A Manufacturing Strategy Utilizing a Continuous Mode Reactor toward Homogeneous PEGylated Bioconjugate Production

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 polyethylene glycol (PEG). The most established methodology for PEGylation utilizes activated ester-incorporating PEG reagents; 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 two seconds. This ultra-rapid 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.

Development and Characterization of a Portable Flow Microreactor for Enhanced Bioconjugation Applications

“…These results were similar to those observed in previous FMR experiments. 15 The aggregation rates assessed by SEC showed that the PEGylation-induced aggregation was insignificant, not exceeding 1% (Figure 5a). 25 In this study, monopegylation was performed on a scale of several hundred milligrams, successfully producing 351 mg of mono-PEGylated IL-6.…”

“…36 Given the high solvent-accessible surface area and abundance of Lys in IL-6, selective PEGylation becomes a challenge compared with proteins such as lysozyme, which have a limited number of Lys residues. 15 Additionally, the tendency of IL-6 to aggregate complicates its handling. 23 We performed PEGylation on such a complex protein substrate to demonstrate the versatility and efficacy of the Portable FMR.…”

“…Yoshida's concept of "flash chemistry," which focuses on microreactors that allow very short residence times, is gaining attention for its potential in bioconjugation. 22 We previously applied flash chemistry to bioconjugation and successfully synthesized practical compounds, including PEGylated proteins 15 and ADCs. 16 These prior study results conclusively show that bioconjugation mediated by flow reactors provides substantial scalability and significantly reduces aggregation levels when compared to traditional batch-mode production methods.…”

Spatiotemporal Control of Protein Refolding through Flash-Change Reaction Conditions

“…17 Recent studies have explored FMR technology for next-generation protein therapeutics, including antibody−drug conjugate production 12 and PEGylation processes. 13 Expanding upon these foundational studies, this study presents an advancement with the successful implementation of a cascaded-mixer system, enabling FMR-mode protein refolding. We studied the conformational changes of the cytokine interleukin (IL-)6 as a model, 18 at different pH and in various organic solvents 19,20 to determine the most effective way to generate the optimal IL-6 conformation.…”