Improving Bioseparations Using 2D-LC

Gregory Staples, Ph.D.

R&D Scientist

Agilent Technologies

August 2015

1

Why 2D Liquid Separations?

2

Resolving power is what it is

all about in analytical

separation science

August 2015

Today’s Examples

1. Additional peak capacity

needed to characterize

samples.

2. Mass measurement

needed from MS-

incompatible separations

One Dimensional LC

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August 2015

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Two Dimensional LC

Page 5

Pump 1

Time % B

0 0

6 0

135 10

200 20

230 30

280 50

295 100

320 100

320.1 0

350 0

Time % B Time % B

0 3 175 3

5 3 201.1 65

26.1 65 201.2 3

26.2 3 210 3

35 3 236.1 65

61.1 65 236.2 3

61.2 3 245 3

70 3 271.1 65

96.1 65 271.2 3

96.2 3 280 3

105 3 306.1 65

131.1 65 306.2 3

131.2 3 315 3

140 3 340 65

166.1 65 340.1 90

166.2 3 345 90

Pump 2

Time Position

0 column 2

26.1 column 1

35 column 2

61.1 column 1

70 column 2

96.1 column 1

105 column 2

131.1 column 1

140 column 2

166.1 column 1

175 column 2

201.1 column 1

210 column 2

236.1 column 1

245 column 2

271.1 column 1

280 column 2

306.1 column 1

315 column 2

345 column 1

Time Position

0 Pos 1

5 Pos 2

30 Pos 1

65 Pos 2

100 Pos 1

135 Pos 2

170 Pos 1

205 Pos 2

240 Pos 1

275 Pos 2

310 Pos 1

6-port valve 10-port valve

Challenges in Implementing 2D-LC

August 2015

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Columns, methods,

checkout samples

2D Acquisition Software

LC and LC/MS

Hardware

2D-LC Specific Valve Kits

2D-LC Primer by

Dwight Stoll and Peter Carr

2D-LC Data Analysis

and Reporting

Heart-Cutting 2D-LC

Heart-cutting 2D-LC (LC-LC):

LC1

LC2

Selected portions of the 1D effluent

are injected onto 2D

Long 2D gradients possible best

possible separations in 2D

Limited 2D information due to

undersampling of 1D

7

August 2015

Comprehensive 2D-LC

LC1

LC2

1st peak from

1st dimension

2nd peak from

1st dimension

3rd peak from

1st dimension

The entire 1D effluent is injected onto

second dimension

Ultra Short 2D gradients required, driven

by fast and low-dispersion pumps

Full (comprehensive) 2D information!

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August 2015

Comprehensive 2D-LC (LCxLC):

Comprehensive 2D-LC

Comprehensive 2D-LC (LCxLC):

LC1

LC2

1st peak from

1st dimension

2nd peak from

1st dimension

3rd peak from

1st dimension

LC1

LC2

min

sec

9

August 2015

Bringing in a 2nd Dimension - How? A unique 2D-LC valve

10

August 2015

MULTIPLE HEART-CUTTING 2D-LC

A new tool closing closing the gap between comprehensive and heart-cutting 2D-LC

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The Shortfall of the Heart-Cutting Approach

12

August 2015

20min

Peaks lost!

1D

2D

4.5min 4.5min

Multiple Heart-Cutting 2D-LC Solution

13

August 2015

20min

1D

2D

4.5min 4.5min 4.5min 4.5min 4.5min

Multiple Heart-Cutting 2D-LC Solution

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Multiple Heart-cutting Valve

Most intuitive software to set

up and edit methods within

seconds:

• 1st dimension gradient

• 2nd dimension gradient

• Time-segments

• Method parameters

• Method set-up calculator

• Time-based or peak-based

mode

• Reference chromatogram

overlay for heart-cutting

• Gradient shift

2D-LC Acquisition Software Easy-to-use software for all operation modes

15

August 2015

Method Optimization using Shifted Gradients Taxanes from taxus extract

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RP (C18)

RP

(P

hen

ylH

ex

yl)

Shifted gradients can be

easily programmed by

drag-and-drop interface

CASE STUDY 1 THERAPEUTIC PROTEIN GLYCAN PROFILING HILIC / ION EXCHANGE 2D-LC SONJA SCHNEIDER, AGILENT (WALDBRONN, GERMANY)

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1D HILIC Separation Agilent AdvanceBio Glycan Mapping Column

min 10 12.5 15 17.5 20 22.5 25 27.5 30 min 10 15 20 25 30 35

EPO Fetuin

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How pure are these peaks?

1D = WAX 2D = HILIC

Fractions collected. Additional hands-

on time for fractionation and re-

analysis.

Previously undetected peaks

identified when using 2D approach.

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Comprehensive 2D-LC

Two possible combinations:

1. WAX / HILIC

1D: Agilent Bio WAX column, 2.1 × 250 mm, 5 μm

2D: Dimension: Agilent AdvanceBio Glycan Mapping column, 4.6 × 50 mm, 2.7

μm

2. HILIC / WAX (Directly Coupled to MS)

1D: Agilent AdvanceBio Glycan Mapping column, 2.1 × 150 mm, 1.8 μm – Total

run time 165 min (Stoptime + Post-time)

2D: Agilent Bio WAX column, 2.1 × 50 mm, 5 μm – 30 second 2D gradients

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Comprehensive 2D HILIC/WAX Fetuin

Tri Sialylated

Tetra Sialylated

Di Sialylated

Neutral

Mono Sialylated

Penta Sialylated

1D – HILIC – 110 minutes

2D

– W

AX

– 3

0 s

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Comprehensive 2D HILIC/WAX EPO

1D – HILIC – 110 minutes

2D

– W

AX

– 3

0 s

Tri Sialylated

Tetra Sialylated

Di Sialylated

Mono Sialylated

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Multiple Heart-Cutting Permits WAX/HILIC Separations

• Using Multiple Heart-

Cutting, adequate time

can be alotted to HILIC

column equilibration in

the second dimension.

• Furthermore, the multiple

heart-cutting loops can

be partially filled, limiting

the amount of aqueous

solvent introduced to the 2D HILIC column.

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Multiple Heart-Cutting

8

min 62 63 64 65

LU

0

0.2

0.4

0.6

0.8

1

1.2 10

min 52 52.5 53 53.5 54 54.5 55 55.5 56

LU

0

2

4

6

8

9

min 56.5 57 57.5 58 58.5 59 59.5 60 60.5

LU

0

1

2

3

4

5 5

min 22.5 23 23.5 24 24.5 25 25.5 26 26.5

LU

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2 4

min 27.5 28 28.5 29 29.5 30 30.5 31 31.5

LU

0

0.25

0.5

0.75

1

1.25

1.5

1.75

1

min 7 8 9 10 11

LU

0

0.05

0.1

0.15

0.2

Multiple Heart-Cutting WAX/HILIC EPO Analysis

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Case study 1: 2D-LC of EPO Glycans – Summary

• Comprehensive WAX/HILIC – Failed due to slow HILIC

equilibration and solvent incompatibility.

• Comprehensive HILIC/WAX – Successful, high peak

capacity.

• Complete automation possible with a run time of about 110 minutes.

• Simplified data interpretation due to the grouping of the glycans

according to their charge in the second dimension.

• Multiple Heart-Cutting WAX/HILIC – Successful, high peak

capacity

• High flexibility – choice of flow rate and gradient length in 2D.

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CASE STUDY 2 INTACT MAB CHARACTERIZATION: HYDROPHOBIC INTERACTION / REVERSED-PHASE 2D-LC/MS

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Hydrophobic Interaction Chromatography (HIC)

• Weakly non-polar phase, typically with

lower bonding density compared to

reversed-phase

• Proteins in lyotropic salts e.g. (NH4)2SO4

separated by hydrophobic interactions with

solid phase. Elution is driven by reducing

salt content.

• Protein higher-order structure is generally

retained, unlike in RP separations.

• Commonly used for purification/polishing of

and analytical scale analysis of mAbs.

August 2015

PO43-, SO4

2-, CH3COO-, Cl-, Br -, NO3-, ClO4

-, I-, SCN-

Chaotropic

NH4+, Rb+, K+, Na+, Cs+, Li+, Mg2+, Ca2+, Ba2+

Lyotropic

Time

[Salt]

27

HIC Buffer

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Example mAb Separations Using HIC

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Oxidized Trp in CDR (+16 Da)

Asp → Succinimide in CDR (-18 Da)

Boyd, D. et. al. J. Chromatography B (2011) 879, 955-960.

Valliere-Douglass, J. et. al. J. Chromatography A (2008) 1214, 81-89.

Oxidized Met in Fc (+16 Da)

Antibody Drug Conjugates (ADCs): mAbs with Missiles

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Linker Chemistry

Cytotoxic Payload

Monoclonal

Antibody

Within the class: different

physicochemical properties

which makes

characterization a

challenge.

HIC for ADC Separation and DAR Calculation

Hamblett, K.J. et al. Clin. Cancer Res. 2004; 10:7063-70.

Wakankar, A. et al. mAbs. 2011; 3:161-172.

Panowski, S. et al. mAbs. 2014; 6: 34-45.

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Sites, linker chemistry,

payload chemistry!

HIC Separation of Cys-linked ADCs with Different Drug Loads

August 2015

Response Units vs. Acquisition Time (min)

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Response Units vs. Acquisition Time (min)

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0.5X

1X

1X

1.5X

2.0X

2.5X

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Detector 2D

Detector 1D

2D LC/MS for Intact mAbs

BioInert Quat

1290 Binary

Multiple Heart-Cutting

1D BioInert Non-MS Compatible Separations:

Hydrophobic Interaction

Cation Exchange

Anion Exchange

Chromatofocusing

Affinity

2D 1290 MS Compatible Separations

Size Exclusion

Reversed Phase

Rapid, automated

mass analysis from

bioseparations!

How can we make this workflow

as efficient as possible?

BioInert

Autosampler

Column

Compartment

TOF or QTOF

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An Improved HIC Buffer for Superior Coupling with MS

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Xiu, L. et. al. Anal. Chem. (2014) 86, 7899-7906.

NH4+

NH4+

Ammonium Tartrate Buffer for HIC

Ammonium sulfate results in +98 Da H2SO4 adducts

that negatively impact protein MS.

HIC/RP-MS of a Force-oxidized mAb

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August 2015

HIC/RP-MS of a mAb Biosimilar

36

Abso

rba

nce

August 2015

HIC/RP-MS of a mAb Biosimilar

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HIC/RP-MS of a Lys-Linked ADC

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HIC/RP-MS of a Lys-Linked ADC

Case study 2: HIC/RP MS of mAbs – Summary

• Multiple heart-cutting 2D-LC/MS platform for bioseparations; complex workflow made simple because of easy to implement hardware and easy to use software.

• Rapid and efficient mass measurement without tedious

collection, desalting, and re-injection of fractions. Great first step in characterizing a given mAb.

• Workflow is also applicable to other bioseparations such as SEC and IEX.

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Thanks for your attention!