Livanos, Maria; (2019) Production of DARPins in the Yeast Pichia pastoris. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text thesis_corrections_merged.pdf - Accepted Version Download (8MB) | Preview

Abstract

Designed Ankyrin Repeat Proteins (DARPins) are industrially relevant small nonimmunoglobulin scaffold proteins that bind their target with high affinity. G3 is a DARPin designed to target the receptor tyrosine kinase protein HER2 (human epidermal growth factor receptor 2). Overexpression of HER2 is prevalent in many cancer types and development of targeting agents for imaging during the course of treatment is an unmet need. The research aims to develop a bioprocess for the production of clinical grade DARPin from Pichia pastoris. In the pilot stage, the effects of the common hexahistidine (His6) purification tag on biodistribution and tumour targeting were assessed in vivo. Three constructs were created; the (His) 6-, the histidine-glutamate (HE) 3- and the regulatory standard, untagged G3 DARPin. The final products were characterised in terms of purity, identity and functionality prior to testing in mice. The (HE) 3-tagged G3 was the superior product in terms of non-target specific uptake and was taken forward as a clinically relevant example for bioprocess development. A regulatory compliant bioprocess for G3 was established in the yeast P. pastoris. The fermentation procedure was transferred to a 20-L bioreactor and a 5 hour induction run implemented. Tangential flow filtration (TFF), immobilised metal affinity (IMAC), size exclusion (SEC) and anion exchange (AEX) chromatography steps were utilised to generate ~60 mg of fully functional homogenous G3. The level of host cell protein (HCP) and endotoxin was within regulatory compliance at <1 ng/mg and <0.05 EU/mg, respectively. The process was then adapted for production of an imaging agent and enabled the addition of maleimide- DOTA via the free cysteine on the C-terminal of the DARPin. This yielded 38-42 mg / run of high purity DOTA_G3 which radiolabelled with 111In at greater than 95% efficiency. Sites of proteolytic cleavage were identified and protein engineering carried out to mitigate breakdown. Mutation of the amino acid methionine (30) to leucine resulted in notable inhibition of proteolytic activity and allowed for increased fermentation times, improving titre by ~75%. The mutation did not alter DARPin binding affinity nor baseline expression. The bioprocess and identified mutation were tested on an independent DARPin (E69) to assess the potential of a ‘universal’ DARPin production platform. As with the G3 the mutated E69 DARPin proved to be far more resistant to proteolytic activity than its wild type counterpart and the bioprocess generated 40 mg of fully functional E69 of equivalent purity at <0.05 EU/mg endotoxin and <3.4 ng/mg HCP. In summary, this study has generated a holistic and innovative end to end development strategy for the production of DARPins in the yeast P. pastoris.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item