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Pharmacokinetic Analysis of the mAb Adalimumab 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

1QPS, 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

than LBA

• High capacity streptavidin coated beads to

achieve high ULOQ

• Immuno enrichment allows enhanced sensitivity

for low abundance samples

• Ready to use vMethods reduce method

development time.

Introduction

The selection of a quantitative protein assay for biologics

bioanalysis in a pre-clinical study depends on what

platform will provide the right data for the drug under

development. Some questions to be considered when

choosing an assay platform include, is the total or free

drug concentration required? Are there in vivo structural

changes that might impact activity? Are these modified

proteins important to measure? Other considerations for

the chosen assay platform include reagents availability,

sensitivity and LDR requirements, sample throughput and

potential interferences.

The popular choice for protein quantitation has been

ELISA because of its high sensitivity, high throughput and

low per sample costs once the assay is developed and

validated. Limitations of the ELISA technique when

considering it as a platform include: lack of specificity in

primary or secondary antibody reagents, limited linear

dynamic range and interference due to ADA cross

reactivity. To avoid this cross reactivity, a more expensive

targeted antibody has to be used for pre-clinical studies.

The reasons for choosing a hybrid LBA and LC-MS assay

include: selectivity, broad LDR which reduces sample

dilution and the ability to multiplex a second analyte or

catabolite. Another reason to choose the hybrid

LBA/LC/MS approach for pre-clinical studies is the quick

method development turnaround time afforded by a

generic method that can be developed where the same

LBA capture reagent can be used across analytes and the

final selectivity of the assay is provided by the LC-MS

system.

The use of hybrid LBA LC/MS assays is growing and

SCIEX has developed the BioBA solution with ready to

use sample prep kits that include all the necessary

reagents and buffers which enables bioanalytical

scientists from all backgrounds to accomplish biologics

bioanalysis. Whether hybrid LBA LC/MS assays are used

BioBA reagents kit and SCIEX QTRAP® 6500 system.

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instead of or as a complement to ELISAs in drug

development, the most important question is do they give

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 adalimumab 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 adalimumab 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 TNFα coated plates was used for analysis.

Hybrid LBA LC/MS Sample Analysis: The samples were

analyzed by SCIEX Labs using the BioBA solution and

following the vMethod protocol for adalimumab using a

generic anti-human IgG capture antibody. The heavy

labeled antibody SILuMAB from was Sigma was added as

internal standard. The signature peptide APYTFGQGTK

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

IonDriveTM

Turbo V source.

Data Processing: MultiQuantTM

software was used for

peak integration, calibration and calculation of unknown

sample and QC concentrations.

Results

The serum concentration of adalimumab was measured

from two different dosing experiments. In dosing

experiment 1 four male Sprague-Dawley rats were used.

The adalimumab concentration from rats in experiment 1

as measured by a TNFα specific ELISA is shown in Figure

1A and the adalimumab serum concentration measured

using the generic hybrid LC-MS/MS assay where a

Figure 1A. Adalimumab serum concentration of Rats 5,6,7 and 8 measured by ELISA.

Figure 1B. Adalimumab serum concentration of Rats 5,6,7 and 8

measured by hybrid LC/MS/MS.

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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 slower

elimination phase of adalimumab in rat 7. The average

adalimumab concentration across all four rats is plotted in

Figure 2 for both platforms and the resulting PK

parameters are in Table 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 extremely similar PK

profiles. Closer inspection of the plot in Figure 2 reveals

the average adalimumab concentration (4 rats) as

measured by the ELISA assay is consistently higher at

each time point than the hybrid LC/MS/MS assay. There is

an approximately 20% bias in the ELISA data compared to

the hybrid LC/MS/MS data. This bias can also be seen in

the PK parameters generated from the two average PK

curves. The Cmax and AUC parameters are higher for the

ELISA measurement than the hybrid LC/MS/MS

measurement. The reason for the 20% bias was

investigated. Selectivity issues with the ELISA assay were

considered but eliminated because of the selective TNFα

ELISA assay used. Next the stock solutions used to

construct the calibration curves in the two labs were

compared. They were compared by LC/MS/MS and a

positive 15% bias was found in the ELISA stock solution

which contributed to the positive bias in the bioanalytical

results.

A second rat dosing study was then performed. The

adalimumab concentration from rats in dosing experiment

2 as measured by a TNFα specific ELISA is shown in

Figure 3A and the adalimumab serum concentration

measured using the generic hybrid LC-MS/MS assay for

the same three rats is shown in Figure 3B.

Figure 3A. Adalimumab serum concentration of Rats 17, 18 and 19 measured by ELISA.

Figure 2. The average adalimumab 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 adalimumab

as determined by ELISA and hybrid LC/MS/MS from dosing experiment 1.

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Figure 3B. Adalimumab serum concentration of Rats 17, 18 and 19 measured by ELISA.

The assay experiments (ELISA and hybrid LC/MS/MS)

from dosing study 2 were carried out in the same lab

(QPS) using the same stock solution. Again it can be seen

that the two different techniques generated similar PK

plots for each rat.

The average adalimumab concentration across all three

rats is plotted in Figure 4 for both platforms and the

resulting PK parameters are in Table 2.

It can be seen from Figure 4 that the two different assay

platforms generated almost identical PK profiles when

working from the same stock solution. Figure 5 shows the

excellent correlation between the two assay platforms for

each rat at each time point. With the smaller bias between

the two platforms in this dosing study the PK parameters

generated from the two average PK curves are the same

within experimental error.

Figure 4. The average adalimumab concentration across all

three rats measured by ELISA (closed circles) and hybrid LC/MS/MS (open triangles) from dosing experiment 2.

Table 2. The PK parameters of a 10 mg/kg dose of adalimumab

as determined by ELISA and hybrid LC/MS/MS from dosing experiment 2.

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Figure 5. The correlation of each rat sample concentration

between each assay platform for dosing study 2. Excellent correlation was seen from dosing study 2 when the same stock solution was used to prepare calibrators for the two platforms

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 2 and 4 that the

two platforms do give similar results. In animal dosing

study 2 both platforms gave 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. In animal dosing study 1 the measured

adalimumab concentrations were comparable within 20%.

After investigation the source of the bias was attributed to

a bias in the two different stock solutions used to prepare

calibrators in the two different labs.

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

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 or subject

samples.

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Document number: RUO-MKT-02-4865-B