Improving sensitive-reactant efficiency in site-specific PEGylation via semi-batch processing: simulation-based evaluation under throughput constraints

Semi-batch reactors are preferred for highly exothermic reactions, consecutive/parallel reaction networks, and sensitive reactants. For instance, site-specific PEGylation can benefit from semi-batch reactions through the feeding of sensitive, activated methoxy maleimide polyethylene glycol (MAL-PEG) esters into the reactor. Batch-mode PEGylation with a MAL-PEG:protein molar ratio of 10:1 considerably affects PEGylated protein’s cost of goods. Herein, we aimed to reduce the total required amount of MAL-PEG for equivalent throughput by feeding MAL-PEG into the reaction. Excess initial MAL-PEG was ineffective because of deactivation and lack of native protein recovery scheme from the discharge. Introducing a native protein recovery and ensuring excess protein notably decreased the required MAL-PEG molar excess. Moreover, a low volumetric flow rate improved the accumulative and cyclic yields while minimizing deactivation, further reducing the required MAL-PEG molar excess. Higher feed and initial reactor concentrations reduced the time to reach the target throughput. The screening phase showed that compared to the batch mode, a 63 % decrease in the required MAL-PEG molar excess was achieved with a 2-mM MAL-PEG feed and a reciprocal time of 1/64 min−1. Subsequently, different discharge percentages and decay profiles of different intensities were tested. Lower discharge percentages resulted in decreased MAL-PEG molar excess but longer reaction time. Similar effects were observed for the decay profiles. A scoring assessment was used to determine the optimal discharge ratio and decay profile based on MAL-PEG molar excess savings and reaction duration. Our findings provide a framework for reactions using sensitive reactants that can benefit from semi-batch reactor operations.