mab aggregates and charge variants...

Shanhua Lin, Ph.D.

mAb Aggregates and Charge Variants Analysis

The world leader in serving science

Structure of IgG and Typical Forms of Heterogeneity

2

Protein and mAb Separation by HPLC

Size Exclusion Chromatography (SEC)Size difference? MAbPac SEC-1

YES

Ion Exchange Chromatography (IEX)Charge difference?

YES

NO

MAbPac SCX-10ProPac WCX-10C ff

NO

Chromatography (IEX)

R PhNO

CX-1 pH gradient Buffer

NO

Reverse Phase Chromatography (RPC)

Hydrophobicity difference?MAbPac HIC 10

YES MAbPac RP

Hydrophobic Interaction Chromatography (HIC)

MAbPac HIC-10MAbPac HIC-20MAbPac HIC-ButylProPac HIC-10

YES

3

mAb Aggregate Analysis

4

MAbPac SEC-1

Overview• Thermo Scientific™ MAbPac™ SEC-1 is a size exclusion chromatography

(SEC) column specifically designed for separation and characterization of(SEC) column specifically designed for separation and characterization of monoclonal antibodies (mAbs).

Features• Proprietary hydrophilic bonded layer

results in minimal non-desired interactions between proteins and stationary phasestationary phase.

• Superior performance for the analysis of monoclonal antibodies, aggregates, and their fragments.

• Stable surface bonding leads to low column bleed and compatibility with MS, ELSD and Corona® CAD detection.

• Rugged reproducible column packing

5

• Rugged, reproducible column packing.

Physical Data

Bonding Chemistry DiolSilica Substrate Spherical high-puritySilica Substrate Spherical, high-purity

porous silica Particle size 5 µmPore size 300 ÅPore size 300 ÅColumn housing PEEK for 4.0 mm I.D.

columnsSST for 7 8 mm and 2 1SST for 7.8 mm and 2.1 mm I.D. columns

Separation range for globular proteins

10,000 - 1,000,000g pExclusion limit for globular proteins

>1,000,000

6

Column Formats Versus Target Applications

Formats Target application Why is it important7 8 × 300 mm Highest resolution separation Accurate quantitation of mAb7.8 × 300 mm Highest resolution separation

of mAb and their aggregates.Accurate quantitation of mAbaggregates. Used in the batch QCrelease assay.

4.0 × 300 mm High resolution separation of The 4.0 × 300 mm column enables4.0 300 mm4.0 × 150 mm

High resolution separation of mAb and their aggregates.

The 4.0 300 mm column enables baseline separation of mAbmonomer and dimer, required ¼ of sample comparing to the 7.8 × 300

lmm column.2.1 × 300 mm2.1 × 150 mm

Designed for SEC-MS application.

Low flow rate and low sample loading makes this format perfect for MS detectionfor MS detection.

7

mAb Aggregate Analysis

mAb monomer

20 0

30.0

40.0

50.0 Column: MAbPac SEC-1, 5 µm, Dimension: a. 7.8 × 300 mm

b. 4.0 × 300 mma. 2.1 × 300 mm

Mobile phase: 50mM Sodium Phosphate pH 6.8,

mAb monomer

Aggregates

(a)

1500.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0

0.0

10.0

20.0 Mobile phase: 50mM Sodium Phosphate pH 6.8,in 300 mM NaCl

Temp.: 30 ºCFlow rate: a. 760 µL/min

b. 200 µL/minc 50 µL/min

gg g

(b)

255075100125

c. 50 µL/minInj. volume: a. 10 µL

b. 5 µLa. 1 µL

Detection: 280 nmSample: mAb (1 mg/mL)

(b)

50 060.070.0

025

0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0

Sample: mAb (1 mg/mL)

(c)

0.010.020.030.040.050.0

8

0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0Retention Time (min)

Impact of Ionic strength: MAbPac SEC-1

Column: MAbPac SEC-1, 5 µmFormat: 7.8 ×300 mmMobile phase: 50 mM sodium phosphate, pH 6.8

and1. 300 mM NaCl2 200 mM NaCl

200

225

250

a b c

de

2. 200 mM NaCl3. 100 mM NaCl4. 50 mM NaCl5. 25 mM NaCl6. 0 mM NaCl

Gradient: IsocraticTemperature: 30 ºC

125

150

175

6. Rs=1.64

5. Rs=1.72

Flow rate: 0.76 mL/minInj. volume: 20 µL Detection: UV (280 nm)Sample: SEC standard

a. Thyroglobulin (bovine): 0.1 mg/mLb. γ-globulin (bovine): 0.1 mg/mLc Ovalbumin (chicken): 0 1 mg/mL

50

75

100 4. Rs=1.80

3. Rs=1.90

2. Rs=2.03c. Ovalbumin (chicken): 0.1 mg/mLd. Myoglobin (horse): 0.05 mg/mLe. Vitamin B12: 0.01 mg/mL

-25

0

25

1. Rs*=2.02

MAbPac SEC-1 column has low secondary interaction its

*Rs is the resolution between γ-globulin and ovalbumin

2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0

-50

Retention Time (min)

secondary interaction, its separation is not sensitive to the mobile phase salt concentration.

9

Rs is the resolution between γ globulin and ovalbumin

Faster Aggregate Analysis

mAb monomer Column: MAbPac SEC-1 5 µm

75

100

140 mAb monomer Column: MAbPac SEC-1, 5 µm, Dimension: a. 4.0 × 300 mm

b. 4.0 × 150 mmMobile phase: 50mM Sodium Phosphate pH 6.8,

in 300 mM NaClTemp : 30 ºC

(a) 4.0 × 300 mm

25

50

75

Aggregates

Temp.: 30 ºCFlow rate: 200 µL/minInj. volume: 2 µLDetection: 280 nmSample: mAb (5 mg/mL)

-20

300

400 (b) 4.0 × 150 mm

Shorter 150 mm MAbPac SEC-1 column enables significant reduction of run times

100

200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 25.0

-50

10

Retention Time (min)

mAb Fragments

(a)

(b)

(c)(c)

11

Separation of mAb Fragments

1Column: MAbPac SEC-1, 5 µmFormat: 7.8 ×300 mmMobile phase: 50 mM sodium phosphate, 300 mM NaCl,

pH 6.8

70.01 mAb

pGradient: IsocraticTemperature: 30 ºCFlow rate: 0.76 mL/minInj. volume: 10 µL

-10.070.0

2

scFc

F(ab)2

Detection: UV (280 nm)Sample: 1. Rituximab (5 mg/mL), inject 2 µL

2. Rituximab + IdeS (2 mg/mL)3. Rituximab + Papain (2 mg/mL)4 Rit i b DTT (2 / L)

-10.070.0

3

Fc

Fab

4. Rituximab + DTT (2 mg/mL)

mAb fragments F(ab)2 and scFc, F d F b HC d LC

-10.0140

4

HC+LC

DTT

Fc and Fab, HC and LC are separated.

0.0 5.0 10.0 15.0 20.0-20

Retention Time (min)

HCLCC C

12

Retention Time (min)

Non-denaturing SEC-MS

monomer(a)Column: MAbPac SEC-1, 5 µm, Dimension: 2.1 × 150 mmMobile phase: 20 mM ammonium formateTemp.: 30 ºC

dimerFlow rate: 50 µL/minInj. volume: 1 µLDetection: Exactive Plus EMRSamples: mAb (1 mg/mL)

(b)+38

+37

+39+40

+26

(c) +27+25

+24+28

13

100(a) mAb

Separation of HC and LC, Fab and Fc

50 Column: MAbPac SEC-1, 5 µm, Dimension: 4.0 × 300 mmMobile phase: 20% acetonitrile

(a)

-1070.0

Mobile phase: 20% acetonitrile,0.1% FA, 0.05% TFA

Temp.: 30 ºCFlow rate: 200 µL/minInj. volume: 5 µLD t ti 280

(b)

mAU

Detection: 280 nmSamples: (a) mAb

(b) mAb reduction by DTT(c) mAb digestion by papain

HC

LC

-10.0100

(c)Fab

50Fc

14

3.0 7.5 12.5 17.5 25.0-10

min

Denaturing SEC-MS

RT:6.88 - 16.56

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

12.71

(a)

7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5Time (min)

0

10

2010.15

8.68 10.818.89 13.7511.008.55 9.527.02 9.17 11.57 14.0114.31 15.037.41 8.117.84

Full MS of heavy chain (HC) Full MS of light chain (LC)

40

50

60

70

80

90

100

ativ

e A

bund

ance

2601.34

2341.31

2926.39

2128.55

40

50

60

70

80

90

100

lativ

e A

bund

ance

2109.962201.64

1947.72 2301.67

2531.731875.652411.25

2664.912812.902978.34

3164.421808.68

3375.33

2323.12

3006 032839.03

Full MS of heavy chain (HC) Full MS of light chain (LC)(b) (d)

+10+9

+8+11

+12

+20+18 +16

1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

10

20

30

40

Rel

a

1951.241801.26

1672.693344.411561.09 2619.36 2946.82

3901.713121.58 3601.602385.901463.66 2169.161988.65 3367.772754.361322.721101.60

1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

10

20

30

Rel

1746.42

1688.25 3616.421557.72

3006.03

3193.701110.59 1340.65 3406.59

3894.423345.21

( ) ( )

12

+7+13

+14

G0

G1G0+Lys

G1+LysG2

(c) (e)

15

G2

Chromatographic Conditions for MAbPac SEC-1

Column ID (mm) 2.1 4.0 7.8

Flow rate (µL/min) 50-75 200-300 760-1 000Flow rate (µL/min) 50-75 200-300 760-1,000

UV flow cell Micro (180 nL) Semi-micro(2.5 µL)

Analytical (11 µL)

Tubing ID (µm) 50-75 100 150-250Tubing ID (µm) 50 75 100 150 250

Sample Loop Size (Pull Loop WPS (µL))

1 5 20

16

Tips and Tricks for SEC Analysis

• If you are getting low resolution separation, check:• System setup: connection tubing, flow rate, flow cell size, injection method.• Use a protein mix standard to QC the system: Bio-rad Gel Filtration Standard p y

#151-1901

160 tB12

75

100

125

mAU

Column: MAbPac SEC-1, 5 µm, Dimension: 4 × 300 mmMobile phase: 0.3 M NaCl in 50mM Sodium Phosphate pH 6.8Temp.: 25 ºCFlow rate: 200 µL/minInj. volume: 5 µLDetection: 280 nmSample: MAb (5 mg/mL)

Thyr

oglo

bulin

gam

ma-

blob

ulin

Ova

lbum

in.

Myo

glob

in

Vi t

0.0 5.0 10.0 15.0 20.0 25.0-10

25

50

min

• If you observe high pressure, check to make sure that the flow cell is not blocked.

17

mAb Charge Variant Analysis

18

Charge Variant Analysis

Untreated mAb

Lysine VariantsYYK

YYProPac WCX-10 Column

10 µm Nonporous Polymeric Beads

Polymeric graftsWCX SCX WAX

YYKK

K

Acidic VariantsBasic Variants

WCX, SCX, WAX,SAX

Boundary(Cross-linked YYCarboxypeptidase B (hydrophilic layer)Core(Highly cross-linkedEVB-DVB)

digested mAb

Acidic VariantsBasic Variants

19

Gold Standard for mAb Analysis

P P WCX 10 C lProPac WCX-10 Column

20

Next-generation CEX Column

YYYYLysine Variants

YYKK

YYK

YY

Acidic YY

YYMAbPacSCX-10 Column

VariantsBasic Variants

YYKK

KLysine Variants

ProPac WCX-10 column, 4×250 mm

Acidic

KK

22

MAbPac SCX-10 column, 4×150Variants

Basic Variants

4×150 mm 15 min. total analysis time

mA

U

MAbPac SCX-10 column, 4×250 mm60 min. total analysis time

0 10

0

5 15Minutes

21

Improve resolution or sample throughput through column chemistry

Thermo Scientific CX-1 pH Gradient Buffers

Buffer A Buffer B

pH 5.6 10.2

Form Liquid Liquid

• Dilute buffers 10-fold with DI water

Form Liquid Liquid

Concentrate 10X 10X

Shi i R R• A linear pH gradient (pH 5.6 - 10.2)

is generated by running a linear pump gradient from 100% Buffer A

Shipping condition

Room Temp

Room Temp

Storage4 ~ 8 C 4 ~ 8 Cp p g

to 100% Buffer B • Generic, fast & high-resolution!

condition4 ~ 8 C 4 ~ 8 C

22

pH gradient platform method for charge variant analysis

Protein and MAb Separation on IEX Columns

Salt Gradient pH Gradient

• Most widely used method • Can predict elution profileMost widely used method

• Relatively simple to make the buffer

Can predict elution profile with pI value

• Lower salt concentration in

• Takes longer to optimize the separation condition (pH,

collected fractions

• In many cases, improved salt concentration) resolution was observed

• Difficult to generate a linear H di tpH gradient

23

pH Gradient Buffers – How Do They Work?

P i El i M h i IEX

+IsoelectricPoint (pI)

COO -

Protein Elution Mechanisms on IEX

Buffer pH typically < pI

Cation-ExchangeChromatography

COOH

NH3R +

Cationic protein binds to

negatively charged cation exchanger

+ ++

++

+ ++

05 6 7 8 9 10 11 12

Buffer/System pHNH3

R +COOH

4

Buffer pH typically > pI

Anion-ExchangeChromatography

COO -

Anionic proteinbinds to

positively chargedanion exchanger

- -- -

-

- - -

pH range covered by CX-1 pH gradient buffers

–Protein net charge vs. pH

NH2R

COO

24

Linear pH Gradient by Zwitterionic Buffer Cocktail

10.510.5

y = 0.1577x + 4.9755R² = 0.9996

9.5

valu

e

y = 0.1577x + 4.9755R² = 0.9996

9.5

valu

e

7.5

8.5

easu

red

pH v

Measured ValueLinear (Measured Value)7.5

8.5

easu

red

pH v

Measured ValueLinear (Measured Value)

6.5

Me

6.5

Me

5.50 10 20 30 40

Retention Time [min]

5.50 10 20 30 40

Retention Time [min]

25

Protein Standard – CX-1 pH Gradient Buffer

90 11

MAbPac SCX-10 Column, 4×250 mmPeak label: protein name – elution pH

pH trace60

10

Lectin-1 - 6.11

p ace

40

60

8

9

Cytochrome C - 10.15

20

6

7Lectin-2 - 6.28

Lectin-3 - 6.45 Trypsinogen - 7.75Ribonuclease A - 8.72

0 5 10 15 20 25 30 35 40-10 5

0

26

Protein Standard – Phosphate-based pH Gradient

70 10.0pHMAbPac SCX-10 Column, 4×250 mm

50

60

8 5

9.0

9.5

pH trace30.

40

7.5

8.0

8.5

10

20

6.5

7.0

2

0 5 10 15 20 25 30 35 40-10

0

5.5

6.012

27

Why pH Gradient Buffers?

• Traditional salt gradient using ion exchange chromatography

• Needs to be tailored for individual charge 10.0

15.0

30.0

gvariants

0 0

5.0 Salt gradient

• Proprietary pH buffer formulations

• Fast robust and reproducible pH gradients

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

min

%B: 10.0

15.0

50.0

• Fast, robust and reproducible pH gradients

• Ready to use with existing CX columns and systems 5.0

10.0

pH gradient

• Simple to optimize and easily automated

• Applicable to the majority of MAbs0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

min

%B: 25.0

25.0gradient

28

• Applicable to the majority of MAbs

pH Gradient Buffers Product Description – Introduction

Buffer A Buffer B

pH 5.6 10.2

F Li id Li idForm Liquid Liquid

Concentrate 10X 10X

• Simple to use• Dilute buffers 1:10 in DI water• A linear pH gradient (pH 5 6 - 10 2) Shipping

conditionRoom Temp

Room Temp

Storage

• A linear pH gradient (pH 5.6 - 10.2) is generated by running a linear pump gradient from 100% eluent A to 100% B. g

condition 4 – 8 C 4 – 8 C• It is generic, fast, and high-res!

29

Benefit of Linear pH Gradient: Compatible with WCX and SCX

• pH gradient can be used with both weak cation exchange (ProPac WCX-10) d t ti h l (MAbP SCX 10)10) and strong cation exchange columns (MAbPac SCX-10)

• There is a pH gradient delay with the weak cation exchange column.

30

ProPac WCX-10 vs MAbPac SCX-10

250 10.50

ProPac WCX 10 4 × 250 mm

100 8.00

U)

ProPac WCX-10, 4 × 250 mm

350 10.50

-50 5.00

banc

e (m

AU

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

125

250

8.00Abs

orb MAbPac SCX-10, 4 × 250 mm

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-50 5.00

R t ti Ti ( i )

31

Retention Time (min)

Benefit of Linear pH Gradient: Generic Approach

• A generic approach to charge variant analysis, covering the pH range 5.6 to 10.2

• The same pH gradients is applicable to majority of MAb charge variants with pI value between 6-10.

• pI value of the unknown MAb can be predicted from the correlation curve

32

Protein Standards Using Linear pH Gradient

0 0

60.0

7 -7

.55

3 -9.9

3

40.0

50.0

mAU

]

-5.8

7 -6

.04

0 6.37 sino

gen

-15.

9 7

A -2

2.00

-8.

5

rom

e C

-31

.55

20.0

30.0

Abs

orba

nce

[m

Lect

in-1

-n-

2 -6

.97

-6.2

ctin

-3 -

8.18

-6

Tryp

Rib

onuc

leas

e

Cyt

ochr

10.0 Lect

inLe

c

0 5 10 15 20 25 30 35 40-5.0

Retention Time [min]

33

Linear Correlation of Elution pH vs pI

Cytochrome Cy = 1.6923x - 7.2914

R² = 0.992910

10.5

Cytochrome Cy = 1.6923x - 7.2914

R² = 0.992910

10.5

Ribonuclease A

R 0.9929

8 5

9

9.5

valu

e

Ribonuclease A

R 0.9929

8 5

9

9.5

valu

e

Trypsinogen

Ribonuclease A

7.5

8

8.5

Mea

sure

d pH

v

Measured pH value

Linear (Measured pH value)

Trypsinogen

Ribonuclease A

7.5

8

8.5

Mea

sure

d pH

v

Measured pH value

Linear (Measured pH value)

Lectin-1Lectin-2

Lectin-3

6

6.5

7M )

Lectin-1Lectin-2

Lectin-3

6

6.5

7M )

5.57.5 8.5 9.5 10.5

pI value

5.57.5 8.5 9.5 10.5

pI value

34

mAb Standards Using Linear pH Gradient

160

180

200

9 50

10.00

10.50

pH trace

120

140

160

8.50

9.00

9.50

U)

60

80

100

7 00

7.50

8.00

sorb

ance

(mAU

20

40

60

6.00

6.50

7.00

Abs

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-20

0

5.00

5.50

35

Retention Time (min)

Linear Correlation of Elution pH vs pI

y = 1.1083x - 1.637R² = 0.9988

9.5

10

8.5

9

pH v

alue

7.5

8

Ab E

lutio

n p

MAb Elution pH value

Linear (MAb Elution pH value)

6.5

7

MA

66.5 7.5 8.5 9.5 10.5

MAb pI value

36

Top-selling mAbs Analyzed by pH Gradient Method

25 0

37.5

50.0

70.0

Rituxan2

25 0

37.5

50.0

70.0

Herceptin

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0-10.0

0.0

12.5

25.0

Retention Time (min)

1 3

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-10.0

0.0

12.5

25.0

Retention Time (min)

15.0

18.0

Humira15 0

20.0

Avastin

5.0

10.0

5.0

10.0

15.0

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-2.0

Retention Time (min)

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-2.0

Retention Time (min)

37

Platform method for mAb charge variant analysis

Benefit of Linear pH Gradient: Simple Optimization

• The method can be simply optimized

• By running a shallower pH gradient a higher resolution separation is obtained• By running a shallower pH gradient a higher resolution separation is obtained (e.g. 50-100%, rather than 0-100%B)

38

mAb Charge Variant Separation, 0–100% B

100% B0% B

40.0 10.50pH trace(a)

20.0

30.0

8.00

9.00

ance

[mA

U]

10.0

6.00

7.00Abs

orba

0 5 10 15 20 25 30 35 40-5.0 5.00

Retention Time [min]

39

*The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity Monitoring Module (PCM-3000)

mAb Charge Variant Separation, 0–50% B

0% B 50% B

20 0

25.0 8.50(b) pH trace

10 0

20.0

7.00

ance

[mA

U]

0 0

10.0

6.00

Abs

orba

0 5 10 15 20 25 30 35 40-5.0

0.0

5.00

Retention Time [min]

40

mAb Charge Variant Separation, 25–50% B

25% B 50% B

16.0 8.00(c) pH trace

10.0 7.50

7.75

ance

[mA

U]

5.0

7.00

7.25

Abs

orba

0 5 10 15 20 25 30 35 40-2.0 6.60

Retention Time [min]

41

Benefit of Linear pH Gradient: Fast Analysis

• By using• A smaller particle (5 µm rather than 10 µm)

• A shorter cation-exchange column (4 × 50 mm)

MAb charge variant profile can be quickly determined within 20 minMAb charge variant profile can be quickly determined within 20 min.

42

mAb Charge Variant Separation With Fast pH Gradient

250 10.50

0% B 100% B

150

200

9.00

U)

pH trace

100

7 00

8.00

bsor

banc

e (m

A

0

50

6.00

7.00Ab

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0-50 5.00

Retention Time (min)

43

Ultra Fast pH Gradient: 10-Minute Run

140 11.00

0% B 100% B

100

120

9.00

10.00

U)

pH trace

60

80

8.00

bsor

banc

e (m

A

20

40

6.00

7.00

1

Ab

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0-20

0

5.00

1

Retention Time (min)

44

Retention Time (min)

Fast Method Development

5 min gradient

10 min gradient

3 steps method development1

0.8 min gradient1. 10 minutes 0100 % B in 10 minutes2. 2040 %B in 5 minutes3. 1827 %B in 0.8 minutes

Number of charge variants and resolution maintained for sub-

45

gminute gradient

Benefit of Linear pH Gradient: High Resolution

• In most cases, we observed improved separation of the charge variants over salt gradient.

46

Salt vs pH Gradient IEC of mAb Sample

15 0

10.0

15.0

30.0

5.0

Salt gradient

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

min

%B: 10.0

g

15.0

50 0

10.0

50.0

0.0

5.0

i

%B: 25.0

25.0pH gradient

47

30 min gradient, Thermo Scientific™ MabPac™ SCX-10, 10 µm, 4 × 250 mm column0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

Benefit of Linear pH Gradient: Great Precision

• The retention times in pH gradient IEC are highly reproducible

• This makes prediction of pI very consistent

48

60 10.50

Repeat Injections of Ribonuclease A: Over 300 Runs

259 00

pH trace

0

8.00

9.00

Run #300

ance

[mA

U]

-50

-25

7.00

Run #200

Abs

orba

-756.00

Run #100

Run #5

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0-100 5.00

Run #5

Retention Time [min]

Retention time reproducibility <0 8% RSD

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Retention time reproducibility <0.8% RSD

CX-1 pH Gradient Buffer Kit (10X): Simple To Use

110

Lot 1 (130523 / 130528)Lot 2 (130524 / 130529)Lot 3 (130531 / 130530)

60

80

mA

U)

Lot 3 (130531 / 130530)

40

60

bsor

banc

e (m

20

Ab

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-10

50

Retention Time (min)

Tips and Tricks for IEC Analysis

• Eluents for ProPac WCX-10 and MAbPac SCX-10 columns should always contain small amount of electrolytes. Never wash the columns with DI water.

• Try to avoid using organic solvent in the mobile phase. If organic solvent is i d d k th i t t i t t!required, do keep the organic content consistent!

• Use inert HPLC system to avoid metal contamination.• If you are want to try out pH gradient method, start with MAbPac SCX-10, 10

4 ×250 lµm, 4 ×250 mm column. • Run a protein standard mix (Lentil Lectin, Ribo A, Trypsinogen, and CytC) to

make sure the system is working properly. Monitoring the pH and Conductivity will be a great PLUS!will be a great PLUS!

• To speed up your analysis, use either shorter format (such as MAbPac SCX-10, 10 µm, 4 ×150 mm column) or smaller particle (such as 5 µm).

• If your sample do not bind and elute immediately check protein pI and pH of theIf your sample do not bind and elute immediately, check protein pI and pH of the sample buffer.

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Thank you!

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