College of Agriculture College of Engineering Bioprocessing Michael Ladisch Laboratory of Renewable Resources Engineering Agricultural and Biological Engineering Weldon School of Biomedical Engineering Purdue University

Outline 1.Bioprocessing Defined 2.Pharmaceutical Industry Trends 3.Unit Operations a. Bioreactors b. Recovery c. Rapid Prototyping 4.Opportunities in Discovery, Learning, and Engagement

Biotechnology Broadly defined in 1991 any technique that uses living organisms (or parts of organisms) to: 1. make or modify products, 2. improve plants or animals, 3. develop microorganisms for specific uses ( Office of Technology Assessment, 1991)

New Biotechnology Technology for manipulating genetic information and manufacturing products that are of biological origin or which impact biological activity. Based on methods introduced since 1970, applied in the laboratory since 1973, used on an industrial scale since 1979.

Biotechnology Broadly defined in 2001 Technology with one or both characteristics: 1. uses organisms, or tissues, cells, or molecular components derived from living things, to act on living things 2. acts by intervening in the workings of cells or the molecular components of cells, including their genetic material. (National Research Council, Opportunities in Biotechnology for Future Army Applications, 2001)

Bioprocess Engineering Subdiscipline within biotechnology responsible for translating life-science discoveries into practical products, processes, or systems capable of serving the needs of society (National Research Council, Putting Biotechnology to Work, 1991)

Pharmaceutical Industry Trends, 2005 Sales: Global $550 billion US $246 billion China $ 8 billion 7 of 30 top pharmaceuticals from biotechnology Biogenerics likely slow to be introduced C and E News, Dec, 2005

Most Major Products in 2005 were Drugs C and E News, Dec, 2005 Sales to June 2005 ($ Billions) Cholesterol & triglyceride reducers$ 31.6 Antiulcerants 26.3 Antidepressants 20.1 Antipsychotics 15.5 Erythropoietins 12.1 Calcium antagonists, plain 11.9 Antiepileptics 11.7 Antirheumatic nonsteroidals 11.4 Oral antidiabetics 10.4 All other antineoplastics 9.9 TOP 10 THERAPY CLASSES$160.9

Blockbusters 86 products with sales in excess of $ 1 billion Potential for huge sales: smoking cessation inhaled insulin cervical cancer/human papilloma virus vaccines hypertension Niche blockbusters for cancer treatment (many monoclonal antibodies) C and E News, Dec, 2005

Metabolic Engineering Redirecting pathways in an organisms to obtain more product Yeast Bacteria Mammalian Cells Plants and Animals

Bioprocess Unit Operations: Bioreactors Microbial fermentation vs cell culture Recovery of Products Centrifugation Protein A (Affinity Chromatography) Ultra-filtration / micro-filtration Ion exchange, Size Exclusion Chromatography

Bioprocessing: Cell Culture Important Produces monoclonal antibodies (MW 150 kD) Bioreactor size of 200 to L Media composition, avoiding contamination, supplying oxygen, removing wastes, operating bioreactor are challenges

PDMS SiO 2 Flat PDMS cover SiO 2, glass, or PDMS substrate Glass fiber (~12 μm diameter) Labeled avidin (green) and BSA (red) liquid mixture; t=0 Glass fiber t= ~3 minutes Glass substrateSiO 2 substrate (a) (b) (c) Flow channel Rapid-prototyping Huang et al., nL/mm channel TAKES LESS THAN 15 minutes

Dip Coating Fibers with Protein or Particles Ultra-sonic cleaning in EtOH for 5 min EtOH Dip-coat in protein or particle slurry Glass fiber coated with particles Glass fiber

Dimethyl-amino microbeads (0.8 µm diameter) Stationary Phase on Fiber OH + N CH 3 H Bare glass fiber

Glass fiber pre-coated with biotin-BSA Biotin Streptavidin microbeads (0.8 µm diameter) + Streptavidin Glass Fiber Coated with Streptavidin Beads

Commercially available Micro / nano Particles TypeSurfaceChemistrySize (µm) Surface charge SilicaHydroxyl -OH PolystyreneHydroxyl -OH PolystyreneCarboxyl -COOH PolystyreneSulfonate -SO PolystyreneAmino -NH PolystyreneDimethylamino -NH(CH 3 ) Data from Spherotec, Inc. Libertyville IL.

TypeSurfacepISize (µm) Surface Affinity PolystyreneAntibody (IgG)NA Binds protein A, G PolystyreneAvdin Binds to biotin PolystyreneStreptavidin Binds to bitoin PolystyreneBiotinNA Binds to strept/avidin PolystyreneProtein A Binds to IgG antibody PolystyreneProtein G Binds to IgG antibody Data from Spherotec, Inc. Libertyville IL. Commercially available Micro / nano Particles

Microscale Chromatography Glass fiber coated with dimethylamino microbeads Labeled avidin (green; 10 µg/ml) and BSA (red; 10 µg/ml) liquid mixture; t=0 Glass fiber coated with biotinylated BSA Glass substrate PDMS t= ~3 minutes t= ~5 minutes (a)(b) Huang et al., 2003

AB Centrifugation Fermentation or cell culture Lysis Centrifugation Micro-filtration Hold Tank Precipitation Tank Membrane Separation Buffer Tank Hold Tank Chromatography Product Tank Elution Buffer Tanks Adsorption Adsorption Chromatography

Bioprocessing Opportunities Biology component needed for designing batch unit operations Rapid Prototyping (microscale) will enable separations development Biorecovery and bioseparations engineering important Learning at P -12 starts with manufacturing cures