Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Abstract

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfidebond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain.

David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments

“…Finally, due to the ability of E. coli to grow at high cell densities, antibody fragments are commonly produced in high cell density cultures grown in a stirred tank reactor using a fed-batch method [61].…”

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

“…In addition to strategies that directly improved Fab production, fundamental studies have greatly contributed to a broader understanding of recombinant protein production in E. coli . For example, some studies have investigated how the expression of selected challenging proteins influenced the host metabolism (cause–effect analyses) . Unfortunately, all these previous strategies have had limited transferability to other host/Fab/process combinations; consequently, we are far from creating a generic platform technology, despite the high degree of similarity within this class of proteins.…”

“…For example, some studies have investigated how the expression of selected challenging proteins influenced the host metabolism (cause-effect analyses). [24][25][26][27][28] Unfortunately, all these previous strategies have had limited transferability to other host/Fab/process combinations; consequently, we are far from creating a generic platform technology, despite the high degree of similarity within this class of proteins. The difficulties in developing general approaches for efficient Fab production are mainly due to our limited understanding of biological and environmental factors that act simultaneously and mutually superimpose their effects on the production of a gene of interest (GOI).…”

Microbials for the production of monoclonal antibodies and antibody fragments

“…Due to the intrinsic high growth rate of E. coli , high cell density cultures are currently used for the production of antibody fragments [48] . Production processes with E. coli are commonly conducted in stirred tank reactors (STR) as limited glucose fed-batch processes because glucose excess induces overflow metabolism and causes the production of the inhibiting metabolite acetate.…”