Proteins as Biotechnology Products Examples of Protein Products

Proteins as Biotechnology Products

Examples of Protein Products? Medications Enzymes to make fuel from hard-to-metabolize biomass Textile processing enzymes Decontamination enzymes for 1 st responders

BIOPROCESSING: The use of biological systems to manufacture (process) a product.

A Protein Product is Made Discovery Research Process Development & Approval Manufacture Verification Quality Control & Quality Assurance Packaging

Discovery Research “ Basic Science” Ongoing scientific study! • Often government funded • Use of model organisms • Communication! • Idea for a bioproduct • Transition to industry: the “start-up company”

Discovery Research Idea for a product, based upon DNA manipulation and cloning….

Process Development Optimizing the manufacturing process, but on a smaller scale. Process Development ends with production of a master cell bank Eventually the process is scaled up for full scale manufacturing

Product Manufacturing Overview Upstream Processing Downstream Processing

Upstream Processing The actual expression of the protein product in the host cell. Photo from Univ. Hawaii

Common Host Cells • Bacterial cells • Fungal cells • Mammalian cells E. coli P. pastoris Chinese Hamster Ovary “CHO”

The Bioreactor • Sterile system! • Nutrient Medium • Process control Ødissolved O 2 Ø pressure Øp. H Øtemperature 4 Liter Bio. Flow 3000, made by New Brunswick Scientific

Large Scale Bioreactor 20, 000 liter bioreactor in Portsmouth, NH

Downstream Processing Isolation of pure, stable, functional protein. Many challenges!

The Harvesting Challenge • Cells: if product is inside the cells, harvesting involves cell lysis which is risky to the protein • Medium: if product is secreted, harvesting involves dealing with huge volumes of liquid.

Harvesting Techniques Lysis by: • Freeze/thawing • Mechanical disruption (beads, blades, sonic) • Detergents

Stabilizing Methods • Cold Temperature • Protease inhibitors protein • Anti-foaming chemicals protease

Purification Get rid of all unwanted substances, but keep as much of the protein as possible…. Don’t forget, it must be perfect !

Typical Product Purification Methods • Filtration • Centrifugation • Chromatography

Filtration Separation of substances by passage through porous membrane. Industrial size filters

Centrifugation Control Panel Protective enclosure Door Separation of particles from liquid by use of centrifugal force. Cut-away view Rotor Motor Basic components of a centrifuge Center of rotation rminimum Drive shaft Centrifugal force Sedimentation path of particles raverage rmaximum Pellet deposited at an angle

Chromatography Molecular separation based on physical or chemical characteristics Example: Gel Filtration separates based on size.

Industrial Chromatography 2 meter column

Verification Monitor for the presence, quantity, and quality of the target protein as it is being produced and purified.

Common Analytical Methods • HPLC - High Performance Liquid Chromatography • SDS PAGE - SDS Polyacrylamide Electrophoresis • IEF - Isoelectric Focusing • Mass Spectrometry

…Analytical Methods… HPLC = High Performance liquid chromatography From LC Resources Inc

…Analytical Methods… SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis http: //fig. cox. miami. edu/~cmallery/255 hist/ecbxp 4 x 4_SDS. jpg

…Analytical Methods… IEF Isoelectric Focusing Similar to SDS PAGE, but higher resolution From Amersham Sci.

…Analytical Methods… • Mass Spectroscopy – a method of analyzing protein structure.

Last Steps 1. Formulation: addition of stabilizing chemicals 2. Filter sterilization 3. Aseptic filling of vials 4. Lyophilization freeze dry 5. Seal, inspect, label

Quality Control - testing Quality Assurance - oversight Throughout all stages to ensure safety and compliance with FDA regulations.

Discovery Research Process Development and Drug Approval Manufacture Verification NEXT!! Quality Control & Quality Assurance Packaging