Abstract
Background: PEGylation of therapeutic antibodies generally decreases immunogenicity, extends systemic circulation, and reduces clearance. Trastuzumab, a monoclonal antibody targeting HER2, is typically administered with a loading dose of 4 mg/kg followed by weekly maintenance doses of 2 mg/kg, with a half-life varying from 10 to 18 days depending on the patient population. In general, site-specific PEGylation has been reported to improve pharmacokinetic behavior, product homogeneity, and analytical tractability in antibody modification.
Objectives: This study aimed to explore and optimize a C-terminal PEGylation approach for trastuzumab using amin-PEG (5 kDa) and to examine its potential effects on the antibody’s physicochemical and hydrodynamic properties.
Methods: PEGylation reactions were performed at pH 3.5 and 7.5 with a PEG: protein molar ratio of 11.2 over a range of reaction times. PEGylated trastuzumab was separated from the unmodified antibody using size-exclusion chromatography (SEC) and subsequently analyzed by SEC, cation-exchange chromatography (CEX), and SDS-PAGE. The physicochemical properties were compared with those of native trastuzumab.
Results: At pH 3.5, PEGylation primarily occurred on the light chain of trastuzumab, whereas at pH 7.5, both light and heavy chains were modified, with a preference for the heavy chain. The hydrodynamic radius (Rh) increased from 4.357 nm for native trastuzumab to 4.519 nm for mono-PEGylated and 4.600 nm for di-PEGylated forms. SDS-PAGE analysis revealed shifts in the apparent molecular weight of PEGylated fragments, likely due to the interaction of PEG moieties with SDS and the increased hydrodynamic radius resulting from conjugation.
Conclusion: Our findings suggest that trastuzumab can be PEGylated at its putative C-terminal site, leading to distinct physicochemical changes. This study provides guidance for future methodological developments and optimizations regarding the site-directed PEGylation of monoclonal antibodies.