large molecule bioanalysis: lc-ms or elisa? a case study
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Large Molecule Bioanalysis: LC-MS or ELISA? A Case StudyZamas Lam, Ph.D.
SVP Preclinical Development
Goal: Compare LC-MS/MS vs. LBA using three mAbs
Compare PK parameters using 2 different bioanalytical methodsPrimary Concern
LLOQ: 10 ng/mLDynamic range: >4 orders if possibleSample volume: <5 µL for serial sampling from a single mouse vs. n = 3/time-pointSelectivity: effectiveness of immunocapture & capacity, capacity, capacity …Robustness with respect to batch sizeAssay throughput: automatableEase of use: well trained analysts vs. Ph.D.-level scientistTimeline: from assay concept to AUC dataCost: reagent and equipment
Secondary ConcernTransferability wrt matrices: serum vs. plasma; other tissues?Transferability wrt species: mice vs. rats vs. NHPs vs. human
2015 ASMS Sciex user Mtg 2
Which biologics to test?
2015 ASMS Sciex user Mtg 3
RIA LOD- 8 µU/mLULOQ- 160 µU/mLMatrix- Human Serum
ELISA or Gyros LOQ-250 ng/mLULOQ-10,000 ng/mLMatrix- Rat, Mouse & Human Serum
ELISA or Gyros LOQ-250 ng/mLULOQ-10,000 ng/mLMatrix- Human Serum
INSULIN ADALIMUMAB INFLIXIMAB
ELISA or Gyros LOQ-500 ng/mLULOQ-100,000 ng/mLMatrix- Rat, Mouse & Human Serum
ELISA or Gyros LOQ-250 ng/mLULOQ-3,000 ng/mLMatrix- Human Serum
ELISA or Gyros LOQ-100 ng/mLULOQ-5,000 ng/mLMatrix- Rat, Mouse & Human Serum
TRASTUZUMAB BEVACIZUMAB RITUXIMAB
In Progress
Validated
Validated
Validated
Validated
Validated
Experimental: in-life
Order Adalimumab, Rituximab, TrastuzumabSingle SC dose mid-scapular area @ 10 mg/kgHousing: metabolism cages; Feeding/Water: ad libitumTime-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 gn = 4/test articleSample 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 gn = 24/test articleSample 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
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
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 YYY
• Streptavidin coated Magnetic Beads binding with target IgG
Y YYYYYYY
YY Y
YY
Y
Y
Wash & Elute
Y YYYY
YYY
InterferencesAnti Fc Anti TNF αAnti InsulinAnti PEGAnti Drug
ExionLC™ QTRAP® 6500 System
Data: Adalimumab in the Rat
2015 ASMS Sciex user Mtg 7
10
100
1000
10000
100000
0 96 192 288 384 480 576 672
Ser
um A
dali
mum
ab C
onc.
(ng
/mL
) .
Hours
Rat # 5
Rat # 6
Rat # 7
Rat # 8
10
100
1000
10000
100000
0 96 192 288 384 480 576 672
Ser
um A
dali
mum
ab C
onc.
(ng
/mL
) .
Hours
Rat # 5
Rat # 6
Rat # 7
Rat # 8
ELISA Immunocapture LC-MS/MS
Data: Adalimumab in the Rat
2015 ASMS Sciex user Mtg 8
100
1000
10000
100000
0 96 192 288 384 480 576 672
Ser
um A
dali
mum
ab C
onc.
(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
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
Ser
um T
rast
uzum
ab C
onc.
(ng
/mL
) .
Hours
Rat # 9
Rat # 10
Rat # 11
Rat # 12
10
100
1000
10000
100000
0 96 192 288 384 480 576 672
Ser
um T
rast
uzum
ab C
onc.
(ng
/mL
) .
Hours
Rat # 9
Rat # 10
Rat # 11
Rat # 12
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
Ser
um T
razt
uzum
ab C
onc.
(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
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)
TimelineSpecific Ab – limiting step (e.g. Rituximab)Target capture – not rate limiting (e.g., Adalimumab , Bevacizumab, Imfluximab)
Shorter initially
Additional Studies in progress/Thoughts
More data set neededAdalimumab – miceRituximab – mice and ratsTrastuzumab – 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/MSRobustness with respect to batch size and ISREase of useAssay throughput and TimelineSerum vs. plasma vs. tissues
2015 ASMS Sciex user Mtg 12
Acknowledgement
SciexSuma RamagiriHua-Fen Liu Witold WoronieckiLei XiongIan MooreChang LiuGary ImpeyDebadeep BhattacharyaJoe FoxElliot Jones
2015 ASMS Sciex user Mtg 13
QPSBruce AungstDanielle GradenErin ReynoldsHelen ShenJamie StefanickSarah Patterson
QPSCaroline BeckerJames McCleanJohn KolmanMelissa CalvareseQuynh-Giao PhamStacey GottschallXun Wang