PEGylation has improved the pharmacological properties of therapeutic proteins. However, polyethylene glycol (PEG) can bring about a negative clinical effect on therapeutic protein due to its immunogenicity. The anti-PEG immune response to PEGylated proteins is probably dependent on the properties of proteins and the conjugated polyethylene glycol (PEG). As a result, the investigation of the effects of protein immunogenicity, the extent of PEGylation, the molecular weight and the branching of mPEG on the anti-PEG immune response is necessary.
Ovalbumin, tetanus toxoid, tetanus toxoid–tetanus toxoid conjugate, and tetanus toxoid–bovine serum albumin conjugate were used as target proteins. Through the fractionation of the PEGylated tetanus toxoid with size exclusion chromatography, the PEGylated proteins with different extent of PEGylation were obtained. In addition, by modifying tetanus toxoid using linear mPEG and branched mPEG, the PEGylated proteins with different molecular weight and branching of mPEG were obtained. The PEGylated proteins triggered high levels of anto-PEG antibodies, and the anti-PEG immune response relied on the immunogenicity of proteins, the extent of PEGylation and the molecular weight of mPEG. The effect of the branching of mPEG on the anti-PEG immune response to the PEGylated proteins is insignificant.