Pharmacokinetic Analysis of the mab Adalimumab by ELISA and Hybrid LBA/LC/MS: A Comparison Study Featuring the SCIEX BioBA Solution Xi Qu 1, Shuyu Hou 1, Meghan McCann 1, Caroline Becker 1, Xun Wang 1, Zamas Lam 1, Susan Zondlo 1, John Kolman 1, Lei Xiong 2, Witold Woroniecki 2, Hua-Fen Liu 2, Ian Moore 3 1 QPS, Delaware, USA, 2 SCIEX, Redwood Shores, USA, 3 SCIEX, 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. BioBA reagents kit and SCIEX QTRAP 6500 system. 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 p 1
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. 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 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 prediluted 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. Figure 1A. Adalimumab serum concentration of Rats 5,6,7 and 8 measured by ELISA. 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 IonDrive TM Turbo V source. Data Processing: MultiQuant TM software was used for peak integration, calibration and calculation of unknown sample and QC concentrations. Figure 1B. Adalimumab serum concentration of Rats 5,6,7 and 8 measured by hybrid LC/MS/MS. p 2
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. 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 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. Figure 3A. Adalimumab serum concentration of Rats 17, 18 and 19 measured by ELISA. The data presented in figure 2 were generated in two different labs, using two different assay platforms and by p 3
Figure 3B. Adalimumab serum concentration of Rats 17, 18 and 19 measured by ELISA. 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. 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. 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. p 4
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. 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. AB Sciex is doing business as SCIEX. 2016 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Biomek Method Launcher may not be compatible with Biomek Accounts and Permissions authentication. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All trademarks are the property of their respective owners. Document number: RUO-MKT-02-4865-B p 5