Israel lectures 4/10-5/10/2010

Quality Control:

Alternatives to Reversed-Phase

Chromatography for Protein

and Peptide Analysis

Andrew Alpert

PolyLC Inc.

Columbia, MD U.S.A.

TOPICS

1) Effect of pore and particle diameter

2) Effect of organic solvents

3) Alternatives to reversed-phase HPLC for

protein variantsa) Size-Exclusion

b) HIC

c) Ion-exchange

d) HILIC

4) Alternatives for peptide QC

min7 8 9 10 11 12 13 14

mAU

0

100

200

300

400

500

600

700

800

Growth Factor on PolyCAT A: Comparison of 3- vs. 5-µm

COLUMNS:

35x4.6-mm; 1000-Å

MINOR VARIANTS

RESOLVED WITH

THE 3-µm COLUMN

DISULFIDE TRISULFIDE

Effect of Pore Diameter

- Resolution is better

with wider pores –

Sample: rec Human Growth

Hormone (21 kDa)

COLUMN: PolyPROPYL A

(Hydrophobic Interaction

Chromatography)

- Data courtesy of Benny

Welinder, Novo Nordisk -

0,0 10,0 20,0 30,0 40,0 55,0

-25

50

100

150

200

250mA

min

3

2

1

WVL:214 nm

40% 2-PrOH

20% 2-PrOH

0% 2-PrOH

Column: 102SE0510(PolySULFOETHYL A)

Mobile Phase:

A: 20mM KH2PO4, pH3.0

B: A + 500mM NaCl

Gradient:

0% B in 5min, 0% to 20% B in 10 min, 20% B to 40% B in 30min, 40% B to 0% B in 5min and 0% B in 5min

Flow:

0.3 ml/min

SCX Analysis of multiPEGylated Protein

- selectivity increases with % solvent -

Effect of Solvents on Protein HPLC

OPTIMUM %

PROTEIN SOLVENT

bFGF 0

Bovine 20Somatotropin

Many growth factors 30-40

Histones, 60-70

Membrane proteins

Optimal concentration must bedetermined on a case-by-case basis

SEC(separation by size)

SEC-TOF-MS of intact proteins: Reduced Antibody (L.J. Brady et al., J. Am. Soc. Mass Spectrom. 19 (2008) 502)

Mobile phase: 0.1% formic acid,0.1 ml/min

Column: PolyHYDROXYETHYL A,250x2.1 mm, 300-Å;

An SEC column was chosen with

pores narrow enough for intact

proteins to elute in the Vo peak,

which is run into the MS. Total

analysis time: 8’.

Light chain MS Heavy chain MS

Screening Combinatorial Libraries for Potential Drug Candidates via ALIS:

Automated Ligand Identification System

(D.A. Annis et al., Int. J. Mass Spectrom. 238 (2004) 77)

Combinatorial Drug Screening via ALIS:

Theory Practice

Target protein

+ bound high

affinity ligands

(sent to mass

spectrometer)

Nonbound low

affinity ligands

(sent to waste)

IMPORTANT: The Vo peak must elute in < 30 seconds or even

high-affinity ligands will start to diffuse off the target protein.

Vo VtCOLUMN:

PolyHYDROXYETHYL A; 60-Å

POLARITY MODES OF CHROMATOGRAPHY

HIC(Hydrophobic Interaction Chromatography)

Gradient: high to low [salt]; nondenaturing

Elution order: Least to most hydrophobic

on the surface of the tertiary (3-D)

structure

TIME (Min)

0.0 5.0 10.0 15.0 20.0 25.0

VO

LT

AG

E

0

100

200

300

400

500Fc

Fab

[O]

[O]

HIC of Fab and Fc Oxidation Products (Met[O])

COLUMN: PolyPROPYL A, 100x4.6-mm; 3-µm, 1500-Å

GRADIENT: Decreasing [(NH4)2SO4]

HIC is a good

way to separate

polarity variants of

proteins

Ion-Exchange

CATION-EXCHANGE OF PEPTIDES (SCX)

PolySULFOETHYL

Aspartamide

SCX of Degraded Peptide: Pramlintide (Amylin)

- Good selectivity for charge variants -

SEQUENCE (37 residues):

KCNTATCATQRLANFLVHSS-

NNFGPILPPTNVGSNTY-NH2

COLUMN: PolySULFOETHYL A

- from Hekman et al., J. Pharmaceut. Biomed. Anal. 20 (1999) 763-772 -

Cyclic

Imide-21

Cyclic Imides

-22 and -35

S-S

TIME (Min)

0.0 5.0 10.0 15.0 20.0

A22

0

0

40000

80000

120000

160000

phosphorylation

variants

Anion-Exchange of Ovalbumin Phosphorylation Variants

COLUMN: PolyWAX LP (#104WX0510) SAMPLE: Sigma Grade VI (99%)

GRADIENT: 10 mM K-PO4, pH 7.0; 60-300 mM NaCl in 20’

Polysuccinimide

Aminopropyl-

silica___

Poly(succinimide)-

silica_____

Poly(aspartic

acid)- silica _

(PolyCAT A)

Preparation of

a Weak

Cation-Exchange

(WCX) material

- from Alpert, J. Chromatogr. 266 (1983) 23-37 -

0

0.01

0.02

0.03

0.04

0.05

10 15 20 25 30

Ab

so

rban

ce

(2

80

nm

)

Time (minutes)

Lys9

Lys65

Lys68

Lys90

Lys22

[Met1+Lys9]

[Met1+Lys22]+

[Met1+Lys90]

Met1

(Lys25:

Not found)

PEGylation Positional Variants of TNF Soluble Receptor Type ICOLUMN: PolyCAT A (204CT0510) (data courtesy Scott Buckel - Amgen, Inc.)

GRADIENT: 30’, 1-160 mM KCl in 20 mM Na-OAc, pH 5.0, with 20% ACN

min0 20 40 60

23.7

90

31.0

32

46.2

34

22.3

77

CEX of Monoclonal AntibodyCOLUMN: PolyCAT A (204CT0510)

The 3-peak pattern is characteristic. Minor peaks usually

reflect additional deamidation, oxidation or sialylation

AFTER

CARBOXY-

PEPTIDASE B

TREATMENT

# Lys or Arg lost

from C-termini

of heavy chains

2

1

0 (native sequence)

Time (min)

0 5 10 15 20

Ab

so

rba

nc

e

0

20

40

60

80

100

CEX of Monoclonal AntibodyCOLUMN: PolySULFOETHYL A (104SE0315)

min0 10 20 30 40 5060

Norm.

0

10

20

30

40

min5 10 15 20 25 30354045

Norm.

0

10

20

30

40

50

min0 2.5 5 7.5 10 12.5 15 17.5

Norm.

0

20

40

60

80

100

PEGylated Growth Factor:

Comparison of three CEX columns

TSK SP-NPR;

selectivity OK

TSK SP-5PW;

capacity OK

unPEGylated

PEGylated

multi-

PEGylated

PolyCAT A (#104CT0315);

Good selectivity and capacity

min10 20 30 40 50 60

mAU

0

5

10

15

20

25

unPEGylated

PEGylated

Degraded PEGylation Reaction Mixture:

Analysis with a PolyCAT A column (item# 104CT0315)

“GIGO”:

“Garbage In,

Garbage Out”

DEGRADATION OF PROTEINS

- DEAMIDATION AND DEHYDRATION -1) Favored if the residue on the C-terminal side is unhindered (Gly; Ala)

2) Consequences for biological activity: Negligible to serious!

- H2O

- NH3

(pKa ~ 4.1)

(pKa ~ 3.1)

CASE STUDY: Degradation of Human Growth Hormone(from Benny Welinder [Novo Nordisk], WCBP ’98)

191 residues

total; pI = 5.0

Asn 149 and

Asn 152 (*):

prone to

deamidation

Asp 130 (**):

prone to

dehydration

Degradation of rec Human Growth Hormone: Effect of pH

High pH:

Deamidation

of Asn-

Neutral/Acid pH:

Dehydration

of Asp-

COLUMN: PolyCAT A

GRADIENT: 130-145

mM NH4–acetate, pH

4.0, with 40% ACN; 30°

DEAMIDATION AND DEHYDRATION OF rHGH

- Effect of pH on Kinetics of Variant Formation -

Cyclic imide-130 Desamido-149+

Desamido-152

isoAsp-130

1) Stability of rec HGH is maximal for formulation ~ pH 6.1.

2) IMPORTANT TO OBTAIN KINETICS DATA LIKE THIS FOR EVERY

BIOPHARMACEUTICAL IF DEAMIDATION IS A PROBLEM!

Method Development:

Analysis of a Growth Factor

via Cation-Exchange

AU

0.00

0.02

0.04

Minutes

16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

Growth Factor on PolyCAT A

- Effects of pore & particle size -

Best selectivity with

small particles &

wide pores

5 µm, 1000 Å

3 µm, 1000 Å

3 µm, 300 Å

Deamidation

variants

Met[O] or

incorrect-S-S-

(biologicallyinactive)

Growth Factor

Variants on

PolyCAT A

(104CT0315)

- batch selectivity

differences -

Lot-to-lot

differences

difficulty of

separation

Test different

lots to find the

selectivity your

protein requires

Lot #1

Lot #2

0% PEG

2% PEG

Analysis of

Growth Factor on

PolyCAT A

- PEG (Polyethylene

Glycol) sometimes

improves selectivity

when added to the

mobile phase -

Growth Factor Variants on

EDTA-treated PolyCAT A

column (104CT0315)

EDTA treatment passivates

metal surfaces

nonspecific protein

interactions (but may let

3-µm silica escape the frits!).

Test on a case-by-case basis.

after

- better separation of variants -

beforetreatment

A different growth factor

yields a similar profile in

cation-exchange HPLC

Structure of a Protein in the TGF-β Family (many growth factors):

Two ~ 120-aa Polypeptide Chains Linked in a Cysteine Knot

three -S-S- links

within each chain one -S-S- link between chains

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mV

olts

0

100

200

300

400

500

mV

olts

0

100

200

300

400

500

Cation-Exchange HPLC of BMP-14 (Bone Morphogenetic Protein 14)COLUMN: PolyCAT A, 100x4.6-mm; 3-µm, 1000-Å (item# 104CT0310; ser# A2562C)

MOBILE PHASE: A) 20 mM K-PO4, pH 6.0, with 20% ACN; B) Same + 0.6 M NaCl

GRADIENT: 20’ linear, 0-100% B, then 5’ at 100% B 1.0 ml/min A220

SAMPLE: 25 µg BMP-14 in 20 µl of (Mobile Phase A:10 mM HCl = 2:1)

Minutes

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0

mV

olts

0

2

4

6

8

10

12

14

mV

olts

0

2

4

6

8

10

12

14

BMP-14: Blowup to show deamidation variants

1

2

3

4

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mV

olts

0

5

10

15

20

25

30

35

mV

olts

0

5

10

15

20

25

30

35

BMP-14: More gradual gradient to improve separation of

variants; resembles preceding growth factor’s profile

QUESTION: Do all recombinant growth factors contain ~ 8%

Met[O] or mismatched -S-S- variants (@ cysteine knot motif)?

New shoulder;

deamidation variant?

Another new

shoulder?

Method Development:

Analysis of a Sialylated,

MultiPEGylated Protein

(GCSF) via Cation-Exchange

SCX Analysis of multiPEGylated Protein

- Effect of sialidase treatment -

COLUMN: 202SE0510 0.1 ml/min

MOBILE PHASE: 5 mM K-P04, pH 3.0, with 30% 1-PrOH + 10% 1-BuOH; 0-50 mM NaCl

0 13 25 38 50 63 75 88 110

-50

100

200

350mA

min

1

WVL:214 nm

20,0

50,0

0,0

20,0

0,0

before sialidase

after sialidase

PER MOLECULE:

4-5 PEGs (at Lys-);

1-2 sialic acid res.

10,0 25,0 37,5 50,0 62,5 75,0 87,5 100,0

-75

0

100

200

300

400

500mA

min

2

WVL:214 nm

Column: PolySULFOETHYL A, 200x2.1mm I.D., 5µm, 1000Å

Mobile Phase:A: 5mM KH2PO4, pH 3.0;

30% 1-PrOH, 10% 1-BuOH

B: A + 50mM NaCl

Flow: 0.1 ml/min

Gradient: 10 – 25 mM NaCl over 80 minInj. 30µg

SDS-PAGEApplication

SCX Analysis of multiPEGylated Protein

- Preliminary characterization before sialidase treatment -

10,0 25,0 37,5 50,0 62,5 75,0 87,5 100,0

-100

0

100

200

300mA

min

2

1

WVL:214 nm

+ Sialidase7.5 µg

- Sialidase7.5 µg

SDS-PAGE

SCX Analysis of multiPEGylated Protein

- Characterization after sialidase treatment; the

PAGE analysis permitted post-sialidase peaks to

be paired with their pre-sialidase versions -

tt Mixed-Bed

Ion-Exchange

HPLC of

Intact Proteins

Mixed-Bed IEX of E. coli Lysate Proteins

COLUMN (WCX): PolyCAT A, 200x4.6-mm; 5-µm, 1000-Å

GUARD CARTRIDGE (WAX): PolyWAX LP, 10x4-mm; 5-µm, 1000-Å

GRADIENT (40’ linear): 0-0.6 M NaCl in 10 mM KH2PO4, pH 6.2,

with 5% ACN

DETECTION: Fluorescence (ex = 280 nm; em = 350 nm)

WCX Column only (big void vol. peak)

WCX Column + WAX Cartridge (* = acidic proteins?)

* ** * *

4 5 6 7 8 9 10 11 12 130

200

400

600

800

1000

1200

# o

f p

rote

ins

pI value

pI Distribution of the Predicted Mouse Proteome

From: H. Wang et al.,

J. Proteome Res. 5

(2006) 361

Acidic proteins; Basic proteins;

Use anion-exchange Use cation-exchange

Minutes0 5 10 15 20 25

mV

olts

0

50

100

150

200

Minutes0 5 10 15 20 25

mV

olts

0

50

100

150

200

Minutes0 5 10 15 20 25

mV

olts

0

50

100

150

200

Anion-Exchange only

(104WX0510)

Cation-Exchange only

(104CT0510)

Mixed-Bed

(204CTWX0510)

Fig. 1. Comparison of regular IEX columns to the mixed-bed column. Sample: Yeast lysate. All

materials were 5-µm, 1000-Å pore diameter. The mixed-bed column was 200x4.6-mm while the others were

100x4.6-mm. Flow rate: 1 ml/min. Detection: A280. Gradient: 0-300 mM NaCl in 20 mM MES, pH 6.0.

A complex mixture of proteins always contains some that elute in the void volume on a single IEX column.

With a mixed-bed column the number that do this is minimal1,2. This helps to insure more uniform

distribution of the proteins for proteomics fractionations.

Minutes0 10 20 30 40 50

mV

olts

0

5

10

15

200

20

40

60

80

Blowup to show

low-abundance

proteins

Full-scale

Fig. 2. Mixed-bed IEX of yeast lysate with a volatile mobile phase.

COLUMN: Same mixed-bed as in Fig. 1. DETECTION: 280 nm

MOBILE PHASE: 20-800 mM ammonium acetate, pH 6.0 FLOW: 1 ml/min

GRADIENT: 0-12’: 0-10%B; 12-30’: 10-60%B; 30-40’: 60-100%B 40-50’: 100%B

Minutes0 10 20 30 40 50

mV

olts

0

200

400

Example of Sample Simplification via General-Purpose

Chromatography: Mixed-bed IEX-SPE of serumHPLC: Mixed-bed column (204CTWX0510) 1 ml/min.

Gradient: 3-segment, 20-800 mM NH4-OAc, pH 7.0 A280

Sample: 25µl serum + 75 µl 10 mM NH4-OAc

Minutes0 10 20 30 40 50

mV

olts

0

100

200

FiltrateFr.1 (50 mM)

Fr.2 (250 mM)

Fr.3 (400 mM)Fr.4 (800 mM)

mV

olts

0

200

400 Serum

IEX-SPE Fractions

Fractionation of

serum via mixed-

bed IEX-SPE

Analysis: Mixed-bed

HPLC column

Gradient: 20-800 mM

NH4-OAc, pH 7.0

Fractionationseems to be

lossless

Fractions from mixed-bed IEX-SPE of serum - blowup

SPE: 100 µg TopTip™ of PolyCAT A & PolyWAX LP; TT1000CATWAX-2015

HPLC: Mixed-bed column (204CTWX0510) 1 ml/min. A280 Gradient: 20-800 mM NH4-OAc, pH 7.0

Samples: 100µl of 200µl collected fraction (Filtrate: 100µl of 400µl)

Minutes0 10 20 30 40 50

A2

80

0

Filtrate

Fr.1 (50 mM)

Fr.2 (250 mM)

Fr.3 (400 mM)

Fr.4 (800 mM)

Result: reproducible

fractions with

minimal overlap

that elute in

predictable ranges

SPE-IEX Fraction 4 on Mixed-Bed IEX Column (fast flow):

Control, Gout and Rheumatoid Arthritis Serum Samples

Sample: 6 mg. of each serum 300 µl each Fraction 4; 100 µl analyzed per run

Gradient: 300-800 mM NH4-OAc, pH 7.0 (load at 20 mM) Column: 102CTWX0510

Flow: 0.8 ml/min Detection: 280 nm UHPLC System: SSI Corp.

MINUTES

0 2 4 6 8 10 12 14 16 18 20

AB

SO

RB

AN

CE

(m

AU

)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

GOUT

RA

CONTROL

CONTROL

(elevated)

(elevated)

HILIC(Hydrophilic Interaction Chromatography)

RPC-HILIC Purification of Variant Glycopeptides

from a Tryptic Digest of -Interferon

HILIC of Glycopeptide Asn-97 HILIC of Glycopeptide Asn-25

PolyHYDROXYETHYL A

(150x1.0-mm)

RPC (Vydac C-18) of

total digest from CHO

batch culture

ADJACENT PEAKS

DIFFER BY ONE

CARBOHYDRATE

RESIDUE OR

POSITION

Data courtesy of J. Zhang

and D. Wang (MIT)

HILIC of Intact Mitochondrial Membrane Proteins

Column: PolyHYDROXYETHYL A (100 x 2.1 mm)

Gradient: 20 mM ammonium formate, pH 3.7, + 0.5% HFIP; (63% 2-PrOH + 22.5% ACN) to 30% 2-PrOH

Sample: 2-PrOH/ACN (pH 3.7) extract of bovine heart mitochondria

From: Carroll et al (2006) PNAS 103, 16170-16175.

Courtesy of John Walker, Medical Research Council Dunn Human Nutrition Unit, Cambridge, U. K.

(ND3)

ND1

ND4

F-ATPase-a +

F-ATPase-A6L

ND2

CI-B9

CIII-IX

CIV-VIII

F-ATPase-g

+ CIV-VIIa+ CIV-VIIc

CIV-VIb

Usmg5

BRP44LBRP44

CIV-VIc + 6.8 KDa

proteolipid

F-ATPase-f

-20

30

80

130

180

0 5 10 15 20 25 30

Time [min]

Ab

so

rba

nc

e 2

80

nm

[a

.u.]

-20

30

80

130

0 5 10 15 20 25 30

Time [min]

Ab

so

rba

nc

e 2

80

nm

[a

.u.]

-20

80

180

280

0 5 10 15 20 25 30

Time [min]

Ab

so

rba

nc

e 2

80

nm

[a

.u.]

HILIC of Intact Apolipoproteins:Separation of Glycation Variants

Mobile Phase A: 50 mM NH4-formate, pH 3.7, in 50% 2-PrOH, 25% ACN, & 0.5% HFIP

Mobile Phase B: 50 mM formic acid + 10% 2-PrOH + 0.5% HFIP + ~ 5 mg/L Trp

Flow: 1 ml/min. Temp: 24°C Gradient: 0-5’: 0% B; 5-35’: 0-100% B

apoA-I (human)

apoA-I

Cyt C (equine)

Cyt C

apoM

(human)

apoM

COLUMN: PolyHYDROXYETHYL A,

200x4.6-mm; 5µm, 300-Å

SAMPLE: 100 µg

COLUMN: TSK Amide-80, 250x4.6-mm; 5µm, 80-Å

SAMPLE: 100 µg

COLUMN: TSK Amide-80, 250x4.6-mm; 5µm, 80-Å

SAMPLE: 1 mg apoM

aglyco-

complex

biantennary,

no Gal (20.5 KDa)

complex

non-biantennary,

2 Gal, 2 Neu5Ac

(20.9 KDa)

complex

non-biantennary,

2 Gal, 2 Neu5Ac

(21.2 KDa)

HILIC on an Ion-Exchange Column~ 60-70% organic solvent present

during the salt gradient

RESULTHydrophilic interactions superimposed

on electrostatic effects

Minutes

0 2 4 6 8 10 12 14

Volts

0

100

200

300

400

500

Volts

0

100

200

300

400

5000

.080

1.9

97

2.3

57

2.5

70

2.9

46

3.0

93

3.3

05

3.4

78

3.9

58

4.1

98

4.4

55

4.6

88

5.0

60

5.5

03

6.4

56

7.6

77

8.2

57

9.8

13

10.1

63

10.7

71

12.7

63

12.8

78

13.1

60

13.3

61

13.9

93

14.5

35

14.6

17

14.9

48

Detector 1-220nm

Retention Time

Minutes

0 2 4 6 8 10 12 14

Volts

0

50

100

150

200

250

Volts

0

50

100

150

200

250

0.0

78

0.2

44

2.0

58

2.2

53

2.5

88

3.1

22

3.3

80

3.7

51

4.1

98

4.4

75

5.2

08

5.3

90

5.8

62

7.4

95

7.9

98

8.4

78

9.1

06

9.6

55

10.3

68

10.9

55 1

1.7

96

12.5

98

13.2

38

14.3

80

Detector 1-220nm

Retention Time

ERLIC of synthetic peptide:

LIFAGKQLEDGR

COLUMN: PolyWAX LP,

200x4.6-mm, 5µm, 300Å

MOBILE PHASE:

20 mM sodium methylphosphonate,

pH 2.0, with %ACN as noted

70%

75%

min0 10 20 30 40 50 60 70

mAU

-20

0

20

40

60

80

100

120

140

DAD1 B, Sig=215,4 Ref=off (K:\HPCHEM\4\DATA\20516A5\003-0401.D)

SCX-HILIC of Lung

Surfactant Protein

(lipid:protein = 500:1)

COLUMN: PolySULFOETHYL A,

200x4.6-mm; 5-µm; 1000-Å

MOBILE PHASE (1 ml/min):

5’ hold, then 0-100% B in 60’

A) 0.1% methylphosphonic acid +

5 mM NaClO4, pH 3, with 70% ACN

B) Same but 100 mM NaClO4

DETECTION: A215

SAMPLE: 24 µg protein/80 µl

Surfactant

Protein

Variants

Lecithins,

steroids, &

other lipids

Histone H4 Acetylation & Methylation VariantsCOLUMN: PolyCAT A (104CT0315; HILIC mode)

27.0 32.0 37.0 42.0 47.0 52.0 57.0 62.0

VO

LT

AG

E0 Acetyl

1 Acetyl

2 Acetyl

H2AMETHYLATION

TIME (Min)20 30 40 50 60 70

VO

LT

AG

E

0

4-Acetyl3-Acetyl

2-Acetyl

1-Acetyl

0-Acetyl

H2A

METHYLATION

(courtesy James Pesavento - U. of Ill.)

MITOSIS

INTERPHASE

The most minor

variants can be

the most critical

TIME (Min)

0 10 20 30 40 50 60

AB

SO

RB

AN

CE

Histone H3(1-50) ex HeLa Cells; Acetylation & Methylation VariantsHILIC on PolyCAT A; [NaClO4] and [ACN]

3,4Ac

2Ac

2Ac

1Ac

1Ac

0Ac

0Ac

butyrate-

treated

control

(courtesy Benjamin

Garcia - U. of Ill.)

Chicken Erythrocyte Histone H1 Isoforms on PolyCAT A

C-18

CEX:

0%

ACN

CEX:

40%

ACN

CEX: 70%

ACN

- much

better

selectivity!

S

Minutes

0.0 0.4 0.8 1.2 1.6 2.0 2.4

mA

U

0

100

200

300

400

500

F

A

A2

C

A1c

Hemoglobin FASC Standard: Fast Flow AnalysisCOLUMN: PolyCAT A, 100x3.0-mm; 5-µm, 1000-Å FLOW: 3.5 ml/min A415 Pressure: 5379 psi

GRADIENT: 0-2’: 11-45% B; 2-2.5’: 45-100% B

A) 20 mM Bis-tris + 2 mM KCN, pH 6.96 B) 20 mM Bis-tris + 2 mM KCN + 200 mM NaCl, pH 6.55