Harnessing the Power of Chemometric Modeling and Plate Readers for High-Throughput Analysis of Biocatalytic Reactions

High-throughput experimentation is a critical enabler in the pharmaceutical process discovery and development. This is especially true when exploring areas with many degrees of freedom such as enzyme evolution campaigns, where it is common to screen tens of thousands of samples to optimize and direct evolution toward the desired product. Importantly, high sample throughput must be complemented by advances in analytics to fully exploit its potential. Herein, we present the development and application of a new high-throughput chemistry (HTC) methodology that complements traditional high-throughput approaches such as liquid chromatography (LC), mass spectrometry, and spectroscopic techniques, each of which comes with its own challenges and advantages. Utilizing HTC, we demonstrate its effectiveness and efficiency by analyzing the directed evolution of cytidine deaminase for enzymatic hydroxyamination ∼10× faster than the comparable LC method. This approach combines the simplicity of plate readers with the power of chemometrics to deliver equivalent results required by LC methods while being faster, greener, less expensive, and user-friendly. Notably, the HTC methodology is generalizable to a variety of chemical systems and spectroscopic techniques, making it a valuable addition to the high-throughput analysis toolkit.