the use of living systems and organisms to develop or make useful products Biomanufacturing industry within biotechnology that is responsible for making biopharmaceuticals biologics and other ID: 1043896
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1. Protein is CA$H!Biotechnology - the use of living systems and organisms to develop or make useful productsBiomanufacturing – industry within biotechnology that is responsible for making biopharmaceuticals (biologics) and other bioproducts such as biofuels and human tissues
2. The Expression Vector - The Basis of Biotechnology Manufacturingwww.bio.davidson.edu/.../plasmid_inducible.html
3. Transformation and Cloning
4. Central Dogma of Biology
5. RNA Codons specify Amino Acids
6. The Twenty Amino Acids
7. Four Levels of Organization of Protein Structure
8. ProteinsThe Structure and Function of LifeHormones (human growth hormone and insulin)Enzymes (lipase, protease,)Immunoglobulins (antibodies)Blood Proteins (hemoglobin, albumin)Muscle contraction proteins (actin and myosin)Structural proteins (collagen, elastin, reticulin, spectrin)Fluorescent proteins (GFP)
9. Upstream Processing- develop equipment and processes used in production to culture the cells that make the biopharmaceuticalDownstream Processing –recovery and purification of drug from cell culture media Formulation/Distribution –product is filled, tested for purity and sterility and distributed for clinical use Stages of Biomanufacturing
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11. Upstream Processing OverviewMain objective – to create the environment necessary for cells to make a protein product (recombinant protein or biologic) Product of interestCalled API’s- Active Pharmaceutical IngredientProduced by mammalian cell culture (Chinese Hamster Ovary - CHO cells, mouse myeloma NSO cells) or bacterial fermentation (E.Coli)Mammalian cells excrete product into mediacan modify proteins (post translation modifications)harvest can be made without cell lysis CHO cells most commonly used Bacterial cells do not have the machinery to secrete the desired product into the mediacells must be lysed during harvest
12. YeastInsectTransgenic PlantsTransgenic AnimalsOther Expression Systems
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14. Provides all the nutrition cells need within a narrow window of environmental conditions for optimal expression of the target proteinMajor media components Carbohydrate energy source – glucoseNitrogen source such as amino acidsLipids – often in the form of fatty acidCells also requireTrace minerals in the form of electrolytes (salts)fetal bovine serum supplements – not so common due to risk of animal virusesChemically defined, serum free media commonly used – reduces threat of adventitious animal virus contaminants Selective agents that cells require for optimal expression of the target protein Culture Media
15. Sterilizing Media/SolutionsGoal: To remove microbial contamination (bioburden)AutoclaveSterile Filtration (.22u pores remove bacteria)
16. InoculumFrozen vials of cells from cell bank thawed and added ( “inoculated”) to spinner or shaker flasks ( 100 – 500 mLCell culture expanded to meet cell density and volume requirements to inoculate larger volume bioreactorBioreactor Seed Cell cultures from cell banks spinner or shaker flasks are “seeded” or scaled up into larger volume bioreactors ( up to 20,000L)Bioreactors are either stainless steel or disposable Cells grow to high densities producing API in the culture media Primary Recovery ( Harvest) main purpose is to separate the cells from the media containing the APICentrifugation to separate cells from media Filtration to remove large debrisUpstream Processing Stages
17. Inoculation- Cell BanksTwo-tiered cell banking system consisting of a master cell bank (MCB) and a working cell bank (WCB) is recommended Cells from the MCB are expanded to form the WCB WCB is characterized for cell viability prior to use in the manufacturing process recommended that production cell banks be stored in two or more widely separated areas within a production facility as well as at a distant site in order to avoid loss of the cell line.
18. Inoculum stage involves the thawing of a frozen vial of cells (ampoule) and its introduction into a bioreactorampoule of frozen cells is generally released from the cell bank per batch of APIthawed cells are then added to prepared media in either shaker flasks or spinner flasksDepending on the type of cells may take a few hours or a few weeks to reach the required volume and cell density volume expansionInoculum
19. Scale Up- Culture FlasksShaker Flasks-bacteria culturesSpinner Flasks- mammalian cultures
20. Cultures grown in a temperature-controlled incubator at approximately 35–38 CMonitor pH and gases (C02, O2)Monitor cell growth/proliferation- growth rate and doubling time help to determine when to feed, when to harvestlive cell counts ( trypan blue assay) optical density (OD) readingsAseptic technique is critical to prevent contamination of the culture cleaning of all materials, gowning (e.g., gloves, face masks, hair nets, etc.) aseptic techniques in the Biological Safety CabinetCell Culture Monitoring
21. Cell Growth
22. Cell Proliferation Curve
23. ParameterMicrobial Cell CultureMammalian Cell CultureGrowth Rate (u)Doubling Time = minutes to hoursDoubling Time = daysTemperature (T)Great diversity: -0 to +100 degrees (plus/minus 1) C often by coolingMost: 37 to 42 degrees CControl to plus or minus .1 degree CpHGreat 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 CO2Dissolved Oxygen (DO)Air or Oxygen spargedRobust cell walls allow rigorous spargingAir spargedExtremely shear sensitive use sintered spargerAgitation RateAgitation rates can be >800rpm Use Rushton impellerAgitation rates <150rpm Use maine impellerFoamFoam probe, anti-foam agent requiredNo foam probe or anti-foam required Characteristics of Microbial and Mammalian Cell Culture
24. Upstream Processing EquipmentLab-Scale Bioreactor-3 litersLarge-Scale Bioreactor25,000 liters
25. Disposable (WAVE) Bioreactor
26. Cell Harvest Main objective is to separate the cells from the media containing the target APIDuring culture samples taken at pre-determined intervals to monitor progress Time to end culture and begin harvest depends on cell line and is pre-determined based on the quality and quantity of the product accumulated in the bioreactor generally highest amount of quality product is reached when cell number drops
27. Cell Harvest- Centrifugation & Filtration2 steps:1. Centrifugation separate cells from culture mediaRapid spinning of the culture from bioreactor; cells sink to bottom of centrifugeFor microbial harvest lysing step prior to centrifugation to release protein product contained inside cell wall 2. Filtration – remove large debrisDepth filtration – API passes through as the filtrate along with other proteins and cell particlesSterile grade membrane filtration – remove smaller particles and potential microbial contaminationfilters with 0.22 micron pore size
28. Cell Harvest Centrifuge
29. Types of Harvest Filters
30. Transformation and Upstream Processing
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32. Escherichia coli Transformed to Produce GFP Escherichia coliGreen Fluorescent Protein
33. Green Fluorescent Protein (GFP) Expression Vector
34. Transformation-Upstream Processing
35. pGLO Transformation – Upstream Processing “Selection”
36. “Inoculation/Scale up”
37. “Cell Harvest”
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