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U P S T R E A M

B I O P R O C E S S I N G. www.integra-biosciences.com. U P S T R E A M. D O W N S T R E A M. Protein Biochemistry - Part I Background Up-Stream Processing  Scale-up Production Down-Stream Processing  Isolation Purification Characterization Finishing / Fill. BIOMAN – 2011 K. Lampe, MC3.

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U P S T R E A M

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  1. B I O P R O C E S S I N G www.integra-biosciences.com U P S T R E A M D O W N S T R E A M BIOMAN 2009 - K. Lampe, MC3

  2. Protein Biochemistry - Part IBackgroundUp-Stream ProcessingScale-upProduction Down-Stream Processing IsolationPurificationCharacterizationFinishing / Fill BIOMAN – 2011 K. Lampe, MC3 BIOMAN 2011 - K. Lampe, MC3

  3. Proteins are EVERYTHINGgenes are just a means to an end BIOMAN 2009 - K. Lampe, MC3

  4. Proteins => Traits Single-celled Simple multi-cellular Complex multi-cellular organism organism organism characteristics characteristics characteristics Organ characteristics Tissue characteristics Set of cellular structural and functional characteristics Set of cellular products Genes BIOMAN 2009 - K. Lampe, MC3

  5. Proteins => Targets • Nearly ALL therapeutic compounds, manufactured or natural, exert their effects by interacting with one or another form of protein… Enzymes Receptors Channels Transporters Antigens BIOMAN 2009 - K. Lampe, MC3

  6. Protein Basics - 1 • 1 Gene 1 protein • Translation  polypeptide • Maturation / Modification  protein BIOMAN 2009 - K. Lampe, MC3

  7. Protein Basics – 2a • Maturation / Modification  protein • Folding (1o -> 2o -> 3o agg -> 4o) • Largely spontaneous • Hydrophobic/hydrophilic interactions • H-bonding • R-group ionic interactions • Modification of in-chain A.A.’s • Cleavage • Addition of non-protein groups • Glycosylation, acylation, phosphorylation, etc. BIOMAN 2009 - K. Lampe, MC3

  8. Protein Basics – 2b • Sorting / Transport / Insertion • Activation / Inactivation • Degradation • Structure determines function BIOMAN 2009 - K. Lampe, MC3

  9. What is Protein Biochemistry ? -- After the molecular biology -- • Expression / Synthesis / PTranslM / Sort & Transport / Activate or Inactivate / Degrade • Structure / Function • Methods • Identify / isolate / purify / modify • Characterize BIOMAN 2009 - K. Lampe, MC3

  10. Why do Protein Biochemistry ? A)Biotech. / Biopharm. / Manufacturing • Product isolation • Product purification • Product modification • Product characterization • Product stability / storage BIOMAN 2009 - K. Lampe, MC3

  11. Why do Protein Biochemistry ? B) Research • Product isolation • Product purification • Product modification • Product characterization • Product stability / storage BIOMAN 2009 - K. Lampe, MC3

  12. Methods for Identifying & Localizing • Study mutants • Ligand binding • In situ hybridization • Chimeric (tagged) proteins (made GFP famous) BIOMAN 2009 - K. Lampe, MC3

  13. Methods for Isolating & Purifying -1 • Primary difference between industrial and research procedure is simply scale(methods and steps generally comparable) • Difficult at best… May be impossible (want to isolate in native/active form) • Scheme depends upon location and character of protein BIOMAN 2009 - K. Lampe, MC3

  14. Methods for Isolating & Purifying – 2a • Scheme depends upon location and character of protein • Location, e.g… • Cytoplasmic free • Cytoplasmic inclusions/vesicles • Membrane-bound • Cell wall • Secreted BIOMAN 2009 - K. Lampe, MC3

  15. Methods for Isolating & Purifying – 2b • Scheme depends upon location and character of protein • Character, e.g… • Hydrophobic / hydrophilic • Globular / fibrous • Large / small • Non-protein components ? • Secreted BIOMAN 2009 - K. Lampe, MC3

  16. Methods for Isolating & Purifying – 2c • Denaturing • Is it likely to occur ? • Does it matter at this point ? BIOMAN 2009 - K. Lampe, MC3

  17. Methods for Isolating & Purifying – 2d • Basic Steps • #1 Crude preparation • Cell homogenate • Cell media • #2 Initial fractionation / “clarification” / concentration • #3 Subsequent purification / “polishing” BIOMAN 2009 - K. Lampe, MC3

  18. #1 - Crude Preparation • Cell homogenate (cell lysate / lysed cells) • Freeze thaw • Lysozyme • Sonication • Homogenization • Osmotic Pressure / Pressure • Shearing • Detergent / Organic Solvent • Cell media • Centrifugation / filtration BIOMAN 2009 - K. Lampe, MC3

  19. #2 - Fractionation / Clarification / Concentration • Differential precipitation / de-solublization • Salts vs. organics • Differential re-solublization • Filtration • Direct to chromatography BIOMAN 2009 - K. Lampe, MC3

  20. #3 - Subsequent Purification • Filtration / Dia-filtration • Chromatography (elaboration to follow) • Size (GF / SEC) • Hydrophobic Interaction (HIC) • Ion Exchange (IEC) • Affinity (AC) • Ligand • Antibody • Antigen BIOMAN 2009 - K. Lampe, MC3

  21. How Pure ? • “Protein Biochemistry – Part II” • QC and Characterization • How pure does it need to be ? • Depends on use • Depends on type of impurity(ies) • FDA allows some level of impurity in biopharm. product BIOMAN 2009 - K. Lampe, MC3

  22. GFP as a model for classroom activities • GFP is ideal • Easily expressed • Easily scaled up • Relatively easy to “purify” • Stable • Amendable to further study (struct. / funct.) • Looks very cool ! BIOMAN 2009 - K. Lampe, MC3

  23. Chromatography • Separation of molecules from masses of others based on differences in characteristics of the molecules in the mixture • With chromatography… Everything is RELATIVE • Many variations, but all are based on same concept • Force mix of molecules to “race” along a narrow, restrained path • Molecules interact with the path differentially… Some move along the path faster than others • Allow the molecules to move along the path far/long enough so that the one you want is well separated from (most of) the rest • Collect the molecule of interest BIOMAN 2009 - K. Lampe, MC3

  24. Chromatography Variations • The Path (“Stationary Phase”) • A column (tube) filled (packed) with some retentive material (“resin”) • A glass plate covered with a “thin layer” of retentive material • Paper • The Carrier (“Mobile Phase”) • Liquid (“LC”) vs. Gas (“GC”) • Flow Rate and Pressure of “Mobile Phase” • Gravity vs. Low Pressure (“LP”) vs. High Pressure (“HP”) • Properties of the retentive material BIOMAN 2009 - K. Lampe, MC3

  25. Chromatography Variations • Properties of the retentive material – continued • Sieve size (“size-exclusion” / “gel-filtration”) • Charge attraction (“ion exchange”) • Hydrophobic / Hydrophilic • “Affinity”; a specific size, shape, charge “fit” between the molecule of interest and another (natural or synthetic) • Antibody – Antigen • Receptor – Ligand • Enzyme – Substrate • Molecule of Interest – Synthetic “binder” • Properties of retentive material and those of mobile phase are customized (matched) to optimize separation of molecule of interest from the rest in the mixture BIOMAN 2009 - K. Lampe, MC3

  26. Chromatography Principle • Properties of retentive material and those of mobile phase are customized (matched) to optimize separation of molecule of interest from the rest in the mixture • With Chromatography, everything is RELATIVE • With SEC… • Pore size of resin is selected to either allow molecule of interest to pass through resin “beads” (move relatively slowly through stationary phase) or go around resin (move relatively quickly through stationary phase) • Because some molecules will be larger, and others smaller than the molecule of interest, a single pass through a SEC resin can not “purify” the molecule of interest • A single mobile phase can be run continuously through / over the stationary phase • Molecule of interest is collected as it washes off of (“elutes” from) the stationary phase BIOMAN 2009 - K. Lampe, MC3

  27. Chromatography Principle • With IXC, HIC, and AC… • Molecules in the mixture have a relative affinity (attractiveness) for the stationary phase and the mobile phase • Higher affinity for stationary phase – move along path slowly • Higher affinity for mobile phase – move along path quickly • Conditions may be established such that the molecule of interest is selectively (and relatively) retained on the stationary phase OR be allowed to pass freely through the stationary phase with the mobile phase • Load  Wash  Elute BIOMAN 2009 - K. Lampe, MC3

  28. Chromatography Abbreviations • STATIONARY PHASE • TLC • PC • CC • MOBILE PHASE • GC • LC • FLOW RATE / PRESSURE • LP (e.g. LPLC) • HP (e.g. HPLC) • RETENTIVE MATERIAL PROPERTY • SEC / GFC • IEC (IXC) / AEC (AXC) / CEC (CXC) • HIC • AC BIOMAN 2009 - K. Lampe, MC3

  29. Chromatography Abbreviations • STATIONARY PHASE • TLC thin layer (plate) chromatography • PC paper chromatography • CC column chromatography • MOBILE PHASE • GC gas chromatography • LC liquid chromatography • FLOW RATE / PRESSURE • LP low pressure (e.g. LPLC) • HP high pressure (e.g. HPLC) • RETENTIVE MATERIAL PROPERTY • SEC / GFC size exclusion / gel filtration chromatography • IEC (IXC) / AEC (AXC) / CEC (CXC) ion exchange chromatography • HIC hydrophobic interaction chromatography • AC affinity chromatography BIOMAN 2009 - K. Lampe, MC3

  30. Gravity-Flow Liquid Column Chromatography • Mini columns packed with resin (stationary phase) • AX resin • CX resin • HI resin • Mobile phase(s) depends upon particular stationary phase • Load sample • Allow mobile phase to run through column by gravity to “wash away” unwanted molecules • “Elute” molecule of interest BIOMAN 2009 - K. Lampe, MC3

  31. Size Exclusion / Gel Filtration Chromatographyhttp://www.science.fau.edu/chemistry/Mari/biochemlab/05_012.jpg BIOMAN 2009 - K. Lampe, MC3

  32. Preparative vs. analytical • LPLC usually preparative • HPLC usually analytical • Isocratic, step gradient, continuous gradient BIOMAN 2009 - K. Lampe, MC3

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