Skip to Content
Merck
  • Transfer of engineered biophysical properties between different antibody formats and expression systems.

Transfer of engineered biophysical properties between different antibody formats and expression systems.

Protein engineering, design & selection : PEDS (2012-07-06)
Jonas V Schaefer, Andreas Plückthun
ABSTRACT

Recombinant antibodies and their derivatives are receiving ever increasing attention for many applications. Nevertheless, they differ widely in biophysical properties, from stable monomers to metastable aggregation-prone mixtures of oligomers. Previous work from our laboratory presented the combination of structure-based analysis with family consensus alignments as being able to improve the properties of immunoglobulin variable domains. We had identified a series of mutations in the variable domains that greatly influenced both the stability and the expression level of single-chain Fv (scFv) fragments produced in the periplasm of Escherichia coli. We now investigated whether these effects are transferable to Fab fragments and immunoglobulin G (IgG) produced in bacteria, Pichia pastoris, and mammalian cells. Taken together, our data indicate that engineered mutations can increase functional expression levels only for periplasmic expression in prokaryotes. In contrast, stability against thermal and denaturant-induced unfolding is improved by the same mutations in all formats tested, including scFv, Fab and IgG, independent of the expression system. The mutations in V(H) also influenced the structural homogeneity of full-length IgG, and the reducibility of the distant C(H)1-C(L) inter-chain disulfide bond. These results confirm the potential of structure-based protein engineering in the context of full-length IgGs and the transferability of stability improvements discovered with smaller antibody fragments.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Human Lambda Light Chains (Bound and Free)−Alkaline Phosphatase antibody produced in goat, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-c-Myc antibody, Mouse monoclonal, clone 9E10, purified from hybridoma cell culture