BioHPLC columns

Tim Rice

Biocolumn Technical Specialist

Typical Application Areas

Size Exclusion: Aggregation Analysis

Reversed Phase:

Separation of light and heavy chains

Ion Exchange: Charge Isoform Analysis

Peptide Mapping:

Post-translational modifications

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Dimer

Monomer

2

Size Exclusion

BioHPLC Columns

NEW Size Exclusion Columns

• 5 m Particle

• 100Å, 150Å, 300Å, 500Å, 1000Å,

2000Å pore sizes

• High stability and long lifetime

• Great reproducibility

• Unique, 3 m particle

• 100Å, 150Å, 300Å pore sizes

• Highest resolution

• Highest efficiency

• Faster SEC separations

Smaller Particles for Higher Resolution

5

Min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Agilent Bio SEC-3, 300Å,

7.8x300mm

Agilent Bio SEC-5, 300Å,

7.8x300mm

Column Pressure

Bio SEC-3

(7.8x300mm) 1350 psi

Bio SEC-5 (7.8x300mm)

650 psi

Peak Protein SEC-3, 300Å

(7.8x300mm)

SEC-5, 300Å

(7.8x300mm)

1 Thyroglobulin 2460 1120

2 BSA Dimer 5100 2720

3 BSA 13090 6590

4 Ribonuclease A 22000 11160

5 Uracil 38500 27860

Technology: Small Particle SEC Columns

Results: Higher Resolution SEC Separations

Use Higher Resolution to Achieve Faster

Separations

6

Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mAb (2mg/ml)

Injection: 5ul

Flow rate: 1.0, 1.5 and 2ml/min (56 bar , 75 bar, 105 bar)

Eluent: 150mM sodium phosphate + 100mM Na-sulfate

Detection: 220nm

4 Minutes

monomer

dimer

2.0ml/min 1.5ml/min 1.0 ml/min

Increase flow rate and use a shorter column for

faster separations.

Technology:

Small Particle SEC Columns

Results:

Faster SEC separations

Mechanism of SEC Separation – Pore Size

Determines Linear Separation Range

Page 7

FLOW

Peptides Proteins Globular

proteins

Column Choice: Resolving Ranges

100D 1kD 10kD 100kD 1,000kD 10,000kD

Agilent Bio SEC 100Å

Agilent Bio SEC 150Å

Agilent Bio SEC 300Å

ProSEC 300S

Zorbax GF-250

Thyr

oglo

bulin

IgG

Ova

lbum

in

BS

A

Myo

glo

bin

Zorbax GF-450

Agilent Bio SEC 500Å

Agilent Bio SEC 1000Å

Agilent Bio SEC 2000Å

Insu

lin

Ion Exchange

BioHPLC Columns

NEW Ion Exchange Columns

• Non-porous PS/DVB particles

(polystyrene divinylbenzene)

• Uniform polymeric coating with SCX,

WCX, SAX, WAX layers, designed for

protein and peptide separations

• Available in 10 µm, 5 µm, 3 µm, 1.7 µm

particle sizes

• High surface area

• High capacity

Agilent Bio IEX Columns Comparing Separations on Each Particle Size

Min

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

1.7 m

3 m

5 m

10 m

Column: Bio WCX-NP, 4.6x50mm

Buffer A: 20 mM PBS

Buffer B: A+1.0 M NaCl

Gradient: 0-100%B (20 min)

Flow rate: 1.0 mL/min for NP10, NP5, NP3

0.75 mL/min for NP1.7

Sample

1) Ribonuclease A

2) Cytochrome C

3) Lysozyme

Concentration: 1.0 mg/mL

Detector: 280 nm

Average N ~80,000 for WCX-NP1.7

Peak N

Peak N

Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies

12

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

Column: Agilent Bio MAb, NP5, 4.6mm x 250mm

Buffer A: 10 mM Sodium Phosphate, pH 7.50

Buffer B: A + 100 mM NaCl, pH 7.50

Gradient: 15-95% B in 60 min

Flow rate: 0.8 mL/min.

Sample: 5 L, 5 mg/mL, mAb

(Acidic Isoforms)

weakly bound

(Basic Isoforms)

Strongly bound

Min 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

A

B

C

D

E

Optimization of method conditions for the isoform characterization of a

monoclonal antibody. Changes in the buffer conditions, pH and gradient

conditions sharpen peaks and increase resolution of acidic and basic isoforms.

Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies

Columns: Agilent Bio MAb, NP10, 4.6x250 mm

Mobile phase: A, 10 mM phosphate, pH 7.5

B, A + 0.1M NaCl

Gradient: A) 15-75%B in 30 min

B) 15-65%B in 30 min

C) 15-55%B in 30 min

D) 15-47.5%B in 30 min

E) 15-40%B in 30 min

Flow rate: 0.8 mL/min

Sample: Monoclonal Antibody

Injection: 10 L (1.5 mg/mL)

Temperature: 25 oC

Detection: UV 214 nm

Agilent Bio WCX: pH 7.0 – 5.8

14

Increased resolution

of early peaks

An increase in pH reduced the retention time.

An increase in pH increased resolution of earlier peaks (circled).

Reverse Phase

BioHPLC Columns

Zorbax Stablebond for

Reverse Phase Separations of Proteins

300 A pore size

available in C-3, C-8, C-18, and CN

non-endcapped

particle sizes: 3.5, 5, and 7

16

O

R RSi

OH

Si

O

SiR R R

OOH

R

R1 R1 R1

NEW! Zorbax 300 SB RRHD for Proteins and Peptides!

• Stablebond 300 silica

• C-18, C-8, C-3, HILIC and diphenyl bonded phase

• 1.8 um particle size

• 1200 Bar pressure limit for uHPLC

• 2.1 x 50 mm and 2.1 x 100 mm

UHPLC Columns Increase Resolution and Increase Speed

Column

Length (mm)

Resolving

Power

N(5 µm)

Resolving

Power

N(3.5 µm)

Resolving

Power

N(1.8µm)

150 12,500 21,000 32,500

100 8,500 14,000 24,000

75 6000 10,500 17,000

50 4,200 7,000 12,000

30 N.A. 4,200 6,500

15 N.A. 2,100 2,500

Analysis Time

Peak Volume

Analysis

Time*

-33%

-50%

-67%

-80%

-90%

Solvent Usage

* Reduction in analysis time compared to 150 mm column

19

Column: Zorbax 1.8um 300SB-C18 RRHD, 2.1x100mm

A : .1% TFA in water, B: .085% TFA in ACN

Gradient: 20%B 1min, 2-45%B 8.8min, 45-95%B .2min, 95%B 2min, 98-2%B .2min, 20%B 1.8min

Flow rate: .5 ml/min, Temp: 50C, Pressure: ~640bar

Sample: Protein digest, 5ul inj vol, 1 mg/ml

300SB-C18 RRHD Protein Digest TB# 5990-7989EN

Fast Separation of Reduced and Alkylated MAb

20

min 1 2 3 4 5

mAU

0

20

40

60

80

100

120

0.4

54

0

.50

1

0.5

69

0.7

66

3.8

97

4.0

10

Light chain

Heavy chain 1 Heavy chain 2

Columns: ZORBAX RRHD 300SB-C3, 2.1 x 100 mm, 1.8 µm

Sample: Reduced MAb (IgG1) (1.0 mg/mL)- BioCreative IgG1

Sample injection: 2 µL

Mobile phase A: 0.1% TFA in water

Mobile phase B: 80% n-propyl alcohol, 10% ACN, 9.9% water and 0.1% TFA

Temperature: 74 °C

Flow rate: 0.5 mL/min

Detection: UV, 280

Conditions optimized for the ZORBAX RRHD 300SB-C3 Column

CHO Cell Derived MAb

Time

(min)

%B

0 1

2 20

5 50

6 90

6.1 1

TB# 5990-9667EN

Comparison C3 and Diphenyl Phases

21

min 1 1.2 1.4 1.6 1.8 2 2.2 2.4

mAU

2

4

6

8

10

12

1.3

37

1.5

87

1.6

66

1.7

54

1.8

11

min 1 1.2 1.4 1.6 1.8 2 2.2 2.4

mAU

2

4

6

8

10

12 1.3

45

1.5

64

1.6

46

1.7

87

1.8

46

ZORBAX RRHD 300SB-C3, 1.8 µm

ZORBAX RRHD 300-Diphenyl, 1.8 µm

Ribonuclease A, Cytochrome C and Lysozyme (3 mg/mL)

Time

(min)

%B

0 10

2.5 70

Arrows indicate better separation

resolution of ZORBAX RRHD

300-Diphenyl

TB# 5990-9668EN

NEW! AdvanceBio Peptide Mapping Column for

HPLC and UHPLC:

• 2.7um Superficially Porous

• 120A pore size

• 600 bar pressure limit

• 2um frit to reduce clogging

22

Save Time: 2 to 3 times faster than fully porous particles

Increased Flexibility: Highly compatible with TFA and formic acid mobile

phases for efficient LC/MS analysis

Greater analytical confidence: Each batch is tested with a rigorous peptide

mix to ensure suitability and reproducibility

Decrease the diffusion time for macromolecules and limit the diffusion path!

The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path

Reduces secondary interactions & enables selective separation for a wide range of

peptides.

.

Superficially Porous Particle Technology

Chromatographic Comparison BSA tryptic digest

24

min 0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

10

20

30

40

50

60

70

min 0 2 4 6 8 10 12

mAU

0

10

20

30

40

2.7um AdvanceBio Peptide Mapping, 2.1 x 150mm, gradient adjusted for length

104 peaks

319 Bar

Vendor B, 1.7um, C18, 2.1 x 100mm, gradient adjusted for length

97 peaks

476 Bar

Greater Rs, increased sensitivity

Less Rs, decreased sensitivity

Conditions: water/ACN (TFA), 40C, .3 mL/min

25

Peptide Mapping for LC/MS

min 0 5 10 15 20 25 30

mAU

0

50

100

150

200

250

300

Agilent LC/MS

100% sequence coverage

Digested EPO (recombinant humanized) separated on a 2.1 x 250mm AdvanceBio Peptide Mapping Column

Mobile phase: A-water (0.1%FA), B- ACN (0.08%FA), temp: 55C, Flow: 0.5mL/min

Excellent separation capability for generating peptide maps of highly desired proteins, i.e.,

glycopeptide mapping during fast analysis times

Provides compatibility with TFA and Formic acid mobile phases for LC/MS analyses

Pressure maintained below 600bar for 250mm column length

Digested EPO Protein

Glycopeptide mapping

TB# 5991-1813EN

Poroshell 300

300 A pore size

Stablebond chemistry

available in C-3, C-8,C-18, and C-18 Extend

5 um particle size

Porous

Shell

SOLID CORE

5 m

High Flow Rates with 2.1 mm ID Poroshell for High

Resolution and Fast Separations

1

2

34

5

67

8

0 0.5 1.0

Time (min)

Columns: Poroshell 300SB-C18

2.1 x 75 mm, 5 m

MP: A: 0.1% TFA

B: 0.07% TFA in ACN

Gradient: 5 – 100% B in 1.0 min.

Flow Rate: 3.0 mL/min.

Temperature: 70

C

Pressure: 250 bar

Detection: UV 215 nm

Sample:

1. Angiotensin II

2. Neurotensin

3. Rnase

4. Insulin

5. Lysozyme

6. Myoglobin

7.Carbonic

Anhydrase

8.Ovalbumin

TB# 5989-9899EN

Reversed Phase

Heavy and light chain analytical characterization

Use of 1200 LC, Poroshell 300SB

columns and UV detection to

characterize antibodies

28

TB# 5989-0604EN

More Poroshell Bonded Phases Provide Selectivity

Options to Enhance Resolution:

min0 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

DAD1 A, Sig=215,16 Ref=360,100 (05080223.D)

Poroshell SB-C18, 2.1 x 75 mm

1

2 3 4

5, 6

7

8

9 0.9

76

1.0

77

1.2

89

1.3

95

1.4

63

1.5

23

1.7

41

1.8

60

3.0

12

min0 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

DAD1 A, Sig=215,16 Ref=360,100 (05080225.D)

51

23

4

6

7

8

9

Poroshell SB-C3, 2.1 x 75 mm

0.9

01

1.0

36

1.2

64

1.3

54 1.4

30

1.4

49

1.4

97

1.7

39

1.8

37

Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 70 0C; Flow: 0.5 mL/min; Det: UV 215 nm

Mobile Phase: A= 0.1% TFA/H2O, B= 0.07% TFA/ACN; Gradient: 5-100% B in 3.0 min

1.5

5

4 6

7

1.5

5

4 6

7

1.5

4

5, 6

7

1.5

4

5, 6

7

Samples:

1. Angiotensin II

2. Neurotensin

3. RNase A

4. Insulin B Chain

5. Insulin

6. Cytochrome C

7. Lysozyme

8. Myoglobin

9. Carbonic

Anhydrase

Poroshell 300SB-C18

2.1 x 75 mm

Poroshell 300SB-C3

2.1 x 75 mm

•Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3 min!

PLRP-S

Polystyrene divinylbenzene bead

Available in 100, 300, 1000, and 4000A pore sizes

Particle sizes 3, 5, 8, 10, and higher

Various geometries from Capillary to preparative

pH 2-14 stable

25-bp Ladder Double Stranded DNA Ladder

1

1 1

12% PAGE

25

25

50

25

25

50

50

100

100

100

100

50

1 1

1 1

time

HPLC

PLRP-S 100Å

PLRP-S 300Å

PLRP-S 1000Å

PLRP-S 4000Å

Column: PLRP-S 150x2.1mm ID

Eluent A: 100mM TEAA

Eluent B: 100mM TEAA, 50% ACN

Gradient: 12.5%-50%B in 150 mins

Flow Rate: 200µl/min

Pore Size Resolving Range

100Å 50-70 bp

300Å Up to 250-300 bp

1000Å Up to 400-450 bp

4000Å >500 bp

In Summary

Agilent has many options for the analysis of bio-molecules and

will continue to develop new products to address this growing

market.

NEED ASSISTANCE?

LC Column help desk

1-800-227-9770

• orders

• customer service

(option 1,1)

• technical support

• applications assistance

(option 3,3,2)

33

lc-column-support@agilent.com

BioHPLC Columns on the Agilent Website

To learn more and order online visit

www.agilent.com/chem/BioHPLC