Multi-Dimensional Nano-Scale Chromatography for Regulated ......Hamilton Microlab STAR S.T.A.R. – Sequential Transfer Automation Robot

Copyright ©2015 Q2 Solutions. All rights reserved.

COMPANY CONFIDENTIALBarry R. Jones

Multi-Dimensional Nano-Scale Chromatography for Regulated Peptide/Protein Bioanalysis

2COMPANY CONFIDENTIAL

• 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


The Tools


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


• 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


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.


Case Study #1:

Human Insulin in Pig Serum


• 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


Insulins - Workflow

SampleProtein

PrecipitationPeptide Affinity

ChromatographyNano-LC

Reversed Phase Trap/Elute

HRMS

Internal Standard


• 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



Multidimensional Chromatography

Acclaim PepMap300 µm x 5 mm

Trap column

EASY-Spray Column75 µm x 150 mm

Antibody Column2.1 mm x 30 mm


60°COven

Nano Flow LC pumpFlow Rate: 0.6 µL/min

AutosamplerConventional Flow Pump 1

Flow 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


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.


XIC

Spectrum

Human Insulin - SensitivityXIC of Human Insulin at 100 pg/mL in Pig Serum and Corresponding Spectrum


Human Insulin – Linearity, Accuracy and Precision


Case Study #2 – Atrial Natriuretic Peptide


• 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


Atrial Natriuretic Peptide - Workflow

SampleBead-Based

IPPeptide Affinity

ChromatographyNano-LC

Reversed Phase Trap/Elute

HRMS

Internal Standard

Same Antibody Reagent


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


Case Study #3 – Beta Nerve Growth Factor


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


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)


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


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).


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


High-Throughput Nanoelectrospray?Nanofluidic Plumbing

Thermo nanoViperTM

Simple, fingertight connections– removes the “Art” of nanofluidic connections

Cutting Glass Capillaries for Fluid Connections


• 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


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.


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


Β-NGF - Representative Biasy and PrecisionAssayed using Q-Exactive Plus (EASY-Spray source)


Case Study #4 – Example Antibody Biotherapeutic (Formic Acid Digestion)

CPSA 2015


• 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


Protein Therapeutic - Workflow

SampleBead-Based Protein IP

Nano-LC Reversed Phase

Trap/EluteQqQ

Internal Standard

Digestion


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



Control Blank

LLOQ

ULOQ

Internal Standard


Inter-Run A&P, Inter-Lot A&P and SelectivityTwo A&P test batches


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


• 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


• 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

Documents

Multi-Dimensional Nano-Scale Chromatography for Regulated ......Hamilton Microlab STAR S.T.A.R. – Sequential Transfer Automation Robot