(176s) Improving Bioprocess Yield of Laparaxin – a Promising Therapeutic Peptide

Laparaxin is a proprietary 5-kD bioactive peptide produced exogenously by a strain of L. paracasei. This peptide has been shown to have significant activity against a number of clinically important pathogens such as L. monocytogenes, Enterococcus faecalis and S. aureus 1316 P-M+ V5 MRSA. In view of the current global antibiotic resistance crisis, antimicrobial peptides (AMPs) like Laparaxin have gained more attention since they may be suitable substitutes for current therapeutic practices. However, in order for an entity to be classified as a therapeutic drug, the efficacy and safety of the product must first be established through several stages, moving from in vitro studies to animal studies and finally culminating in human clinical trials. Thus, it is apparent that appropriate quantities of the active peptide must be readily available for every stage of the drug development process. In this study we have focused our efforts on improving yields of Laparaxin by modulating parameters in the fermentation broth of L.paracasei to produce the optimal set of the factors for the maximal yield of Laparaxin. Substate requirements and process parameters were optimized using OFAT (One factor at a time) and Box-Behken/ Response Surface Methodology (RSM) methods. Static flask cultures based on reported growth parameters were initially used to obtain a base line for Laparaxin yield in the fermentation broth. Subsequently, substrate optimization was carried out by screening various carbon, nitrogen and phosphates sources using OFAT and RSM, followed by titration of their concentrations for optimal Laparaxin yield. Physical parameters such as incubation temperature, pH and agitation were also optimized using flask cultures with previously determined optimum substrate over a 24-hour period. Finally with the determined optimal substrate and process parameters, the fermentation of L. paracasei was scaled up to 4L in a 5L bioreactor where further adjustments to the process was implemented. Antimicrobial activity of Laparaxin observed was nearly 3-fold over baseline levels, at 24-hr timepoint, indicating a substantial improvement in the production process of this peptide.