NOVEL PROCESSES AND PRODUCTS FOR RECOMBINANT PRODUCTION OF BIOPHARMACEUTICALS

• Main Author: Giuliani, Maria
• Format: Doctoral or Postdoctoral Thesis
• Language: Italian; English
• Published: 2009
• Subjects: Antarctic; The Antarctic; Antarc*
• Online Access: http://www.fedoa.unina.it/4104/; http://www.fedoa.unina.it/4104/1/Thesis_M._Giuliani.pdf; https://doi.org/10.6092/UNINA/FEDOA/4104

The monoclonal antibody market represents the fastest-growing segment within the biopharmaceutical industry (Evans and Das 2005). Indeed, recombinant antibodies and antibody fragments are widespread tools for research, diagnostics and therapy (Joosten et al., 2003). Large-scale production of recombinant antibodies and antibody fragments requires a suitable expression system which has to be cheap, accessible for genetic modifications, easily scaled up for greater demands and safe for use in consumer applications. However, the established eukaryotic systems are expensive, time consuming and sometimes inefficient (Farid, 2007). Although prokaryotic expression systems can reduce production costs, recombinant antibody production in conventional bacterial hosts, such as E. coli, often results in inclusion bodies formation (Baneyx and Mujacic 2004). Since the lowering of the expression temperature can increase product solubility facilitating its correct folding (Sahdev et al., 2008), a novel process for recombinant antibody fragments production at low temperatures was developed based on the use of the Antarctic Gram-negative bacterium P. haloplanktis TAC125 as recombinant expression host. To test the versatility of the new developed process, the production of three aggregation prone model proteins was evaluated corresponding to the most common formats of antibody fragments: Fab, ScFv and VHH. The construction of an ad hoc genetic expression system for each model protein followed a rational design where several critical aspects were considered including the selection of molecular signals for periplasmic protein addressing and the choice of optimal gene-expression strategy. For Fab fragment production in heterodimeric form an artificial operon was designed and constructed. Moreover, a new defined minimal medium was developed to maximise bacterial growth parameters and recombinant production yields. The production of model antibody fragments has been evaluated in lab-scale bioreactor and the effect of different ...