lc/ms on-line training - bioanalysis of biotherapeutics · 2016. 3. 9. · •lc/ms platforms...
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Biologics and Future Technologies for the Clinical Laboratory
Jack Henion
Quintiles Bioanalytical and ADME Labs
LECTURE 8
Lecture 8, Page 1
•LC/MS platforms continue to grow and diversify › LC/MS/MS systems in a regulated environment
» 2 AB SCIEX API 3000™ triple quadrupoles
» 15 AB SCIEX API 4000™ triple quadrupoles
» 10 AB SCIEX API 5000™ triple quadrupoles
» 1 AB SCIEX Triple Quad™ 5500
» 1 AB SCIEX Triple Quad™ 6500
» 3 Thermo Fisher Scientific TSQ Vantage™ triple quadrupoles
» 1 Thermo Fisher Scientific Q-Exactive™ (validated system)
» 1 Thermo Fisher Scientific Q-Exactive™ Plus (validate Winter 2014)
› UPLC, Micro- and Nano-LC systems » 3 Waters Acquity UPLC I-Class systems
» 4 Shimadzu Nexera UHPLC systems
» 2 Dionex Ultimate 3000 RSLC systems
» 4 Dionex Ultimate 3000 multidimensional nano-LC systems
› Hamilton STAR Liquid Handling Workstations
New and Developing LC/MS Bioanalytical Capabilities: A Representative Modern Laboratory
Lecture 8, Page 2
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.com
Dr. Barry Jones, Quintiles Lecture 8, Page 3
• Digestion and Surrogate Peptide Assays
› Extensive experience in establishing multiple methods to meet regulated bioanalysis standards » Regulatory expectations and trends for protein therapeutics by LC/MS seem to meet small
molecule LC/MS criteria rather than LBA criteria
› Employs the proven triple quadrupole platform
› Microspray (1 – 50 µL/min) often suitable – and most practical
› Nanospray (0.050 – 0.800 µL/min) for maximum sensitivity » Possibilities: – Thermo EasySprayTM – Advion Chip-MateTM – Waters ionKeyTM
– AB SCIEX NanoSpray IIITM
– New Objective PicoChipTM and PicoViewTM
› Multidimensional LC to add selectivity and step down flow regimes has proven the practical way to implement nanospray LC/MS
› Multidimensional LC is becoming a standard approach for delivering selectivity and robustness to our large molecule LC/MS assays » Selectivity is key » Sample prep is also an important factor in the quest for selectivity
New and Developing LC/MS Bioanalytical Capabilities
Large Molecule Specific
Lecture 8, Page 4
Immunoaffinity-LC/nanoLC-MS/MS
Autosampler
Loading Pump
MPA load sample
MPB Ab column wash
30°C Oven
6
7
8
9
10 1
2
3
4
5
= not used Valve 1, Position 10_1
Ab
Ab column Protein G
30 mm x 2.1 mm
waste
Dr. Gary Schultz, Quintiles Lecture 8, Page 15
Immunoaffinity-LC/nanoLC-MS/MS
75°C Oven
Micropump 1
MPA Ab Elution
MPB Trap column equilibration
MPC Trap column wash
Trap column Pepmap C18
5 mm x 0.3mm
30°C Oven
= not used Valve 2, Position 10_1
Trap
6
7
8
9
10 1
2
3
4
5
6
7
8
9
10 1
2
3
4
5
= not used Valve 1, Position 1_2
Ab
Ab column Protein G
30 mm x 2.1 mm
waste
waste
Lecture 8, Page 6
Acclaim Pepmap C18
150 mm x 75µm
Immunoaffinity-LC/nanoLC-MS/MS
75°C Oven
Nano LC pump
EASY Spray
Nano LC column
Trap column Pepmap C18
5 mm x 0.3mm
= not used Valve 2, Position 1_2
Trap
6
7
8
9
10 1
2
3
4
5
+
Lecture 8, Page 7
Thermo EASY-Spray
Lecture 8, Page 8
In support of pharmacokinetic studies of therapeutic insulin analogs, it is necessary to determine the concentration of intact insulin in biological matrix samples. Traditional LB assays suffer from specificity challenges.
Example #1: Insulin by LC/MS/MS
5.8 kDa Endogenous Peptide
Lecture 8, Page 9
Extraction Procedure
insulin
Anti-
insulin
• 350 μL of sample is incubated overnight at 4 C with 2 μg of biotinylated anti-insulin antibody.
• Sample preparation using a Hamilton MicroLab® STAR and custom-made magnet plates.
• Washed and blocked streptavidin-coated magnetic beads are combined with the sample containing antibody and incubated for 1 hour.
• Beads are collected and washed with buffer.
• Insulin-antibody complex is eluted from beads using 30 mM hydrochloric acid and subsequently neutralized.
• Internal standard (labeled insulin analog) is added post extraction.
Lecture 8, Page 10
Insulin Quantitation
Immunoaffinity-LC/nanoLC-MS/MS
Lecture 8, Page 11
• Nerve growth factor (NGF) is a small secreted protein (13.5 kDa - monomer) that is important for the growth, maintenance, and survival of certain target neurons.
• NGF prevents or reduces neuronal degeneration in animal models of neurodegenerative diseases and these encouraging results in animals have led to several clinical trials in humans.
• An important biomarker for a variety of therapeutic treatments.
Example #2: β - Nerve Growth Factor
Lecture 8, Page 12
• Intact NGF is isolated from 600 µL serum by means of magnetic bead-based immunoaffinity extraction. › Offline capture antibody has affinity for intact NGF
› Internal Standard is added post extraction
› Extended, cleavable SIL analog of the signature peptide
• Immuno-purified extract is reduced, alkylated, and digested with trypsin
• Digested extract is loaded to anti-peptide column (anti-signature peptide antibody, immobilized to cross-linked protein-G substrate) on Dionex Ultimate 3000 › Loaded at 300 µL/min
• Eluted from A-peptide column with acid, focused onto C18 trap column › Eluted at 300 µL/min
• Trap column switched in-line with nano-column, eluted with gradient › Flow rate = 600 nL/min
• Ionization via Thermo EASY-Spray on Thermo Vantage QQQ
Extraction, LC, and MS
Lecture 8, Page 13
LLOQ Standard 7 pg/mL β-NGF
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 3.6
Time (min)
3,632
78,704
1.64E3
2.73E4
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4
Time (min)
4,425
4.97E3
5.30E3
18-fold improvement
Lecture 8, Page 14
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