ABSTRACT
Under stressed conditions such as prolonged exposure to high pH, the C-terminal disulfide bridge in bovine somatotropin (bST) is susceptible to a base catalyzed beta-elimination reaction. This reaction converts the disulfide bond to a dehydroalanine residue with loss of a sulphur atom. Two altered species were isolated in pure form and determined to be generated from this dehydroalanine intermediate. One is a monomeric lanthionyl bST (L-bST) with a thioether linkage, and the other is an inter-molecular disulfide linked dimer containing a lysinoalanine. These two novel structures were unambiguously determined using various techniques including enzymatic digestion, amino acid sequencing and analysis, and mass spectrometry. The monomeric L-bST was demonstrated to be equipotent to normal bST in a hypox rat assay, thus showing that formation of lanthionine in place of this disulfide bond does not affect it bioactivity.
Subject(s)
Alanine/analogs & derivatives , Growth Hormone/chemistry , Alanine/chemistry , Amino Acid Sequence , Animals , Cattle , Chromatography, Ion Exchange , Growth Hormone/isolation & purification , Hydrogen-Ion Concentration , Lysinoalanine/chemistry , Molecular Sequence Data , Peptide Mapping , Protein Conformation , Protein Multimerization , Sequence Analysis, Protein , Sulfides/chemistry , Tandem Mass SpectrometryABSTRACT
Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.
