LC/MS On-Line Training - Bioanalysis of Biotherapeutics · 2016. 3. 9. · •LC/MS platforms continue to grow and diversify ›LC/MS/MS systems in a regulated environment » 2 A

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



» 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


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


Trap

6

7

8

9

10 1

2

3

4

5

6

7

8

9

10 1

2

3

4

5


Ab

Ab column Protein G

30 mm x 2.1 mm

waste

waste

Lecture 8, Page 6

Acclaim Pepmap C18

150 mm x 75µm


75°C Oven

Nano LC pump

EASY Spray

Nano LC column

Trap column Pepmap C18

5 mm x 0.3mm


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


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

Documents

LC/MS On-Line Training - Bioanalysis of Biotherapeutics · 2016. 3. 9. · •LC/MS platforms continue to grow and diversify ›LC/MS/MS systems in a regulated environment » 2 A