Ranibizumab is a biotherapeutic expressed in E.coli and is used for the treatment of age related macular degeneration and macular oedema. A major issue in commercial production of Ranibizumab is its low expression level. This can be increased by manipulating nutritional requirements (such as carbon source, nitrogen source, buffering agent), physical parameters (such as pH, inducer concentration, agitation, temperature), and genetic make-up of the producing strain. Assessment of these parameters can enhance our process understanding and help us to build a QbD based process. In our study, the effects of pH, inducer concentration, agitation, temperature, carbon source, nitrogen source and buffering agent were monitored during time course fermentation of BL21 (DE3) strain in Reisenberg medium using one-factor-at-a-time (OFAT) approach. Consequently, the optimized parameters were used for cultivation to maximize the target product. Among the screened parameters, agitation did not impact biomass production. Maximum product titer was observed after 8 h of induction at 30oC. Based on the preliminary screening, eight nutrients were identified to form the modified Reisenberg medium. These components were further analyzed by Plackett-Burman experimental design to understand the interactions among the nutrients at varying concentrations. The expressed product was characterized by Liquid Chromatography–Mass Spectrometry (LC-MS). However, the OFAT study was unable to explain the interaction effect between these parameters. To overcome this issue, QbD based method will be followed using the screened variables. Amongst chosen parameters- pH, inducer concentration and temperature were found to be critical and will be considered for building QbD based process development. A five-fold enhancement of the target protein titer was obtained using the screened medium components vis-a-vis basal medium.

Quality by Design (QbD) based process development for production of ranibizumab in E. coli

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.