Downstream Processing in Biopharmaceuticals

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  • Continuous multichamber disc-stack centrifuge. The bowl contains a
    number of parallel discs providing a large clarifying surface with a small sedimentation distance. The sludge (cells) is removed through
  • This flow chart is more simplified than the other one. What do you think?
    For “separation graphic” show image of the chromatographic apparatus instead of a box.
    Combine fractions box: Graphic of little tubes pouring into a bigger beaker.
    We need to fit in a short text block with a description of the steps in order using numbers to label the steps.
  • ANIMATION.
    Show cartoon of microbial cells or tissue.
    Break the cells
    Have colors which show cellular membranes and junk contrasting with the proteins into solution.
  • User clicks the PLOT VALUES button.
    Animation: A graph grows.
  • After the animation ends
    The next appears and the user is prompted to discard the fractions that don’t belong by clickin on the tubes.
    Go to next screen to see what it might look like.
    Should the A280 value table go away? And just leave the profile to “declutter” the screen.
    Peter feels strongly that they should not be encouraged to throw away at this point. Perhaps set aside would be a better term, or eliminate altogether.
  • they are prompted to perfom an enzyme activity assay on the remaining tubes.
    They are prompted to click the “Enzyme Assay” button.
    Go to next screen to see results.
  • Show the color of activity tapers off in intensity.
    Go to next screen.
    Since so few assays use color, Peter would like these to be absorbance values.
  • Creates a gentle squeezing action to move fluid through flexible tubing.
    In this example three rollers on rotating arms pinch the tube against an arc and move the fluid along. There are usually three or four sets of rollers
  • Should these link to a Glossary? I don’t think so.
    The student should not worry if they don’t understand the techniques at this point. This is just to introduce some terminology in context.
    Is the table a good idea? If so, it needs to be more complete.
    JB Table is a good idea - we don’t want the students to consider SDS PAGE to be a purification technique, though, so I removed it from the table.
  • More background content needed? Theory of it?
    We need the info on the proteins.
    Should the proteins all be the same size?
  • Students click on “run column”
    Go to next slide to see results.
  • Just a next button?
    I might have gotten the charges mixed up. Let’s make sure we get this straight.
    We might need a legend (or something of the kind) on every page???
  • Do we need more content on what is size? How is it measure? MW.
    Pick real numbers is Kd correct?
    Show them all repeated together.
    Should these proteins be more blob like than perfect circles?
    Each color represents a different size/charge.
    Perhaps we should develop a little “protein” profile next to each blob.
    Protein 1.
    60 kD
    I think that we should make it so that we make one of the proteins the one that the student wants to purify.
    In both Gel filtration and Ion exchange, let’s make it so that neither method works.
    The final point we can make is that you have to do both separation methods in sequence.
  • ANIMATION on next.
  • Downstream Processing in Biopharmaceuticals

    1. 1. Downstream ProcessingDownstream Processing in Biopharmaceuticals; anin Biopharmaceuticals; an IntroductionIntroduction Angel L. Salaman, PhDAngel L. Salaman, PhD
    2. 2. Know the Characteristics of Your ProteinKnow the Characteristics of Your Protein Ex. Human Serum AlbuminEx. Human Serum Albumin Sequence of AminoSequence of Amino AcidsAcids MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGEMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVADEHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEPESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGKERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGACYLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVALLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDRRLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDAADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRRIPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKLHPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQKHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLILVSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETYNRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKPVPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLVKATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV WSTQTALAWSTQTALA Tertiary StructureTertiary Structure
    3. 3. Know the Characteristics of YourKnow the Characteristics of Your ProteinProtein Human Serum Albumin:Human Serum Albumin:  MW (molecular weight = 69,000MW (molecular weight = 69,000 Daltons (69 kD)Daltons (69 kD)  pI (isoelectric point) = 5.82pI (isoelectric point) = 5.82  Hydropathicity (=hydrophobicity) =Hydropathicity (=hydrophobicity) = -.395-.395
    4. 4. LARGE SCALE PROTEIN PRODUCTION Transfected cells grown to confluence in 10 x T175 flasks Wash with sterile PBS to remove contaminant proteins from serum (BSA) Culture cells in serum free medium (growth arrest) 3 x medium exchange every 48/76 hours CONDITIONED MEDIUM READY FOR PURIFICATION
    5. 5. EASY 2 STEPS PROTEIN PURIFICATION AFFINITY CHROMATOGRAPHY GEL FILTRATION 0 500 Absorptionat280nm(mAU) 1000 1500 2000 2500 500 mM Imidazole -45kDa Elution volume (ml) Vo 10 15 20 25 0 500 1000 1500 Absorptionat280nm(mAU) 2000 -45kDa
    6. 6. GLYCOSYLATIONGLYCOSYLATION – Mammalian sugar chains have highlyMammalian sugar chains have highly complex structurescomplex structures – Good for functional studiesGood for functional studies – Big problem for protein crystallizationBig problem for protein crystallization SOLUTIONSSOLUTIONS – Mutagenesis of glycosylation sitesMutagenesis of glycosylation sites – Enzymatic deglycosylationEnzymatic deglycosylation – Engineered cell lines (CHO Lec strains)Engineered cell lines (CHO Lec strains) – Chemical inhibitors of glycosylationChemical inhibitors of glycosylation pathwaypathway – Insect cells (simple sugars)Insect cells (simple sugars)
    7. 7. DDR2 Receptor Tyrosine KinaseDDR2 Receptor Tyrosine Kinase – 3 N-linked glycosylation sites in3 N-linked glycosylation sites in ectodomainectodomain – Predicted MW = 42 kDaPredicted MW = 42 kDa Mutagenesis Enzymatic deglycosylation CHO Lec 3.2.8.1 Stable transfectant -50kDa -40kDa -40kDa -50kDa -50kDa -40kDa wt wtmut deg Lec
    8. 8. Typical Protein Production Process FlowTypical Protein Production Process Flow (Feed 2) (Feed 3) (Feed 4) Chrom 1 Chrom 3 Cryo-preservation Concentration / Diafiltration Centrifuge Viral Removal Filtration (Feed1)Inoculum Expansion (Spinner Bottles) Ampule Thaw Chrom 2
    9. 9. Media Prep 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 Collection Centrifuge 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 Filtering Ultra Filtration Diafiltration Bulk Fill Purification 24 days 31 days 8 days 1 day Mfg Process OverviewMfg Process Overview
    10. 10. cGMP Pilot Plant Manufacturing FacilitycGMP Pilot Plant Manufacturing Facility Small Scale Manufact. DSP 1 DSP 2 DSP 3 DSP 4 Small Scale Manufact. Medium Scale Manufact. Large Scale Manufacturing Media/ Buffer Prep. Equipment Wash Inoc. Break Room Toilets/ Lockers Toilets/ Lockers Toilet Toilet Toilets/ Lockers Toilets/ Lockers Return Clean Street / Plant Employee Entrance Visitors/ Admin. Entrance Waste Dock Shipping & Receiving Lab Dock Support Lobby Support QC Lab Future Expansion Warehouse Dispensary Warehouse Shipping & Receiving Offices Building Utilities Maintenance Waste Staging Cylinder/Solvent Staging Filling Suite Waste Stage Process Utilities
    11. 11. Clarification orClarification or Removal of Cells andRemoval of Cells and Cell DebrisCell Debris Using CentrifugationUsing Centrifugation (Using Depth Filtration)(Using Depth Filtration)
    12. 12. Continuous CentrifugationContinuous Centrifugation Media and Cells In & Clarified Media OutMedia and Cells In & Clarified Media Out
    13. 13. Separation of particles from liquid by applying a pressure to the solution to force the solution through a filter. Filters are materials with pores. Particles larger than the pore size of the filter are retained by the filter. Particles smaller than the pore size of the filter pass through the filter along with the FiltrationFiltration
    14. 14. Uses cross flow to reduce build up of retained components on the membrane surface Allows filtration of high fouling streams and high resolution Tangential Flow FiltrationTangential Flow Filtration
    15. 15. Tangential Flow Filtration – TFFTangential Flow Filtration – TFF Separation of Protein of InterestSeparation of Protein of Interest Using TFF with the right cut off filters, the protein ofUsing TFF with the right cut off filters, the protein of interest can be separated from other proteins andinterest can be separated from other proteins and molecules in the clarified medium.molecules in the clarified medium. HSAHSA has a molecular weight of 69KD. To make surehas a molecular weight of 69KD. To make sure that the protein of interest is retained, a 10KD cut-that the protein of interest is retained, a 10KD cut- off filter is used.off filter is used. After we concentrate or ultrafilter our protein, we canAfter we concentrate or ultrafilter our protein, we can diafilter, adding the phosphate buffer at pH 7.1diafilter, adding the phosphate buffer at pH 7.1 that we will use to equilibrate our affinity columnthat we will use to equilibrate our affinity column to prepare for affinity chromatography ofto prepare for affinity chromatography of HSAHSA..
    16. 16. Overview of TFF SOPOverview of TFF SOP  Prepare buffer:Prepare buffer: Sodium phosphate buffer pH 7.1Sodium phosphate buffer pH 7.1  Set up the apparatus-Set up the apparatus-CAUTION Stored in NaOHCAUTION Stored in NaOH  Flush with water-Flush with water-CAUTION Stored in NaOHCAUTION Stored in NaOH Adjust flow rate to 30-50ml/minAdjust flow rate to 30-50ml/min Flush retentate lineFlush retentate line Flush permeate lineFlush permeate line  Precondition with buffer (just the permeate line)Precondition with buffer (just the permeate line)  Perform TFFPerform TFF  Prepare cleaning solution (NaOH)Prepare cleaning solution (NaOH)  Flush with waterFlush with water  Flush with NaOH to clean and storeFlush with NaOH to clean and store
    17. 17. Downstream Processing EquipmentDownstream Processing Equipment Lab-Scale TFF SystemLab-Scale TFF System Large-Scale TFFLarge-Scale TFF SystemSystem
    18. 18. Lab-Scale TFF Filter = Pall’sLab-Scale TFF Filter = Pall’s PelliconPellicon
    19. 19. How TFF Concentrates andHow TFF Concentrates and DiafiltersDiafilters the Protein of Interestthe Protein of Interest
    20. 20. Low PressureLow Pressure ProductionProduction ChromatographyChromatography The System: Components andThe System: Components and ProcessesProcesses The Media: Affinity, IonThe Media: Affinity, Ion Exchange, HydrophobicExchange, Hydrophobic Interaction ChromatographyInteraction Chromatography and Gel Filtrationand Gel Filtration
    21. 21. 22 Sample Separation technique FractionationFractionation Purification is a Multi-Step Procedure. Is there activity?Set aside NN oo Combine Fractionsyesyes Monitor purityMonitor purity Assay total protein Assay enzyme activity Pure? Prepare for analytical technique yesyes NN oo Repeat with anotherRepeat with another separationseparation technique until puretechnique until pure
    22. 22. 23 General Protein PurificationGeneral Protein Purification SchemeScheme • Grow cells in media (vector+tag) •Bacteria Suspension •Bioreactor Purification Strategy Expression SDS PAGE Assay Solubility Aggregation Recombination Characterization Mass Spectroscopy X-ray Crystallography Functional Assay Lyse the cells (appropriate buffer) Centrifuge Collect the pellet
    23. 23. 24 1. Evaluate an assay for the protein of interest 2. Shortlist a method to have a reasonable source for that activity Set Protein Purification Strategy
    24. 24. 25 Preparing the samplePreparing the sample (Crude Extract)(Crude Extract) Protein from cells or tissueProtein from cells or tissue Microbial cellsMicrobial cells or tissueor tissue Break cells,Break cells, Blender,Blender, homogenizer,homogenizer, sonication,sonication, pressurepressure osmoticosmotic Pellet with intactPellet with intact cells, organelles,cells, organelles, membranes andmembranes and membrane proteinsmembrane proteins Supernatant withSupernatant with Soluble proteinSoluble protein
    25. 25. 26  As the column separates the proteins inAs the column separates the proteins in the mixture, the “the mixture, the “ effluenteffluent ” drips into a” drips into a series of fraction tubes that are movingseries of fraction tubes that are moving at a specific rate of speed. These tubesat a specific rate of speed. These tubes are calledare called fractionsfractions..  Here we are showing 20 tubes. FractionHere we are showing 20 tubes. Fraction collectors in most labs have about 75-collectors in most labs have about 75- 200 tubes.200 tubes.  How do we know which fractions containHow do we know which fractions contain protein? Total protein a can be estimatedprotein? Total protein a can be estimated by taking the absorbance at 280 nm in aby taking the absorbance at 280 nm in a spectrophotometer. Aromatic aminospectrophotometer. Aromatic amino acids absorb light at this wavelengthacids absorb light at this wavelength causing all proteins to have absorbancecausing all proteins to have absorbance at 280nm. Many fraction collectorsat 280nm. Many fraction collectors measure the A280 as the column ismeasure the A280 as the column is running.running. Collect fractions.Collect fractions.
    26. 26. 27 A280 Plot valuesPlot values 00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # Question 1. How do we knowQuestion 1. How do we know which fractions contain protein?which fractions contain protein?
    27. 27. 28 • Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. • The values can be plotted against the fraction number in is what is called an elution profile. • Notice the peaks on the graph. These indicate where the fractions are that contain protein. Question 1. How do we know which fractions contain protein? A280 00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fraction # A280 Fraction # PeaksPeaks
    28. 28. 29 • Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein. Which fractions contained the desired protein? A280 00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A280 Fraction # Enz. Assay.Enz. Assay.Enz. Assay.Enz. Assay. Fraction #
    29. 29. 30 • Enzyme activity can beEnzyme activity can be determined by performing andetermined by performing an enzyme assay on each fractionenzyme assay on each fraction that contains protein.that contains protein. • Notice the results of the enzymeNotice the results of the enzyme assay. The highest activityassay. The highest activity corresponds to one of the peaks.corresponds to one of the peaks. • Now we can have them discardNow we can have them discard tubes that don’t have enzymetubes that don’t have enzyme activity.activity. Which fractions contained the desired enzyme? A280 00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A280 Fraction # EnzAssay Results
    30. 30. Downstream Processing EquipmentDownstream Processing Equipment Lab ScaleLab Scale ChromatographyChromatography SystemSystem Large ScaleLarge Scale ChromatographyChromatography SystemSystem
    31. 31. Peristaltic PumpPeristaltic Pump  Creates a gentleCreates a gentle squeezing action tosqueezing action to move fluid throughmove fluid through flexible tubing.flexible tubing.
    32. 32. 33 The Way to ChromatographyThe Way to Chromatography  In order to isolate sufficient quantities ofIn order to isolate sufficient quantities of protein, you may need to start with kilogramprotein, you may need to start with kilogram quantities of source (i.e. bacteria, tissues,quantities of source (i.e. bacteria, tissues, etc.) These amounts can best be handledetc.) These amounts can best be handled using precipitation methods (e.g.using precipitation methods (e.g. ammonium sulfate precipitation). Later inammonium sulfate precipitation). Later in the purification, large columns can be usedthe purification, large columns can be used to handle gram to milligram quantities.to handle gram to milligram quantities. Amounts handled on gels are typically inAmounts handled on gels are typically in microgram quantities.microgram quantities.
    33. 33. Liquid Column ChromatographyLiquid Column Chromatography ProcessProcess  Purge Air from System with Equilibration BufferPurge Air from System with Equilibration Buffer  Pack Column with Beads (e.g. ion exchange, HIC,Pack Column with Beads (e.g. ion exchange, HIC, affinity or gel filtration beads)affinity or gel filtration beads)  Equilibrate Column with Equilibration BufferEquilibrate Column with Equilibration Buffer  Load Column with Filtrate containing Protein ofLoad Column with Filtrate containing Protein of Interest in Equilibration BufferInterest in Equilibration Buffer  Wash Column with Equilibration BufferWash Column with Equilibration Buffer  Elute Protein of Interest with Elution Buffer of HighElute Protein of Interest with Elution Buffer of High or Low Salt or pHor Low Salt or pH  Regenerate Column or Clean and StoreRegenerate Column or Clean and Store
    34. 34. LP LC ComponentsLP LC Components  Mixer for Buffers, Filtrate with Protein ofMixer for Buffers, Filtrate with Protein of Interest, Cleaning SolutionsInterest, Cleaning Solutions  Peristaltic PumpPeristaltic Pump  Injector to Inject Small Sample (in ourInjector to Inject Small Sample (in our case for HETP Analysis)case for HETP Analysis)  Chromatography Column and MediaChromatography Column and Media (Beads)(Beads)  Conductivity MeterConductivity Meter  UV DetectorUV Detector
    35. 35. UV DetectorUV Detector Detects proteins coming out of theDetects proteins coming out of the column by measuring absorbancecolumn by measuring absorbance at 280nmat 280nm
    36. 36. Conductivity MeterConductivity Meter  Measures the amount of salt in theMeasures the amount of salt in the buffers – high salt or low salt arebuffers – high salt or low salt are often used to elute the protein ofoften used to elute the protein of interest from the chromatographyinterest from the chromatography beads.beads.  Also measures the bolus of salt thatAlso measures the bolus of salt that may be used to determine themay be used to determine the efficiency of column packing (HETP)efficiency of column packing (HETP)
    37. 37. 38 PropertyProperty MethodsMethods SolubilitySolubility PrecipitationPrecipitation with ammoniumwith ammonium sulfate (saltingsulfate (salting out)*out)* Size / shapeSize / shape Size-exclusionSize-exclusion chromotographychromotography IsoelectricpoIsoelectricpo int (charge)int (charge) Ion exhangeIon exhange chromatographychromatography binding tobinding to smallsmall moleculesmolecules AffinityAffinity chromatographychromatography Common methods of protein purification *Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme.  Purification procedures attempt toPurification procedures attempt to maintain the protein in native form.maintain the protein in native form. Although some proteins can beAlthough some proteins can be re-natured, most cannot!re-natured, most cannot!  To purify a protein from a mixture,To purify a protein from a mixture, biochemists exploit the ways thatbiochemists exploit the ways that individual proteins differ from oneindividual proteins differ from one another. They differ in:another. They differ in:  Thermal stabilityThermal stability: For most protein: For most protein purifications, all steps are carriedpurifications, all steps are carried out at ~5°C to slow downout at ~5°C to slow down degradation processes.degradation processes.
    38. 38. 39 Picture of protein gelPicture of protein gel with lanes showingwith lanes showing sequential purificationsequential purification stepssteps ProcedProced ureure FractioFractio n voln vol (ml)(ml) TotalTotal ProtProt (mg)(mg) ActivityActivity (units)(units) SpecificSpecific activityactivity Units/Units/ mgmg CrudeCrude cellularcellular extractextract 14001400 1000010000 100,000100,000 1010 Size-Size- exclusioexclusio nn 9090 400400 80,00080,000 200200 IonIon exchangexchang ee 8080 100100 60,00060,000 600600 Note: The type and order of steps are customized for each protein to be purified. An effective purification step results in a high yield (minimal loss of enzyme activity) and large purification factor (large increase in specific activity). Purification YieldPurification Yield
    39. 39. 40 Chromatographic ModeChromatographic Mode AcronymAcronym Separation PrincipleSeparation Principle Non-interactive modes of liquid chromatographyNon-interactive modes of liquid chromatography Size-exclusion chromatographySize-exclusion chromatography SECSEC Differences in molecular sizeDifferences in molecular size Agarose chromatography (forAgarose chromatography (for DNA) for DNA binding proteinsDNA) for DNA binding proteins -- Diff. in length and flexibilityDiff. in length and flexibility Interactive modes of liquid chromatographyInteractive modes of liquid chromatography Ion-exchange chromatographyIon-exchange chromatography IECIEC Electrostatic interactionsElectrostatic interactions Normal-phase chromatographyNormal-phase chromatography NPCNPC Polar interactionsPolar interactions Reversed-phase chromtographyReversed-phase chromtography RPCRPC Dispersive interactions*Dispersive interactions* Hydrophobic interactionHydrophobic interaction chromatographychromatography HICHIC Dispersive interactions*Dispersive interactions* Affinity chromatographyAffinity chromatography ACAC Biospecific interactionBiospecific interaction Metal interactionMetal interaction chromatographychromatography MICMIC Complex w/ an immobilizedComplex w/ an immobilized metalmetal Chromatographic Modes of Protein Purification * Induced dipole – induced dipole interactions
    40. 40. 41 Column Selection
    41. 41. 42 Affinity Chromatography Surface bound with Epoxy, aldehyde or aryl ester groups Metal Interaction Chromatography Surface bound with Iminodiacetic acid + Ni2+ /Zn2+ /Co2+ Affinity Chromatography
    42. 42. 43 Metal Interaction Chromatography (AC) Points to Note: 1. Avoid chelating agents 2. Increasing incubation time 3. Slow gradient elution
    43. 43. 44 Affinity Chromatography Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa) Depends on Molecular weight Degree of substitution /ml medium ~15mmol Ni2+ Backpressure ~43psi Change the guard column filter
    44. 44. 45 Biopolymer (phenyl agarose - Binding Surface) Driving force for hydrophobic adsorption Water molecules surround the analyte and the binding surface. When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy. Temperature plays a strong role Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer Hydrophobic Interaction Chromatography Hydrophobic region
    45. 45. 46 ION –EXCHANGE 1ION –EXCHANGE 1  First, to determine theFirst, to determine the charge on a protein, given itscharge on a protein, given its pI and the pH.pI and the pH.  Ion-exchange columnIon-exchange column chromatography separateschromatography separates proteins on the basis ofproteins on the basis of charge.charge.  We will start with 4 proteins.We will start with 4 proteins.  pH 7.2pH 7.2  Positive charged columnPositive charged column 60 Kd Low pI (6) 20 Kd Low pI (7) 20 Kd Medium pI (7) 5 Kd Hi pI (8)
    46. 46. 47 pos • The matrix of an ion exchange is positively charged. • What do you think will happen?pos pos pos pos pos pos Run columnRun column pos pos pos pos pos pos
    47. 47. 48 • The matrix of an ion exchange is positively charged. • Only the pos charged proteins run through the pos charged column. The others “stick” to the column. pos pos pos pos pos pos pos pos pos pos pos pos pos
    48. 48. 49 Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen) Ion Exchange Chromatography Globular Protein Deformation due to interaction with conventional ion exchanger Maintenance of conformation while interacting with tentacle ion exchanger
    49. 49. 50 Gel Filtration
    50. 50. 51  Gel filtration columnGel filtration column chromatography separateschromatography separates proteins on the basis of size.proteins on the basis of size.  We will start with 4 proteins.We will start with 4 proteins.  You will want to purify theYou will want to purify the “yellow one”“yellow one” 60 Kd Low pI (6) 20 Kd Low pI (7) 20 Kd Medium pI (7) 5 Kd Hi pI (8) Gel Filtration
    51. 51. 52  The matrix of a size-exclusionThe matrix of a size-exclusion chromatography column ischromatography column is porous beads.porous beads. Run columnRun column
    52. 52. 53  The matrix of a gel filtrationThe matrix of a gel filtration column are beads withcolumn are beads with pores.pores.  The largeThe large graygray proteinsproteins can’t fit in pores so flowscan’t fit in pores so flows faster.faster.  TheThe redred // yellowyellow mediummedium sized proteins get trapped insized proteins get trapped in the pores.the pores.  TheThe blackblack small proteinssmall proteins stay trapped in pores longer.stay trapped in pores longer.
    53. 53. 54 Immune Affinity Chromatography
    54. 54. 55 ATP immobilized on polyacrylamide resin DNA Binding Proteins Heparin Sepharose Negatively charged proteins (pI >7) are not captured/separated effectively.
    55. 55. 56 Capillary Electrochromatography • CEC is an electrokinetic separation technique • Fused-silica capillaries packed with stationary phase • Separation based on electro-osmotically driven flow • Higher selectivity due to the combination of chromatography and electrophoresis Fused silica tube filled with porous methacrylamide-stearyl methacrylate-dimethyldiallyl ammonium chloride monolithic polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000 Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N number of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline
    56. 56. 58 CEC columns AC, IEC columns CEC column NP, RP columns
    57. 57. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
    58. 58. ComponentComponent Culture HarvestCulture Harvest LevelLevel Final Product LevelFinal Product Level Conventional MethodConventional Method Therapeutic AntibodyTherapeutic Antibody 0.1-1.5 g/l0.1-1.5 g/l 1-10 g/l1-10 g/l UF/CromatographyUF/Cromatography IsoformsIsoforms VariousVarious MonomerMonomer ChromatographyChromatography Serum and host proteinsSerum and host proteins 0.1-3.0 g/l0.1-3.0 g/l < 0.1-10 mg/l< 0.1-10 mg/l ChromatographyChromatography Cell debris and colloidsCell debris and colloids 101066 /ml/ml NoneNone MFMF Bacterial pathogensBacterial pathogens VariousVarious <10<10-6-6 /dose/dose MFMF Virus pathogensVirus pathogens VariousVarious <10<10-6-6 /dose (12 LRV)/dose (12 LRV) virus filtrationvirus filtration DNADNA 1 mg/l1 mg/l 10 ng/dose10 ng/dose ChromatographyChromatography EndotoxinsEndotoxins VariousVarious <0.25 EU/ml<0.25 EU/ml ChromatographyChromatography Lipids, surfactantsLipids, surfactants 0-1 g/l0-1 g/l <0.1-10 mg/l<0.1-10 mg/l ChromatographyChromatography BufferBuffer Growth mediaGrowth media Stability mediaStability media UFUF Extractables/leachablesExtractables/leachables VariousVarious <0.1-10 mg/l<0.1-10 mg/l UF/ ChromatographyUF/ Chromatography Purification reagentsPurification reagents VariousVarious <0.1-10mg/l<0.1-10mg/l UFUF Common Process Compounds and Methods of Purification or Removal
    59. 59. 61 REFERENCESREFERENCES  Christian G. Huber, Biopolymer Chromatography, EncylcopediaChristian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000in analytical chemistry, 2000  www.qiagen.comwww.qiagen.com  www.novagen.comwww.novagen.com  http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromhttp://lsvl.la.asu.edu/resources/mamajis/chromatography/chrom atography.htmlatography.html  http://www.cellmigration.org/resource/discovery/discovery_protehttp://www.cellmigration.org/resource/discovery/discovery_prote omics_approaches.htmlomics_approaches.html  http://www.capital-hplc.co.ukhttp://www.capital-hplc.co.uk  http://www.ls.huji.ac.il/~purificationhttp://www.ls.huji.ac.il/~purification  www.biovectra.comwww.biovectra.com  http://www.ls.huji.ac.il/~purificationhttp://www.ls.huji.ac.il/~purification

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