July Land O' Lakes 2019 -- A study assessing the feasibility of performing quantitative analysis of adalimumab in human whole blood collected using Mitra® (Neoteryx) volumetric adsorptive microsampling (VAMS) devices was conducted. Adalimumab (Humira®) was chosen as a representative monoclonal antibody therapeutic to assess the practicality of protein analysis via LC-MS/MS analysis coupled with VAMS technology.

1. Presented at July Land O’ Lakes Conference 2019
Analysis of the Monoclonal Antibody Adalimumab in Human Blood Collected via Volumetric
Adsorptive Microsampling (VAMS) Technology and Utilizing LC-MS/MS Detection
Mark Leahy1, Aaron Ledvina1, Brendan Powers1, Mathew Ewles2 and Stephanie Cape1; Covance Laboratories, 1Madison, WI, 2Harrogate, UK
Introduction
A study assessing the feasibility of performing quantitative analysis of
adalimumab in human whole blood collected using Mitra® (Neoteryx) volumetric
adsorptive microsampling (VAMS) devices was conducted. Adalimumab
(Humira®) was chosen as a representative monoclonal antibody therapeutic to
assess the practicality of protein analysis via LC-MS/MS analysis coupled with
VAMS technology.
Materials
▶ Mitra® microsampling devices (20 µL) in 96-Autorack format, Neoteryx
▶ SMART Digest™ Kit, Thermo Scientific
▶ SOLA™ HRP 10 mg/2 mL SPE Plate, Thermo Scientific
▶ Various protein Lo-bind labware, Eppendorf
▶ Adalimumab, provided as Humira®, AbbVie
▶ Adalimumab surrogate peptide: GLEWVSAITWNSGHIDYADSVEGR
(Heavy chain position 44-67)
▶ Isotopically labeled surrogate peptide internal standard,
GLEWVSAITWNSGHIDYADSVEGR^ (13C6,15N4-labeled arginine), New
England Peptide
Results
▶ Experiments were performed to optimize recovery of adalimumab from the Mitra®
tip, including impact-assisted extraction. Best results were obtained through a
combination of vortex-mixing and sonication in a buffer solution.
▶ Adalimumab peak area responses obtained from blood collected using the Mitra®
VAMS device were comparable to those obtained from a direct 20 µL aliquot of
whole blood.
▶ Significant matrix effects (signal suppression) were noted in whole blood during
LC-MS/MS detection. Optimization of the SPE procedure appears to mitigate
these effects. For comparison, human serum was evaluated in parallel, with
fewer matrix effects noted.
▶ Initial experiments suggest that a 0.500 µg/mL lower limit of quantitation is
achievable in either serum or whole blood.
▶ A precision and accuracy batch in serum produced a calibration curve that was
linear (1/x2 weighted) from 0.500 to 100 µg/mL, with acceptable precision and
accuracy at each of 5 QC concentrations.
HPLC Parameters
Parameter Details
LC System Shimadzu, Prominence, 30 Series
Analytical Column Waters, Acquity BEH C18, 100 x 2.1 mm, 1.7 µm
Column Temperature 45°C
Mobile Phase A Water: Formic Acid (100:0.5)
Mobile Phase B Acetonitrile: Formic Acid (100:0.5)
Gradient
0.00 – 6.00 min, 15→35% B, 0.4 mL/min
6.00 – 7.00 min, 35→90% B, 0.4 mL/min
7.00 – 8.50 min, 90→90% B, 0.4 mL/min
8.50 – 8.60 min, 90→15% B, 0.4 mL/min
Injection Details 4-6 µL
MS/MS System Sciex API 6500 in Positive TurboIonSpray® (ESI+)
Gas and Voltages
CAD = 8, CUR = 30, GS1 = 50, GS2 = 50, ISV = 4500, EP = 10, DP = 130, CE = 36
Temperature = 650°C, CAD gas = Nitrogen
Mass Spectrometer Parameters
Compound Name Transition Retention Time (min)
Analyte
Surrogate Peptide
GLEWVSAITWNSGHIDYADSVEGR
886.5 → 1040.0 5.12
Internal standard
Stable labeled peptide
GLEWVSAITWNSGHIDYADSVEGR^ (13C6,15N4)
891.5 → 1044.4 5.12
Photograph of VAMS sample collection, used by
permission of Neoteryx, LLC.
Photograph of Mitra® Autorack used by permission of Neoteryx, LLC
Human Whole Blood
or Serum Applied to
VAMS Device
Resuspend in Buffer
Addition of Labeled
Internal Standard
SPE Sample Cleanup
Evaporation/
Reconstitution
Surrogate Peptide
Analysis by
LC-MS/MS
On-Tip
Trypsin Digest
▶ Mitra® microsampling device (20 µL) is allowed to
completely adsorb matrix sample. Sample is dried
overnight at ambient temperature.
▶ Dried tip is removed from the applicator and placed
in a well of a 96-well collection plate. 300 µL of
Trypsin SMART Digest™ Buffer is added to each
well and the plate is vortex-mixed for 10 minutes
followed by 5 minutes of sonication.
▶ 5 µL of Soluble SMART Digest™ Trypsin Enzyme is
added to each well. Digest the sample on a heated
mixer at 70°C for 180 minutes.
▶ 25 µL of 13C6,15N4-labeled surrogate peptide is
added to each sample.
▶ Each sample is diluted with 100 µL of water:formic
acid (100:1) solution and loaded onto a SPE plate
(SOLA™ HRP, 10 mg/2 mL plate, Thermo
Scientific). Sample elution is achieved using
methanol:ammonium hydroxide (95:5).
▶ Samples are evaporated to dryness using a stream
of nitrogen at 40°C.
▶ 200 µL of water:acetonitrile:formic acid (85:15:0.5) is
used to reconstitute the samples.
Sample Preparation Procedure
Method
Figure 1. Human whole blood: 1 µg/mL adalimumab (and internal standard) using on-tip digestion.
Figure 2. Calibration curve from adalimumab in human serum precision and accuracy batch.
Figure 3. Human serum: 0.5 µg/mL adalimumab (and internal standard) using on-tip digestion.
Figure 4. Human serum blank using on-tip digestion.
Conclusions
▶ As proof of concept, the work performed demonstrates the feasibility of using samples collected with a VAMS
device, coupled with LC-MS/MS detection, for protein analysis in monoclonal antibody therapy.
▶ A therapeutically relevant calibration curve range (0.500-100 µg/mL) has been obtained using this procedure.
▶ Additional work will be performed to improve sample cleanup to reduce matrix effects, and investigate
selectivity and hematocrit effects.
▶ VAMS technology allows for remote and at-home sampling, dramatically simplifying the lives of patients who
find themselves on strict drug regimens.
Quality Control Sample Data for Adalimumab in Human Serum
Theoretical Concentration (µg/mL)
0.500 1.50 8.00 40.0 80.0
n 6 6 6 6 6
Intra-Assay Mean 0.459 1.5 7.58 39.7 74.3
SD 0.0571 0.132 1.45 6.6 8.17
RSD (%) 12.4 8.8 19.1 16.6 11.0
Accuracy (%) 91.8 100.0 94.8 99.3 92.9
Calibration Curve Data for Adalimumab in Human Serum
Theoretical Concentration (µg/mL)
0.500 1.00 2.50 5.00 15.0 50.0 85.0 100
Back-Calculated Calibrator
Concentration (µg/mL) 0.487 1.03 2.60 5.21 13.5 53.0 83.4 96.9
Accuracy (%) 97.4 103.0 104.0 104.2 90.0 106.0 98.1 96.9