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

512 549 584 642 694 698 47 56 65 75 87 110

100 200 300 400 500 600 700 800 900 Worldwide Sales (billion USD) Pharma Biopharma

Source: IMS Health (accessed August 3, 2010)

While pharmaceuticals have higher sales, biopharmaceuticals are growing at a faster rate worldwide > 14 %

Global Market for Recombinant Protein Drugs

Source: Martı´nez et al., Current Opinion in Biotechnology 2012;23:965–971

Top Selling Biopharmaceuticals Worldwide

2004 2005 2006 2007 2008 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% Insulin Diabetes 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. Etanercept Enbrel Amgen, Wyeth Takeda Rheumatoid arthritis, Psoriasis 2.6 3.7 4.4 5.2 7.7 31% Infliximab Remicade J&J, Schering Plough, Mitsubishi Tanabe Rheumatoid arthritis, Psoriasis, Crohn's 2.1 3.5 4.2 5.1 6.2 31% Rituximab Rituxan Roche Lymphoma, Rheumatoid arthritis 3.2 4.7 5.0 5.5 20% Trastuzumab Herceptin Roche Breast Cancer 1.7 3.1 4.4 4.7 40% GCSF 3.9 4.5 3.9 4.0 1% PEG-filgrastim Neulasta Amgen 2nd Generation 1.7 2.3 2.7 2.7 17% Filgrastim Neupogen Amgen 1.2 1.2 1.2 1.3 3% Adalimumab Humira Abbott Rheumatoid arthritis, Psoriasis, Crohn's 1.4 2.0 3.1 4.5 48% Bevacizumab Avastin Roche Colon cancer 1.3 2.4 3.9 4.8 55% Generic Name Brands Companies Indications Worldwide (WW) Sales excluding biosimilars (Bil. USD) Average Growth Source: KNOL

Insulins, EPO, GCSF, and therapeutic monoclonal antibodies

Advance in Biotechnological Process Scheme

Advantages

• Fastest expression method (days)
• Inexpensive bioproduction media
• High density biomass
• Simple process scale-up
• Well characterized genetics
• Limited posttranslational

modifications

• Unsoluble proteins and not

correctly folded

Cell Factory for Recombinant Protein Production

Mammalian

Disadvantages

• Rapid expression method (days - weeks)
• Inexpensive bioproduction media
• High density biomass
• Most posttranslational modifications**
• High folding capacity
• N-linked glycan structures

different from mammalian forms

Transient-transfection

• Moderate rapid expression method

(weeks)

• All posttranslational modifications
• High folding capacity
• Low density biomass and

expensive bioproduction media

• Difficult process scale-up

Stable-transfection

• Low density biomass and expensive

bioproduction media

• Difficult process scale-up
• Longest expression method (months)
• All posttranslational modifications
• High folding capacity

Bacteria Yeast Mammalian

7

Number and Percentage of Recombinant Proteins Approved as Biopharmaceuticals in Different Cell Factories

Source: Ferrer-Miralles, Microbial Cell Factories 2009;8:17-25

 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: GE Healthcare

Posttranslational Protein Modification: Glycosylation

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

Source: GE Healthcare

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)

Platform of Humanized Glycosylated Proteins Production in Yeast 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
• 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

Yeast system Limitation of mammalian system

Production Level of Recombinant Therapeutic Proteins in Different Production System

Y = yeast F = filamentous fungi M = mammalian

Source: Martı´nez et al. , Current Opinion in Biotechnology 2012;23:965–971

Therapeutic Proteins Developed in Pichia pastoris

Source: Gerngross, Nature Biotechnology 2004;22:1409-1414

 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

Advantages of Pichia pastoris Expression System

• Mannose type glycan
• Complex glycan

Genetic engineering

• f strain

Engineering of glycosylation site

 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 in silico metabolic models for strain engineering

Milestones and Recent Accomplishments for Biopharmaceutical Production in Pichia pastoris

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

Source: Pourcq et al., Appl Microbiol Biotechnol 2010;87:1617–1631

1st 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

2nd Approach: deletion

• f yeast-specific genes

involved in hypermannosylation, especially OCH1 gene

Och1p = mannosyltransferase

Complementation with several glycosyltransferases and glycosidases from various species

Source: Hamilton et al., Glycobiology 2013;1:1–12

Production of Sialylated O-Linked Glycans in Pichia pastoris

Source: Lee et al., Nature Biotechnology 2006;24:210-215

Elucidation of Humanized IgG in Glycoengineered Pichia pastoris Characterization of antibody

Elucidation of Humanized IgG in Glycoengineered Pichia pastoris

Source: Lee et al., Nature Biotechnology 2006;24:210-215

Wild type Engineered

N-Glycan analysis

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

Source: Thai FDA

Year

2000 2002 2004 2006 2008

Drug Expenditure (Billion THB)

10 20 30 40 50 60 70

Domestic Import

• Thailand has become more dependent on drug imports
• All the biopharmaceuticals (except vaccines) in Thailand are imported as finished

products

• About 20% of the import drug expenditure are biopharmaceuticals
• Thailand needs own production to serve increasing demand with affordable price

Biopharmaceuticals in Thailand

Current Research and Development of Biopharmaceutical Using Microbial Cell Factories in Thailand Vaccines

Bacterial system

Biologics

Bacterial system

Through collaboration with Department of Biotechnology, Faculty of Science and others

Future Research and Development of Biopharmaceutical Using Microbial Cell Factories at Mahidol University

• Bacterial system (E. coli, B. subtilis)
• Yeast system (P. pastoris)

Thank you