Zwitterlation mitigates protein bioactivity loss in vitro over PEGylation.

Conjugation with poly(ethylene glycol) (PEG) or PEGylation is a widely used tool to overcome the shortcomings of native proteins, such as poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity. However, PEGylation is often accompanied by an unwanted detrimental effect on bioactivity, particularly, resulting from the amphiphilic nature of PEG. This is especially true for PEGylated proteins with large binding targets. Pegasys, a PEGylated interferon alpha-2a (IFN-alpha 2a) bearing a 40 kDa branched PEG, is a typical example that displays only 7% in vitro activity of the unmodified IFN-alpha 2a. In this work, by employing IFN-alpha 2a as a model protein, we demonstrated that a protein conjugated with zwitterionic polymers (or zwitterlation) could significantly mitigate the antiproliferative bioactivity loss in vitro after polymer conjugation. The retained antiproliferative activity of zwitterlated IFN-alpha 2a is 4.4-fold higher than that of the PEGylated IFN-alpha 2a with the same polymer molecular weight, or 3-fold higher than that of the PEGylated IFN-alpha 2a with a similar hydrodynamic size. It is hypothesized that nonspecific interactions between zwitterionic polymers and IFN-alpha 2a/IFN-alpha 2a receptors can be mitigated due to the super-hydrophilic nature of zwitterionic polymers. This, in turn, reduces the 'nonspecific blocking' between IFN-alpha 2a and IFN-alpha 2a receptors. In addition, we demonstrated that zwitterlated IFN-alpha 2a showed a prolonged circulation time and a mitigated accelerated blood clearance after repeated injections in rats.