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 highly activated ester group linking 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 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.

Lipoic Acid Ligase A‐Mediated Ligation: Mechanism, Applications, and Emerging Innovations in Bioconjugation

“…[85] Recently, continuous operation has become a key approach for biomolecule applications. [48,86,87] This tunable and scalable system could pave the way for new applications in enzyme immobilization, including the LplA method.…”

“…For example, polyethylene glycol (PEG) chain ligation to therapeutic proteins can improve their pharmacokinetic properties, increase their circulation time, and reduce their immunogenicity. [47,48] LplA can ligate PEG chains to specific sites on a protein, ensuring that its activity is not disrupted. In addition, the ability of LplA to accept a wide range of carboxyl-bearing molecules as substrates has allowed the development of methods for metabolic labeling of proteins.…”

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.…”

“…The continuous-mode flow reaction, which allows chemical reactions to take place in dedicated systems consisting of tubes, mixers, and pipes, − holds promise for achieving high-throughput protein refolding. However, its application in the biotherapeutic field is limited.…”

“…FMRs offer rapid mixing on a millisecond scale and precise control of residence time, enabling the control of low molecular weight intermediates. − FMR technology takes advantage of microscale phenomena within continuous-mode reaction devices . Rapid mixing and precise residence time control have enabled previously challenging reactions, such as generating unstable lithium species for protecting group-free reactions .…”