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Transformation and Upstream Processing

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.

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Transformation and Upstream Processing

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  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 coliTransformed to ProduceGFP Escherichia coli Green 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 coliTransformed to ProduceGFP Escherichia coli Green Fluorescent Protein

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

  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 Tryptone or Peptone Arabinose – The Inducer

  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/SolutionsGoal: To remove microbial contamination (bioburden) Autoclave Sterile Filtration (.22u pores remove bacteria)

  24. Placed in a CO2 incubator to provide a controlled environment for CHO cell scale-up Temperature: 37oC CO2: 5% pH: 7.2 Agitation via Magnetic Stir Plate: 75 rpm Scaling Up in Spinner Flasks

  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 (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.

  29. Growth Rate and Doubling TimeCalculations Growth Rate u = (lnOD2-lnOD1)/T2-T1 Or u = (lnX2 – lnX1)/T2-T1 (where X=live cell count) Doubling Time Td = 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 Monitoredby 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. Characteristics of Microbial and Mammalian Cell Culture

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

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

  37. PID Control No Control PID Control

  38. Virtual Biomanufacturing Production

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

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