Pharmacokinetic Analysis of the mAb Trastuzumab by

ELISA and Hybrid LBA/LC/MS: A Comparison Study

Featuring the SCIEX BioBA Solution

Xi Qu1, Shuyu Hou1, Meghan McCann1, Caroline Becker1, Xun Wang1, Zamas Lam1, Susan Zondlo1, John
Kolman1, Lei Xiong2, Witold Woroniecki2, Hua-Fen Liu2, Ian Moore3
1
QPS, Delaware, USA, 2SCIEX, Redwood Shores, USA, 3SCIEX, Concord, Canada

Key Challenges in Biologics Quantitation

• Achieving wide linearity across the expected PK
sample range to minimize sample >ULOQ
reanalysis
• Hybrid LBA/LC-MS sample work-up requires
sourcing reagents from different vendors
• Lengthy and complicated procedures for
measuring active or free circulating drug are more
challenging in complex matrices
Key Features of the SCIEX BioBA Solution
• A complete solution for biologics bioanalysis by
immuno-affinity sample preparation
o BioBA kits provide all the reagents
necessary from high capacity streptavidin
beads to digestion enzyme
• Assays with mass spec detection offer wider LDR BioBA reagents kit and SCIEX QTRAP® 6500 system.
than LBA
The popular choice for protein quantitation has been
• High capacity streptavidin coated beads to ELISA because of its high sensitivity, high throughput and
achieve high ULOQ low per sample costs once the assay is developed and
• Immuno enrichment allows enhanced sensitivity validated. Limitations of the ELISA technique when
for low abundance samples considering it as a platform include: lack of specificity in
primary or secondary antibody reagents, limited linear
• Ready to use vMethods reduce method
dynamic range and interference due to ADA cross
development time. reactivity. To avoid this cross reactivity, a more expensive
Introduction targeted antibody has to be used for pre-clinical studies.
The reasons for choosing a hybrid LBA and LC-MS assay
The selection of a quantitative protein assay for biologics include: selectivity, broad LDR which reduces sample
bioanalysis in a pre-clinical study depends on what dilution and the ability to multiplex a second analyte or
platform will provide the right data for the drug under catabolite. Another reason to choose the hybrid
development. Some questions to be considered when LBA/LC/MS approach for pre-clinical studies is the quick
choosing an assay platform include, is the total or free method development turnaround time afforded by a
drug concentration required? Are there in vivo structural generic method that can be developed where the same
changes that might impact activity? Are these modified LBA capture reagent can be used across analytes and the
proteins important to measure? Other considerations for final selectivity of the assay is provided by the LC-MS
the chosen assay platform include reagents availability, system.
sensitivity and LDR requirements, sample throughput and
potential interferences.

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The use of hybrid LBA LC/MS assays is growing and
SCIEX has developed the BioBA solution with ready to Results
use sample prep kits that include all the necessary
reagents and buffers which enables bioanalytical In dosing experiment 1 four male Sprague-Dawley rats
scientists from all backgrounds to accomplish biologics were used. The trastuzumab concentration from rats in
bioanalysis. Whether hybrid LBA LC/MS assays are used experiment 1 as measured by a HER2 specific ELISA is
instead of or as a complement to ELISAs in drug shown in Figure 1A and the trastuzumab serum
development, the most important question is do they give concentration measured using the generic hybrid LC-
equivalent results?
In this tech note we have compared the PK parameters
generated from two different bioanalytical assays using
the two different platforms. Samples from a PK study of
the mAb drug trastuzumab were split and tested with both
assays and the resulting sample concentrations and PK
parameters generated by the two assays were compared.

Methods
Dosing Study: Male Sprague-Dawley rats were given a
sub-cutaneous dose of trastuzumab at 10 mg/kg and
samples were collected by tail-snip at: predose, 0.5, 2, 6,
24 h, 2, 3, 6, 8, 10, 14, 17, 21, 24 and 28 days, processed
into serum, split into 2 samples then frozen for ELISA and
LBA LC/MS analysis.

ELISA Sample Analysis: The samples were analyzed by

QPS using a previously validated ELISA assay in the
range of 250 to 10 000 ng/mL and samples were pre-
diluted 5 or 10 fold prior to analysis. A target specific
ELISA using HER2 coated plates was used for analysis.
Figure 1A. Trastuzumab serum concentration of Rats 9, 10, 11
Hybrid LBA LC/MS Sample Analysis: The samples were and 12 measured by ELISA.
analyzed by SCIEX Labs using the BioBA solution and
following the vMethod protocol for trastuzumab using a
generic anti-human IgG capture antibody. The heavy
labeled antibody SILuMAB from was Sigma was added as
internal standard. The signature peptide FTISADTSK was
used for quantitation and the heavy labeled signature
peptide DTLMIS[R] from SILuMAB was used as the
internal standard.

Chromatography: Separation of the signature peptides of

the digested samples was performed on a Shimadzu
Prominence system using a Phenomenex 2.6 µm, Kinetex
C18 Column, (50 x 3.0 mm). A 7.0 minute gradient was
used and 20 µL of sample was injected onto the column.

Mass Spectrometry: The MRM analysis was performed

on a SCIEX QTRAP 6500® system equipped with an
TM
IonDrive Turbo V source.
TM
Data Processing: MultiQuant software was used for
peak integration, calibration and calculation of unknown
sample and QC concentrations.

Figure 1B. Trastuzumab serum concentration of Rats 9, 10, 11p 2

and 12 measured by hybrid LC/MS/MS.
MS/MS assay where a generic anti-human IgG antibody
was used for the same rats is shown in Figure 1B. The
sample analysis was carried out by QPS and SCIEX Labs
separately as a blinded study. It can be seen from
inspecting both plots that the two different techniques
generated similar PK plots for each rat. Furthermore the
uniqueness of each animal’s PK profile from the ELISA
plot is also apparent using the hybrid LC/MS/MS assay,
e.g., the faster elimination phase of trastuzumab in rat 12.
The average trastuzumab concentration across all four
rats is plotted in Figure 2 for both platforms and the
resulting PK parameters are in Table 1.
Figure 2. The average trastuzumab concentration across all four
rats measured by ELISA (closed circles) and hybrid LC/MS/MS
(open triangles) from dosing experiment 1.
Table 1. The PK parameters of a 10 mg/kg dose of trastuzumab
as determined by ELISA and hybrid LC/MS/MS from dosing
experiment 1.
The data presented in figure 2 were generated in two
different labs, using two different assay platforms and by
two different analysts yet generate very similar PK
profiles. Figure 3 shows the excellent correlation between
the two assay platforms for each rat at each time point.
In this dosing study the PK parameters generated from
the two average PK curves are the same within
experimental error. There was a small 10% positive bias in
the ELISA data compared to the hybrid LC/MS/MS data. A
similar positive bias was seen in a similar study with the
mAb adalimumab and most likely reflects the specificity of
the LC/MS assay.
Figure 3. The correlation of each rat sample concentration
between each assay platform for dosing experiment 1.
Conclusions
The objective of the animal dosing study was to determine
if the same samples tested using a generic hybrid
LC/MS/MS assay where a generic anti-human IgG
antibody was used would yield similar results to an ELISA
assay where a target specific immunocapture was used. It
can be seen from the PK plots in figures 1 and 2 that the
two platforms do give similar results and the same PK
parameters (Cmax and AUC) which indicates that a
generic hybrid LC/MS/MS assay can be used as a
selective method for each antibody drug even before a
target specific assay is available.
It is important to know when choosing an assay platform
that it will deliver accurate results that the user can be
confident in. The reasons for choosing a hybrid LBA and
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LC-MS assay over an ELISA assay include: selectivity,
wider LDR which reduces ULOQ sample dilutions, the
ability to multiplex a second analyte or catabolite and the
faster sample turnaround time afforded by a generic
method that can be applied to multiple studies. With the
BioBA solution SCIEX has provided a ready-to-use kit with
reagents that provide a generalized approach for the
immuno-capture and signature-peptide quantitation of
monoclonal antibody therapeutics that allow bioanalytical
scientists to easily benefit from the advantages of hybrid
LC/MS/MS assays. With the results of this tech note
bioanalytical scientists can be confident that these assays
will deliver accurate results from real animal and subject
samples.

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Document number: RUO-MKT-02-4866-A

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