Transformation and Upstream Processing DNA is the flash

Transformation and Upstream Processing

DNA is the flash Protein is the cash The natural history of a commercial protein.

The Expression Vector: The Basis of Biotechnology Manufacturing

Expression Vector for Green Fluorescent Protein (GFP)

Transformation and Cloning

Escherichia coli Transformed to Produce GFP Escherichia coli Green Fluorescent Protein

Central Dogma of Biology

Transcription

Translation

Proteins = Amino Acid Polymers

RNA Codons specify Amino Acids

The Twenty Amino Acids

• Nonpolar Amino Acids (hydrophobic) glycine Gly G alanine Ala A valine Val V leucine Leu L isoleucine Ile I methionine Met M phenylalanine Phe F tryptophan Trp W proline Pro P • Polar (hydrophilic) serine Ser S threonine Thr T cysteine Cys C tyrosine Tyr Y asparagine Asn N glutamine Gln Q • Electrically Charged (negative and hydrophilic) aspartic acid Asp D glutamic acid Glu E • Electrically Charged (positive and hydrophilic) lysine Lys K arginine Arg R histidine His H

Non-Polar Amino Acids (Hydrophobic)

Four Levels of Organization of Protein Structure

Proteins are the Machines and form the Structure of Life • • • Hormones (human growth hormone and insulin) Enzymes (lipase, protease, cellobiase) Receptors (for neurotransmitters, hormones, and transferrin) Signal transduction proteins (produce cascades; cause transcription of DNA into RNA) Carrier proteins (HDL and LDL for cholesterol; transferrin for iron) Membrane proteins (ion channels, receptors) Immunoglobulins (antibodies) Blood Proteins (hemoglobin, albumin, transferrin, factor VIII) DNA Transcription Factors Muscle contraction proteins (actin and myosin) Structural proteins (collagen, elastin, reticulin, spectrin) Fluorescent proteins (GFP, RFP, others)

Escherichia coli Transformed to Produce GFP Escherichia coli Green Fluorescent Protein

Upstream/Downstream Manufacturing Overview Large Scale Bioreactor 1 day Media Prep Seed Bioreactors Working Cell Bank 150 L Bioreactor Sub. Culture 750 L Bioreactor 26, 000 L Bioreactor Centrifuge Depth Filtration Wave Sub. Culture Bag Sub. Culture Collection Inoculum Fermentation 24 days 31 days Filter Harvest Collection Tank 5, 000 L Bioreactor Column Chromatography Skid Eluate Hold Tank Harvest/Recovery Viral Inactivation Eluate Hold Tank 20, 000 L Column Chromatography Skid 8, 000 L 1, 500 L Anion Exchange Chromatography (QXL) Hydrophobic Interaction Chromatography (HIC) Purification Filter Column Chromatography Skid Filter Eluate Hold Tank Post-viral Hold Vessel 3, 000 L 6, 000 L Protein A Chromatography Viral Filtering 8 days Column Chromatography Skid Eluate Hold Tank 5, 000 L Anion Exchange Chromatography (QFF - Fast Flow) Ultra Filtration Diafiltration Bulk Fill

Media Preparation for Cell Growth and Protein Expression Feeding Doubling of Cells and Synthesis of Protein

Media for Growing Cells and Producing Protein • E. coli media requires some chemicals and nondefined components (hydrolyzed protein and yeast extract) to grow a batch and an inducer to produce the protein of interest. This is the cheapest medium. • Mammalian (CHO) cells require complex medium containing all 20 amino acids, fatty acids, and carbohydrates. Growth media requires 10% fetal bovine serum (FBS) but can be weaned to a serumfree medium. Most expensive medium.

Escherichia coli (Prokaryot)Media LB Broth with Arabinose Na. Cl Yeast Extract Arabinose – The Inducer Tryptone or Peptone

Yeast Extract The main components of yeast extract are: – total nitrogen content : 8 to 12 %, corresponding to a protein content of 50 to 75 % – amino nitrogen content : 3. 0 to 5. 2 % – total carbohydrate content : 4 to 13 % – lipid content : none or very little. Click here to see how yeast extract is made: http: //www. eurasyp. org/public. levure. extrait. screen

Sterilizing Media/Solutions Goal: To remove microbial contamination (bioburden) Autoclave Sterile Filtration (. 22 u pores remove bacteria)

Scaling Up in Spinner Flasks Placed in a CO 2 incubator to provide a controlled environment for CHO cell scale-up • Temperature: 37 o. C • CO 2: 5% • p. H: 7. 2 • Agitation via Magnetic Stir Plate: 75 rpm

Scaling Up in Shake Flasks in Shaking Incubator

Scaling Up Using a Disposable (WAVE) Bioreactor

Upstream Processing Equipment Lab-Scale Bioreactor 3 liters – Process Controlled Large-Scale Bioreactor 25, 000 liters – Process Controlled

Monitoring Growth • The importance – The growth rate (u) and doubling time (Td) help to determine when to feed, when to harvest and such. • The assays for cell growth and reproduction – live cell counts, optical density (OD) readings, and WCW measurements give you the data needed to determine the growth rate and doubling time.

Growth Rate and Doubling Time Calculations Growth Rate u = (ln. OD 2 -ln. OD 1)/T 2 -T 1 Or u = (ln. X 2 – ln. X 1)/T 2 -T 1 (where X=live cell count) Doubling Time Td = ln 2/u

Optical Density (OD) Measurements using a Spectrophotometer

Live Cell Count: Bacteria Spread Plate Method

Monitoring The Product (Protein) • Via absorption at 280 nm to determine the amount of protein • Via ELISAs to specify and quantify the protein • Via SDS-PAGE to determine the purity of the protein

Parameters Monitored by Process Control via Feedback Loops • • p. H (via addition of base or acid) Temperature (via jackets that heat or cool) Oxygen (via sparging air or oxygen and agitation) Rate of Agitation (via need for oxygen) Carbon Dioxide (via sparging) Feed (via addition of appropriate nutrients) OD (via spectrophotometer) Analytes (via biolyzer or nova)

Characteristics of Microbial and Mammalian Cell Culture Parameter Microbial Cell Culture Mammalian Cell Culture Growth Rate (u) Doubling Time = minutes to hours Doubling Time = days Temperature (T) Great diversity: -0 to +100 degrees (plus/minus 1) C often by cooling Most: 37 to 42 degrees C Control to plus or minus. 1 degree C p. H Great diversity: p. H 2 -10 (Pichia = Narrow range: p. H 6. 8 to 7. 2 p. H 5. 8) (CHO = p. H 7. 2) Control by adding acid or base Control by sparging CO 2 Dissolved Oxygen (DO) Air or Oxygen sparged Robust cell walls allow rigorous sparging Air sparged Extremely shear sensitive use sintered sparger Agitation Rate Agitation rates can be >800 rpm Use Rushton impeller Agitation rates <150 rpm Use maine impeller Foam probe, anti-foam agent required No foam probe or anti-foam required

Temperature Control for Mammalian Cell Culture For mammalian cell culture heating is more critical than cooling due to slow metabolic rates (doubling time) Temperature Probe Heating Blanket on single wall vessel

Process-Control Loops p. H Process-Control Loop DO Process-Control Loop

PID Control No Control PID Control

Virtual Biomanufacturing Production

Virtual Biomanufacturing Production See www. atelearning. com/Bio. Testbed/Upstream User Name (use your email address) Password is sonwal