LECTURE 8. Biologics and Future Technologies for the Clinical Laboratory

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1 LECTURE 8 Biologics and Future Technologies for the Clinical Laboratory Jack Henion Quintiles Bioanalytical and ADME Labs Lecture 8, Page 1

2 New and Developing LC/MS Bioanalytical Capabilities: A Representative Modern Laboratory 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 Lecture 8, Page 2

3 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 Dr. Barry Jones, Quintiles Lecture 8, Page 3

4 New and Developing LC/MS Bioanalytical Capabilities Large Molecule Specific 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 ( µl/min) for maximum sensitivity» Possibilities: Thermo EasySpray TM Advion Chip-Mate TM Waters ionkey TM AB SCIEX NanoSpray III TM New Objective PicoChip TM and PicoView TM 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 Lecture 8, Page 4

5 Immunoaffinity-LC/nanoLC-MS/MS waste 30 C Oven Ab 10 9 Ab column Protein G 30 mm x 2.1 mm Autosampler Loading Pump MPA load sample MPB Ab column wash = not used Valve 1, Position 10_1 Dr. Gary Schultz, Quintiles Lecture 8, Page 15

6 Immunoaffinity-LC/nanoLC-MS/MS 75 C Oven waste Trap 10 9 Trap column Pepmap C18 5 mm x 0.3mm = not used Valve 2, Position 10_1 waste 30 C Oven Ab column Protein G 30 mm x 2.1 mm 4 Ab 8 Micropump 1 MPA Ab Elution = not used Valve 1, Position 1_2 MPB Trap column equilibration MPC Trap column wash Lecture 8, Page 6

7 + Immunoaffinity-LC/nanoLC-MS/MS 75 C Oven Trap column Pepmap C18 5 mm x 0.3mm Nano LC pump 4 5 Trap Acclaim Pepmap C18 Nano LC column 150 mm x 75µm = not used Valve 2, Position 1_2 EASY Spray Lecture 8, Page 7

8 Thermo EASY-Spray Lecture 8, Page 8

9 Example #1: Insulin by LC/MS/MS 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. 5.8 kda Endogenous Peptide Lecture 8, Page 9

10 Extraction Procedure 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. Antiinsulin insulin Lecture 8, Page 10

11 Insulin Quantitation Immunoaffinity-LC/nanoLC-MS/MS Lecture 8, Page 11

12 Example #2: β - Nerve Growth Factor 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. Lecture 8, Page 12

13 Extraction, LC, and MS 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 Lecture 8, Page 13

14 LLOQ Standard 7 pg/ml β-ngf With Anti-peptide Antibody Column (3D-LC) Without Anti-peptide Antibody Column (2D-LC) 3, E3 4.97E3 78, E4 4, E Time (min) Time (min) 18-fold improvement Lecture 8, Page 14