Pichia pastoris as a platform for the production of therapeutic glycoproteins

Sammanfattning: Recombinant protein therapeutics is a growing market within the human medical biotechnology industry. The majority of all approved biopharmaceuticals are protein based and includes for example blood factors, anticoagulants, hormones, vaccine and monoclonal antibodies. Some of these protein based drugs are glycoproteins which require special carbohydrate structures attached to certain amino acids for correct folding, biological activity and/or stability in the circulation. The biosynthesis and covalent attachment of these oligosaccharides to the polypeptide core, the glycosyaltion, is species and tissue specific. Eukaryotic cells can attach the glycoproteins either to the side chain of aspargine (N-linked) or through the side chains of threonine or serine (O- linked). Controlling the synthesis of these carbohydrate structures, the glycosylation, is of prime concern when developing therapeutic glycoproteins and today mammalian cell culture systems are the preferred systems due to their ability to perform human-like glycosylation. However, mammalian systems are often hampered by disadvantages such as long production times, low protein titres, product heterogeneity and viral containment issues. These factors complicate large scale production of therapeutic glycoproteins and consequently there is a continual search for alternative expression systems with improved performance. The methylotrophic yeast Pichia pastoris (P. pastoris) has a number of attractive characteristics for heterologous protein production, including the ability to perform post-translational modifications, such as N- and O- linked glycosylation, and secrete large amounts of recombinant protein. Recombinant protein antigens glycosylated by P. pastoris have shown enhanced immunogenicity compared to their non-glycosylated counterparts. The high mannose content of yeast derived N- and O-glycans is proposed to target the recombinant antigens to immunoregulatory, mannose specific receptors which upon binding promotes the enhanced immune responses. These findings suggest P. pastoris as a platform for production of recombinant vaccines. However, structural and functional characterizations of the glycans involved are poor, specifically for O-glycans. PSGL-1/mIgG2b, is a chimeric mucin-like protein with the potential to carry 106 O-glycans and six N-glycans, and AGP-1/mIgG2b is a chimeric protein with the potential to carry twelve N-glycans. In the context of mannose specific receptor targeted vaccines, glycoproteins with this type of glycosylation expressed by P. pastoris have not been studied before. The objective of this study was to develop bioprocesses for efficient production of PSGL-1/mIgG2b and AGP-1/mIgG2b The main purpose of this was to supply enough material for characterisation and functional studies of PSGL-1/mIgG2b and AGP-1/mIgG2b in the context of binding three mannose specific receptors, MR, DC-SIGN and MBL, but also to identify important bioprocess parameters for large scale production of the recombinant glycoproteins. Methanol feed, pH and certain media components were found to be critical for productivity and homogeneity of the recombinant proteins. During the course of this study a bioprocess which improved productivity from 10 mg/L to around 200 mg/L for PSGL- 1/mIgG2b and from 3.5 mg/L to 21 mg/L for AGP-1/mIgG2b along with significantly reduced proteolytic activity was developed. To relate a certain glycan structure with biological activity, characterization of the PSGL-1/mIgG2b O-glycans in combination with binding studies to the recombinant mannose specific receptors MR, DC-SIGN and MBL was conducted. Biacore analysis revealed high affinity binding of both PSGL-1/mIgG2b and AGP-1/mIgG2b to all receptors. MS of O-glycans released from PSGL-1/mIgG2b indicated Hex2-9 structures. For fast, on-line optimization of recombinant protein production an optimization system based on the intrinsic fluorescence of the green fluorescent protein (GFP) was developed. Recombinant strains of P. pastoris secreting the GFP fusion protein PSGL-1/mIgG2b/GFP weregenerated and the fluorescence system was applied to follow on-line fluorescence under various induction conditions. Subsequently, P. pastoris secreting PSGL- 1/mIgG2b was induced under the same conditions. Correlations between the on- line fluorescence and the secreted amount of PSGL-1/mIgG2b were investigated. It was concluded that the on-line system had the potential to reflect the translational rate of PSGL-1/mIgG2b/GFP. However, due to different secretion properties of PSGL-1/mIgG2b/GFP and PSGL-1/mIgG2b in combination with potential genetic instability no correlations were found. The system may still have a value for recombinant proteins expressed intracellularly.

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