Abstract
Protein PEGylation is a traditional bioconjugation technology that enhances the therapeutic
efficacy and in vivo half-life of proteins by the formation of covalent bonds with highly activated ester
group linked polyethylene glycol (PEG). However, the high reactivity of these reagents
induces a random reaction with lysine residues on the protein surface, resulting in
a heterogeneous mixture of PEGylated proteins. Moreover, the traditional batch-mode
reaction has risks relating to scalability and aggregation. To overcome these risks
of traditional batch-mode PEGylation, a manufacturing strategy utilizing structural
analysis and a continuous-flow-mode reaction was examined. A solvent exposure analysis
revealed the most reactive lysine of a protein, and the continuous-flow mode modified
this lysine to achieve the mono-PEGylation of two different proteins within 2 seconds.
This ultrarapid modification reaction can be applied to the gram-scale manufacturing
of PEGylated bioconjugates without generating aggregates. A similar trend of the exposure
level of protein lysine and mono-selectivity performed by continuous-flow PEGylation
was observed, which indicated that this manufacturing strategy has the potential to
be applied to the production of a wide variety of bioconjugates.
Key words
continuous-flow synthesis - microreactor - PEGylation - cytokines - bioconjugates
