The document compares the use of LC-MS/MS versus ELISA for the bioanalysis of three monoclonal antibodies (adalimumab, rituximab, and trastuzumab) in rat and mouse pharmacokinetic studies. The LC-MS/MS method showed higher sensitivity, dynamic range, and selectivity compared to ELISA, though it had lower throughput and higher costs. Results from the two methods showed good concordance for adalimumab and trastuzumab in rats, demonstrating LC-MS/MS as a viable alternative to ELISA for bioanalysis of large molecules.
2. Goal: Compare LC-MS/MS vs. LBA using three mAbs
Compare PK parameters using 2 different bioanalytical methods
Primary Concern
LLOQ: 10 ng/mL
Dynamic range: >4 orders if possible
Sample volume: <5 µL for serial sampling from a single mouse vs. n = 3/time-point
Selectivity: effectiveness of immunocapture & capacity, capacity, capacity …
Robustness with respect to batch size
Assay throughput: automatable
Ease of use: well trained analysts vs. Ph.D.-level scientist
Timeline: from assay concept to AUC data
Cost: reagent and equipment
Secondary Concern
Transferability wrt matrices: serum vs. plasma; other tissues?
Transferability wrt species: mice vs. rats vs. NHPs vs. human
2015 ASMS Sciex user Mtg 2
3. Which biologics to test?
2015 ASMS Sciex user Mtg 3
RIA
LOD- 8 µU/mL
ULOQ- 160 µU/mL
Matrix- Human Serum
ELISA or Gyros
LOQ-250 ng/mL
ULOQ-10,000 ng/mL
Matrix- Rat, Mouse & Human Serum
ELISA or Gyros
LOQ-250 ng/mL
ULOQ-10,000 ng/mL
Matrix- Human Serum
INSULIN ADALIMUMAB INFLIXIMAB
ELISA or Gyros
LOQ-500 ng/mL
ULOQ-100,000 ng/mL
Matrix- Rat, Mouse & Human Serum
ELISA or Gyros
LOQ-250 ng/mL
ULOQ-3,000 ng/mL
Matrix- Human Serum
ELISA or Gyros
LOQ-100 ng/mL
ULOQ-5,000 ng/mL
Matrix- Rat, Mouse & Human Serum
TRASTUZUMAB BEVACIZUMAB RITUXIMAB
4. Experimental: in-life
Order Adalimumab, Rituximab, Trastuzumab
Single SC dose mid-scapular area @ 10 mg/kg
Housing: metabolism cages; Feeding/Water: ad libitum
Time-points: Pre-dose, 0.5 h, 2 h, 6 h, 1, 2, 3, 6, 8, 10, 14, 17, 21, 24, 28 d
2015 ASMS Sciex user Mtg 4
Male Sprague Dawley rats: 250-300 g
n = 4/test article
Sample collection
individual time-points;
tail-snip @ 150 µL/time-point processed to serum split to 2 samples for LBA
and immunocapture LC-MS/MS
Male CD-1 mice: 20-22 g
n = 24/test article
Sample collection
3 animals/group/time-point
retro-orbital and cardiac puncture @ 150 µL/time-point processed to serum
split to 2 samples for LBA and immunocapture LC-MS/MS
5. Experimental: LBA
2015 ASMS Sciex user Mtg 5
Coat with Target
or Capture Ab
Sample Dilution
and Addition
Detection Ab
Substrate
Read 450nM
96-well plate
6. Experimental: Immunocapture LC-MS/MS
2015 ASMS Sciex user Mtg 6
Antibody Enrichment Tryptic Digestion LC/MS Analysis
Animal Serum
Add SIL Whole Protein
Y Y Y Y
Y
Y
• Streptavidin coated Magnetic Beads binding with target IgG
Y Y
YYY
YY
Y
Y
Y Y
YY
Y
Y
Wash & Elute
Y Y
Y
Y
Y
Y
Y
Y
Interferences
Anti Fc
Anti TNF α
Anti Insulin
Anti PEG
Anti Drug
ExionLC™ QTRAP® 6500 System
7. Data: Adalimumab in the Rat
2015 ASMS Sciex user Mtg 7
10
100
1000
10000
100000
0 96 192 288 384 480 576 672
SerumAdalimumabConc.(ng/mL).
Hours
Rat # 5
Rat # 6
Rat # 7
Rat # 8
10
100
1000
10000
100000
0 96 192 288 384 480 576 672
SerumAdalimumabConc.(ng/mL).
Hours
Rat # 5
Rat # 6
Rat # 7
Rat # 8
ELISA Immunocapture LC-MS/MS
8. Data: Adalimumab in the Rat
2015 ASMS Sciex user Mtg 8
100
1000
10000
100000
0 96 192 288 384 480 576 672
SerumAdalimumabConc.(ng/mL).
Hours
ELISA
MS
Adalimumab (Humira) 10 mg/kg SC
Mean SD Mean SD
Cmax (ng/mL) 68200 7970 49800 4860
tmax (h) 102 49.5 78.0 45.4
AUClast (h*ng/mL) 21900000 4000000 16100000 3210000
t1/2 (h) 108 139 132 186
ELISA LC-MS/MS 100
1000
10000
100000
100 1000 10000 100000
LC-MS/MS
ELISA
Rat # 5
Rat # 6
Rat # 7
Rat # 8
Unity
~20% bias
9. Data: Trastuzumab in the Rat
2015 ASMS Sciex user Mtg 9
ELISA Immunocapture LC-MS/MS
100
1000
10000
100000
0 96 192 288 384 480 576 672
SerumTrastuzumabConc.(ng/mL).
Hours
Rat # 9
Rat # 10
Rat # 11
Rat # 12
10
100
1000
10000
100000
0 96 192 288 384 480 576 672
SerumTrastuzumabConc.(ng/mL).
Hours
Rat # 9
Rat # 10
Rat # 11
Rat # 12
10. Data: Trastuzumab in the Rat
2015 ASMS Sciex user Mtg 10
~10% bias
100
1000
10000
100000
0 96 192 288 384 480 576 672
SerumTraztuzumabConc.(ng/mL).
Hours
ELISA
MS
1000
10000
100000
1000 10000 100000
LC-MS/MS
ELISA
Rat # 9
Rat # 10
Rat # 11
Rat # 12
Unity
Trastuzumab (Herceptin) 10 mg/kg SC
Mean SD Mean SD
Cmax (ng/mL) 72800 3880 71496 6873
tmax (h) 60 13.9 66.0 12.0
AUClast (h*ng/mL) 26900000 5020000 29600000 5570000
t1/2 (h) 258 150 268 160
ELISA LC-MS/MS
11. Comparison: LBA vs. Immunocapture LC-MS/MS
2015 ASMS Sciex user Mtg 11
Parameters LBA Immuno-LC-MS/MS
LLOQ (mAb usually high exposure) High Sensitivity (~10 ng/mL) vs. Standard Sensitivity (~150 ng/mL) ~10 ng/mL
Dynamic Range
~2-3 orders for colourmetric
≥3 for Gyros and ECL
~4 orders
Sample Volume <5 µL (Gyros ~0.1 µL) ~25 µL
Selectivity Single dimension, but manageable Higher
Robustness
Singlet vs. Duplicate
≥90% ISR
Potential Stable label IS
mAb & catabolites, but Why Depends on the epitopes TBD
Transferability High Higher
Multiplexing, but Why Not really Potentially
Assay Throughput High (120-180 samples/analyst/day) Medium
Equipment Cost Low (USD$10K) High (USD$500K)
Reagent Cost Low Medium (SILmAb)
Timeline
Specific Ab – limiting step (e.g. Rituximab)
Target capture – not rate limiting (e.g., Adalimumab , Bevacizumab,
Imfluximab)
Shorter initially
12. Additional Studies in progress/Thoughts
More data set needed
Adalimumab – mice
Rituximab – mice and rats
Trastuzumab – mice
Understand the AUC differences, if any, and what is “acceptable”
Understand realistic LLOQ and dynamic range
What is the approach for new mAb
Discovery screening (generic IgG assays) vs. development (selectivity)
Confirm feasibility of single mouse PK (≤5 µL per time-point): LBA vs. LC-MS/MS
Robustness with respect to batch size and ISR
Ease of use
Assay throughput and Timeline
Serum vs. plasma vs. tissues
2015 ASMS Sciex user Mtg 12
13. Acknowledgement
Sciex
Suma Ramagiri
Hua-Fen Liu
Witold Woroniecki
Lei Xiong
Ian Moore
Chang Liu
Gary Impey
Debadeep Bhattacharya
Joe Fox
Elliot Jones
2015 ASMS Sciex user Mtg 13
QPS
Bruce Aungst
Danielle Graden
Erin Reynolds
Helen Shen
Jamie Stefanick
Sarah Patterson
QPS
Caroline Becker
James McClean
John Kolman
Melissa Calvarese
Quynh-Giao Pham
Stacey Gottschall
Xun Wang
Editor's Notes
LLOQ: Typically required sensitivity for a mAb drug is 50 to 200 ng/mL. Some ADC drug may require higher sensitivity, particularly with high potent payload.