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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.

  • p 2

    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 TNF 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 m, Kinetex

    C18 Column, (50x3.0 mm). A 7.0 minute gradient was

    used and 20 L 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 TNF 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.

  • p 3

    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

    animals 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 TNF

    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 TNF 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.

  • p 4

    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