title: Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').
abstract: We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.
authors.affiliation: Bioprocess Research & Development, Sanofi Pasteur , 1755 Steeles Avenue West, Toronto, Ontario M2R 3T4, Canada.
authors.forename: Cristopher
authors.initials: C
authors.lastname: Roque
authors.forename: Anthony
authors.initials: A
authors.lastname: Sheung
authors.forename: Nausheen
authors.initials: N
authors.lastname: Rahman
authors.forename: S Fernando
authors.initials: SF
authors.lastname: Ausar
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002942:Circular Dichroism; D000067493:Dynamic Light Scattering; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011092:Polyethylene Glycols
keywords: diffusion interaction parameter; monoclonal antibody; phase diagram; physical stability; stability