Phenylketonuria (PKU), a rare inherited disease caused by a defect in phenylalanine hydroxylase activity, is characterized by the accumulation of systemic phenylalanine (Phe) that can lead to severe neurological deficits unless patients are placed on a strict low-Phe diet. As an alternative treatment, Escherichia coli Nissle, a well-characterized probiotic, was engineered to efficiently import and degrade Phe (SYNB1618). The coupled expression of a Phe transporter with a Phe ammonia lyase allows rapid conversion of Phe into trans-cinnamic acid, which is then further metabolized by the host to hippuric acid and excreted in the urine. In this work, we describe the design, characterization, and scale-up of an upstream process to produce live and active SYNB1618 material for the treatment of phenylketonuria. Fast, high cell density <i>E. coli </i>biomass increase is balanced against protein expression levels as a function of induction conditions and available nutrients. Due to the use of this product as a live oral biotherapeutic, key product quality attributes assessed include cell health and potency as a function of production conditions.

Process development for production of a live biotherapeutic drug product for the treatment of Phenylketonuria

2019 Spring National Meeting

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