Universitat Autònoma de Barcelona. Facultat de Biociències

Vasile, Simona

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  USE OF SYNTHETIC BIOLOGY TO HUMANIZE THE YEAST PICHIA PASTORIS APPLIED IN MONOCLONAL ANTIBODIES PRODUCTION Simona Vasile                  Biotechnology Degree, Universitat Autònoma de Barcelona Pre-program Terminations  INTRODUCTION Therapeutic proteins are the fastest-growing source of new therapies in the biopharmaceutical industry resulting in a demand for more efficient protein expression systems. Yeast-based expression systems offer a variety of advantages: shorter fermentation times, cheaper operating costs, chemically defined media, no viral contamination, higher protein titers, and much shorter development times from gene to protein. However, for the production of glycoproteins for human use, native highmannose yeast glycosylation is not suitable and therefore represents a major limitation for yeast based protein expression systems.          OBJECTIVES  obtaining a humanized Pichia pastoris strain able to produce N-gycosilated proteins with proper folding, pharmacokinetic stability, and efficacy.   application in the production of monoclonal antibodies. REFERENCES [1] Nicole Ballew. Revolutionising protein production in fungi. Innovation in pharmaceutical technology. Glycofi. [2] [6] SR. Hamilton, TU. Gerngross. Glycosylation engineering in yeast: the advent of fully humanized yeast. 2007, Current Opinion in Biotechnology, 18:387–392 [3] FW. Krainer et al. Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris. 2013, Nature. [4] Stefan Wildt and TU. Gerngross. The humanization of the N-glycosylaton patways in yeast. 2005, Nature Publishing Group, Vol 3. 119-128. [5] Davidson RC.,et al. Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. 2004, Glycobiology, 4:757–766 [7] Bösch Peter. Humanization of posttranslational modifications in yeasts. 2005. [8] Dongxing Zha, et al. Production of Monoclonal Antibodies in Glycoengineered Pichia pastoris. Antibody Expression and Production. Cell engineering Vol.7.  N-GLYCOSYLATION IN YEAST AND HUMAN Fig.8.Glycoengineered yeast cultivation platform.[8] Fig.1. Pichia pastoris yeast cells. [1] Fig.3. Schematic workflow of the knockout of OCH1 using a flipper cassette. [3] REBUILDING THE HUMAN SECRETORY PATHWAY Fig.4. A working model for the cellular distribution of glycosyltransferases throughout the secretion pathway. [4] • Screening of a combinatorial library of α-1,2 mannosidases. • Screening of a library of catalytic domains encoding N-acetyl-glucosaminyltransferase I (GnTI) fused to a library of yeast localization signals. • Use of the combinatorial library approach to introduce mannosidase II and GnTII. A galactosyl transferase should add one galactose sugar onto every branch. The mutant strain capable of producing complex human N-glycans was accomplished through deletion of PpALG3 and  expression of GnT II and GalT.  GALACTOSYLATION SIALIC ACID TANSFER The transfer of sialic acid onto terminal β-1,4 galactose sugars of complex glycoproteins requires a library with catalytic domains, a library with several N-terminal fragments for the location and a pool of CMP-sialic acid available (not available in yeast).  Therefore a pathway to produce CMP-sialic acid and a CMP-sialic acid transporter have to be introduced to the system.   Fig.2. N-glycosylation pathway in: (a) human and yeast (b) humanized Pichia pastoris [2]  Fig.5. P.pastoris glycoengineering to produce complex human N-glycans. [5] CONSTRUCTION OF EXPRESSION STRAINS APPLICATION IN MONOCLONAL ANTIBODIES PRODUCTION • the insertion of the gene into an expression vector. • introduction of the expression vector into the P. pastoris genome. • examination of potential expression strains for the foreign gene product.    Alternative promoters non induced by methanol: GAP, FLD1, PEX8, and YPT1 were found.  HUMANIZING GLYCOSYLATION PATHWAY IN PICHIA PASTORIS EARLY APPROACHES Creation of och1 knockout strain Fig.6. Glycoengineering steps required for sialic acid transfer in the yeast Golgi.[6] Expression vectors Expression of any foreign gene in P. pastoris requires three basic steps: Fig.7. General diagram of a P. pastoris expression vector. YFG (Your Favorite Gene) sites for cassette amplification. [7]  CONCLUSIONS  Compared to CHO cells, the glycoengineered yeast produced antibody with similar motilities on SDS-PAGE, comparable size exclusion chromatograms and antigen binding affinities but with highly uniform N-linked glycans of the type Man5GlcNAc2.   The production of monoclonal antibodies in P. Pastoris offers a robust and economically viable alternative to mammalian cell expression.  Expression vectors are integrated into the P. pastoris genome to maximize the stability of expression strains. This can be done in two ways: • restricting the vector at a unique site in either the marker gene or the AOX1 promoter fragment and then to transform it. • digesting certain P. pastoris expression vectors in such a way that the expression cassette and marker gene are released, flanked by 5’ and 3’ AOX1 sequences. Multicopy strains can be approached: • Vector with multiple head-to-tail copies of an expression cassette flanked by restriction sites with complementary termini. • Expression vectors that contain the P. pastoris HIS4 and kanamycin resistance gene. • Vector with resistance to the antibiotic zeocin. Selectable markers • biosynthetic markers: HIS4, ARG4, ADE1, URA3 • drug-resistance markers: kanR gene, Sh ble gene. Combinations of these different markers are employed for efficient selection of true transformants. Antibodies often achieve therapeutic benefit through two binding events: • the variable domain of the antibody binds a specific protein on a target cell. • the effector cells  bind to the constant region (Fc) of the antibody and destroy cells to which the antibody has bound. Construction of the antibody expression vector The plasmid pGLY2988 (containing IgG1 heavy and light chain under control of AOX1 promoter) was transformed into YGLY638 by electroporation to yield YGLY4140. The host strain has been modified to secrete recombinant proteins with highly uniform N-linked glycans of the type Man5GlcNAc2. 

Use of synthetic biology to humanize the yeast Pichia pastoris applied in monoclonal antibodies production

http://ddd.uab.cat/pub/tfg/2014/119216/TFG_simonavasile.pdf

Use of synthetic biology to humanize the yeast Pichia pastoris applied in monoclonal antibodies production

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