Sotiriadis, Alexandros; (2000) Factors affecting the production of a single-chain antibody fragment (SCFV) by Aspergillus awamori in stirred tank reactors. Doctoral thesis (Ph.D), UCL (University College London).

Text Factors_affecting_the_producti.pdf Download (10MB)

Abstract

Recombinant antibodies have in recent years proved their potential to be an important class of therapeutics. Potential applications demand large doses for administration and therefore production of antibody fragments in microbial or fungal systems has become important. In this study, a recombinant strain of Aspergillus awamori, producing anti- lysozyme scFv under the control of the xylanase promoter, was used. A fermentation strategy was created for the production of the antibody fragments by a fed-batch process, based on the investigation of parameters which affected production, namely growth medium composition, induction regime and protease production. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 hours after inoculation) improving the titre of the product from 14.5 mg L-1, obtained in the early exponential phase (7 hours after inoculation) to 16.2 mg L-1. A 100% increase of the carbon (fructose) and nitrogen (ammonium sulphate) sources in the growth medium resulted in an increase in product concentration from 16.2 mg L-1 to 108.9 mg L-1 and an increase in maximum dry cell weight from 7.5 g L-1 to 11.5 g L-1. A 50% reduction in the concentration of the inducer resulted in an increase in the specific product yield from 10 mg g-1 dry cell weight to 12 mg g-1 Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L-1 trypsin but storage experiments showed that they had no detrimental effect on the concentration of the antibody fragment. The process was scaled-up to 75 L where maximum scFv concentration reached 160 mg L-1 after 42 hours of fermentation. Specific yield and specific productivity were increased by 100% and 50% respectively, while volumetric productivity decreased by 500%. Pellet dimensions were the same. A comparison between production of the same scFv antibody fragment in A. awamori and E. coli was carried out.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item