromatography (IMAC) as a primary capture step. A hexa-histidine (His6) tagged single-chain antibody shMFELL2cys was used for the study. The protein is secreted from P. pastoris strain X-33 and has an affinity towards the carcinoembryonic antigen (CEA), an onco-foetal tumour antigen overexpressed in cancer cells used for therapeutic as well as diagnostic purposes. A high cell density culture of P. pastoris, which had been induced to express shMFELL2cys was directly applied to (i) Cellthru™ resin in a radial bed and (ii) STREAMLINE™ chelating resin in expanded bed adsorption chromatography. Copper-mediated IMAC capture of the tagged protein occurred while cells and cell debris were allowed to flow through the matrix bed. To conduct this study, first, the binding conditions were optimised at a small scale using 1 mL IDA chelating Cellthru™ and STREAMLINE™ chelating resins used in RBA and EBA chromatography columns, respectively. Second, a comparison of the static and dynamic binding capacity of the RBA and EBA resins was performed at a small scale using the purified His6 tagged scFv antibody. Third, an investigation of the effect of the residence time on the recovery of the His6 tagged scFv antibody using the RBA column was performed using a fermentation culture fluid. The His6 tagged scFv antibody shMFELL2cys was successfully expressed in the high cell density P. pastoris fermentation at a concentration of 680 to 600 mg/L, and captured using the RBA and EBA chromatography processes, respectively. The calculated step recovery for RBA and EBA chromatography was 30% and 47%, respectively. A total amount of 801 mg and 611 mg of the scFv antibody shMFELL2cys was purified from the EBA and RBA chromatography processes, respectively. The scFv antibody shMFELL2cys purified using RBA and EBA chromatography has specificity for carcinoembryonic antigens (CEA). A single monomer peak of the purified scFv antibody was seen on the analytical SEC column and a scFv antibody band at ~ 27 kDa was present on the SDS-PAGE for the scFv which was purified using RBA and EBA chromatography. The HCP concentration in the final purified scFv antibody using EBA chromatography was 161.8 ng/mL, equivalent to 39 ng/mg of scFv antibody. The HCP concentration in the final purified scFv antibody using RBA chromatography was 83 ng/mL, equivalent to 22.3 ng/mg of scFv antibody. The high HCP concentration in the EBA eluted protein in comparison to the RBA eluted protein was also calculated. Cost of goods analysis of the RBA and EBA chromatography processes were performed from 8L lab scale to 2000L large production scale using Excel-based BioSolve software®. At 2000L production scale, factors affecting the CoG/g and CoG/batch of the His6 tagged scFv antibody were analysed using DoE software for the RBA and EBA chromatography processes. BioSolve software® cost comparisons between the capture steps revealed a significant decrease in the total CoG/g of the product manufactured at 2000L scale. At best-case scenario of 6 g/L fermentation yield, 40 mg/mL resin binding capacity, 60 resin CIP cycles and 60% step recovery, calculated CoG/g were £54 and £197, respectively for the RBA and EBA processes. In the worst-case scenario of 1 g/L fermentation yield, 20 mg/mL resin binding capacity, 20 resin CIP cycles and 20% step recovery, calculated CoG/g were £556 and £1400, respectively for the RBA and EBA processes. Primary chromatography process RBA out performed the EBA chromatography process, at both best-case and worst-case scenario levels. Therefore, preference is given to introducing RBA chromatography as a primary capture step to purify the His6 tagged scFv antibody from a high cell density P.pastoris fermentation run, following the evaluation of quality, quantity and cost of goods to manufacture the antibody.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Evaluation of Primary Capture Procedures for Recombinant Antibodies Manufactured in Pichia pastoris: Method, Viability and Economics
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10167810
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