Optimisation and intensification of the production of recombinant scFv fragments to the \(escherichia\) \(coli\) periplasm: an insight into host physiology and cell surface characterisitics

Kasli, Ikhlaas Makbul (2018). Optimisation and intensification of the production of recombinant scFv fragments to the \(escherichia\) \(coli\) periplasm: an insight into host physiology and cell surface characterisitics. University of Birmingham. Ph.D.

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Abstract

The single chain variable fragment (scFv) is an emerging class of immunoglobulin G (IgG) fragment for potential biopharmaceutical application. The scFv is more suitable than the IgG for production in bacterial hosts due to its small size and requirement for less post-translational modification. Shorter doubling times, relatively simple genetic tractability, cheaper media and the ability to form disulphide bonds in the periplasm make Escherichia coli a suitable host for recombinant production of scFvs.

This study optimised production of the model scFv, 13R4, to the E. coli periplasm via the co-translational SRP pathway and post-translational SecB pathway using a Design of Experiments approach with the temperature, concentration of inducer and OD\(_6\)\(_0\)\(_0\) for induction as factors. The latter system was intensified and an optimal concentration of inducer per OD\(_6\)\(_0\)\(_0\) was determined. The optimal growth conditions, and variations on these conditions, were scaled-up in four fed-batch fermentations where an OD\(_6\)\(_0\)\(_0\) of 119 and yield of 3.517 μg of active periplasmic 13R4 per millilitre of culture were achieved.

Finally, difficulties in measuring the cellular adhesion and Young's modulus during recombinant protein production using atomic force microscopy led to identification and quantification of differences in surface charge and hydrophobicity of transformed and untransformed bacteria.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):