Engineering aggregation resistance in IgG by two independent mechanisms: lessons from comparison of Pichia pastoris and mammalian cell expression.
Aggregation is an important concern for therapeutic antibodies, since it can lead to reduced bioactivity and increase the risk of immunogenicity. In our analysis of immunoglobulin G (IgG) molecules of identical amino acid sequence but produced either in mammalian cells (HEK293) or in the yeast Pichia pastoris (PP), dramatic differences in their aggregation susceptibilities were encountered. The antibodies produced in Pichia were much more resistant to aggregation under many conditions, a phenomenon found to be mainly caused by two factors. First, the mannose-rich glycan of the IgG from Pichia, while slightly thermally destabilizing the IgG, strongly inhibited its aggregation susceptibility, compared to the complex mammalian glycan. Second, on the Pichia-produced IgGs, amino acids belonging to the α-factor pre-pro sequence were left at the N-termini of both chains. These additional residues proved to considerably increase the temperature of the onset of aggregation and reduced the aggregate formation after extended incubation at elevated temperatures. The attachment of these residues to IgGs produced in cell culture confirmed their beneficial effect on the aggregation resistance. Secretion of IgGs with native N-termini in the yeast system became possible after systematic engineering of the precursor proteins and the processing site. Taken together, the present results will be useful for the successful production of full-length IgGs in Pichia, give indications on how to engineer aggregation-resistant IgGs and shed new light on potential biophysical effects of tag sequences in general.
Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Jonas V
JV
Schaefer
Andreas
A
Plückthun
D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia; D015202:Protein Engineering; D055550:Protein Stability
D007074:Immunoglobulin G