Kinna, AW;
(2017)
Improved production and purification of recombinant proteins from mammalian expression systems.
Doctoral thesis , UCL (University College London).
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Abstract
The biopharmaceutical industry is becoming increasingly reliant on recombinant proteins as therapeutic agents. The work presented here details the development of rapid mammalian expression systems and novel capture methods for use in early recombinant protein development. A key aim was to investigate expression of recombinant proteins via cost effective production methods and to compare the resultant products at small scales of manufacture. A model single chain variable fragment Fc conjugate (scFv-Fc) targeted against a clinically relevant glycoprotein, the carcinoembryonic antigen (CEA), was expressed and characterised using both transient and stable-based expression of transgenic DNA. Transient gene expression in suspension HEK293 cells produced a maximum scFv-Fc level of 72μg/mL, which was used for initial protein characterisation. A stable pool of transfected CHO cells was also generated using a ubiquitous chromatin opening element (UCOE)-based vector achieving increased protein expression and culture periods when combined with a fed-batch regimen. Characterisation of the resulting proteins showed that stability and effector function was maintained across transient and stable production methods at all scales, indicating that preliminary data generated from transient expression in HEK293F cells could be generalised to predict that of protein stably expressed in CHO cell populations. A significant bottleneck in harvesting and purifying proteins from cell containing feed streams is the requirement for solid-liquid separation prior to capture. Therefore, a technique was proposed for direct capture of recombinant protein from unclarified feed streams that can integrate directly into the bioreactor harvest line. The process was demonstrated using immobilised metal affinity chromatography (IMAC) capture of recombinant CEA with a polyhistidine (His6) tag from a bioreactor culture. This provides a basis for direct primary capture of recombinant proteins from unclarified mammalian cell feed streams that could be generalized to other capture methods and proteins.
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