Enzymes immobilized on solid supports are increasingly used for greener, more sustainable chemical transformation processes. Here, we used microreactors to study enzyme-catalyzed ring-opening polymerization of ε-caprolactone to polycaprolactone. A novel microreactor design enabled us to perform these heterogeneous reactions in continuous mode, in organic media, and at elevated temperatures. Using microreactors, we achieved faster polymerization and higher molecular mass compared to using batch reactors. While this study focused on polymerization reactions, it is evident that similar microreactor based platforms can readily be extended to other enzyme-based systems, for example, high-throughput screening of new enzymes and to precision measurements of new processes where continuous flow mode is preferred. This is the first reported demonstration of a solid supported enzyme-catalyzed polymerization reaction in continuous mode.

An enzyme packed microreactor was compared with a batch reactor system to determine effects of reaction water content on immobilized Candida antarctica Lipase B (Novozyme 435, N435) reusability, efficiency of end-group functionalization, apparent monomer conversion rate constants (k app), average molar mass (M n), and leaching of Candida antarctica Lipase B (CALB) from Lewatit macroporous beads. Conversion of ε-caprolactone to poly(ε-caprolactone), PCL, using benzyl alcohol for end-functionalization, was the model system for studies conducted herein. The apparent rate constant in microreactor (k app = 0.027 s–1) was 27 times larger than in batch reactor (k app = 0.001 s–1). Furthermore, in microreactor, the M n vs conversion plots for “dry” and “water saturated” conditions were similar. In contrast, under “water saturated” conditions in a batch reactor, M n is much lower. Moreover, at both high and low water content, higher end-group functionalization was achieved for polymerizations in microreactor (0.75 to ≥0.98) as compared to batch reactor (0.2). Also, microreactors run for 30 cycles under “dry” or “water saturated” conditions gave product where the fraction of benzyl ester groups on chains remains high, between 80 and 90%. In contrast, in the “water saturated” batch system, the fraction of benzyl ester terminal groups remains at about 0.30 throughout all 11 reaction cycles. These results led to the conclusion that the microreactor design results in effectively “dry” conditions even when reactants are “water saturated”. CALB leaching during 7, 18, and 25 reaction cycles in microreactor steadily increased from 10 to 15 and 22%. However, at 30 cycles, CALB leaching disproportionaly increased to 76% without apparent physical deterioration of beads. Comparative results in batch reactors for all experiments above are reported and discussed.

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A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.