Downstream Processing in Biopharmaceuticals
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Transcript of Downstream Processing in Biopharmaceuticals
Downstream Processing Downstream Processing in Biopharmaceuticals; an in Biopharmaceuticals; an
IntroductionIntroduction
Angel L. Salaman, PhDAngel L. Salaman, PhD
Know the Characteristics of Your Protein Know the Characteristics of Your Protein Ex. Human Serum AlbuminEx. Human Serum Albumin
Sequence of Amino AcidsSequence of Amino AcidsMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGEMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE
EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVADEHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD
ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP
ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGKERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK
YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGACYLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC
LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA
RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDRRLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR
ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDAADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA
IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR
HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKLHPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL
KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQKHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ
VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL
NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETYNRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY
VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKPVPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP
KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLVKATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV
WSTQTALAWSTQTALA
Tertiary StructureTertiary Structure
Know the Characteristics of Your Know the Characteristics of Your Protein Protein
Human Serum Albumin:Human Serum Albumin: MW (molecular weight = 69,000 Daltons MW (molecular weight = 69,000 Daltons
(69 kD)(69 kD) pI (isoelectric point) = 5.82pI (isoelectric point) = 5.82 Hydropathicity (=hydrophobicity) = -.395Hydropathicity (=hydrophobicity) = -.395
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
EASY 2 STEPS PROTEIN PURIFICATION
AFFINITY CHROMATOGRAPHY
GEL FILTRATION
0
500
Ab
sorp
tio
n a
t 28
0 n
m (
mA
U)
1000
1500
2000
2500
500 mM Imidazole
-45kDa
Elution volume (ml)Vo 10 15 20 25
0
500
1000
1500
Ab
sorp
tio
n a
t 28
0 n
m (
mA
U)
2000
-45kDa
GLYCOSYLATIONGLYCOSYLATION
– Mammalian sugar chains have highly Mammalian 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 glycosylation Chemical inhibitors of glycosylation
pathwaypathway– Insect cells (simple sugars)Insect cells (simple sugars)
DDR2 Receptor Tyrosine KinaseDDR2 Receptor Tyrosine Kinase
– 3 N-linked glycosylation sites in 3 N-linked glycosylation sites in ectodomainectodomain
– Predicted MW = 42 kDaPredicted MW = 42 kDa
Mutagenesis Enzymaticdeglycosylation
CHO Lec 3.2.8.1Stable transfectant
-50kDa-40kDa -40kDa
-50kDa
-50kDa-40kDa
wt wtmut deg Lec
Typical Protein Production Process FlowTypical Protein Production Process Flow
(Feed 2)
(Feed 3)
(Feed 4)
Chrom 1Chrom 3
Cryo-preservation
Concentration / Diafiltration
Centrifuge
Viral Removal Filtration
(Feed1)Inoculum Expansion(Spinner Bottles)Ampule Thaw
Chrom 2
Media PrepMedia 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
CollectionCollection
CentrifugeCentrifuge
Harvest/Recovery
HarvestCollection
Tank
1,500L
HarvestCollection
Tank
1,500L
FilterChromatography
Skid
Anion Exchange Chromatography (QXL)
ColumnEluateHold Tank
8,000L
EluateHold Tank
8,000L
EluateHold Tank
6,000L
EluateHold Tank
6,000L
FilterChromatography
Skid
Protein A Chromatography
Column
Chromatography Skid
Column
EluateHold Tank
20,000L
EluateHold Tank
20,000L
Hydrophobic Interaction Chromatography (HIC)
EluateHold Tank
20,000L
EluateHold Tank
20,000L
Viral Inactivation
EluateHold Tank
5,000L
EluateHold Tank
5,000L
FilterChromatography
Skid
Anion Exchange Chromatography
(QFF - Fast Flow)
Column
Post-viralHold
Vessel3,000L
Post-viralHold
Vessel3,000L
Viral Filtering Ultra FiltrationDiafiltration
Bulk Fill
Purification
24 days 31 days
8 days
1 dayMfg Process OverviewMfg Process Overview
cGMP Pilot Plant Manufacturing FacilitycGMP Pilot Plant Manufacturing Facility
SmallScale
Manufact.
DSP 1 DSP 2 DSP 3 DSP 4
SmallScale
Manufact.
MediumScale
Manufact.
LargeScale
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
SupportLobby
Support
QC Lab
Future Expansion
WarehouseDispensary
Warehouse
Shipping & Receiving
Offices Building Utilities
Maintenance
Waste Staging
Cylinder/Solvent Staging
Filling Suite
Waste Stage Process Utilities
Clarification or Clarification or Removal of Cells and Removal of Cells and
Cell DebrisCell DebrisUsing CentrifugationUsing Centrifugation
(Using Depth Filtration)(Using Depth Filtration)
Continuous CentrifugationContinuous CentrifugationMedia and Cells In & Clarified Media OutMedia and Cells In & Clarified Media Out
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 thefilter are retained by the filter.
Particles smaller than the pore size of the filter pass through the filter along with the
liquid.
FiltrationFiltration
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 Filtration Tangential Flow Filtration
Tangential Flow Filtration – TFFTangential Flow Filtration – TFFSeparation of Protein of InterestSeparation of Protein of Interest
Using TFF with the right cut off filters, the protein of Using TFF with the right cut off filters, the protein of interest can be separated from other proteins and interest 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 sure has 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 can After we concentrate or ultrafilter our protein, we can diafilter, adding the phosphate buffer at pH 7.1 diafilter, adding the phosphate buffer at pH 7.1 that we will use to equilibrate our affinity column to that we will use to equilibrate our affinity column to prepare for affinity chromatography of prepare for affinity chromatography of HSAHSA..
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/minFlush retentate lineFlush retentate lineFlush 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
Downstream Processing EquipmentDownstream Processing Equipment
Lab-Scale TFF SystemLab-Scale TFF System
Large-Scale TFF SystemLarge-Scale TFF System
Lab-Scale TFF Filter = Pall’s Lab-Scale TFF Filter = Pall’s PelliconPellicon
How TFF Concentrates and How TFF Concentrates and DiafiltersDiafilters
the Protein of Interestthe Protein of Interest
Low Pressure Low Pressure Production Production
ChromatographyChromatographyThe System: Components and The System: Components and
ProcessesProcesses
The Media: Affinity, Ion The Media: Affinity, Ion Exchange, Hydrophobic Exchange, Hydrophobic
Interaction Chromatography Interaction Chromatography and Gel Filtration and Gel Filtration
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Sample Separationtechnique
FractionationFractionationFractionationFractionation
Purification is a Multi-Step Procedure.Purification is a Multi-Step Procedure.
Is there activity?Set asideNNooNNoo
CombineFractionsyesyesyesyes Monitor purityMonitor purityMonitor purityMonitor purity
Assay total protein
Assay enzyme activity
Pure?
Prepare for analytical technique
yesyesyesyes
NNooNNoo
Repeat with Repeat with another another
separationseparationtechnique until technique until
purepure
Repeat with Repeat with another another
separationseparationtechnique until technique until
purepure
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General Protein Purification General Protein Purification SchemeScheme
General Protein Purification General Protein Purification SchemeScheme
• Grow cells in media (vector+tag)•Bacteria Suspension•Bioreactor
Purification Strategy
ExpressionSDS PAGE Assay
SolubilityAggregation
Recombination
CharacterizationMass Spectroscopy
X-ray CrystallographyFunctional Assay
Lyse the cells (appropriate buffer)Centrifuge Collect the pellet
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1. Evaluate an assay for the protein of interest2. Shortlist a method to have a reasonable source for that activity
Set Protein Purification StrategySet Protein Purification Strategy
25
Preparing the sample Preparing the sample (Crude Extract)(Crude Extract)
Preparing the sample Preparing the sample (Crude Extract)(Crude Extract)Protein from cells or tissueProtein from cells or tissueProtein from cells or tissueProtein from cells or tissue
Microbial cells Microbial cells or tissueor tissueMicrobial cells Microbial cells or tissueor tissue
Break cells,Break cells,
Blender, Blender, homogenizer, homogenizer, sonication,sonication,pressurepressureosmoticosmotic
Break cells,Break cells,
Blender, Blender, homogenizer, homogenizer, sonication,sonication,pressurepressureosmoticosmotic Pellet with intact Pellet with intact
cells, organelles, cells, organelles, membranes and membranes and membrane proteinsmembrane proteins
Pellet with intact Pellet with intact cells, organelles, cells, organelles, membranes and membranes and membrane proteinsmembrane proteins
Supernatant withSupernatant withSoluble protein Soluble protein Supernatant withSupernatant withSoluble protein Soluble protein
26
As the column separates the proteins As the column separates the proteins in the mixture, the “in the mixture, the “effluenteffluent” drips ” drips into a series of fraction tubes that are into a series of fraction tubes that are moving at a specific rate of speed. moving at a specific rate of speed. These tubes are called These tubes are called fractionsfractions..
Here we are showing 20 tubes. Here we are showing 20 tubes. Fraction collectors in most labs have Fraction collectors in most labs have about 75-200 tubes.about 75-200 tubes.
How do we know which fractions How do we know which fractions contain protein? Total protein a can be contain protein? Total protein a can be estimated by taking the absorbance at estimated by taking the absorbance at 280 nm in a spectrophotometer. 280 nm in a spectrophotometer. Aromatic amino acids absorb light at Aromatic amino acids absorb light at this wavelength causing all proteins to this wavelength causing all proteins to have absorbance at 280nm. Many have absorbance at 280nm. Many fraction collectors measure the A280 fraction collectors measure the A280 as the column is running.as the column is running.
Collect fractions.Collect fractions.Collect fractions.Collect fractions.
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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 20Fraction #
Question 1. How do we know Question 1. How do we know which fractions contain protein?which fractions contain protein?Question 1. How do we know Question 1. How do we know which fractions contain protein?which fractions contain protein?
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.
• 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?
Question 1. How do we know which fractions contain protein?
A28000 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 #
PeaksPeaksPeaksPeaks
29
• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.
• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.
Which fractions contained the desired protein?
A28000 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 20Fraction#
A280
Fraction #
Enz. Assay.Enz. Assay.Enz. Assay.Enz. Assay.
Fraction#
30
• Enzyme activity can be Enzyme activity can be determined by performing an determined by performing an enzyme assay on each fraction enzyme assay on each fraction that contains protein.that contains protein.
• Notice the results of the enzyme Notice the results of the enzyme assay. The highest activity assay. The highest activity corresponds to one of the peaks.corresponds to one of the peaks.
• Now we can have them discard Now we can have them discard tubes that don’t have enzyme tubes that don’t have enzyme activity.activity.
• Enzyme activity can be Enzyme activity can be determined by performing an determined by performing an enzyme assay on each fraction enzyme assay on each fraction that contains protein.that contains protein.
• Notice the results of the enzyme Notice the results of the enzyme assay. The highest activity assay. The highest activity corresponds to one of the peaks.corresponds to one of the peaks.
• Now we can have them discard Now we can have them discard tubes that don’t have enzyme tubes that don’t have enzyme activity.activity.
Which fractions contained the desired enzyme?Which fractions contained the desired enzyme?
A28000 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 20Fraction#
A280
Fraction #
EnzAssayResults
Downstream Processing EquipmentDownstream Processing Equipment
Lab Scale Lab Scale Chromatography SystemChromatography System Large Scale Large Scale
Chromatography SystemChromatography System
Peristaltic PumpPeristaltic Pump
Creates a gentle Creates a gentle squeezing action to squeezing action to move fluid through move fluid through flexible tubing.flexible tubing.
33
The Way to The Way to Chromatography Chromatography
The Way to The Way to Chromatography Chromatography
In order to isolate sufficient quantities of In order to isolate sufficient quantities of protein, you may need to start with protein, you may need to start with kilogram quantities of source (i.e. kilogram quantities of source (i.e. bacteria, tissues, etc.) These amounts bacteria, tissues, etc.) These amounts can best be handled using precipitation can best be handled using precipitation methods (e.g. ammonium sulfate methods (e.g. ammonium sulfate precipitation). Later in the purification, precipitation). Later in the purification, large columns can be used to handle large columns can be used to handle gram to milligram quantities. Amounts gram to milligram quantities. Amounts handled on gels are typically in handled on gels are typically in microgram quantities.microgram quantities.
Liquid Column Chromatography Liquid 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 of Load Column with Filtrate containing Protein of
Interest in Equilibration Buffer Interest in Equilibration Buffer Wash Column with Equilibration BufferWash Column with Equilibration Buffer Elute Protein of Interest with Elution Buffer of High Elute 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
LP LC ComponentsLP LC Components
Mixer for Buffers, Filtrate with Protein of Mixer for Buffers, Filtrate with Protein of Interest, Cleaning SolutionsInterest, Cleaning Solutions
Peristaltic PumpPeristaltic Pump Injector to Inject Small Sample (in our case Injector to Inject Small Sample (in our case
for HETP Analysis)for HETP Analysis) Chromatography Column and Media Chromatography Column and Media
(Beads)(Beads) Conductivity MeterConductivity Meter UV DetectorUV Detector
UV DetectorUV Detector
Detects proteins coming out of the Detects proteins coming out of the column by measuring absorbance column by measuring absorbance at 280nmat 280nm
Conductivity MeterConductivity Meter
Measures the amount of salt in the Measures the amount of salt in the buffers – high salt or low salt are buffers – high salt or low salt are often used to elute the protein of often used to elute the protein of interest from the chromatography interest from the chromatography beads.beads.
Also measures the bolus of salt that Also measures the bolus of salt that may be used to determine the may be used to determine the efficiency of column packing (HETP)efficiency of column packing (HETP)
38
PropertyProperty MethodsMethods
SolubilitySolubility Precipitation Precipitation with ammonium with ammonium sulfate (salting sulfate (salting out)*out)*
Size / shapeSize / shape Size-exclusion Size-exclusion chromotography chromotography
IsoelectricpoIsoelectricpoint (charge)int (charge)
Ion exhange Ion exhange chromatographychromatography
binding to binding to small small moleculesmolecules
Affinity Affinity chromatographychromatography
Common methods of protein purificationCommon 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 Purification procedures attempt to maintain the protein in native to maintain the protein in native form. Although some proteins form. Although some proteins can be re-natured, most cannot!can be re-natured, most cannot!
To purify a protein from a To purify a protein from a mixture, biochemists exploit the mixture, biochemists exploit the ways that individual proteins ways that individual proteins differ from one another. They differ from one another. They differ in: differ in:
Thermal stabilityThermal stability: For most : For most protein purifications, all steps protein purifications, all steps are carried out at ~5°C to slow are carried out at ~5°C to slow down degradation processes.down degradation processes.
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Picture of protein Picture of protein gel with lanes gel with lanes showing sequential showing sequential purification stepspurification steps
ProcedProcedure ure
FractioFraction voln vol
(ml)(ml)
Total Total ProtProt
(mg)(mg)
ActivityActivity
(units)(units)Specific Specific activityactivity
Units/Units/mgmg
Crude Crude cellular cellular extractextract
14001400 1000010000 100,000100,000 1010
Size-Size-exclusioexclusionn
9090 400400 80,00080,000 200200
Ion Ion exchangexchangee
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 YieldPurification YieldPurification Yield
40
Chromatographic ModeChromatographic Mode AcronymAcronym Separation PrincipleSeparation Principle
Non-interactive modes of liquid chromatographyNon-interactive modes of liquid chromatography
Size-exclusion Size-exclusion chromatographychromatography SECSEC Differences in molecular sizeDifferences in molecular size
Agarose chromatography (for Agarose chromatography (for DNA) for DNA binding DNA) for DNA binding proteinsproteins
-- Diff. in length and flexibilityDiff. in length and flexibility
Interactive modes of liquid chromatographyInteractive modes of liquid chromatography
Ion-exchange Ion-exchange chromatographychromatography IECIEC Electrostatic interactionsElectrostatic interactions
Normal-phase Normal-phase chromatographychromatography NPCNPC Polar interactionsPolar interactions
Reversed-phase Reversed-phase chromtographychromtography RPCRPC Dispersive interactions*Dispersive interactions*
Hydrophobic interaction Hydrophobic interaction chromatographychromatography HICHIC Dispersive interactions*Dispersive interactions*
Affinity chromatographyAffinity chromatography ACAC Biospecific interactionBiospecific interaction
Metal interaction Metal interaction chromatographychromatography MICMIC Complex w/ an immobilized Complex w/ an immobilized
metalmetal
Chromatographic Modes of Protein PurificationChromatographic Modes of Protein Purification
* Induced dipole – induced dipole interactions* Induced dipole – induced dipole interactions
41
Column SelectionColumn Selection
42
Affinity Chromatography
Surface bound with
Epoxy, aldehyde or aryl ester groups
Metal Interaction Chromatography
Surface bound with
Iminodiacetic acid + Ni2+/Zn2+/Co2+
Affinity ChromatographyAffinity Chromatography
43
Metal Interaction Chromatography (AC)Metal Interaction Chromatography (AC)
Points to Note:
1. Avoid chelating agents
2. Increasing incubation time
3. Slow gradient elution
44
Affinity ChromatographyAffinity Chromatography
Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa)
Depends on Molecular weight
Degree of substitution /ml medium~15mmol Ni2+
Backpressure ~43psiChange the guard column filter
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Biopolymer (phenyl agarose - Binding Surface)
Driving force for hydrophobic adsorptionWater 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 ChromatographyHydrophobic Interaction Chromatography
Hydrophobic region
46
ION –EXCHANGE 1 ION –EXCHANGE 1 ION –EXCHANGE 1 ION –EXCHANGE 1 First, to determine the First, to determine the
charge on a protein, charge on a protein, given its pI and the pH.given its pI and the pH.
Ion-exchange column Ion-exchange column chromatography chromatography separates proteins on the separates proteins on the basis of charge.basis of charge.
We will start with 4 We will start with 4 proteins.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)
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 columnpos
pos pos
pos
pos
pos
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
49
Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen)
Ion Exchange ChromatographyIon Exchange Chromatography
Globular Protein
Deformation due to interaction with conventional ion
exchanger
Maintenance of conformation while
interacting with tentacle ion exchanger
50
Gel FiltrationGel Filtration
51
Gel filtration column Gel filtration column chromatography separates chromatography 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 the You 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 Gel Filtration
52
The matrix of a size-The matrix of a size-exclusion chromatography exclusion chromatography column is porous beads.column is porous beads.
Run columnRun column
53
The matrix of a gel The matrix of a gel filtration column are filtration column are beads with pores.beads with pores.
The large The large graygray proteins proteins can’t fit in pores so flows can’t fit in pores so flows faster.faster.
The The redred / / yellowyellow medium sized proteins medium sized proteins get trapped in the pores.get trapped in the pores.
The The blackblack small proteins small proteins stay trapped in pores stay trapped in pores longer.longer.
54
Immune Affinity ChromatographyImmune Affinity Chromatography
55
ATP immobilized on polyacrylamide resin
DNA Binding ProteinsDNA Binding Proteins
Heparin SepharoseNegatively charged proteins (pI >7) are not captured/separated effectively.
56
Capillary ElectrochromatographyCapillary 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/Nnumber of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline
58
CEC columns AC, IEC columns
CEC columnNP, RP columns
HIGH PERFORMANCE LIQUID CHROMATOGRAPHYHIGH PERFORMANCE LIQUID CHROMATOGRAPHY
ComponentComponent Culture Harvest Culture 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
61
REFERENCESREFERENCESREFERENCESREFERENCES
Christian G. Huber, Biopolymer Chromatography, Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000Encylcopedia in analytical chemistry, 2000
www.qiagen.comwww.qiagen.com www.novagen.comwww.novagen.com http://lsvl.la.asu.edu/resources/mamajis/http://lsvl.la.asu.edu/resources/mamajis/
chromatography/chromatography.html chromatography/chromatography.html http://www.cellmigration.org/resource/discovery/http://www.cellmigration.org/resource/discovery/
discovery_proteomics_approaches.html discovery_proteomics_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