Giuliani, Maria (2009) NOVEL PROCESSES AND PRODUCTS FOR RECOMBINANT PRODUCTION OF BIOPHARMACEUTICALS. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||Psychrophilic bacteria, recombinant proteins production, antibody fragments|
|Date Deposited:||02 Dec 2009 10:53|
|Last Modified:||30 Apr 2014 19:40|
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 cultivation operational strategies on production yields has been investigated. All model proteins were produced in soluble and biologically competent form in optimised conditions. About 5 mg L-1 of biologically active ScFv were obtained in P. haloplanktis TAC125 batch fermentation and up to 4 mg L-1 of soluble Fab in C-limited chemostat cultivation with a volumetric productivity of 0,2 mg L-1 h -1. Moreover, a comparison of Fab fragment production by different microbial hosts including yeasts, filamentous fungi and bacteria was performed revealing that the psychrophilic expression system leads to the highest Fab specific productivity and best quality of recombinant product (Dragosit et al., submitted). In order to further optimise the novel process for recombinant production of biopharmaceuticals by the psychrophilic expression system, new genetic tools for recombinant gene expression and protein addressing in P. haloplanktis TAC125 were searched out and characterised. Transcriptional analysis of several target genes was performed in a cheap defined medium in the presence of inexpensive compounds of physical stimuli as inducers in order to identify new psychrophilic promoters for regulated expression of recombinant proteins at low temperatures. The analysis led to the identification of the gluconate permease PSHAb0479 and the alginate lyase PSHAa1748 promoters, strongly up-regulated by the presence of D-gluconic acid and alginic acid respectively. In order to improve extracellular secretion of recombinant proteins in psychrophilc expression system a deep investigation was performed on protein secretion machineries evolved by P. haloplanktis TAC125. Molecular characterisation of canonical T2SS was carried out and a deep study was performed on the still uncharacterised psychrophilic secretion system (PSS) responsible for the secretion of the recombinant alpha-amylase used as carrier for recombinant protein secretion in the psychrophilic bacterium. The function of pssA gene was related to the new secretion system. Data collected have provided important information which can be used for the construction of engineered P. haloplanktis TAC125 strains with improved ability in recombinant proteins extracellular secretion.
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