Microbial Cell Factories for Recombinant Therapeutic Proteins - PowerPoint PPT Presentation

Microbial Cell Factories for Recombinant Therapeutic Proteins Assoc.Prof. Chuenchit Boonchird Department of Biotechnology Faculty of Science Mahidol University Reserch Expo 2013 27-28 January 2014, Srivarindhira Building, Siriraj Hospital

Platform of Microbial Cell Factories  Biopharmaceuticals/Biotherapeutics/Biologics are cost intensive  Biotechnological process using microbial cells can serve high demand with industrial scale-up, low cost, and rapid production  Microbial cell factories was fully established in the 80’s through the intensive public and private investment  Since then, the commercial biopharmaceuticals industry has grown substantially  Track record of providing safe and efficacious clinical benefit.

Growth of Biopharmaceuticals Worldwide 900 > 14 % Pharma Biopharma 800 Worldwide Sales (billion USD) 110 87 700 75 65 600 56 47 500 400 698 694 642 300 584 549 512 200 100 0 While pharmaceuticals have higher sales, biopharmaceuticals are growing at a faster rate worldwide Source: IMS Health (accessed August 3, 2010)

Global Market for Recombinant Protein Drugs Source: Martı ´nez et a l ., Current Opinion in Biotechnolog y 2012;23:965 – 971

Top Selling Biopharmaceuticals Worldwide Insulins, EPO, GCSF, and therapeutic monoclonal antibodies Worldwide (WW) Sales excluding biosimilars (Bil. USD) Average Generic Name Brands Companies Indications 2004 2005 2006 2007 2008 Growth EPO Anemia 10.4 10.8 11.6 12.5 6% Darbepoetin Aranesp Amgen 2nd Generation 2.4 3.3 4.1 4.2 3.1 7% Epoetin alfa Epogen Amgen 2.6 2.5 2.5 2.5 -1% Epoetin alfa Procrit/Eprex J&J 3.6 3.3 3.2 3.9 3% Epoetin beta Recormon Roche 1.8 1.7 1.8 1.9 2% Diabetes Insulin 6.1 7.1 9.0 10.8 21% Lantus Sanofi Aventis 1.0 1.4 2.2 2.4 34% Novulin Novo Nordisk 0.9 1.2 1.7 2.7 44% Humlin Novo Nordisk 2.1 2.4 2.5 2.5 6% Humalog Lilly 1.1 1.0 1.3 1.5 11% Humulin Lilly 1.0 1.1 0.9 1.0 0% Byetta Lilly 0.4 0.7 N.A. Rheumatoid Etanercept � Enbrel Amgen, Wyeth 2.6 3.7 4.4 5.2 7.7 31% arthritis, Psoriasis Takeda Infliximab Remicade J&J, Schering Rheumatoid 2.1 3.5 4.2 5.1 6.2 31% Plough, arthritis, Psoriasis, Mitsubishi Crohn's Tanabe Rituximab Rituxan Roche Lymphoma, 3.2 4.7 5.0 5.5 20% Rheumatoid arthritis Trastuzumab Herceptin Roche Breast Cancer 1.7 3.1 4.4 4.7 40% GCSF 3.9 4.5 3.9 4.0 1% 2nd Generation PEG-filgrastim Neulasta Amgen 1.7 2.3 2.7 2.7 17% Filgrastim Neupogen Amgen 1.2 1.2 1.2 1.3 3% Rheumatoid Adalimumab Humira Abbott 1.4 2.0 3.1 4.5 48% arthritis, Psoriasis, Crohn's Bevacizumab Avastin Roche Colon cancer 1.3 2.4 3.9 4.8 55% Source : KNOL

Advance in Biotechnological Process Scheme

Cell Factory for Recombinant Protein Production Advantages Disadvantages • • Fastest expression method (days) Limited posttranslational • Inexpensive bioproduction media modifications • • High density biomass Unsoluble proteins and not • Simple process scale-up correctly folded Bacteria • Well characterized genetics • • Rapid expression method (days - weeks) N-linked glycan structures • Inexpensive bioproduction media different from mammalian forms • High density biomass • Most posttranslational modifications** Yeast • High folding capacity Transient-transfection • Moderate rapid expression method • Low density biomass and Mammalian (weeks) expensive bioproduction media • All posttranslational modifications • Difficult process scale-up • High folding capacity Mammalian Stable-transfection • • Low density biomass and expensive All posttranslational modifications • bioproduction media High folding capacity • Difficult process scale-up • Longest expression method (months) 7

Number and Percentage of Recombinant Proteins Approved as Biopharmaceuticals in Different Cell Factories  70% are glycoproteins >annual growth rate of app.26%  Nonglycosylated protein >annual growth rate of app.12% Mammalian cells: CHO, BHK, and HEK cell lines murine Source: Ferrer-Miralles, Microbial Cell Factories 2009; 8 :17-25

Posttranslational Protein Modification: Glycosylation Source: GE Healthcare

Humanized Proteins are Glycosylated Impact of glycosylation:  Biological activity – maintaining the integrity - Enhance thermal stability - Provide protection from proteolysis - Improve solubility - Inhibit aggregation of proteins  Receptor binding Recognition, signaling and interaction events within and between cells and proteins  Pharmacokinetics, clearance, and serum half-life  Antibody function  Immunogenecity Interferon- β ( top) and glycosylated interferon- β with glycosylation highlighted in blue (bottom). Source: Gerngross, Nature Biotechnology 2004;22:1409-1414

Problem of Incorrect Glycosylation • Most humanized proteins are highly glycosylated • Accumulated product titer of high producer cells is in range 1,000 – 2,000 mg/ml and beyond: protein biosynthesis exceeds significantly the capacity for full / correct glycosylation • Incorrectly glycosylated proteins may not be (fully) functional and / or might exhibit non-desired pharmaceutical behavior (e.g. receptor-mediated protein resorption) Source: GE Healthcare

Platform of Humanized Glycosylated Proteins Production in Yeast System Limitation of mammalian system  Shortages of growth factors supplies and prohibition of using animal derived medium for animal cell culture  Limitation of secretion capacity and low protein yields  High cost production Yeast system  Common steps of glycosylation process as in mammalian  More resistance to shear stress during fermentation process due to rigid cell wall structure  High protein yield  Lower cost of production

Production Level of Recombinant Therapeutic Proteins in Different Production System Y = yeast F = filamentous fungi M = mammalian Source: Martı ´nez et a l . , Current Opinion in Biotechnolog y 2012;23:965 – 971

Therapeutic Proteins Developed in Pichia pastoris Source: Gerngross, Nature Biotechnology 2004;22:1409-1414

Advantages of Pichia pastoris Expression System  Cheap methanol as a sole carbon source / inducer - Strong inducible alcohol oxidase 1 ( AOX1 ) promoter  Expression proteins at high levels, intracellularly or extracellularly  Very high cell densities (>130 g/l DCW) - Cheap medium and lowering the cost  Less hypermannosylation compared to Saccharomyces cerevisiae Engineering of • Mannose type glycan Genetic engineering glycosylation site • Complex glycan of strain

Milestones and Recent Accomplishments for Biopharmaceutical Production in Pichia pastoris  FDA GRAS (Generally Regarded as Safe) status in 2006 for food industry (Phospholipase C by Diversa Corp., for degumming vegetables oils for food)  FDA approved biopharmaceutical production processes in 2009 (Kalbitor by Dyax Corp., a Kallikrein inhibitor) and 2012 (Jetrea by ThromoGenics NV, for the treatment of vitreomascular traction)  Glycoengineered strain providing humanized, uniform N-glycosylation patterns  Synthetic promoters for fine-tuning expression levels  Efficient strategies for knockouts of multiple genes an over-expression of entires pathways  High quality genome sequence  Establishment of i n silico metabolic models for strain engineering Source: Vogl et al. Current Opinion in Biotechnology 2013;24:1094 – 1101

N-Glycan Type In Human and P. pastoris • A dolichol-linked glycan precursor is synthesized in cytoplasm via sequential addition of N-acetylglucosamine (GlcNAc) and mannose (Man) residues into a dolichol • Addition of sugar to dolichol-linked glycan precursor is occurred in ER via sequential activity of the glycosyltransfereases and glycosidases • Oligosaccharides are transferred to the nascent proteins in ER • Sequential addition of sugar is occurred in Golgi apparatus via activity of glycosyltransferases and glycosidases Source: Gerngross, Nature Biotechnology 2004;22:1409-1414

Step of N-Glycosylation and Engineering in ER 1 st Approach: Deletion of genes involved in earlier steps especially the ALG3 gene Source: Pourcq et al., Appl Microbiol Biotechnol 2010;87:1617 – 1631

Step of N-Glycosylation and Engineering in the Golgi Och1p = mannosyltransferase 2 nd Approach: deletion of yeast-specific genes involved in hypermannosylation, especially OCH1 gene Complementation with several glycosyltransferases and glycosidases from various species Source: Pourcq et al., Appl Microbiol Biotechnol 2010;87:1617 – 1631

Production of Sialylated O-Linked Glycans in Pichia pastoris Source: Hamilton et al., Glycobiology 2013;1:1 – 12

Elucidation of Humanized IgG in Glycoengineered Pichia pastoris Characterization of antibody Source: Lee et al., Nature Biotechnology 2006;24:210-215

Elucidation of Humanized IgG in Glycoengineered Pichia pastoris N-Glycan analysis Engineered Wild type Source: Lee et al., Nature Biotechnology 2006;24:210-215

Elucidation of Humanized IgG in Glycoengineered Pichia pastoris Receptor binding assay Source: Lee et al., Nature Biotechnology 2006;24:210-215

Advances Through Metabolic Engineering Engineering of secretion pathway to improved yield of protein secretion Bioengineered Source: Nelson, Bioengineered 2013;2:207-211