Copyright ©2015 Q2 Solutions. All rights reserved.
COMPANY CONFIDENTIALBarry R. Jones
Multi-Dimensional Nano-Scale Chromatography for Regulated Peptide/Protein Bioanalysis
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• The tools
• Case Studies– Insulin
• PPT online immunoaffinity C18 Nano-LC HRMS
– ANP• Offline IP online immunoaffinity C18 Nano-LC HRMS
– NGF• Offline IP digestion online immunoaffinity C18 Nano-LC HRMS
– Protein Therapeutic example• Offline IP Digestion C18 Nano-LC QqQ
• Conclusion
OutlineOur approach to peptide and protein bioanalysis by LCMS
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The Tools
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Hamilton Microlab STAR
S.T.A.R. – Sequential Transfer Automation Robot
• Advanced Liquid Handling:> Monitored Air Displacement> Liquid Level Detection (cLLD / pLLD)
• Integrated Third-Party Devices:> Tele-shakers> Inheco heater/cooler modules> Magnetic bead plates
• “Walk Away” Methods:> Error Handling> Network Capability
www.hamiltonrobotics
For Bead-Based IP
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• Allows for multi-dimensional liquid chromatography
• Low flow rates
• Low system volumes
• Large injection volume possible, use multi-dimensionality to step-down flow rates and column sizes
• Allows for 10-12 minute injection-injection cycle time with sub µL/min analytical flow rates
Dionex RSLC Ultimate 3000 Nano LC Systems
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High Resolution Accurate Mass SpectrometryThermo QExactive+
HCD Cell
Orbitrap MassAnalyzer
S-lensIon Source
Enhanced FT
C-Trap Quadrupole Mass Filter
Sturm RM, Jones BR, Mulvana DE, Lowes S (2016), “HRMS Using a Q-Exactive Series Mass Spectrometer for Regulated Quantitative Bioanalysis: How, When, & Why to Implement.” Bioanalysis. 8(16): 2343-2356.
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Case Study #1:
Human Insulin in Pig Serum
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• Need to measure human insulin in pig serum to 100 pg/mL levels
• LBA specificity challenges– Need to leverage LC-MS
• LCMS sensitivity and selectivity challenges– Human insulin does not fragment well for MS/MS acquisitions
The Bioanalytical Challenge
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Insulins - Workflow
SampleProtein
PrecipitationPeptide Affinity
ChromatographyNano-LC
Reversed Phase Trap/Elute
HRMS
Internal Standard
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• 2.1 mm ID
• Packed with protein G slurry
• Anti-insulin antibody loaded to column onsite
• Cross-linked with Dimethyl pimelimidate (DMP)
Affinity Chromatography
Conventional Flow Pump 2Flow Rate: 300 µL/min
30°COven
67
8
9
101
2
3
4
5
= not used
Ab
Human Insulin Ab column
30 mm x 2.1 mm
wasteAutosampler
Conventional Flow Pump 1Flow Rate: 300 µL/min
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Multidimensional Chromatography
Acclaim PepMap300 µm x 5 mm
Trap column
EASY-Spray Column75 µm x 150 mm
Antibody Column2.1 mm x 30 mm
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60°COven
Nano Flow LC pumpFlow Rate: 0.6 µL/min
AutosamplerConventional Flow Pump 1
Flow Rate: 300 µL/min
Conventional Flow Pump 2Flow Rate: 300 µL/min
Trap column Pepmap C18
5 mm x 0.3 mm
30°COven
= not used
Trap
67
8
9
101
2
3
4
5
67
8
9
101
2
3
4
5
= not used
Ab
Human Insulin Ab column
30 mm x 2.1 mm
waste
waste
Pepmap C18150 mm x 75µm
… Followed by Trap/elute Reversed Phase nanoLC
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HCD Fragment
Insulin precursor survived at HCD cell is monitored in orbitrap mass analyzer to quantify insulin
Mass SpectrometryHRMS – Survivor SIM*
*Ciccimaro E, Ranasinghe A, D'Arienzo C, Xu C, Onorato J, Drexler DM, Josephs JL, Poss M and Olah T (2014) Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides. Analytical chemistry86:11523-11527.
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XIC
Spectrum
Human Insulin - SensitivityXIC of Human Insulin at 100 pg/mL in Pig Serum and Corresponding Spectrum
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Human Insulin – Linearity, Accuracy and Precision
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Case Study #2 – Atrial Natriuretic Peptide
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• Need to measure atrial natriuretic peptide in human urine to sub-pg/mL levels
• LBA specificity and sensitivity challenges
• LCMS sensitivity challenges– Low endogenous levels
– ANP does not fragment well
The Bioanalytical Challenge
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Atrial Natriuretic Peptide - Workflow
SampleBead-Based
IPPeptide Affinity
ChromatographyNano-LC
Reversed Phase Trap/Elute
HRMS
Internal Standard
Same Antibody Reagent
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MS/MS scan mode, quantify on molecular ion
100 fg/mL extracted from 1 mL synthetic
matrix
+ Sequential immunoprecipitation
and nanoelectrospray
*Ciccimaro E, Ranasinghe A, D'Arienzo C, Xu C, Onorato J, Drexler DM, Josephs JL, Poss M and Olah T (2014) Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides. Analytical chemistry86:11523-11527.
ANP – Survivor SIM
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Case Study #3 – Beta Nerve Growth Factor
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Bioanalytical Challenge
• β-NGF is a 13.5 kDa protein (dimer) that is endogenous in human serum
• Important role in the modulation of pain through its interaction with p75 and TrkA receptors on neurons> Important drug target
• LBA approaches challenged by presence of antibody therapeutic bound to target > Requires hybrid LBA/LCMS approach
• Endogenous levels ~20-50 pg/mL> Need to drive sensitivity
• To be applied to large clinical studies> Robustness concern
Hefti et al., Novel class of pain drugs based on antagonism of NGF. Trends Pharmacol. Sci. 27, 85-91 (2006).
β-Nerve Growth Factor
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Beta NGF- LBA/LCMS Assay Workflow
SampleBead-Based Protein IP
Peptide Affinity Chromatography
Nano-LC Reversed Phase
Trap/Elute
QqQ or HRMS
Internal Standard
Digestion
Sequential Immunoprecipitation (Protein Peptide)
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Sequential Immunoaffinity Required to Enable Sensitivity
With Anti-peptide Antibody Column (3D-LC)
Without Anti-peptide Antibody Column (2D-LC)
2.4 2.6 2.8 3.0 3.2 3.4 3.6Time (min)
3,632
78,704
1.64E3
2.73E4
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4Time (min)
4,425
4.97E3
5.30E3
18-fold improvement
LLOQ Standard 7 pg/mL β-NGF
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The NGF Method• Previously presented development and validation of an immunoaffinity nano-LC-
MS/MS assay for quantification of total human β-NGF in serum• Application to clinical phase I and II programs, including analysis of several thousand clinical
samples • Major technological advancement
- Neubert H, et al. Sequential protein and peptide immunoaffinity capture for mass spectrometry-based quantification of total human β-nervegrowth factor. Anal. Chem. 85(3), 1719-1726 (2013).
- Porter KM, et al. Validation of an immunoprecipitation, digestion and immunoaffinity LC/MS/MS assay for human β-NGF biomarker andimplementation in support of clinical trials. Presented at ASMS Conference 2011. Denver, CO (2011).
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Improvements?
• High degree of expertise and attention required• Robustness and ease-of-use could be improved• Simplification needed
• Technological improvements available to address such issues without compromising assay performance• Nanofluidic plumbing• Nanospray source design
• Thermo EASY-Spray• Mass spectrometric detection technologies
• High Resolution Accurate Mass Spectrometry
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High-Throughput Nanoelectrospray?Nanofluidic Plumbing
Thermo nanoViperTM
Simple, fingertight connections– removes the “Art” of nanofluidic connections
Cutting Glass Capillaries for Fluid Connections
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• Advion Triversa NanoMate– Pro: Highly sensitive
– Pro: Robust against sprayer (nozzle) failure
– Con: Added complexity from additional software
– Con: Post column mixing
– Con: Can’t use nanoViper for connection to column
– Con: Open air source
Considerable expertise required to setup and maintain system
NanoSpray Sources for Bioanalysis
Improvements?
• Thermo EASY-Spray – Pro: Integrated column/Sprayer
– Pro: All connections can be nanoViper
– Pro: No additional software
– Pro: Closed source
– Con: Integrated column/Sprayer • Sprayer failure means column replacement
• Turns out to be robust
Does not require considerable expertise to setup, maintain and operate system
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Results7 pg/mL LLOQ Extracted Ion Chromatograms
• Tested on several different occasions, using the EASY-Spray ion source coupled to a TSQ Vantage MS, the signal-to-noise ratio for the β-NGF SRM transition was too low to accurately quantify β-NGF at a 7 pg/mL level.
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High Resolution Accurate Mass SpectrometrySelectivity Advantage
• The orthogonal selectivity offered by accurate mass measurements using a Q Exactive Plus MS enabled the EASY-Spray ion source to accurately quantify β-NGF at a 7 pg/mL level
PRMSRM
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Β-NGF - Representative Biasy and PrecisionAssayed using Q-Exactive Plus (EASY-Spray source)
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Case Study #4 – Example Antibody Biotherapeutic (Formic Acid Digestion)
CPSA 2015
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• Large Molecule mAb Biotherapeutic– Difficulty with human plasma LB Assay
• Hybrid IA-LC/MS method feasibility initiated.– Bottom-up digestion approach.
• No suitably selective tryptic signature peptides.– Required alternative digestion.
• Putative unique peptides were identified from in-silico formic acid chemical digestion (cleavage at aspartic acid residues)
Bioanalytical Challenge
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Protein Therapeutic - Workflow
SampleBead-Based Protein IP
Nano-LC Reversed Phase
Trap/EluteQqQ
Internal Standard
Digestion
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Representative XIC for Formic Acid Digestion Assay
RT: 0.00 - 6.00 SM: 5B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
1004.57 4.58 5.44
4.554.823.99 4.834.19 4.81
4.223.89 5.285.054.46 5.653.783.50 5.833.743.433.032.941.17 1.850.99 1.180.12 0.66 2.32 2.731.981.560.33 2.53
RT: 3.89AA: 10205
5.444.554.53 4.913.95 4.57 4.93 5.423.80 5.57 5.763.763.470.52 2.851.520.98 3.100.23 1.86 2.080.74 2.46 2.741.08 1.43 2.28RT: 3.90AA: 2215870
5.394.04 4.534.19 4.75 4.83 5.113.79 5.541.94 3.06 5.823.633.270.54 1.170.970.31 0.83 2.57 2.731.830.15 1.34 2.441.52 2.16RT: 3.91AA: 1677018
4.494.00 4.16 4.58 5.825.474.91 5.082.110.26 5.26 5.613.783.11 3.20 3.511.090.57 0.67 2.00 2.74 2.992.401.18 1.49 1.75
NL: 3.95E2TIC F: + c NSI SRM ms2 687.300 [648.995-649.005] MS DBM2930-BM_DHPL002_150601111054
NL: 3.99E3TIC F: + c NSI SRM ms2 687.300 [648.995-649.005] MS ICIS dbm2930-bm_dhpl003_150601111054
NL: 1.20E6TIC F: + c NSI SRM ms2 687.300 [648.995-649.005] MS ICIS dbm2930-bm_dhpl010_150529131808
NL: 9.43E5TIC F: + c NSI SRM ms2 694.000 [655.695-655.705] MS ICIS dbm2930-bm_dhpl011_150529131808
Control Blank
LLOQ
ULOQ
Internal Standard
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Inter-Run A&P, Inter-Lot A&P and SelectivityTwo A&P test batches
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CarryoverRelative to mean LLOQ (100 ng/mL) response
STD 1 rep 1 0.111389STD 1 rep 2 0.120343Mean LLQ response (ratio) 0.1159
STD 1 rep 1 61364.37STD 1 rep 2 40520.47Mean LLQ response (area) 50942
First primary carryover zero 0Second primary carryover zero 0.004932Mean response 0.002466% carryover response 2.1
First post carryover zero 0Second post carryover zero 0.012715Mean response 0.006358%post carryover response 5.5
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• Nanospray ionization allows for highly sensitive LC-MS quantitation
• Technological improvements have increased– Ease of use
– Robustness
• Flexible format– Adjust to meet the bioanalytical challenge
• Multidimensional trapping enables short cycle times– 10-12 minutes is common
• Also allows for introduction of orthogonal retention– Additional purification for robustness and sensitivity
• Can achieve high-volume, “high-throughput” sample analysis…– …to current standards for regulated bioanalysis
Conclusions
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• Steve Lowes• Jack Henion• Robert Sturm• Gary Schultz• Kathlyn Tilley
• Lian Shan• Miaoqing Shen• Raymond Biondolillo• Zhe Xu• Colleen Dwyer• Jake Usery• Jacob Edelman• Scott Ruane• Lauren Binns• Cindy Yong• Chelsea Maceda
Acknowledgments
LC-MS Biologics TeamQ2 Solutions