A novel zwitterionic copolymer with a short poly(methyl acrylic acid) block for improving both conjugation and separation efficiency of a protein without losing its bioactivity

Abstract

PEGylation of protein drugs has thus far been the most important method used in improving the stability and the circulation time, while lowering the immunogenicity of protein drugs. However, the loss of bioactivity of PEGylated protein drugs and the low recovery through the complicated separation process are the most challenging issues. To overcome these problems, a zwitterionic block copolymer poly(methyl acrylic acid-b-sulfobetaine methacrylate) (PMAA-b-PSBMA or abbreviated to PMS) has been chosen to modify a protein drug (uricase) since zwitterionic materials are superior to polyethylene glycol (PEG) in resistance to nonspecific protein adsorption, chemical stability and also low cytotoxicity. As indicated by the results, the conjugation between PMS and uricase could be achieved under 0.03 mg mL⁻¹ of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 0.018 mg mL⁻¹N-hydroxysuccinimide (NHS) through weak charge-induced adsorption of PMS on uricase, which is about 0.3% of the concentration for conventional protein–polymer conjugation. The conjugates, efficiently separated from the reaction solution and re-suspended in physiological solution through pH adjustment from pH 5.35 to 7.4, show about 133% of the original activity and better anti-trypsin digestion than the native uricase. These results demonstrate that the conjugation of a zwitterionic copolymer with a short poly(methyl acrylic acid) block could improve the stability of an enzyme without destructively affecting its bioactivity. This may open a new way for the delivery of therapeutic protein drugs.