Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody improves homogeneity, stability, and biological activity.
The antibody MAB007 was recently shown to be cysteinylated on an unpaired cysteine residue in the CDR3 variable region. Cysteinylation at this position was not complete and resulted in heterogeneous lots of MAB007 with respect to this posttranslational modification. In this report, a mild redox step was used that effectively removed cysteinylation while keeping native inter and intra-molecular disulfide bonds intact. Biophysical methods were employed to determine what consequences cysteinylation of the variable region had by directly comparing cysteinylated and de-cysteinylated MAB007 antibodies. No differences were detected in secondary structure; however, several pieces of evidence indicated that cysteinylation may result in tertiary or quaternary structural perturbations. These included differences in the cation-exchange chromatography and fluorescence-emission spectra of the cysteinylated and de-cysteinylated antibodies as well as differences in the solvent accessibility of the unpaired cysteine residue determined by labeling experiments. Such structural changes induced by cysteinylation were shown to increase the rate of MAB007 aggregation and to decrease the melting temperature of the Fab region by as much as 6 degrees C. The bioactivity of MAB007 was also shown to be adversely affected by cysteinylation and a direct correlation was made between the percent cysteinylation and biological activity.
Banks
Douglas D
DD
Department of Pharmaceutics, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119, USA. dbank@amgen.com
Gadgil
Himanshu S
HS
Pipes
Gary D
GD
Bondarenko
Pavel V
PV
Hobbs
Vipa
V
Scavezze
Joanna L
JL
Kim
Jun
J
Jiang
Xu-Rong
XR
Mukku
Venkat
V
Dillon
Thomas M
TM
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002850:Chromatography, Gel; D003545:Cysteine; D004355:Drug Stability; D007074:Immunoglobulin G / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry