Expression, purification and characterization of B72.3 Fv fragments

Abstract

The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.

Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli

“…Due to the short doubling time, stability and the possibility to grow in inexpensive minimal media, this bacterial expression system becomes an affordable way to produce large amounts of recombinant proteins, in case that post-translational modifications of the target protein can be neglected (Miller et al, 2005;King et al, 1993). Using high cell density cultivation (HCDC), various pharmaceutical antibody fragments have been already produced (Premsukh et al, 2011;Casey et al, 1995).…”

A secretory system for bacterial production of high‐profile protein targets

“…Periplasmic signal peptides are widely used in phage display and scFv antibody production exploiting the favorable oxidizing environment in the periplasm of E. coli for folding of the antibody fusion proteins 17. However, their toxicity to the cell and accumulation in the periplasm may lead to a leaky phenotype with increased outer membrane permeability,18 in some cases providing the opportunity to retrieve recombinant antibodies from the culture medium 19, 20. The use of a signal peptide for a type I secretion pathway such as the hemolysin system was shown to circumvent such problems associated with periplasmic over‐expression 21, 22…”

Evaluation of the effects and interactions of mixing and oxygen transfer on the production of Fab’ antibody fragments in Escherichia coli fermentation with gas blending

“…Fed-batch fermentations are preferable to batch operation for the production of antibody fragments since batch methods usually result in low biomass concentrations with reported titres from 40 mg/L [7] to 450 mg/L [25]. On the other hand, fed-batch fermentations using a highly defined medium can result in levels up to 50 g/L of dry cell weight (DCW) [18] and titres up to 1-2 g/L [10].…”