1. Transformation and Upstream Processing

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

3. The Expression Vector: The Basis of Biotechnology Manufacturing

4. Expression Vector for Green Fluorescent Protein (GFP)

5. Transformation and Cloning

6. Escherichia coli Transformed to Produce GFP Escherichia coliGreen Fluorescent Protein

7. Central Dogma of Biology

8. Transcription

9. Translation

10. Proteins = Amino Acid Polymers

11. RNA Codons specify Amino Acids

12. The Twenty Amino Acids

13. 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

14. Non-Polar Amino Acids (Hydrophobic)

15. Four Levels of Organization of Protein Structure

16. 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)

17. Escherichia coli Transformed to Produce GFP Escherichia coliGreen Fluorescent Protein

18. Media Prep Working Cell Bank Working Cell Bank Sub- Culture Sub- Culture Inoculum Sub- Culture Sub- Culture Sub- Culture Sub- Culture Sub- Culture Sub- Culture Sub- Culture Sub- Culture Large Scale Bioreactor Wave Bag Wave Bag Seed Bioreactors Fermentation 150L Bioreactor 750L Bioreactor 5,000L Bioreactor 26,000L Bioreactor Depth Filtration Depth Filtration Collection Centrifuge Harvest/Recovery Harvest Collection Tank 1,500L Harvest Collection Tank 1,500L Filter Chromatography Skid Anion Exchange Chromatography (QXL) Column Eluate Hold Tank 8,000L Eluate Hold Tank 8,000L Eluate Hold Tank 6,000L Eluate Hold Tank 6,000L Filter Chromatography Skid Protein A Chromatography Column Chromatography Skid Column Eluate Hold Tank 20,000L Eluate Hold Tank 20,000L Hydrophobic Interaction Chromatography (HIC) Eluate Hold Tank 20,000L Eluate Hold Tank 20,000L Viral Inactivation Eluate Hold Tank 5,000L Eluate Hold Tank 5,000L Filter Chromatography Skid Anion Exchange Chromatography (QFF - Fast Flow) Column Post-viral Hold Vessel 3,000L Post-viral Hold Vessel 3,000L Viral FilteringUltra Filtration Diafiltration Bulk Fill Purification 24 days31 days 8 days 1 day Upstream/Downstream Manufacturing Overview

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

20. Media for Growing Cells and Producing Protein E. coli media requires some chemicals and non- defined 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 serum- free medium. Most expensive medium.

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

22. 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

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

24. 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% pH: 7.2 Agitation via Magnetic Stir Plate: 75 rpm

25. Scaling Up in Shake Flasks in Shaking Incubator

26. Scaling Up Using a Disposable (WAVE) Bioreactor

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

28. Monitoring Growth The importance – The growth rate (u) and doubling time (T d ) 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.

29. Growth Rate and Doubling Time Calculations Growth Rate u = (lnOD 2 -lnOD 1 )/T 2 -T 1 Or u = (lnX 2 – lnX 1 )/T 2 -T 1 (where X=live cell count) Doubling Time T d = ln2/u

30. Optical Density (OD) Measurements using a Spectrophotometer

31. Live Cell Count: Bacteria Spread Plate Method

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

33. Parameters Monitored by Process Control via Feedback Loops pH (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)

34. ParameterMicrobial 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 pHGreat diversity: pH 2-10 (Pichia = pH 5.8) Control by adding acid or base Narrow range: pH 6.8 to 7.2 (CHO = pH 7.2) 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 RateAgitation rates can be >800rpm Use Rushton impeller Agitation rates <150rpm Use maine impeller FoamFoam probe, anti-foam agent required No foam probe or anti-foam required Characteristics of Microbial and Mammalian Cell Culture

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

36. Process-Control Loops pH Process-Control LoopDO Process-Control Loop

37. PID Control No Control PID Control

38. Virtual Biomanufacturing Production

39. See www.Atelearning.com/BioTestbed/Upstream User Name (use your email address) Password is sonwal