Droplet-Based Microfluidics for Measuring Enzymatic Activities: Application to L-Asparaginase used in Antileukemic Therapy

Abstract

Our work aims to ameliorate catalytic properties of L-asparaginase (L-ASNase) which is a protein drug used in antileukemic therapy. Bacterial L-ASNases are FDA-approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn. Their therapeutic efficacy is based on the fact that several hematological malignancies, in particular Acute Lymphoblastic Leukemia (ALL), depend for growth on the extracellular supply of L-Asn. To avoid various side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. In order to find variants of improved specific activities in mutated enzyme libraries, we developed a droplet-based microfluidic platform for high throughput and miniaturization of kinetic assays that can be performed not only on purified enzymes, but also in single cells. When analyzing enzyme activity in bacterial cells, critical assumptions are: (i) protein expression levels are uniform in the population, and (ii) the reaction rate is not limited by the diffusion of small-molecule substrates/products to/from the enzyme. We demonstrate here a three-step coupled-enzyme assay for measuring ASNase activity on its natural substrate Asn, using water-in-oil droplets of nL to pL reaction volumes, at throughput rates of up to 104 per sec. Catalytic rates were found to vary by a factor of less than two within a population of more than 108 bacterial cells, following the same kinetics as the purified enzyme free in solution. To increase serum half-life of the enzyme, we loaded L-ASNases onto calcium carbonate nanoparticles serving as core templates, and manufactured biocompatible microcapsules under mild conditions to preserve catalytic activity, thereby improving protein stability and preventing exposure of the enzyme to the immune system. Encapsulated and free enzymes were able to kill leukemic cells with similar efficacy.