Downstream Processing in Biopharmaceuticals

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Downstream Downstream Processing in Processing in Biopharmaceuticals; Biopharmaceuticals; an Introduction an Introduction Angel L. Salaman, PhD Angel L. Salaman, PhD

description

Downstream Processing

Transcript of Downstream Processing in Biopharmaceuticals

Page 1: Downstream Processing in Biopharmaceuticals

Downstream Processing Downstream Processing in Biopharmaceuticals; an in Biopharmaceuticals; an

IntroductionIntroduction

Angel L. Salaman, PhDAngel L. Salaman, PhD

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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

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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

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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

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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

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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)

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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

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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

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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

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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

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Clarification or Clarification or Removal of Cells and Removal of Cells and

Cell DebrisCell DebrisUsing CentrifugationUsing Centrifugation

(Using Depth Filtration)(Using Depth Filtration)

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Continuous CentrifugationContinuous CentrifugationMedia and Cells In & Clarified Media OutMedia and Cells In & Clarified Media Out

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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

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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

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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..

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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

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Downstream Processing EquipmentDownstream Processing Equipment

Lab-Scale TFF SystemLab-Scale TFF System

Large-Scale TFF SystemLarge-Scale TFF System

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Lab-Scale TFF Filter = Pall’s Lab-Scale TFF Filter = Pall’s PelliconPellicon

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How TFF Concentrates and How TFF Concentrates and DiafiltersDiafilters

the Protein of Interestthe Protein of Interest

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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

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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

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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?

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• 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

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• 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#

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• 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

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Downstream Processing EquipmentDownstream Processing Equipment

Lab Scale Lab Scale Chromatography SystemChromatography System Large Scale Large Scale

Chromatography SystemChromatography System

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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.

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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.

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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

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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

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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

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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)

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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

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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

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Column SelectionColumn Selection

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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

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Metal Interaction Chromatography (AC)Metal Interaction Chromatography (AC)

Points to Note:

1. Avoid chelating agents

2. Increasing incubation time

3. Slow gradient elution

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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

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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)

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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

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• 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

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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

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Gel FiltrationGel Filtration

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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

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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

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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.

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Immune Affinity ChromatographyImmune Affinity Chromatography

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ATP immobilized on polyacrylamide resin

DNA Binding ProteinsDNA Binding Proteins

Heparin SepharoseNegatively charged proteins (pI >7) are not captured/separated effectively.

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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

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CEC columns AC, IEC columns

CEC columnNP, RP columns

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHYHIGH PERFORMANCE LIQUID CHROMATOGRAPHY

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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

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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