Crystallization kinetics of ampicillin were estimated from induction-times and seeded and unseeded experiments. The concentration of ampicillin in solution was monitored by polarimetry and refractometry, while crystals were analyzed by focused beam refractive measurements and light microscopy. Variations of solute concentration with time, along with measured induction times were used to estimate parameters in kinetic expressions for primary nucleation, secondary nucleation, and crystal growth rate. Experimental data were obtained in runs involving different rates of change of pH, mass of seed crystals, and initial supersaturation. These data were used in parameter-estimation routines consisting of a stochastic minimization to localize a set of parameters spanning all the experiments, followed by a deterministic minimization to refine the parameters. The result was a set of parameters that fit a range of conditions of seeded and unseeded crystallizations and gave good estimates of desupersaturation rates.

Seeded reactive crystallization in the manufacture of semi-synthetic β-lactam antibiotics is described and the beneficial effects on yield are discussed. Conventional enzymatic synthesis of β-lactam is limited by secondary hydrolysis reactions that consume the desired product as it is being produced. Recent work in this area has pointed to the potential advantage of performing reactions at conditions that allow product crystallization to reduce the rate of secondary hydrolysis by protecting ampicillin in the solid phase. However, these approaches led to crystallization of both D-phenylglycine and ampicillin, which will greatly increase downstream processing. In the work described here, seeded crystallization is used to promote secondary nucleation of the desired ampicillin while it is being produced by the synthesis reaction, thereby selectively crystallizing ampicillin. Quantification of the solid phase confirmed selective crystallization of ampicillin with purities greater than 95% wt in all runs.React. Chem. Eng. This journal is

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The assessment of the operational stability of biocatalysts by conventional direct determination of the total turnover number (TTN), a useful indicator of lifetime biocatalyst productivity, via continuous isothermal experiments tends to be time‐consuming, material‐intensive, and prone to disturbances, especially in case of rather stable catalysts. In the present work, we present and validate two alternative methods for estimating the TTN of a biocatalyst for any desired operating temperature. The first method is a mechanistic approach, built upon mathematical derivation of enzyme deactivation models derived from first principles, in which TTN can be calculated from two straightforward isothermal biochemical batch measurements. The second method relies on a few non‐isothermal, continuous‐mode experiments in conjunction with mathematical modeling to determine the intrinsic deactivation parameters of the biocatalyst. We verify both methods on the test case of TEM‐1 β‐lactamase‐catalyzed penicillin G (Pen G) hydrolysis. Both alternative methods provide estimates of TTN which are typically within a factor of two to five or less of the values measured directly via lengthy, costly, and error‐prone conventional isothermal aging tests. Therefore, both the mechanistic approach and the non‐isothermal continuous approach are extremely valuable tools to enable calculation of catalyst cost contribution in continuous processing and to eliminate underperforming candidates in search of the most stable biocatalyst.