lc/ms workflows for biosimilars & antibody drug …...caroline s. chu, ph.d application...
TRANSCRIPT
June 22, 2015
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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
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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
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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
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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
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2x10
0.1
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0.5
0.6
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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
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0.9
1
Counts (%) vs. Mass-to-Charge (m/z)
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
Counts (%) vs. Mass-to-Charge (m/z)
5000 5200 5400 5600 5800
2x10
0
0.1
0.2
0.3
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0.6
0.7
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0.9
1
Counts (%) vs. Mass-to-Charge (m/z)
5000 6000 7000 8000 9000
2x10
0
0.1
0.2
0.3
0.4
0.5
0.6
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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
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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
Counts vs. Mass-to-Charge (m/z)
5000 5200 5400 5600 5800 6000
2x10
0
0.1
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0.3
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1
Counts (%) vs. Mass-to-Charge (m/z)
5000 6000 7000 8000 9000
1x10
0
1
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4
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6
7
Counts vs. Mass-to-Charge (m/z)
5000 6000 7000 8000 9000
2x10
0
0.1
0.2
0.3
0.4
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0.6
0.7
0.8
0.9
1
Counts (%) vs. Deconvoluted Mass (amu)
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
Counts vs. Deconvoluted Mass (amu)
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
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-1.2
-1.1
-1
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-0.2
-0.1
0
0.1
0.2
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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
Counts vs. Deconvoluted Mass (amu)
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
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-1.8
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
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1.4
1.6
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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
Counts vs. Deconvoluted Mass (amu)
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
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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
Counts vs. Deconvoluted Mass (amu)145500 146000 146500 147000 147500 148000 148500 149000 149500
Both glycans removed
4x10
0
1
2
3
4
5
6
149089.58
Counts vs. Deconvoluted Mass (amu)145500 146000 146500 147000 147500 148000 148500 149000 149500
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
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3x10
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7
Counts vs. Mass-to-Charge (m/z)
2200 2400 2600 2800 3000 3200 3400
3x10
0
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Counts vs. Mass-to-Charge (m/z)
2710 2720 2730 2740 2750 2760 2770 2780
4x10
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4x10
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Counts vs. Deconvoluted Mass (amu)
143500 144000 144500 145000 145500 146000
Full Spectrum Zoomed In Deconvoluted-Max Entropy
Deglycosylated mAbmAb ProtID Chip
Intact mAbC18 Chip
-3
-2.5
-2
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He
xose
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ose
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Ma
nn
ose
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He
xose
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Ga
lact
ose
- 0
.05
Ma
nn
ose
- 0
.05
He
xose
- 0
.05
Ga
lact
ose
- 0
.05
Ma
nn
ose
- 0
.05
Counts vs. Deconvoluted Mass (amu)
143600 143800 144000 144200 144400 144600 144800 145000 145200 145400 145600 145800 146000 146200
Intact mAb vs. Deglycosylated mAb
June 22, 2015
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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
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-1.2
-1.1
-1
-0.9
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-0.7
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-0.4
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-0.1
0
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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
Counts vs. Deconvoluted Mass (amu)
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
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-1x10
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9
Counts (%) vs. Mass-to-Charge (m/z)
2400 2600 2800 3000 3200
-1x10
1
2
3
4
5
6
7
8
9
Counts (%) vs. Mass-to-Charge (m/z)
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
Counts (%) vs. Deconvoluted Mass (amu)
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
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0
0.1
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1
Counts (%) vs. Mass-to-Charge (m/z)
2400 2600 2800 3000
0
0.1
0.2
0.3
0.4
0.5
0.6
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0.8
0.9
1
Counts (%) vs. Mass-to-Charge (m/z)
2830 2840 2850 2860
2x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
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0.45
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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
Counts (%) vs. Deconvoluted Mass (amu)
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
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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
Counts (%) vs. Deconvoluted Mass (amu)
20000 40000 60000 80000 100000 120000 140000
Intact AFC Batch 2Online Deglycosylation
Intact AFC Batch 1Online Deglycosylation
Intact AFC Batch 3Online Deglycosylation
mAb Glyco Workflow: Automated N-Glycan Analysis
June 22, 2015
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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
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Molecular Feature Extraction SpectrumRaw Spectrum
Extracted Ion Chromatogram Structure Viewer
Glycans Identified
MassHunter Qualitative Analysis B.06.00:mAb Glycan Analysis
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4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
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0.8
0.9
1
1.1
1.2
1.3
1.4
893.8445([C68H115N5O49]+2H)+2
Counts vs. Mass-to-Charge (m/z)
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
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0.7
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0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
596.2311([C68H115N5O49]+3H)+3
Counts vs. Mass-to-Charge (m/z)
594 595 596 597 598 599 600 601
4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
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0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
596.2311([C68H115N5O49]+3H)+3
Counts vs. Mass-to-Charge (m/z)
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
Counts vs. Mass-to-Charge (m/z)
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
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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
Counts vs. Acquisition Time (min)
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
Counts vs. Acquisition Time (min)
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
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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
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
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
Injection #1 Injection #2 Injection #3
Microfluidic HPLC-Chip Workflow
June 22, 2015
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• 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
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Ning Tang
Andy Gieschen
Greg Staples
Jose Meza
Michael Yap
Gurmil Gendeh