Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies

Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.