College of AgricultureCollege 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 Operationsa. Bioreactorsb. Recoveryc. 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, 2005Sales: Global $550 billion

US $246 billion

China $ 8 billion

7 of 30 top pharmaceuticals from biotechnology

Biogenerics likely slow to be introducedC 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

Blockbusters86 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 10000 L

Media composition, avoiding contamination, supplying oxygen, removing wastes, operating bioreactor are challenges

PDMS

SiO2

Flat PDMS cover

SiO2, 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 substrateSiO2 substrate

(a) (b) (c)

Flow channel

Rapid-prototyping

Huang et al., 20031 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 OH OH+N CH3CH3

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

Type Surface Chemistry Size

(µm)

Surface charge

Silica Hydroxyl -OH 0.1-7.9 -

Polystyrene Hydroxyl -OH 0.7-7.9 -

Polystyrene Carboxyl -COOH 0.7-7.9 --

Polystyrene Sulfonate -SO30.7-7.9 ---

Polystyrene Amino -NH30.7-7.9 +

Polystyrene Dimethylamino -NH(CH3)20.7-7.9 ++

Data from Spherotec, Inc. Libertyville IL.

Type Surface pI Size

(µm)

Surface Affinity

Polystyrene Antibody (IgG) NA 0.7-7.9 Binds protein A, G

Polystyrene Avdin 10.0 0.7-7.9 Binds to biotin

Polystyrene Streptavidin 5.0 0.7-7.9 Binds to bitoin

Polystyrene Biotin NA 0.7-7.9 Binds to strept/avidin

Polystyrene Protein A 4.9 0.7-7.9 Binds to IgG antibody

Polystyrene Protein G 5.0 0.7-7.9 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

Labeled avidin (green; 10 µg/ml) and BSA (red; 10 µg/ml) liquid mixture; t=0

Glass fiber coated with biotinylated BSA

Glass substrate Glass substrate

PDMSPDMS

t= ~3 minutes t= ~5 minutes(a) (b)

Huang et al., 2003

A B

CentrifugationCentrifugation

Fermentation or cell culture

Lysis

Centrifugation

Micro-filtrationMicro-filtration

Hold TankHold Tank

Precipitation TankPrecipitation Tank

Membrane SeparationMembrane Separation

Buffer TankBuffer Tank

Hold TankHold Tank

ChromatographyChromatography

Product Tank

Elution Buffer Tanks

AdsorptionAdsorptionAdsorption

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