bio-columns products, applications, and principles … · bio-columns products, applications, and...

Bio-Columns Products,

Applications, and Principles

Paul Dinsmoor

BioHPLC Columns Specialist

Sep. 27, 2012

BioChromatography

2012 EMEA Seminar Series

2

Size Exclusion

BioHPLC Columns

Define the Separation Goals

For SEC, what is the molecular weight range of the proteins you

wish to separate?

The answer to this questions helps you determine the

appropriate pore size to achieve the best resolution of your

protein.

Pore volume is a key factor in separation.

Size Exclusion Process

13-17 February 2012

Bio-applications Training Program

5

Larger molecules spend less time

in the pores and elute sooner.

Smaller molecules spend longer

in the pores and elute later.

Mechanism of SEC Separation – Pore Size

Determines Linear Separation Range

Agilent Bio SEC-5 Column Characteristics – Pore Size/ Separation Range

Some General Guidelines for SEC

1. When methods are to be validated, test for ruggedness

with several different column lots, mobile phase

preparations, and operators.

2. As a rule of thumb, SEC will only provide baseline

separation of molecules with more than a 2 fold difference

in MW.

3. Sample volume should be limited to below 5% of the total

column volume. Max resolution .5 – 2% CV.

Loaded Sample Volume / Resolution

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

Min

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Injection volume: 10 L

BSA Concentration

(from low to high):

1 mg/mL

2 mg/mL

5 mg/mL

10 mg/mL

20 mg/mL

50 mg/mL

Column: Bio SEC-3, 150Å, 7.8x300mm

Mobile phase: 150 mM Phosphate, pH 7.0

Flow Rate: 1.0 mL/min

Injection volume: 10 µL

Detector: 214 nm

Column Selection Criteria

Pore size

depends on molecular weight range of sample

avoid exclusion of sample components

maximize pore volume in required separation region

Particle size

smaller particles for higher resolution

Number of columns/ length

compromise between resolution and analysis time

Column ID

smaller column ID can be used for reduced solvent

consumption and smaller injection volume

What does Exclusion look like ?

Exclusion: very sharp

peak with almost

vertical take-off !

Normal: somewhat

broad, generally

gaussian in shape

11

Pore Size Comparison

September 30, 2012

Agilent Confidential

12

Eluent: 50mM NaH2PO4 + 0.15M NaCl, pH6.8

Columns: Agilent Bio SEC-3 ,4.6x300mm

Flow: 0.35ml/min

Detector: UV@220nm

System: Agilent 1260 Infinity Bio-Inert LC System

Sample: BioRad Gel Filtration Standards Mix

Mouse IgG

1. Thyroglobulin Aggregates

2. Thyroglobulin

3. IgA

4. g-globulin

5. Ovalbumin

6. Myoglobin

7. Vitamin B12

300Å

150Å

100Å

1

2

3

4

5 6

7

300Å

150Å

100Å

1

2

3

4

5

1. Dimer

2. Monomer

3. Monomer Fragment

4. Azide

5. Retained Molecule

Peptides Proteins Globular

proteins

Column Choice: Resolving Ranges

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

Agilent Bio SEC 100Å



ProSEC 300S

Zorbax GF-250

Th

yro

glo

bu

lin

IgG

Ova

lbu

min

B

SA

Myo

glo

bin

Zorbax GF-450




Insu

lin

0 2 4 6 8 10 12 14

Time (min)

020

040

0

mAu

214

nm

0 2 4 6 8 10 12 14

Time (min)

020

040

0

mAU

214

nm

Agilent Bio SEC-5

150 Å

TSK

G2000SWxl 1. thyroglobulin, 5.64 min

2. BSA dimer, 6.23 min

3. BSA monomer, 7.02 min

4. ribonuclease A, 9.22 min

5. poly-DL-alanine 10.02 min

6. uracil, 11.81 min

1. thyroglobulin, 5.43 min

2. BSA dimer, 6.19 min

3. BSA monomer, 6.93 min

4. ribonuclease A, 8.74 min

5. poly-DL-alanine 9.90 min

6. uracil, 12.13 min

Resolution is affect by pore volume

14

Effects of Particle Size on Resolution

Decreasing the Particle Size of the GPC media in a column improves

the resolution

The smaller the particle the more particles that can be packed into a

column. Therefore the number of pores increases

The smaller the particles, the faster equilibrium is reached in the

column between the stationary phase (the pore volume) and the

mobile phase (interstitial volume)

Improved Efficiency With Smaller Particles

Column: Bio SEC-3 300Å and Bio SEC-5 300Å

Buffer: 150 mM Phosphate buffer, pH 7

Flow rate: 1.0 mL/min for 7.8x300 mm

Temperature: Ambient (~23° C)

Detection: UV 214nm

Injection: 10 µL (3 L for 4.6x300 mm)

Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA

dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4)

Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil

(2.5 g/mL), 120D.

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) 93 bar

Bio SEC-5

(7.8x300mm) 45 bar

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

Determine Optimal Mobile Phase Conditions

What conditions give you the best results?

What additives may be required to reduce non-specific

interactions?

What is the optimal flow rate?

1. 150mM Sodium phosphate buffer

2. 150mM Sodium phosphate buffer + 50mM NaCl



Agilent Bio SEC-3, 300A 7.8 x 300mm MAb separation, Sodium phosphate buffer and different NaCl concentrations

Size Exclusion Chromatography


19

• Impurity testing, aggregation analysis (separation of monomer/dimer/aggregates)

• Molecular weight characterization – over wide MW range (1000 – 10M) possible

• Expression and folding studies

• Separation of Reaction Components and Products, (conjugates)

• Purification

• Desalting and salt exchange

• Collection of fractions under non-denaturing conditions

Non-Denaturing Conditions

Separations Based on Size in Solution

NEW Size Exclusion Columns

• 5m Particle

• 100Å, 150Å, 300Å, 500Å, 1000Å,

2000Å pore sizes

• High stability and long lifetime

• Great reproducibility

20

• Unique, 3m particle

• 100Å, 150Å, 300Å pore sizes

• Highest resolution

• Highest efficiency

• Faster SEC separations

Agilent Biopharma Training, AFO NPT 2010

Aggregation Studies


21

ICH (Q6B):

• “The category of aggregates include dimers and higher multiples of

the desired product. These are generally resolved from the desired

product and product-related substances, and quantitated by appropriate

analytical procedures (e.g., size exclusion chromatography, capillary

electrophoresis)” (ICH Q6B)

• “Multimers and aggregates should also be appropriately characterized

using a combination of methods. The formation of aggregates, sub-visible

and visible particulates in the drug product is important and should be

investigated and closely monitored on batch release and during stability

studies.

Agilent Bio SEC-5 Monoclonal Antibody Aggregation Monitoring

Columns: Bio SEC-5, 300Å, 7.8x300mm

Buffer: 150 mM sodium phosphate, pH 7


Sample: Mab (2.5 mg/mL)

Injection: 10 L

Concentration: 10, 2.5, 0.5 mg/mL

(from high to low)

Temperature: Ambient

AU

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

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

Bio SEC-5 columns can easily handle higher flow rates, this separation

was done at 1 mL/min, resolution between dimer and monomer can be

improved by decreasing the flow rate

Agilent Bio SEC-5 Monoclonal Antibody Aggregation Monitoring

MAb Dimer

Mab Aggregates

Columns: Bio SEC-5, 300Å, 7.8x300mm

Buffer: 150 mM Phosphate, pH 7


Sample: Mab


AU

-0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

Min 1.0 2.00 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Buffer Peak

Low molecular

weight forms

Monitoring Aggregation and Impurities


24

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

Column: Bio SEC-3 300Å, 7.8x300mm

Mobile phase: 150 mM Phosphate, pH 7

Flow rate: 1.0 mL/min


Sample: Monoclonal antibody (10 L, 5 mg/mL)

Buffer/Excipients

MAb

Dimer MAb

Aggregates

Low Molecular

Weight Impurities

Phosphate buffer to control pH and provide ionic strength

Optimized to eliminate interactions – for this MAb

Agilent Bio SEC-5 High Molecular Weight Proteins

Proteins:

1.Thyroglobulin (670kD)

2. Uracil (120D)

Column: Bio SEC-5, 1000Å, 4.6x300mm

Buffer:150 mM phosphate buffer, pH 7.0


Detection: 214 nm


1

Possible Dimer

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Min

0 2 4 6 8 10 12 14

2

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

Columns: Bio SEC-5, 7.8x300mm

Buffer: 150 mM Phosphate, pH 7.0


Detector: 214 nm

Injection: 10 µL (2.5 mg/mL)

SEC-5, 1000Å

SEC-5, 500Å

SEC-5, 300Å

SEC-5, 150Å

Agilent Bio SEC-5 Size Exclusion Separations of E. coli Lysate

Columns: Bio SEC-5, 1000Å, 7.8x300mm + Bio SEC-5, 150Å, 7.8x300mm

Buffer: 150 mM Phosphate, pH 7.0


Detector: 214 nm

Injection: 10 µL (2.5 mg/mL)

AU

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Agilent Bio SEC-5 Size Exclusion Separations of E. coli Lysate

(Two Columns In Series)

Column: Column: Bio SEC-3 150Å, 7.8x300mm & Bio SEC-3 300Å, 7.8x300mm

Mobile phase: 0.15 M Phosphate, pH 7.0


Detection: UV 214 nm


Injection: 10 µL

Sample: E. coli lysate (2.5 mg/mL)

Min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Bio SEC-3 150Å, 7.8x300mm

Bio SEC-3 300Å, 7.8x300mm

Agilent Bio SEC-5

Separations of E. coli Lysate

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

Min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

3

1

2

Column: Bio SEC-3, 150Å (3µm particle, 7.8x300mm)

Sample: 1, Protein with MW 70KD (from an undisclosed drug company)

2, Protein 1 attached with 1 PEG (MW 40KD) molecule

3, Protein 1 attached with 2 PEG (MW 40KD) molecules

Mobile phase: 150 mM Phosphate pH=7.0; Flow rate: 1.0 mL/min

Wavelength: 214 nm; Temperature: Ambient

Sample concentration: 2.0 mg/mL; Injection volume: 20 µL

Column backpressure: 110 bar

Size Exclusion Separation of Protein and

PEGylated Proteins

30

Fast separation of dimer from monomer

using the Agilent Bio SEC-3 300Å

7.8 x 150 mm column

31

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

Detection: 220nm

Flow Rate Resolution

Monomer/Dimer

Monomer

Efficiency

Percentage

Dimer

1.0ml/min 1.53 3,510 0.64

1.5ml/min 1.43 2,502 0. 47

2.0ml/min 1.13 1,917 0.64

monomer

dimer

2.0 1.5 1.0

ml/m ml/min ml/min

Fast separation of dimer from monomer using the

Agilent Bio SEC-3 300 Å 7.8 x 150 mm column

Fast SEC – TB5990-8613EN

Calibration-free Molecular Weight Determination ? Agilent 1260 Infinity Multi Detector Suite for Protein SEC

September 30, 2012 32

Helps to differentiate aggregates of monoclonal antibodies

Gamma Globulin (MW 150kD) Agilent Bio SEC-5 500Å, 7.8 x 300 mm


33

Monomer

Dimer High MW

aggregates

RI imbalance

(fresh eluent) UV, 254nm

LS 90

RI

Ion Exchange

BioHPLC Columns

Ion Exchange Chromatography Separation based on charge

Ion Exchange Chromatography of Proteins

Ion-exchange chromatography (IEC) discriminates between proteins on the basis of accessible surface charges and their corresponding electrostatic interaction with the column’s stationary phase.

The degree of protein retention is dependent on the strength and number of interactions.

The 3-D structure of the protein determines which surface residues will be available to contact the column’s stationary phase.

Secondary interactions/ non-specific interactions (particularly hydrophobic interactions) can also impact chromatography

Some Guidelines for IEX

1.The General Rule for choosing a Bio IEX column

– Acidic proteins: SAX or WAX

– Basic proteins: SCX or WCX

2. Consider the isoelectric point (pI) of your protein when choosing the pH of your mobile phase:

– if pH>pI, your protein will have a net negative charge

– If pH<pI, your protein will have a net positive charge

3. The pH of your starting buffer should be 0.5 to 1 pH unit

– above pI for Anion Exchange

– below pI for Cation Exchange

Proteins have a net charge, dependant on pH

+

+

+

+

+ -

-

-

-

-

+

+

+ +

At their isoelectric point, pI, the net charge is zero and the molecule will not be

retained on an ion-exchange column.

If pH < pI, the net charge will be positive and the molecule can be analyzed by

cation-exchange. If pH > pI, the net charge will be negative and the molecule can

be analyzed by anion-exchange.

13-17 February 2012

Bio-applications Training Program

38

Protein Isoelectric Point - Charge


39

Pro

tein

Ne

t C

ha

rge

+

_

pH 4 6 8 10 12

Iso

ele

ctirc

Po

int (p

I)

+

- - - - - -

- - - - - - - -

- -

Cation

Exchan

ge

Particle

pH lower than

the protein pI

pH greater than

the protein pI

I

Basics of Bio-Chromatography Ion Exchange Chromatography

• Sample is injected in a mobile phase buffer with a low

salt – this binds proteins to the column

• Analytes are typically eluted at constant pH with

increasing salt (ionic strength) to displace proteins

from stationary phase

• Higher charge proteins bind more strongly, more salt is

needed to elute them

• A typical mobile phase will contain NaCl

40

Ion Exchange Chromatography How it works

Bio IEX Columns Target Applications

• Antibodies

• Peptides in volatile buffers

• Glycans (glycosylated bio-molecules, not sugar analysis)

• Proteins

• Polynucleotides

• Cell lysates

• 2D separations

Ion Exchange Columns

• Non-porous PS/DVB particles

• Uniform polymeric coating and WCX

layer, specifically designed for antibody

separations

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

particle sizes

Agilent Bio MAb Column Characteristics

44

Charge Variant Analysis


45

Light Chain

Fc

Fab

Antigen

binding

Hinge

Glycosylation

site Truncation

(lysine)

Pyroglutamate Heavy Chain

Disulfide

shuffling

Deamidation/ Oxidation

Charge Variants (charge heterogeneity) are translational modifications

Sites where charge variants can occur

Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies

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 (Method) 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


Sample: Monoclonal Antibody

Injection: 10 L (1.5 mg/mL)

Temperature: 25 oC


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

Agilent Bio IEX High Resolution Protein Separations

min 0 2 4 6 8 10 12 14

BSA

Ovalbumin Column : Bio SAX, NP3 , 4.6x50mm

Buffer: 20 mM Tris, pH 8.0

Gradient: 0-0.3 M NaCl (15 min)


Backpressure: 110 bar

Detector: 214nm

Agilent Bio IEX Columns Peptide Separations

ELSD

@280nm

C1 C2

C3

C4

Column: Bio SCX, NP3, 4.6x50mm

Buffer A: 5 mM CH3COONH4

Buffer B: 0.5 M CH3COONH4:ACN=4:1 (v/v)

Gradient: 0-50% B (20 min)


Detector: ELSD & UV 280 nm

Injection volume: 5 µL (0.1 mg/mL)

Peptide Sequence Net Charge

C1 Ac-Gly-Gly-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide + 1

C2 Ac-Lys-Tyr-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide + 2

C3 Ac-Gly-Gly-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide + 3

C4 Ac-Lys-Tyr-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide + 4

Agilent Bio IEX Columns High Resolution Glycan Separations

min 0 10 20 30 40 50 60 70

LU

0

100

200

300

400

500

G0F G1F

G2F SA1F (NGNA)

SA2F (NGNA)

2-AA (anthranilic acid) labeled N-linked oligosaccharide profiling of an IgG1 sample

G0F: asialo, agalacto, core-fucosylated biantennary glycan

G1F: asialo, mono-galacto, core-fucosylated biantennary glycan

G2F: asialo, di-galacto, core-fucosylated biantennary glycan

SA1F: mono-sialylated, galactosylated, core-fucosylated biantennary glycan

SA2F: di-sialylated, galactosylated, core-fucolylated biantennary glycan

NGNA: N-glycolylneuraminic acid

Column: Bio SAX, NP5, 4.6x150mm

Buffer A: 2.5% (v/v) acetic acid, 0.5% TEA in H2O

Buffer B: 0.5% acetic acid in ACN



Fluorescence detector: Ex/Em=360/425nm,

2-AA labeled N-linked oligosaccharide profiling of an IgG1 sample

min 20 30 40 50 60

LU

0

100

200

300

400

Isomer of G1F Isomers of SA1F (NGNA)

G0F G1F

G2F SA2F

(NGNA)

min 20 30 40 50 60 70 80

0

50

100

150

200

250

No Isomer of G1F

Separated Isomers of SA1F (NGNA)

Not Separated

G0F G1F

G2F SA2F

(NGNA)

Bio SAX, NP5, 2.1x150mm

Asahipak Amino NH2P-50 2D,

5 µm, 2.1x150 mm

Agilent Bio SAX Comparison to Column Commonly Used For Glycans

min 20 22.5 25 27.5 30 32.5 35 37.5 40

Bio SAX, NP5

Competitor SAX Column

Sample: 50-mer Oligo

Buffer A: 0.025 M TRIS pH9

Buffer B: A+1.0 M LiCl

Gradient: 30 to 75%B in 45min

Flow Rate: 1mL/min

System: Agilent 1100

Injection: 50 μL

Detection: UV260nm

Agilent Bio IEX Columns Oligonucleotides, Comparison of Non-Porous vs. Porous

min 0 5 10 15 20 25 30 35 40

mAU

0 5

10 15 20 25

Bio SAX, NP5

min 0 5 10 15 20 25 30 35 40

mAU

0 2.5

5 7.5 10

12.5 15

17.5 Competitor SAX Column

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

Bio SAX, NP10

4.6x50 mm

Peak Capacity > 40

Bio SAX, NP5

4.6x50 mm

Peak Capacity > 60

Bio SAX, NP3

4.6x50 mm

Peak Capacity > 70

Buffer A: 20 mM Tris, pH 9.0

Buffer B: A + 0.5 M NaCl



Injection: 10 µL/min (2.5 mg/mL)

Agilent Bio IEX Columns E. Coli Lysate Separation:

Particle Size Effect on Resolution/Efficiency

Confidentiality Labelwww.sepax-tech.com

10 11 12 13 14 15 16 17 18 19 20 21 22

Bio SAX, NP10

Bio SAX, NP5

Bio SAX, NP3

Agilent Bio IEX Column E. Coli Lysate Separation:

Particle Size Effect on Peak Capacities

Agilent Bio IEX Columns E. Coli Lysate Separation:

Particle Size Effect on Peak Capacities

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

Bio SAX, NP3, 4.6x50mm

E. coli Lysate (2.5 mg/mL)

Min 18 19 20 21

Injection

50 µL

20 µL

10 µL

The Agilent 1260 Infinity Bio-inert Quaternary LC


58

100% Bio-inert Precious sample does not touch metal surfaces

pH range 1-13 (shortterm 14)

2 M salt, 8 M urea

No stainless steel inmobile phase flow path

New capillary technology

UHPLC capability 600 bar

Superior Ease of Use and Robustness Highly corrosion resistant

Active seal wash

Quaternary buffer mixing

Agilent proven quality

Agilent warranty and service quality

Superior Bio-HPLC columns for biotherapeutic

characterization

The choice for both, bioanalytical and biopurification up to 10 ml/min

Metal Free

Sample Flow Path

New Capillary Design

Metal cladded PEEK capillary

Newest fitting design: Hybrid technique

New capillary technolgy enables

600 bar AND is completely

metal-free!!

Buffer Advisor Software Quaternary Buffer Mixing with Buffer Advisor and 1260

Infinity Bio-inert LC System

September 30, 2012

Introduction to Agilent Buffer Advisor

60

• Recommends buffer stock solutions

• Shows recipes for stock solutions

• Calculates optimized gradient steps

• Enables automation and unified methods throughout a company

Instead of setting up different

bottles with different pH…

…you use a quaternary Bio-

inert LC with Buffer

Advisor software to

instantly run salt- and pH

gradients

…Saving Time and Resources….

September 30, 2012

Introduction to Agilent Buffer Advisor

61

Agilent Bio WCX: Data Analysis


62

Step 1

• pH 7.0 to 5.0

• In 0.2 unit increments

Step 2

• Buffer concentration

• 10, 15, 20, 25 mM

Step 3

• Gradient slope

• Rate of [salt] increase

Robust IEX Method

11 pH values

4 concentrations

at each pH

3 gradients

For each chromatogram calculate resolution between peaks 1 and 2

1

2

Reverse Phase

BioHPLC Columns

Characterization of Primary Structure


64

Levels of Characterization

Improve

accuracy

and

resolution

Intact MAb

Reduction/Alkylation

Light and Heavy Chain

Enzymatic Digestion

Peptide Map

Basics of Bio-Chromatography Strategy for RP-HPLC Method Development of

Proteins and Peptides

• Start at low pH (acidic mobile phase) and choose the initial column and conditions

• Initial selection parameters include: pore size (300Å), mobile phase, bonded phase,

particle size, column length and internal diameter

• Obtain best resolution by optimizing:

• Gradient steepness, bonded phase, temperature, column configuration

• Obtain best recovery by optimizing:

• Bonded phase, temperature, sample solubility

• Evaluate alternate columns/technologies for improved selectivity and efficiency

• Try ZORBAX 300Extend-C18 at higher pH for different selectivity; LC/MS

• Try Poroshell 300SB for improved efficiency and shorter analysis times; LC/MS

February 2010

Agilent Confidential 65

300Å Reverse Phase Columns Reverse Phase of Proteins

Requirements

• Wide pore - 300Å for unrestricted

access to bonded phase

• Multiple bonded phases for

selectivity optimization

• Bonded phases with different pH

ranges for LC/MS and method

confirmation (WCBP)

• Many configurations for LC/MS

compatibility, small sample sizes

and proteomics

Columns available

• 300StableBond – C18, C8, C3, CN

• Poroshell 300SB – C18, C8, C3

• 300 Extend-C18

• Configurations from nano (0.075

mm i.d.) to prep (21.2 mm i.d.)

Basics of Bio-Chromatography Strategy for RP-HPLC Method Development of

Proteins and Peptides

• Initial selection parameters include:

- pore size (300Å), mobile phase, bonded phase, particle size, column length and ID

• Obtain best resolution by optimizing:

- Gradient steepness, bonded phase, temperature, column configuration

• Obtain best recovery by optimizing:

- Bonded phase, temperature, sample solubility

• Evaluate alternate columns/technologies for improved selectivity and efficiency

- Try ZORBAX 300Extend-C18 at higher pH for different selectivity; LC/MS

- Try Poroshell 300SB for improved efficiency and shorter analysis times; LC/MS

68

StableBond 300Å

• Four different bonded-phases,

300SB-C18, C8, CN, and C3 for

selectivity optimization

• Extremely stable at low pH

• Use with TFA, Formate and

Acetate mobile phases

• Stable at high temperature – up

to 80 - 90°C

• Particle Sizes: 3.5, 5, and 7

O

R RSi

OH

SiO

SiR R R

OOH

R

R1 R1 R1

300Å Reverse Phase Columns ZORBAX 300SB

69

• Uses unique bidentate-C18 bonded phase for long lifetime at high pH

• Double endcapped

• Can also be used at low pH

• Ammonium hydroxide mobile phase good for high pH LC and LC/MS

Si

O

Si

O

Silica Support

C18C18

ZORBAX 300Extend-C18 Columns for the Analysis of Proteins and Peptides at High pH

Comparison of Small Peptide Selectivity

Differences on 300SB Bonded Phases

Conditions:

Columns: ZORBAX 300SB, 4.6 x 150 mm, 5 mm

Mobile Phase: Gradient, 0 - 26% B in 30min.

A = 0.1% TFA in Water

B = 0.1% TFA in Acetonitrile

Temperature: 40°C

Sample: 2 µg of each peptide

Flow Rate: 1.0 mL / min.

Detection: UV-210nm

300SB-C18

300SB-C8

300SB-C3

300SB-CN

NEW! Zorbax 300 SB RRHD for Proteins and Peptides!

•Stablebond 300 silica

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

•1.8 um particle size

•1200 Bar pressure limit for uHPLC

•Other phases to follow

•2.1 x 50 mm and 2.1 x 100 mm

Four Rapid Resolution High Definition Phases

Reversed-Phase RRHD

72

C18 C8 C3 Diphenyl

Peptide Maps Increasing protein size/hydrophobicity

Small Proteins

Ligand Application

C18 Small intact proteins/peptide maps

C8 Intact proteins

C3 Larger /hydrophobic proteins, including MAbs

Diphenyl Unique selectivity

ZORBAX

300Å, 1.8 µm

Agilent 1290 Infinity

Family of Four Reversed-Phase Ligands

Peptide Mapping

Reversed-Phase RRHD

73

min 0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

10

20

30

40

50

60

min 0 2 4 6 8 10 12 14 16 18

mAU

0

5

10

15

20

25

30

35

TFA

formic acid

5990-8244EN

Monoclonal Antibody Tryptic Digest

ZORBAX RRHD 300SB-C18

Fast Separation of Reduced and Alkylated MAb

Reversed-Phase RRHD

74

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


Detection: UV, 280

Conditions optimized for the ZORBAX RRHD 300SB-C3 Column

CHO Cell Derived MAb

5990-9667EN

Time

(min)

%B

0 1

2 20

5 50

6 90

6.1 1

Comparison C3 and Diphenyl Phases

Reversed-Phase RRHD

75

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

Column: 2.1 x 50 mm columns

Sample injection: 1 µL

Mobile phase A: 0.1% TFA in d. water

Mobile phase B: 0.01% TFA in ACN

Flow rate: 1 mL/min (~700bar)

HPLC instrument: 1290 Infinity Series

Detection: UV, 280

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

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

Poroshell Particles Provide More Efficient Peaks

for Peptides and Proteins

Poroshell 300 is for the separation of large peptides and proteins

Poroshell particles have a solid silica core with a superficially porous surface with a 300Å pore size

This results in more efficient mass transfer and sharper peaks with large proteins and peptides

Comparison of Diffusion Distance Totally porous silica vs. superficially porous silica

0.25 µm

5 µm

Totally Porous Particle

2.5 µm Required diffusion distance

for a macromolecule

reduced 10 fold !

5 µm

Superficially Porous Particle

Flow Rates for Poroshell Columns

Column Internal Diameter Porous Particle

Flow Rate Range

Poroshell

Flow Rate Range

2.1 mm 0.1 – 0.3 mL/min 0.3 – 3 mL/min

1.0 mm 30 – 60 L/min 0.08 - 0.75 mL/min

• Very high flow rates can be used effectively with Poroshell columns

1 2 3

4

Sustained Efficiency and Resolution

Poroshell 300 Reverse Phase Effect of Increasing Flow Rate in Protein Analysis with

Poroshell

1 2 3 4

Effect of Increasing Flow Rate in Protein

Analysis using Totally Porous Silica

Lost Efficiency and Resolution

1 2 3 4

Lost Efficiency and Resolution

Effect of Increasing Flow Rate in Protein

Analysis using Totally Porous Silica

• Poroshell technology facilitates ultra-fast HPLC analysis of very large protein subunits. Note the improved peak shape and recovery on Poroshell, in <2 min. Dramatic example of the lower surface area advantage.

Improved Recovery with Poroshell Analysis of High-Molecular Weight Proteins - Thyroglobulin 660 kDa

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 mm

Mobile Phase: 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


Sample:

1. Angiotensin II

2. Neurotensin

3. Rnase

4. Insulin

5. Lysozyme

6. Myoglobin

7.Carbonic Anhydrase

8.Ovalbumin

Reversed Phase

Heavy and light chain analytical characterization

Use of 1200 LC, Poroshell 300SB

columns and UV detection to

characterize antibodies

February 2010

Agilent Confidential 86

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 Microbore to Preparative

PLRP-S Continued:

Features

pH 1-14

Extreme buffer concentrations

High temperature stability

Durable and Resilient

Inherently hydrophobic so does not

require a bonded alkyl chain to confer

hydrophobicity

Benefits

Acid and base cleanup

Typically < 8M

200°C

Long Lifetimes

Avoids typical silica problems of

silanol group

Analysis of very large biomolecules

or high speed separations 1000A & 4000A pores

PLRP-S & RP-Silica Comparison

Eluent A :1% ACN (0.1% TFA) Flow rate: 1.0 mL/min

Eluent B :99% ACN (0.1% TFA) Detector: UV, 210 nm

Gradient :35-80% B, 0-20 mins

Key

1 H-(Phe)1-OH

2 H-(Phe)2-OH

3 H-(Phe)3-OH

4 H-(Phe)4-OH

5 H-(Phe)5-OH

PLRP-S 300Å 5 µm RP-Silica C18 300Å 5 µm

mins

1

2

3 4

5

0 20 mins

1

2 3

4

5

0 20

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.

New BioHPLC SEC New Zorbax SB 300A

and IEX Brochure 1.8um RRHD Brochure

#5990-5195 #5990-8124EN

BioHPLC Columns Navigator Poster #5990-5526EN

94

New BioHPLC Column

Selection Guide

#5990-9384

95

New LC Handbook

#5990-7595

September 30, 2012

Agilent Bio-inert LC Seminar

96

Biomolecule Purification

Brochure

#5990-8335

New Protein Identification

And Impurity Profiling

#5991-0625EN

New Applications on the bio-inert LC and

Bio-IEX Columns: Usage of Buffer Advisor

# 5990-9628EN # 5990-9629EN

Further Information: App Notes

Reversed-Phase RRHD

98

Pub Number Title

5990-9628EN Optimizing Protein Separations with Weak Cation-Exchange Columns

5990-9629EN pH Gradient Elution for Improved Separation of Monoclonal Antibody Charge Variants

5990-9894EN Optimum Pore Size for Characterizing Biomolecules with Agilent Bio-SEC Columns

5990-9931EN Fast Separations Using Agilent Weak Cation Exchange Columns

5990-9631EN Rapid UHPLC analysis of reduced monoclonal antibodies using Agilent ZORBAX Rapid Resolution High Definition (RRHD)

300SB-C8 column

5990-9667EN Reversed-phase optimization for ultra fast profiling of intact and reduced monoclonal antibodies using Agilent Rapid Resolution

High Definition 300SB-C3 column

5990-9668EN Ultra high speed and high resolution separations of reduced and intact monoclonal antibodies with Agilent ZORBAX RRHD

sub-2 µm 300 Diphenyl UHPLC column

Further Information: App Notes & Primer

Reversed-Phase RRHD

99

Pub

Number

Title

5990-

7989EN

Agilent ZORBAX 300SB-C18 1.8 µm Rapid

Resolution High Definition columns for proteins

5990-

8244EN

Analyze MAb and BSA digests by UHPLC with UV

detection and Agilent ZORBAX RRHD 300SB-C18

5990-

7988EN

Analysis of oxidized insulin chains using reversed-

phase Agilent ZORBAX RRHD 300SB-C18

5990-

9248EN

Fast separation of recombinant human erythropoietin

using reversed-phase Agilent ZORBAX RRHD

300SB-C18, 1.8 µm

5990-

9016EN

Reversed-phase separation of Intact Monoclonal

Antibodies using Agilent Zorbax RRHD 300SB

5990-

9544EN

Separation of recombinant Human Erythropoietin

(rEPO) using Agilent Bio-SEC 3

5990-

9270EN

Separation of Protein Standards on Agilent 3um Ion

Exchange Columns by Cation Exchange

Chromatography

New Primer: Recombinant

Protein Characterization

#5990-8561

Further Information: App Notes & Posters

Reversed-Phase RRHD

100

Pub

Number

Title

5989-

9899EN

Fast Protein Separations Using Agilent Poroshell 300

5990-

6414EN

Characterization of monoclonal antibodies on Agilent

1260 Bio-inert Quaternary LC by SEC using Bio-SEC

columns

5990-

6192EN

Physicochemical characterization of a therapeutic

protein by peptide mapping, SEC and IEX using Agilent

1260 LC

5990-

8613EN

Fast Separation of Monoclonal Antibody Monomer and

Dimer by SEC with Agilent Bio-SEC

5990-

8895EN

Defining the optimum parameters for efficient size

separations of proteins

5989-

6840EN

Comparison of Zorbax Stablebond 300A columns to

optimize selectivity for antibody separations using

LC/MS

5989-

0604EN

Optimization of rapid HPLC analysis of monoclonal

Intact Antibodies. Light Chains, and Heavy Chains

using Zorbax Poroshell

Further Information: App Notes & Posters

PUB # Title

5989-

0030EN

Rapid HPLC Analysis of Monoclonal Antibody IgG1

Light Chains Using Zorbax Poroshell 300SB-C8

5990-

9940EN

Charge Variant Analysis of Monoclonal Antibodies by

pH Gradient Separation on Cation-exchange Columns

5990-

0691EN

Increasing Throughput: The Advantages of pH

Gradients in High Speed Ion-Exchange Analysis of

Proteins

5990-

0680EN

Protein Aggregation: A Multiple Detector Investigation

into Size Exclusion Chromatography

6989-

9733EN

Rapid Human Polyclonal IgG Quantification using the

Agilent Bio-Monolith HPLC Column

5989-

9674EN

Rapid IgM Quantification in Cell Culture Production

and Purification Process Monitoring Using the Agilent

Bio-Monolith QA Column

5990-

3247EN

Fast Monitoring of Bacteriophage Production During

Fermentation Using the Agilent Biomonolith HPLC

Column

5990-

5524EN

Rapid Analysis of Adenovirus Type 5 Particles with

Bio-Monolith Anion-Exchange HPLC Columns to

Support the Development of a High Titre

Manufacturing Platform

101

Agilent Biopharma Training, AFO NPT 2010

BioHPLC Columns on the

Agilent Website

103

NEED ASSISTANCE?

LC Column help desk

1-800-227-9770

• orders

• customer service

(option 1,1)

• technical support

• applications assistance

(option 3,3,2)

[email protected]

Thank you !

Questions?

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