LC/MS Workflows for Biosimilars & Antibody Drug …...Caroline S. Chu, Ph.D Application Scientist Agilent Technologies, Inc. Santa Clara, CA Comprehensive Portfolio of Analytical Instrumentation

June 22, 2015

Confidentiality Label

1

LC/MS Workflows for Biosimilars & Antibody Drug Conjugates

Caroline S. Chu, Ph.D

Application ScientistAgilent Technologies, Inc.Santa Clara, CA

Comprehensive Portfolio of Analytical Instrumentation

June 22, 2015

Biosimilar & Antibody-Drug-Conjugate Analysis Workflow:

June 22, 2015Agilent Confidential

3

Intact

• Analysis of the intact mAb(s)

Reduced• Reducing the mAb(s) to their Heavy and Light Chains

Peptide Mapping

• Tryptic Digests of the mAb(s) for sequence coverage from the peptide map

Glycan Analysis

• Using microfluidic technology to do online deglycosylation of the mAb(s) and characterizing the released N-Glycans

On-Chip Glycan Characterization

M.A. Bynum, H. Yin, K. Felts, Y.M. Lee, C.R. Monell, K. Kileen, Anal.Chem. 2009, 81, 8818-8825

mAb-Glyco Chip

Immobilised PNGase F Enzyme Reactor

PGC - Separation Column

PGC - EnrichmentColumn

Nano-pump in

Cap-pump in

Inner Rotor:Turns enrichment column between separation and loading path

Outer Rotor:Turns enzyme reactor into/out of the loading path

Rotor-In-Rotor Design

Agilent MassHunter BioConfirm for Data Analysis

BioConfirm

Intact protein analysis

Peptide mapping

Glycan analysis

Synthetic peptide

Oligo-nucleotide

June 22, 2015


8

Native MS

Cysteine Conjugated AFC

Online Deglycosylation/Denaturing MS

Microfluidic ADC Characterization

Lysine Conjugated ADC

Online Deglycosylation

Online Deglycosylation:

N-Glycans

mAb Glyco HPLC Chip

mAb GlycoPersonal

Compound Database/Library

Intact mAb Workflow: Automated Intact Analysis

June 22, 2015


10

Acquire Data• HPLC Chip Interfaces with high resolution Q/TOF• Native MS: Capillary Flow

Integrate Chromatogram & Extract MS• MassHunter’s BioConfirm

Deconvolute MS & Match Sequences• BioConfirm Provides Two Deconvolution Algorithmns• Maximum Entropy (MaxEnt)• Peak Modeling (pMod)

Custom Reports• Summary of Accurate Intact Mass Measurements• Post Translational Modifications including N-Glycans

Native MS: Deglycosylated mAb vs. AFCTriplicate Overlays

June 22, 2015


11

2x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Counts (%) vs. Mass-to-Charge (m/z)

5000 5200 5400 5600 5800

2x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


5000 6000 7000 8000 9000

2x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


5000 5200 5400 5600 5800

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


5000 6000 7000 8000 9000

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Counts (%) vs. Deconvoluted Mass (amu)

142000 143000 144000 145000 146000 147000 148000 149000

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Counts vs. Deconvoluted Mass (amu)

142000 144000 146000 148000

DAR 0

DAR 3

DAR 2DAR 1 DAR 4

DAR 5 DAR 6DAR 7

mAb

DAR 0

DAR 3

DAR 2

DAR 1DAR 4

DAR 5 DAR 6

DAR 7

DAR 8

10 µg of deglycosylated mAb on column15 µg of deglycosylated AFC on column

mAbSpectrum

AFCSpectrum

Zoom

Zoom

Deconvoluted

Native MS: Glycosylated mAb vs. AFCTriplicate Overlay

June 22, 2015


12

2x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

Counts vs. Mass-to-Charge (m/z)

5350 5360 5370 5380 5390 5400 5410 5420

1x10

0

1

2

3

4

5

6

7


5000 5200 5400 5600 5800 6000

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


5000 6000 7000 8000 9000

1x10

0

1

2

3

4

5

6

7


5000 6000 7000 8000 9000

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


145000 146000 147000 148000 149000 150000 151000 152000

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1


145000 146000 147000 148000 149000 150000 151000 152000 153000

DAR 0DAR 3

DAR 2DAR 1 DAR 4DAR 5

DAR 6 DAR 7

mAb

DAR 0 DAR 3DAR 2

DAR 1

DAR 4

DAR 5

DAR 6 DAR 7

1 µg of glycosylated mAb on column5 µg of glycosylated AFC on column

mAbSpectrum

AFCSpectrum

Zoom

Zoom

Deconvoluted

Online Deglycosylation of mAb vs. AFC1 µg on column

June 22, 2015


13

-1.2

-1.1

-1

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

70946.9

23906.8

23907.3

141894.550969.5 119959.3

45850.5

98020.1

52576.974522.8

91702.859979.736461.0 137545.0

91701.2

108508.3

104825.8

127415.167442.3 80411.2

144783.5119625.3 137553.5


20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000

Intact AFC

Intact AFCOnline Deglycosylation

Light Chain

2 Light Chains

Light Chain +Heavy Chain

2 Heavy Chains

Light Chain +2 Heavy Chains

2 Light Chains +2 Heavy Chains

Intact Antibody Fluorescence Conjugate, AFC vs. Deglycosylated Intact AFC (Heavy Chain)

June 22, 2015


14

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

He

xose

- 0

.05

Ga

lact

ose

- 0

.05

Ma

nn

ose

- 0

.05

He

xose

- 0

.05

Ga

lact

ose

- 0

.05

Ma

nn

ose

- 0

.05

98

1.0

0

98

1.0

0

98

1.0

0


48200 48400 48600 48800 49000 49200 49400 49600 49800 50000 50200 50400 50600 50800 51000 51200 51400 51600 51800 52000 52200 52400 52600 52800

Intact AFCC18 Chip

Deglycosylated AFCCustom mAb Glyco Chip

Glycans Released

Addition of Fluorescence Tag(s)

Microfluidic HPLC-Chip Workflow

June 22, 2015


15

ProtID Chip: Reverse Phase Chip • Intact & Reduced mAb Analysis• Peptide Mapping

mAb ProtID Chip: Deglycosylated mAb Analysis• Online Deglycosylation of an Intact mAb or a

mAb Digest

mAb Glyco Chip: N-Glycan Characterization• Online Deglycosylation of an Intact mAb or a

mAb Digest

Porous Graphitized Carbon Chip: Glycan Analysis• Offline Deglycosylation Glycan Analysis

Custom On-chip Deglycosylation and Analysis of Intact Antibodies

4x10

012345678

146202.21

147645.93

Counts vs. Deconvoluted Mass (amu)145500 146000 146500 147000 147500 148000 148500 149000 149500

1 glycan removed

147806.94

147962.53

4x10

0

1

2

3

4

5

6Deglycosylated mab


Both glycans removed

4x10

0

1

2

3

4

5

6

149089.58


149253.47

149415.31

3 sec. deglycosylation

6 sec. deglycosylation

Intact Antibody Prior to Deglycosylation

4 minutes

1 minute

Q/TOF MS Detector

mAb sample

2 minutes

PNGase F, enzymatic N-glycan release

N-Glycan concentration

Deglycosylated mAbAnalysis

Intact mAb vs. Deglycosylated Intact mAb

June 22, 2015


17

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7


2200 2400 2600 2800 3000 3200 3400

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7


2710 2720 2730 2740 2750 2760 2770 2780

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6


143500 144000 144500 145000 145500 146000

Full Spectrum Zoomed In Deconvoluted-Max Entropy

Deglycosylated mAbmAb ProtID Chip

Intact mAbC18 Chip

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

He

xose

- 0

.05

Ga

lact

ose

- 0

.05

Ma

nn

ose

- 0

.05

He

xose

- 0

.05

Ga

lact

ose

- 0

.05

Ma

nn

ose

- 0

.05

He

xose

- 0

.05

Ga

lact

ose

- 0

.05

Ma

nn

ose

- 0

.05


143600 143800 144000 144200 144400 144600 144800 145000 145200 145400 145600 145800 146000 146200

Intact mAb vs. Deglycosylated mAb

June 22, 2015


18

Intact mAbC18 Chip

Deglycosylated mAbmAb ProtID Chip

Glycans Released

Hexose Differences, 0.05ppm

Microfluidic HPLC-Chip Workflow:Intact ADC vs. Online Deglycosylated ADC

June 22, 2015


19

-1.2

-1.1

-1

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

151096.2

148044.4147087.2

152053.5

146129.3

150138.6

149001.9

153011.4

149180.5

145174.2 149957.9

153968.1

155089.2

150916.4

147512.1 148223.0

155092.1152807.3151913.0

156182.5146670.9

154143.3


145000 146000 147000 148000 149000 150000 151000 152000 153000 154000 155000 156000

DAR 0

DAR 3DAR 2DAR 1

DAR 4

DAR 5

DAR 6 DAR 7

DAR 1

DAR 2

DAR 3

DAR 4

DAR 5

DAR 6DAR 7

DAR 8

DAR 8

Intact ADC

Intact ADCOnline Deglycosylation

6 7 8 9 1011121314151617Retention Time (min)

HIC

Triplicate Injections of 1 µg of intact Lysine conjugate ADC using the ProtID HPLC Chip

June 22, 2015


20

-1x10

1

2

3

4

5

6

7

8

9


2400 2600 2800 3000 3200

-1x10

1

2

3

4

5

6

7

8

9


2710 2715 2720 2725

Spectrum

Zoom of a DAR z state

2x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1 151096.4

152053.5

150139.7

153011.9

149180.7

153969.4

154926.3

148223.6 156181.7

Dru

g-L

inke

r +

6.6

Dru

g-L

inke

r -

5.4

Dru

g-L

inke

r -

3.0

Dru

g-L

inke

r -

5.4

Dru

g-L

inke

r -

5.4

Dru

g-L

inke

r +

2.6

Dru

g-L

inke

r +

1.0


148000 148500 149000 149500 150000 150500 151000 151500 152000 152500 153000 153500 154000 154500 155000 155500 156000 156500

DeconvolutedMaxEnt

Triplicate Injections of 1 µg of intact Lysine conjugate ADC using the ProtID HPLC Chip

June 22, 2015


21

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


2400 2600 2800 3000

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


2830 2840 2850 2860

2x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1 147086.2148044.0

149002.2

146130.3

149961.9

145172.7

150917.7

151916.9

Drug

-Lin

ker -

3.0

Drug

-Lin

ker -

0.6

Drug

-Lin

ker -

4.6

Drug

-Lin

ker -

3.0

Drug

-Lin

ker +

1.8

Drug

-Lin

ker +

3.4

Drug

-Lin

ker -

3.0


145000 145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500 152000

DAR 1DAR 2 DAR 3 DAR 4

DAR 5

DAR 0DAR 6

DAR 7

Spectrum

Zoom of a DAR z state

DeconvolutedMaxEnt

Batch to Batch Comparison of AFC: HPLC Chip

June 22, 2015


22

2x10

0

0.2

0.4

0.6

0.8

170954.822927.9

49996.2

98021.3 143871.3119972.9

81683.235973.4

2x10

0

0.2

0.4

0.6

0.8

1 70946.8

23906.7 45849.9

141893.8119959.6

98021.681281.859980.1

2x10

0

0.2

0.4

0.6

0.8

1 70946.9

23907.3

141894.550969.5 119959.3

98020.1

59979.736461.0


20000 40000 60000 80000 100000 120000 140000

Intact AFC Batch 2Online Deglycosylation



mAb Glyco Workflow: Automated N-Glycan Analysis

June 22, 2015


26

Acquire Data•HPLC Chip Interfaces with high resolution Q/TOF

Extract Compounds•MassHunter’s Molecular Feature Extraction (MFE) Algorithmn (analysis method provided with Chip)

•MassHunter’s Find By Formula (FBF)

Identify Glycans with mAb Glyco Database•Personal Compound Database/Library (provided with Chip)

Custom Reports•Summary of N-Glycans Identified•Percent Distribution

mAb-Glyco Chip Kit – A Comprehensive Workflow Solution

mAb-Glyco chipReagent Pack

Structure Viewer

Total workflow solution

Glycan Database

Reporting Templates

MassHunter Qualitative Analysis B.07.00:mAb Glycan Analysis

6/22/2015Agilent Restricted

28

Molecular Feature Extraction SpectrumRaw Spectrum

Extracted Ion Chromatogram Structure Viewer

Glycans Identified

MassHunter Qualitative Analysis B.06.00:mAb Glycan Analysis

6/22/2015

Agilent Restricted

29

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

893.8445([C68H115N5O49]+2H)+2


892 893 894 895 896 897 898 899 900

Zoomed SpectrumTheoretical Isotopic Overlaid onto Experimentalz = 2+

Raw Spectrum4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

596.2311([C68H115N5O49]+3H)+3


594 595 596 597 598 599 600 601

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

596.2311([C68H115N5O49]+3H)+3


594 595 596 597 598 599 600 601

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

893.8445([C68H115N5O49]+2H)+2


892 893 894 895 896 897 898 899 900

Molecular Feature Extraction Spectrumz = 2+

Zoomed SpectrumTheoretical Isotopic Overlaid onto Experimentalz = 3+

Molecular Feature Extraction Spectrumz = 3+

mAb Glyco Chip: Glycan Analysis of mAb,1 µg on column

6/22/2015

Agilent Restricted

31

Injection #1 Injection #2 Injection #35x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8 8.6

8.7

8.7

8.7

Counts vs. Acquisition Time (min)

8.5 8.55 8.6 8.65 8.7 8.75 8.8 8.85

5x10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

8.7

8.7

8.7

8.7


8.5 8.55 8.6 8.65 8.7 8.75 8.8 8.85

5x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.48.7

8.7

8.7

8.7


8.5 8.55 8.6 8.65 8.7 8.75 8.8 8.85

N

N N

N

N

N N

N

N

N N

N

mAb Glyco Chip: Glycan Analysis of Lysine Conjugated ADC, 2 µg on column

June 22, 2015


32

2x10

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

Counts (%) vs. Acquisition Time (min)8.42 8.44 8.46 8.48 8.5 8.52 8.54 8.56 8.58 8.6 8.62 8.64 8.66 8.68 8.7 8.72

2x10

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1


2x10

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1


Injection #1 Injection #2 Injection #3

Microfluidic HPLC-Chip Workflow

June 22, 2015


33

• Intact & Reduced mAb Analysis

• Peptide Mapping

C18 HPLC-Chip

• Online Deglycosylation

• Intact/Digested mAb Analysis

Custom mAbGlyco Chip

• Online Deglycosylation

• Glycan Analysis

mAb GlycoHPLC Chip

• Offline Deglycosylation

• Glycan Analysis

PGC Chip

Drug-to-Antibody

Ratio (DAR)

Denaturing Mass

Spectrometry

Denaturing Analysis-

HPLC-Chip

Glycan Analysis

HPLC- Chip

Intact Quantitation

Native Mass Spectrometry

Acknowledgments:

June 22, 2015


34

Ning Tang

Andy Gieschen

Greg Staples

Jose Meza

Michael Yap

Gurmil Gendeh

Documents

LC/MS Workflows for Biosimilars & Antibody Drug …...Caroline S. Chu, Ph.D Application Scientist Agilent Technologies, Inc. Santa Clara, CA Comprehensive Portfolio of Analytical Instrumentation