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WatersEssentialsin Bioresearch

rinciplesandpractice of

proteinpurificationby ion exchange

WatersD*wsJon at MI[LIPORE

WATERS ESSENTIALS IN BIORESEARCH

PRINCIPLES AND PRACTICE OFPROTEIN PURIFICATION BY

ION EXCHANGE

' WatersD_visionof MI[[IPORE

EXPERIMENTAL DESIGN

1. Gather Information

2. Select Column and Operating Conditions

3. Perform Initial Experiment

4. Optimize Separation Conditions

5. Isolate Required Mass6. Assess Results and Plan Next Step

INFORMATION USED TO PLAN EXPERIMENTS

1. Specific Protein or Biological Activity• Assay Procedure

• Solubility

• Stability• Size• Isoelectric Point

• Experience and Literature

2. Starting Material and Extraction3. Amount Required

4. Degree of Purity Required

MODES OF PROTEIN CHROMATOGRAPHY

• Ion Exchange• Gel Filtration

• Hydrophobic Interaction• Reversed Phase

• Affinity

ADVANTAGES OF ION EXCHANGE

• Applicable to Virtually All Proteins

• Generally Excellent Recovery of Biological Activity

• Very High Resolving Power Based on Subtle Charge Differences

• Works Well with Large Volumes of Dilute Samples• Highest Mass Capacity

• Numerous Options for Improving Separation

COLUMN SELECTION

1. Anion or Cation Exchanger

2. Strong or Weak Exchanger

3. Functional Group

4. Supporting Material and Particle Size5. Physical Dimensions

6. Capacity and Economics

2

GUIDELINES FOR ION EXCHANGE

CHARGE 0 p l

CATIOI",; ."FXCIIANGER ANION - '

pH

ION EXCHANGE PACKING MATERIALS

ANION CATION

STRONG QUATERNARY AMINES SULFONIC ACID

SULFOPROPYL

WEAK DEAE CARBOXYMETHYL

MODERN PACKING MATERIALS FOR ION EXCHANGE

• Familiar Chemistries on Small, Uniform Particles:5 - 40 p• ReducedBandBroadening

BetterSeparation

BetterSensitivity• FasterSeparations

• Easier to PredictablyControlSeparation- Use of Instrument

- CompleteChemicalEquilibrium

- RapidPhysicalEquilibrium

SELECTION OF PARTICLE SIZE

• Larger Particles

Easy to Pack in Large Volume Columns For High CapacityCan Be Used at Higher Flow Rates or Linear VelocitiesMore Economical

- Often Preferred for Early Purification Steps• Smaller Particles

- Generally Better Resolution

- Generally Smaller Peak Volume

FACTORS IN DEFINING COLUMN DIMENSIONS

• Mass Capacity Is Proportional to Column Volume

• Peak Volume Is Proportional to Column Volume

• Operating Conditions Adjusted to Column Dimensions

- Flow Rate Proportional to Cross-Sectional Area- Gradient Duration as a Constant Number of

Column Volumes

STEPS IN DEFINING COLUMN DIMENSIONS

• Establish Separation Conditions on Small Column

• Define the Mass Capacity of the Small Volume Column

by Injecting Progressively Larger Samples Until the

Separation is No Longer Useful• Transfer Conditions to a Proportionately Larger Volume

Column for the Required Mass Load

SELECTING CONDITIONS FOR FIRST RUN

• Use pH 0.5-1.0 Units from Isoelectric Point if Known

• Select Buffer, Often 20mM, with pK Near Desired pH

- With Anion Exchange, Use Cationic Buffer, e.g., Tris

- With Cation Exchange, Use Anionic Buffer, e.g., Phosphate• Equilibrate with 5-10 Column Volumes of Initial Buffer

• Inject Sufficient Sample to Detect in Specific Activity Assay;

Proteins Will Usually Elute in about 1ml from a 3-8ml Column• Run Linear Gradient to 1000mM NaCI Over 10 Column Volumes

EVALUATE SEPARATION

• Monitor Protein Profile by Absorbance at 280nm; Enhanced

Sensitivity may be obtained at Lower Wavelengths, e.g.,

220nm for Tris or 214nm for Phosphate

• Monitor Specific Activity with Defined Assay• Assess Heterogeneity of Active Fractions with Alternative

Separation Technique

IMPROVING THE PURIFICATION

• Change to a Different Column Using the Same Separation Mode,

e.g., Supporting Material, Functional Group, Brand, etc.

• Change to a Different Separation Mode• Adjust Operating Conditions to Obtain Best Resolution and

Recovery from the Column Chemistry in Use

5

IMPROVING PROTEIN SEPARATIONS

....ELUTE LATER [ RETENTION, K" ]

......CF.IANGF THE RELA'TIVE RETENTION5 { SELECTIVITY,(_{ ]

MAKE THE PEAKS NARROWER ( SHARPER )

REDUCE DILUTION DURING THE SEPARATION{ EFFICIENCY, N]

OPTIMIZING GRADIENT SLOPE

• Determine Ionic Strength that Elutes the Protein of Interest

Measure Conductivity or Estimate from System Volume

• Program Steep Initial Gradient Segment to 5mM less thanElution Point

• Program Shallow Segment, 25mM/col.vol., around Elution Point

• Program Steep Final Segment to 500-1000mM NaCI to Regenerate• Evaluate Separation and Repeat as Necessary

EVALUATION OF GRADIENT CONDITIONS

COLUMN: Protein-Pak'" DEAL 8HR, 10mmX 100mm

GRADIENT: 0 - 200mM NaCI in 20mM Tris-HCI, pH 7.5

FLOW: t .5ml/min

DURATION: TIME COLUMN VOLUMES SLOPE (raM / Col. Vol.)10 2 100

20 4 5040 8 25

80 16 12.5

6

EFFECT OF GRADIENT DURATION ON RETENTION

70 - - i

• Soybean Trypsin Inh_bltc,r60- i

"2 iso- ,_'

¢' 40 OvaibumlnEI-c 30 -ra:_s'errln0 I

C3nalbuIn:r:2(.' i

0 i i _ i i

2 4 5 8 !0 12 I" 15 8

6rad_en_DuraNon In ColJ,Tn Volu'_,es

COMPARISON OF 2 AND 16 COLUMN VOLUME GRADIENTS

0006- S

T. Trarlslerr,_ _ I Io- c.,.,_,,o I}_ II I is. so,_,o ,.,_........ h/l i! i_

o_ ____ _.2 L_21)

M.n_te,s

C S

..... o II

oo(x

70Minutes

COMPARISON OF 2 AND 16 COLUMN VOLUME GRADIENTS

S

0.0061o/S= Soybean Trypsin Inhibitor

A" ° AO.O00_CoL Vol.

ltOc°_v°'. . .........o 70

Minutes

7

EFFECT OF GRADIENT DURATION ON PEAK VOLUME

6.05.5 • Ovalbumin

5.0 •G"

4.5-

E 4.0"

o 3.5>

3.0

_" 2.5

2.0

1.50 2 4 6 8 1; 1'2 1"4 1"6 18

Gradient Duration in Column Volumes

NET CHARGE OF A PROTEIN AS A FUNCTION OF pHAMINO ACID SIDE CHAINS

LYS

t CYS _._,J

INCREASING J

NEGATIVE

CHARGEGLU

1 2 3 4 5 6 _ 8 9 I'0 I'I I'2

pH

SELECTION OF pH FOR OPTIMUM SELECTIVITY

NET0

CHARGE

+

pH

8

EFFECT OF THREE-DIMENSIONAL STRUCTURE ON SELECTIVITY

RETENTION AS A FUNCTION OF BUFFER pHCONDITIONS FORANION EXCHANGE

COLUMN: Protein-Pak'" DEAE 8HR, 10 X 100mmFLOW: 1.5ml/min

GRADIENT: 0 - 200mM NaCI, 8 Column VolumesBUFFER: 20mM Tris-HCI

pH 7.0 / pH 7.5 / pH 8.0 / pH 9.0

EFFECT OF BUFFER pH ON PROTEIN RETENTIONANION EXCHANGE

$

T. Ttansfor_

O- Ova_burn_ O_ !S- Soybean Trypsin Inh/_ltC¢ II

C T

0000

40MJnutes

c'r

40

9

EFFECT OF BUFFER pH ON PROTEIN RETENTIONANION EXCHANGE

I¢, Soybean Trypsin Inhibitor

"tv30

m Ova:_urr.ln

- - 4c T,"ans'err in_o 20 _ ...... * ......

• _ C°nalouiln

Go.

10

7 75 80 8.5 90

Buffer- pH

EFFECT OF BUFFER pH ON PROTEIN RETENTIONCONDITIONS FOR CATION EXCHANGE

COLUMN: Protein-Pak'" SP 8HR, 10mm X 100mm

FLOW: 1.5ml/min

GRADIENT: 0 - 300mM NaCI, 8 Column Volumes

BUFFER: 20mM Sodium Phosphate

pH 6.0 / pH 6.5 / pH 7.0 / pH 7.5 / pH 8.0

EFFECT OF BUFFER pH ON PROTEIN RETENTION

CATION EXCHANGE

51

c

LysozymeEr:31

c C_,tochrome co

•_ oe-Chyrrlotrypsino_:en

II . i . Ribcr.__l.._. A60 65 70 75 80

Buffer F.,I=

10

EFFECT OF BUFFER pH ON PROTEIN RETENTIONCATION EXCHANGE

l C

R- n.bonu_ease A. pl.9 S

A- ALphaChymmq, Ps_o0en. pl.9 I ' L

c:c;,_,Oo7,:.%,0, = . .

i;iio0 5O

M,nu_esC

I

M,nutes

EFFECT OF BUFFER ON PROTEIN RETENTION

COLUMN: Protein-Pak'"SP 8HR, 10mm X 100mmFLOW: 1.5ml/min

GRADIENT: 0 - 300mM NaCI, 8 Column Volumes

BUFFERS: 20mM Sodium Phosphate, pH 5.520mM Sodium Acetate, pH 5.5

EFFECT OF BUFFER ON PROTEIN RETENTION

A. ACha ChymOtryps_nogen CR. R_onuc_ase A A

0012 _ifI i[[I

C- C_OChrOmec

L= LySOZyrne R

hIi

0001

5OM=nu!es

C

II

5OM,nutos

11

EFFECT OF COUNTERION ON PROTEIN RETENTION

COLUMN: Protein-Pak TM DEAE 8HR, 10ram X 100ramFLOW: 1.5ml/min

BUFFER: 20raM Tris-HCI, pH 7.5

GRADIENT: 0-200raM Sodium Chloride, 8 Column Volumes

0-200raM Sodium Acetate, 8 Column Volumes

EFFECT OF COUNTERION ON PROTEIN RETENTION

S C- Conarou_n

0 004 C T. Transfernn

t _ I O-OvaJb ...._,omc,,o,,_B o II s.So_=,ant !!' I_ II "_.........

o 6oC Minutes

°°t I o

Minutes

EFFECT OF TEMPERATURE ON PROTEIN RETENTION

COLUMN: Protein-Pak" DEAE 5PW, 7.5ram X 75ram

FLOW: 1.0ml/min

BUFFER: 20raM Tris-HCI, pH 7.5

GRADIENT: 0-200raM Sodium Chloride, 9 Column Volumes

Hold 200raM Sodium Chloride, 5 Column Volumes

200-800raM Sodium Chloride, Step

12

EFFECT OF TEMPERATURE ON PROTEIN RETENTION

1"

it ....0 030 T. Tfansferr,n

O- Ovall_u_n

So Soyboan

Ec h O T_p$,n Inh,bntor

0_.

M,nut_ 5O

T

M_utes 5O

CONTROL OF ION EXCHANGESEPARATIONS

• Retention Is Optimized by Adjustment of Ionic Strength

• Selectivity Is Most Conveniently Optimized with pH

• Changing Buffer and Counterion May Improve Selectivity

• Methods May Require Adjustment If The Temperature Is Changed

SUGGESTED EXPERIMENTAL APPROACH

FOR PROTEIN ION EXCHANGE

• Select Buffer pH to Minimize Retention

• Equilibrate Column with 5-10 Column Volumes of Initial Buffer

• Inject Aliquot of Sample and Elute with a Gradient to 1000mM

Sodium Chloride Over 5-10 Column Volumes

• Assay Fractions for Specific Activity

• Change pH 0.5 Units, Equilibrate, and Repeat

• Adjust Gradient Duration and End Points

• Final Conditions Based on Such Factors as Recovery,

Specific Activity, and Sample Volume

]3

WATERS AUTO-BLEND" METHOD

FOR OPTIMIZING PROTEIN SEPARATIONS

I

(/_ Acidic Buffer ]Precise combination of stock buffers L

/_ to automatically produce the desired pH Precise generation of linear

! (Curve 6) or non-linear gradientsfor optimal sample resolulion

Basic Buffer andrecoveryGradient Curve Shapes

(/_ Salt Solution 1_1

IPrecise addition ol stock salt soluhon J

/_ when required to effect the separationMilli-Q Water s_arts_,,_._ T_e EnOs,g,,_n_

AUTO-BLEND TM TABLES FOR TRIS AND PHOSPHATE

Auto.Blend'- T_ble Iot Tr,s Belier System

9 A_ 01M "r.s HO

_o 0 IM T.S_

o _ • 6 s Ic Iz i* _6 ,e 2_

PO:ls A where Parts A, ParlS B • 2O

Auto.U._" Table Ior Phospt_lle Butter Syslem_5

5 A 0 _M N3t12PO4

o Bb 0 IM Na2HPO4

S5

o _ • 6 6 '0 17 ,. I_ ,I 2o

Parts A whe,e Parts A • Parts B • 20

MOUSE LIVER EXTRACT

SEPARATION OF LACTATE _me Row°/oA°JoB°/oC°_OCurveIN 1.0 18 2 0 80

DEHYDROGENASE, pH7.2 30 1.o 1B 2 Boo 633 10 18 2 80 0 6

AUFS 35 1.0 18 2 0 80 6I 0- 55 1.0 18 2 0 80 6

Minutes 0

14

MOUSE LIVER EXTRACT

SEPARATION OF LACTATE Time Row %A %B %C %0 CurveIN 1,0 15 5 0 80

DEHYDROGENASE, pH7.7 30 1.o is s 80 o 633 1.0 15 5 80 0 6

AUFS 35 1,0 15 5 0 80 61.0 55 10 15 5 0 80 6

o

_J0 i = = =

Minutes 30

MOUSE LIVER EXTRACT

SEPARATION OF LACTATE Time Row %A %B %C %D CurveIN 1,0 8 12 0 80

DEHYDROGENASE, pH8.4 30 1.0 8 12 80 0 633 1,0 8 12 80 0 6

AUFS 3S 1.0 8 12 0 80 6,.0 ss 1.0 8 12 0 80 6

z

Minutes 30

MOUSE LIVER EXTRACT

SEPARATION OF LACTATE Time Flow %A %B %C %0 CurveIN 1.0 3 17 0 80

DEHYDROGENASE, pH8.6 30 1.o 3 17 80 o 633 1.0 3 17 80 0 6

AUFS 3S 1.0 3 17 0 80 6lO- ss 1.o 3 lz o 80 6

0 i w i0 " Minutes 30

15

SEPARATION OF ASCITES FLUID

CONDITIONS OPTIMIZED FOR IgG

Ilbumin

Initial Separallon

I_1_1 1.0 6 14 0 80 20mMTIL_CJ. pH853O 1.0 19 1 3O 5O 2¢mMTr_.PH?O

_m 03M NaCi

1..50+ 40 1 0 lg 1 3O

x

transferrin Monoclonll IgA

0.00

o.oo ,.'oo _.'oo _.'oo ,.'oox 101 minutes

SEPARATION OF ASCITES FLUID

CONDITIONS OPTIMIZED FOR IgA

Monoclonl[ IgA

Optimized Separation

hl_J 10 6 14 0 80 20mMTr_.C_.pH85

3.00" 10 1+0 12 8 15 65 20mMTtls/CJ. pH80I_th 0 ISM NaCI

20 10 18 1 50 30 20mMTI_I. pHT0w_h0.SM NaCl

3O 1 0 19 1 5O 30

E °2oo

x J _mln

1.00' trans_rrln

o.oo ,.'oo ;,._ +,.oo ' '4,00

• 101 i|nUttl

SUMMARY OF ION EXCHANGE

• Small Particle Packings and Appropriate Instrumentation Increase

Control of the Separation Process

• Gradient Slope Is a Compromise Between Resolution and

Peak Volume

• Ion Exchange Chromatography IsOptimized by Adjusting pH

and Ionic Strength

• Auto-Blend" Method Facilitates Purification

• Ion Exchange Chromatography Can Be Effectively Used at an Early

Stage in the Purification Process

]6

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