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

2. BIOMAN 2011 - K. Lampe, MC3 Protein Biochemistry - Part I Background Up-Stream Processing  Scale-up Production Down-Stream Processing  Isolation Purification Characterization Finishing / Fill BIOMAN – 2011 K. Lampe, MC3

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

4. BIOMAN 2009 - K. Lampe, MC3 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

5. BIOMAN 2009 - K. Lampe, MC3 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

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

7. BIOMAN 2009 - K. Lampe, MC3 Protein Basics – 2a Maturation / Modification  protein –Folding (1 o -> 2 o -> 3 o agg -> 4 o ) 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.

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

9. BIOMAN 2009 - K. Lampe, MC3 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

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

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

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

13. BIOMAN 2009 - K. Lampe, MC3 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

14. BIOMAN 2009 - K. Lampe, MC3 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

15. BIOMAN 2009 - K. Lampe, MC3 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

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

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

18. BIOMAN 2009 - K. Lampe, MC3 #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

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

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

21. BIOMAN 2009 - K. Lampe, MC3 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

22. BIOMAN 2009 - K. Lampe, MC3 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 !

23. BIOMAN 2009 - K. Lampe, MC3 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

24. BIOMAN 2009 - K. Lampe, MC3 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

25. BIOMAN 2009 - K. Lampe, MC3 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

26. BIOMAN 2009 - K. Lampe, MC3 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

27. BIOMAN 2009 - K. Lampe, MC3 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

28. BIOMAN 2009 - K. Lampe, MC3 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

29. BIOMAN 2009 - K. Lampe, MC3 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

30. BIOMAN 2009 - K. Lampe, MC3 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

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

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