Strategy for the Quantitation of a Protein Conjugate via Hybrid

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A Novel Strategy for the Quantitation of a Therapeutic Protein Conjugate via Hybrid Immunocapture-Liquid Chromatography with Sequential HRMS and SRM-Based LC-MS/MS Analyses Yue Zhao, Guowen Liu, Xiling Yuan, Jinping Gan, Jon E Peterson, and Jim X. Shen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00926 • Publication Date (Web): 12 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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Analytical Chemistry

A Novel Strategy for the Quantitation of a Therapeutic Protein Conjugate via Hybrid Immunocapture-Liquid Chromatography with Sequential HRMS and SRM-Based LC-MS/MS Analyses Yue Zhao*, Guowen Liua, Xiling Yuan, Jinping Gan, Jon E. Peterson, Jim X. Shen* Analytical and Bioanalytical Operations, Research & Development, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA.

* Corresponding Authors Yue Zhao Route 206 & Province Line Road, Princeton, NJ, 08543 Tel: 609-252-4566 Email address: [email protected]

Jim X. Shen, PhD Route 206 & Province Line Road, Princeton, NJ, 08543 Tel: 609-252-6768 Email address: [email protected]

a

Current affiliation: DMPK-Clinical Bioanalytical, Agios Pharmaceuticals, Cambridge, MA 02139, USA.

ACS Paragon Plus Environment


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Abstract With the development of modern instrumentation and technologies, mass spectrometry-based assays have played an important role in protein therapeutic bioanalysis. We have developed a novel strategy by combining the “bottom-up” and “top-down” approaches using both high-resolution (HRMS) and selected reaction monitoring (SRM)-based mass spectrometric detection to quantify a positron emission tomography (PET) detection tracer for an oncology marker. Monkey plasma samples were processed by immunocapture purification, followed by liquid chromatography (LC) with HRMS full scan analysis. Summed multiple charge states and multiple isotopes per charge state of the analyte were used during quantitation for optimized sensitivity. After the HRMS analysis, the remaining samples were digested by trypsin, followed by SRM detection. The HRMS approach provided the solution to a unique problem related to stability of the protein conjugate by quantifying the intact protein. The SRM method only measured a signature peptide generated from enzymatic digestion, but had a lower quantitation limit to meet the sensitivity requirement to assess the pharmacokinetics in a toxicology study. Both methods demonstrated good sensitivity, accuracy, precision and robustness, and the results revealed that there was no significant difference between the data sets obtained from both methods, indicating no in vivo or ex vivo degradation occurred in the incurred samples after dosing. This workflow not only provided the quantitative results for pharmacokinetic evaluation, but also revealed valuable in vivo stability information on the intact protein level.


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INTRODUCTION Mass spectrometry is a widely applied analytical technique in drug discovery and drug development, and are commonly used in quantitation, structure elucidation, and identification

1-6

. Triple quadrupole

mass spectrometers, connected with liquid chromatography (LC), are standard platforms for small molecule therapeutic drug quantitation in biological fluid, due to their excellent sensitivity and selectivity

2,7-9

. More recently, triple quadrupole mass spectrometry has been increasingly and

successfully applied toward protein quantitation

10-15

. As triple quadrupole mass spectrometry is most

applicable for analytes between ranges of a few hundred to a few thousand mass/charge units (m/z), the general accepted quantitation procedure for protein involves in the measuring of a surrogate peptide (a unique peptide to represent the entire protein) generated by enzymatic digestion of the protein.14,16,17 The digested bioanalytical work flow, referred to as the “bottom up” approach, includes the following basic steps: isolation of the protein of interest via either protein precipitation or immunocapture (IC) prior to trypsin digestion followed by further isolation of the peptide of interest via traditional sample preparation techniques before chromatographic separation with MS/MS detection11,18-20. One concern with the “bottom up” approach has been the suitability of representing the entire protein via a single peptide. Under some circumstances, a small change in the protein (such as post translational modifications and different protein variants) will not be detected because the signature peptide may not contain the modification sites of the protein. To remediate these concerns, techniques such as selecting a confirmatory peptide from a different region of the molecule has provided a measure of assurance 21. Yet, an orthogonal method that can measure the intact protein is better at providing unequivocal confirmation over the identity of the analyte. The analysis of intact protein, or “top down” analysis, can be performed on high molecular weight capable instruments such as a time of flight (TOF) and orbit trap mass spectrometers 20,22-26. Both of these mass analyzers are equipped to handle a higher m/z generated by an intact analyte as well as provide a higher mass resolution. Unfortunately, the sensitivity for these



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machines are still about a fold less than those can be achieved on a triple quad setup. Therefore, there is an obvious advantage in marrying high resolution and triple quadrupole mass spectrometers to provide both sensitivity and structural confirmation of the analyte. As a non-invasive functional imaging technique, positron emission tomography (PET) has seen increasing applications in oncology disease diagnostic and treatment 27. PET is ideal in assessing the pharmacokinetics and pharmacodynamics of novel therapeutics during treatment through the application of radiolabeled tracer molecules 28-30. BMS has developed an engineered, linked protein conjugate, as the PET imaging tracer which contains an adnectin 17, and a modified maleimide linker with a fluorine18 [18F] tracer (Figure 1, [18F]BMS-986192). The detailed synthesis of the cold molecule BMS986192 and the radiolabeling with [18F] will be summarized elsewhere31. The molecule has a total molecular weight of 11 KDa, which is suitable for intact high resolution mass spectrometry analysis. To support this program, a bioanalytical assay to quantitatively measure BMS-986192 (the cold compound) is needed. In the present study, we developed a “bottom up” LC-MS/MS method which monitored a surrogate peptide generated from the adnectin portion after tryptic digestion. The method was straight forward with good sensitivity and performance. A major limitation of the assay was that it only represented a portion of the protein (the adnectin portion) and might not reveal other components, such as degradation products and post-translational modified species. Particularly, we were interested to know if the maleimide linker and the tracer were still intact with the adnectin both in vivo and ex vivo. For this purpose, we further developed a full scan MS method which allowed us to obtain useful information from the whole protein using the high resolution method as a confirmatory assay. However, while the “top down” approach provided information on the overall stability of the protein, the digested method had a better sensitivity and achieved the LLOQ requirement to capture the concentrations at later time points.

The methods were successfully qualified and applied to a discovery monkey

toxicology study.


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Materials & Methods Chemicals, Reagents and Materials Dynabeads® MyOne™ Streptavidin T1 was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco’s phosphate buffered saline (PBS) was obtained from Lonza Walkersville, Inc. (Walkersville, MD, USA). Monkey plasma was purchased from Bioreclamation, Inc. (Westbury, NY, USA). Reference material of BMS-986192 and the biotinylated anti-adnectin monoclonal antibody was synthesized and purified internally at BMS. Deionized water was generated in house using a NANOpure Diamond ultrapure water system from Barnstead International (Dubuque, IA, USA). HPLC grade acetonitrile and phosphate buffered saline with Tween-20 (PBST), and trypsin from bovine pancreas (TPCK treated) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid (SupraPur grade), acetic acid, and trifluoroacetic acid were purchased from EMD Chemicals (Gibbstown, NJ, USA). LC-MS conditions for HRMS approach The Shimadzu (Columbia, MD, USA) Nexera UHPLC system consists of two LC-30AD pumps, two DGU-20A5 degassers, one SIL-30ACMP autosampler and one CTO-30AS column heater. Mobile phase A was 0.1% trifluoroacetic acid (TFA) and 0.5% acetic acid in water, and mobile phase B was 0.1% TFA , 0.5% acetic acid and 5% DMSO in acetonitrile. An Acquity BEH-C8 column (2.1 x50 mm, 1.7 µm, Waters Corporation, MA, USA) was used as the analytical column with the column temperature set at 80 °C. Gradient elution started at 5% B for 0.5 min, increased linearly to 60% B in 5.5 min; increased to 95% B in 1 min; held at 95% B for 1.9 min and then returned to 5% B in 0.1 min. The UHPLC flow rate was set at 0.6 ml/min and the injection volume was 30 µL. High resolution mass spectrometric (HRMS) detection was carried out using a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer (ThermoFisher, CA, USA). The mass spectrometer was operated using positive ion electrospray ionization (ESI) with the MS parameters set as follows: spray voltage and capillary temperature were set at 3kV and 400 °C. The sheath gas and auxiliary gas were at



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60 and 20 (arbitrary units), respectively. A resolution of 70,000 was applied with maximum ion injection time of 200 ms. Full scan mode was employed for data acquisition with a mass scan range of 1400-2600 m/z. The system was controlled by Xcalibur software (version 2.1, ThermoFisher, CA, USA) for data acquisition and data processing. A weekly external calibration was performed for ESI using the Pierce TM

LTQ ESI positive ion calibration solution (ThermoFisher, CA, USA).

LC-MS conditions for MS/MS approach A Triple Quad 5500 mass spectrometer (AB Sciex, Foster City, CA) was coupled with the Shimadzu Nexera UPLC system. The LC-MS/MS system was controlled by Analyst® 1.6.2 software. The following optimized MS conditions were used: curtain gas and collision gas were set as 30 and 10; the turbo spray voltage was set at 5500 V and ion source gas 1 and gas 2 were both set at 55 psi. The probe temperature was set at 550 ºC. The collision energy, declustering potential, entrance potential and collision cell exit potential were set at 22 V, 60 V, 10 V and 15 V, respectively. The transition monitored for selective reaction monitoring (SRM) for the surrogate peptide EFPI (first four amino acids was used for the abbreviated name for the surrogate peptide) was m/z 570.0 → 431.8. Mobile phases were 0.1% formic acid in water and acetonitrile. The gradient elution was employed on an Acquity HSS T3 column (2.1 x50 mm, 1.7 µm, Waters Corporation, MA, USA) with the following gradient: 0 - 0.5 min, 5% B; 0.5 - 6 min, 50% B; 6 - 6.1 min, 90% B; 6.1 - 8 min, 90% B; 8-8.1 min, 5% B. Run stopped at 9 min and the flow rate was 0.8 mL/min. The column temperature was set at 80 °C.

Monkey Toxicology Study Design Three male cynomolgus monkeys were intravenously administered a single dose of [19F]BMS-986192 (1 mg/kg). Plasma samples were collected at 0m, 2m, 10m, 15m, 30m, 45m, 1h, 2h, 3h, 5h, 7h and 24h post dose.


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Sample Preparation Preparation of calibration standards and quality control samples Stock solution for BMS-986192 was prepared at a concentration of 5.64 mg/ml in PBS. Eleven levels of standards were prepared daily at the concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 750 and 1000 ng/mL in blank monkey plasma. Six levels of QCs (3, 30, 60, 200, 500, 800 ng/mL) were prepared similarly and stored at -70 °C before analysis. Two different analytical ranges were used for the HRMS and MS/MS assays due to the sensitivity limit. All standards and QCs were extracted at once but selected levels were used for quantitation for separate assays. To be specific, QC 60 ng/mL, 200 ng/mL, 500 ng/mL and 800 ng/mL were used in the HRMS assay; and QC 3 ng/mL, 30 ng/mL, 500 ng/mL and 800 ng/mL were used for the MS/MS assay. All standard/QC preparation as well as frozen QC/sample thawing were done in an ice-water bath. Anti-adnectin antibody biotinylation An anti-adnectin scaffold monoclonal antibody was biotinylated using EZ-Link NHS-PEG4-Biotin kit (ThermoFisher, CA, USA) according to manufacturer's instructions. Briefly, the antibody at 2 mg/mL in DPBS was incubated with biotin at 20:1 biotin/protein molar ratio at room temperature for 30 minutes. Free biotin was removed using a Corning® Spin-X® UF20 (50K MWCO) concentrator (Corning, NY). Preparation of magnetic beads The biotinylated anti-adnectin monoclonal antibody was immobilized to the magnetic bead (Dynabeads® MyOne™ Streptavidin T1 10 mg/mL) suspension at a 200 µg antibody per mL of bead ratio by incubating the solution mixture at room temperature for 30 min, followed by a cleaning step with PBST to remove the free antibody. Immuocapture An aliquot of 100 µL of each plasma sample was mixed with 150 µL of PBST buffer and 50 µL of the immobilized magnetic bead suspension (containing 200 µg antibody per mL bead). The solution mixture



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was incubated at room temperature for 1 h, washed 2 times with PBST and 1 time with PBS. After washing, 100 µL of 12 mM HCl was added to elute the analyte off the beads by hard vortexing. The eluents were collected in a clean 96-well plate and then neutralized with 10 µL of 200 mM ammonium bicarbonate. The collected samples were analyzed on the HRMS system. Digestion After the HRMS analysis, the samples were digested by adding 25 µL of the Sigma trypsin (500 µg/mL in 200 mM ammonium bicarbonate), followed by incubation at 60 °C for 15 min. After incubation, the digestion was stopped by the addition of 5 µl of 10% formic acid in water. A volume of 20 µL was injected for LC-MS/MS analysis. LBA assay validation and sample analysis An electrochemiluminescence (ECL) ligand binding PK assay was developed towards BMS-986192 using well characterized non-overlapping anti-adnectin scaffold mAbs for capture (18D8) and detection (34G11). The desirable detection range of the assay, selected from an earlier PK dosing feasibility study as 0.01 – 2 nM (~ 0.12 to 23 ng/mL), was achieved with a minimal required dilution (MRD) of 80. The assay was validated with satisfactory accuracy and precision and was further used to for sample analysis.

Results and Discussion Study Design and Workflow As previously discussed, both “top down” and “bottom up” approaches have their advantages and limitations. The “top down” approach is easier to monitor the entire protein, but is limited by the protein size and sensitivity. In some cases, the “bottom up” approach may also provide useful biotransformation information on the protein level depending on the sample treatment method. For example, for a novel therapeutic protein that has potential in-vivo proteolytic liabilities on the C-terminus, scientists were able


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to use different capture antibodies that bind to either C or N -terminus to provide quantitative results on the intact protein and truncated protein forms.32 In our situation, since the stability concern was on the linker/tracer compartments, we utilized an anti-adnectin capture antibody which allowed us to capture both the intact protein form (with linker/tracer attached) as well as the degraded form (without linker/tracer). The “top down” approach was established for stability evaluation but lacked the desired sensitivity.To obtain a broader picture, we applied both “top down” and “ bottom up” strategies in a monkey toxicology study. Figure 2 showed a schematic study design/work flow for this study. Monkey plasma samples were first purified by immunocapture purification using the anti-adnectin mAb, the resultant samples were first analyzed on the high resolution mass spectrometer directly. After the HRMS analysis, the remaining samples were digested using trypsin, followed by LC-MS/MS analysis. The two data sets were compared to evaluate the protein stability. As BMS-986192 maybe highly reactive, the other goal of the bioanalytical method is to use HRMS to establish the stability of the reference material and ensure no undesirable degradation/reaction occurs in solution or plasma environment. One of the primary concerns is a hydroxylation product that was noted when a solution stored at 4 °C for a prolonged period of time indicated an increase in concentration of the hydroxylation product compared to a fresh sample. As such HRMS monitoring of the hydroxylation product was part of the method which may not otherwise possible using digested approach. We also routinely perform high resolution scan using incurred samples to ensure no other additional in vivo degradation product are identified.

LC-HRMS Detection One of the important aspects of LC-MS chromatography development is the need to balance the overall run time with quality of separation. To this end, we conducted a number of experiments by creating methods using various column stationary phases, mobile phases, and additives, with different HPLC



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column temperatures. Column temperature has been observed as a modulator to peak shape, resolution, and speed in bioanalytical analysis 33,34. Our method development optimization resulted in the selection of BEH C8 column and a water and acetonitrile based mobile phase with both TFA and acetic acid added to improve peak shape. Moreover, the addition of acetic acid could also minimize the negative effect of TFA in ionization, as previously reported.35 The column temperature was set at 80 °C for best balance between the column back pressure, peak resolution, and overall run time and there was no observed negative impact on the column life at this temperature. MS spectra were extracted from the total ion chromatogram (TIC) as shown in Figure 3(A). Under ESI mode, BMS-986192 was positively multiple charged (Figure 3B) with most abundant isotopic ions of m/z 1474.7359 (z=8), 1685.1247 (n=7), 1965.8114 (n=6) and 2358.9714 (n=5). At 70,000 resolution, isotopic peaks were well resolved for each of the charge state (Figure 3C). The mass spectrum also includes the hydroxylation product of BMS-986192, shown as a cluster of ions to the right of the analyte peaks. When the MS spectra were deconvoluted using XCalibur, the resultant two molecular masses represent BMS-986192 at 11782.76 Da and the hydroxylation product of BMS-986192 at 11800.75 Da. To enhance sensitivity, peak integration was conducted by combining four of the most abundant ions for charge state z=5 and z=6 (eg, 2358.5765, 2358.7754, 2358.9745, 2359.1735 for z=5 and 1965.6350, 1965.8114, 1965.9781 and 1966.3110 for z=6). The chromatogram of the combined ions showed a distinct chromatographic peak at the retention time of 4.3 min (Figure 3D). With the effort to further improve sensitivity, more method development work was conducted to optimize the HPLC condition. DMSO has been reported36 as one of the “supercharging reagents” which helped to increase sensitivity by reducing the mass-to-charge (m/z) ratio, and increasing tandem MS efficiency. We tested the impact of using DMSO as an additive into mobile phases in our method. The results reported in Figure 4 demonstrated the shift in the charge states observed upon the addition of 5% of DMSO from a more diversified distribution at higher m/z ratio to lower numbers (Figure 4(B)). As a result of the shift, the peak intensity (peak height) increased from 2.1


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x 105 to 4.2 x 105 and 2.9 x 105 to 4.4 x 105 for charge state 5 and 6, thus increasing our assay sensitivity. Higher or lower concentrations of DMSO did not result in additional improvement in sensitivity. Therefore, 5% DMSO was used in the final method. Although concerns might be raised regarding the use of both DMSO and TFA in the mobile phase, we have tested the method for several months and no obvious loss of sensitivity was observed on the mass spectrometer. LC-MS/MS After digestion, we monitored peptide EFPI (abbreviated) which located in the adnectin area as a surrogate peptide for BMS-986192. As shown in Supporting Information S1, the MS spectrum for EFPI showed the doubly charged [M+2H]2+ protonated ions at m/z 570.0 and a singly charged ion [M+H]+ at m/z 1138.7. Further dissociation yielded a few major fragment ions at m/z 431.8, 652.5 and 862.7, respectively. After method optimization, SRM transition of m/z 570.0 → 431.2 was selected for quantitation with lower background and better signal-to-noise ratio.

Sample Preparation Optimization The endogenous proteins in the biological samples often cause large background interferences for target protein bioanalysis. For a ‘bottom up” approach without appropriate sample cleanup, the highly abundant endogenous proteins will be digested together with the target protein and generate a large amount of background peptides, which could cause severe ion suppression and have negative impact on assay sensitivity and ruggedness. A highly specific sample cleanup is critical for the success to identify the analyte of interest from the highly abundant endogenous components when full scan HRMS is used to acquire data across a wide mass range. In our methods, a super clean extract was achieved by using immuno-capture with a capture antibody very specific to the adnectin portion of BMS-986192. The antibody was biotinylated and immobilized onto the streptavidin magnetic beads, which were then added into the plasma samples to extract BMS-986192 from plasma. Since this antibody targeted the adnectin,



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both the intact BMS-986192 and any degraded products (as long as the binding site on the adnectin was functioning) would be isolated from the matrix. This specific immuno-capture sample cleanup approach made it possible for us to perform full scan HRMS analysis. Assay Performance Evaluation Eleven levels of standards and six levels of quality control samples were prepared as described in the experimental section to cover the analytical range for both assays. Due to the sensitivity limit of the HRMS method, two different curve ranges were selected. A range of 20.0 to 1000 ng/mL, and 1.00 to 1000 ng/mL were selected for the HRMS and MS/MS method, respectively. Therefore, for the HRMS assay, the first four standards at the low end were not used for the quantitation, and different QC concentration levels were selected to better suit the assay range. The standard calibration curves were reported in supporting information Figure S2. A quadratic 1/x2 weighted regression model was chosen for both assays, with R2 of > 0.994. Good assay performance was demonstrated for both assays, and the accuracy and precision results are shown in Table 1. For HRMS assay, the % Dev was within 6.3% and the between run, within run precision and total variation (% CV) were < 6.1%, 6.8% and 6.8%, respectively. For the LC-MS/MS assay, the % Dev was < 9.4% and the between run, within run and total % CV were < 9.5%, 6.6%, and 10.6% respectively. Stability was evaluated using spiked QC at 500 ng/mL and analyzed with three replicates using the HRMS assay. As shown in Figure 5, a less than 20% of degradation was observed for samples stored at room temperature for 4 hr and even less degree of degradation was observed when samples were stored on ice. This experiment demonstrated that BMS986192 was still intact (with adnectin, linker/tracer) when post-spiked into commercial monkey plasma and stored for up to 4 hr at both room temperature and on ice. For the HRMS assay, although a single charge state can be used for quantitation, two charge states (charge state 5 and 6) as previously described were combined to increase sensitivity. To demonstrate that combining two charge states will enhance the overall signal-to-noise (S/N) ratio, rather than


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increasing both signal and noise at the same time, two samples (one at 10 ng/mL and the other one at 1000 ng/mL) were processed with different processing methods. As shown in Figure 6, sample A1 and B1 showed the chromatograms resulted from extracting four of the most abundant 4 ions from both charge state 5 and 6 (2358.5765, 2358.7754, 2358.9745, 2359.1735 for z=5 and 1965.6350, 1965.8114, 1965.9781 and 1966.3110 for z=6), while A2 and B2, A3 and B3 represented results from only charge state 6 and 5, respectively. The results revealed that for the sample tested at 10 ng/mL, the peak intensity (height) increased proportionally when combining two charge states, and S/N ratio also increased to 100 as compared to the S/N ratio of 71 or 67 when extracting charge state 5 or charge state 6 alone. For the high concentration sample, both peak intensity and S/N ratio increased proportionally when comparing B1 to B2 or B3. The results demonstrated the combination of the two most abundant ion peaks by HRMS can further improve sensitivity. It should be noted such improvement is compound dependant and the charge states used should be carefully selected during method development. In the case where there is high background interference in a particular charge state, the inclusion of the charge state will likely decrease the S/N ratio. Representative chromatograms for BMS-986192 in monkey plasma using HRMS full scan are presented in Supporting Information Figure S3. Samples prepared at various concentrations as well as a blank plasma sample were monitored and compared. At the retention time of 4.3 min, no interference peak was observed in the blank sample. Figure S3 demonstrated sample at 20 ng/mL showed sufficient S/N ratio and was established as the LLOQ. For the MS/MS assay, Figure S4 shows the representative chromatograms for the SRM acquisition of the surrogate peptide EFPI after digestion of BMS-986192. The chromatogram obtained from the LLOQ of 1.00 ng/mL showed an S/N ratio of greater than 100. A small peak observed in the blank monkey plasma sample at the retention time of 3.4 min was due to a small carryover issue, however, its impact on the LC-MS assay was considered as insignificant because the LC-MS response was much less than 20% of the LLOQ.



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It was worth mentioning that no internal standard was used for both HRMS and MS/MS assays. For a traditional small molecule LC/MS method, an internal standard which is either a stable-isotopic labeled version of the analyte or a structural analog is always preferred to compensate the sample preparation/instrumentation variabilities. Due to the nature of the analyte, which is an engineered protein, and the required study timeline, no suitable internal standard was available for both assays at the time of development. Since the processed samples were analyzed twice on two different mass spectrometers, the success of the methods would totally rely on good sample preparation and cleanup (immunocapture), accurate injection volume and stable MS instrument performance. Tremendous effort had been put into method development and optimization. Nevertheless, the analytical results demonstrated assay robustness with good accuracy and precisions for both methods. Sample Analysis The developed assays were applied to a discovery monkey toxicology study. Regardless the radio label, both cold BMS-986192 and [18F]BMS-986192 share the same pharmacokinetic and stability properties. The toxicology study was designed to dose only cold compound BMS-986192 to investigate its pharmacokinetics to support the future dosing of [18F]BMS-986192 in the clinics. Another goal of this study was to answer an important question regarding its in vivo stability information. According to the study design, the same set of unknown study sample was analyzed twice using these two methods. The strategy used in the study is similar to a “one extraction, two injection” approach, although after the first analysis on the HRMS, further sample preparation (digestion) step was executed prior to the second analysis. The HRMS analysis could provide us with concentration information regarding the intact protein; while the quantitative results obtained by MS/MS focused on a segment of the protein. The two sets of data were compared and the difference was used to evaluate if there was any degradation of the protein in the incurred samples. Table 2 summarizes the data comparison from 13 representative samples using LC-MS/MS assay and the LC-HRMS assay. The percentage difference (% diff) was


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calculated using: % Diff =100* (A-B)/ [(A+B)/2], where A and B represented the SRM and HRMS results. The results demonstrated a good correlation between the two data sets. And among 64 samples analyzed, 58 samples had the % diff within 10%, providing us with good confidence that BMS-986192 was still intact after dosing. The LC-MS/MS assay did have an improved sensitivity which was 20 times lower than the HRMS method and can meet the LLOQ requirement of the study to provide a complete PK profile. After confirming the stability, the LC-MS/MS results were reported and used for PK calculation. In addition, the same set of samples were also analyzed by a ligand-binding assay (LBA). The LBA assay was first developed which used two non-competing anti-adnectin scaffold mAbs as both capture and detection antibodies. Since both capture and detection are aimed on the adnectin portion, the original concern was that the measured concentration really cannot represent the intact protein conjugate if any modification or instability occurred. After the development of the HRMS and LC-MS/MS assay, we compared the results generated from these two different analytical platforms. As shown in Figure 7, the PK profile obtained using both LC-MS/MS and LBA methods averaged from three animals are compared. LC-MS/MS and LBA results were in good agreement from 0 to 24 hr, further proving the invivo stability of BMS-986192 and demonstrated our assay reliability.

Conclusions Herein we report a strategy of integrating two MS methodologies for the quantitative bioanalysis of an engineered protein therapeutic. An immunocapture purification procedure was utilized for sample cleanup, followed by high resolution MS detection to monitor the intact protein. The major advantage of the HRMS method is that it could provide additional information on the intact protein level, which might be lost using the “bottom-up” approach. The feasibility of extracting and integrating the two most abundant ion peaks by HRMS in order to improve the sensitivity of the assay has also been



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demonstrated. MS/MS measurement of a surrogate peptide from the digested protein was carried on afterwards which only monitored a portion of the protein but provided a better assay sensitivity (20x lower LLOQ). By comparing results from these two methodologies, valuable information was obtained in regards to protein stability, and the final quantitative results were reported using the MS/MS data set. Both methods were quantitative and demonstrated good accuracy, precision and reproducibility, and were applied in a monkey toxicology study. The workflow and strategy used in this study demonstrated the different values from both MS methodologies, and each of the method serves as a complementary method to the other. The full scan HRMS approach could provide more comprehensive information on the intact molecule level than targeted MS/MS detection. Although an improved sensitivity is desired, HRMS could be used to develop quantitative bioanalytical assays for wider applications in protein bioanalysis to guide drug candidate selection.

Supporting Information Available: Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Notes: The authors declare no competing financial interests. Acknowledgements The authors would like to thank Mr. Huidong Gu, Dr. Jianing Zeng and Dr. Naiyu Zheng for their scientific discussion and critical review of this manuscript.


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(31) Donnelly, D. J.; Morin, P.; Adam Smith, R.; Lipovšek, D.; Gokemeijer, J.; Cohen, D.; Lafont, V.; Tran, T.; Cole, E. L.; Wright, M.; Henley, B.; Pena, A.; Kukral, D.; Hayes, W.; Cao, K.; Kim, J.; Bonacorsi Jr, S. J. In preparation 2017. (32) Hager, T.; Spahr, C.; Xu, J.; Salimi-Moosavi, H.; Hall, M. Anal Chem 2013, 85, 2731-2738. (33) Cunliffe, J. M.; Dreyer, D. P.; Hayes, R. N.; Clement, R. P.; Shen, J. X. J Pharm Biomed Anal 2011, 54, 179-185. (34) Shen, J. X.; Merka, E. A.; Dreyer, D. P.; Clement, R. P.; Hayes, R. N. J Sep Sci 2008, 31, 242-254. (35) Shou, W. Z.; Naidong, W. J Chromatogr B Analyt Technol Biomed Life Sci 2005, 825, 186-192. (36) Sterling, H. J.; Prell, J. S.; Cassou, C. A.; Williams, E. R. J Am Soc Mass Spectrom 2011, 22, 11781186.


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Figure 1. Structure of protein conjugate [18F]BMS-986192. The green rectangle shows the HRMS measurement of the intact protein including adnectin and the tracer with the 18F label, and the red square shows the SRM measurement of the adnectin part only. Note: the reported method in this manuscript only measures the cold version of the protein conjugate [19F]BMS-986192.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Plasma Samples (STD, QC and incurred samples) Immunocapture Purification

LC-HRMS analysis Remaining samples (Tryptic digestion)

LC-MS/MS analysis

Data analysis

Data Comparison

Data analysis

Figure 2. Schematic workflow of the monkey toxicology study.


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Page 21 of 28

100

(A) Total Ion Chromatogram (TIC) 50 0.0

1.0

2.0

100

Time (min)

1685.1247 1474.7359 z=7 z=8 800

1000

1200

1400

2358.7754 2358.5765

100

(C)

1600 2358.9745

2358.3732 2358.1718 2357.9690

50 0

4.0

5.0

2355

2356

2357

z=5

1965.8114

(B) HRMS Spectra

0

3.0

2358.9714

50

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2358

2359

100

z=6

1800 2000 m/z

2948.1959 z=4 2200

2400

2600

100

2359.1735

2800 11782.76

50

2359.3800 2359.5784 2359.7783 2359.9787 2360.1743 2360 2361 2362 m/z

11800.75 11760

2363

3000

2364

2365

11780 m/z

11800

2366

(D)

50 0.0

1.0

2.0

3.0

4.0

5.0

Time (min)

Figure 3. High resolution MS analysis of BMS-986192 in monkey plasma after immunocapture. (A) Total ion chromatogram (TIC) obtained from full MS scan. (B) MS spectra at the selected retention time at 4.3 min. (C) The enlarged spectrum at charge state of 5 and the deconvoluted molecular masses. (D) Chromatographic peak of the combined ions of charge state 5 and 6.



2358.9714 z=5 100

1685.1247

50 1474.7359 1310.9833 z=8 z=9 0

800

100

1000

1200

(B)

1400

1600

50

z=7 2948.1959 z=4 1800

1474.7359 1685.1247 z=8

0

Max Intensity: 1.96E4

1965.8114 z=6

(A)

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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z=7

2000

2200

2400

2600

2800

1965.8114

Max Intensity: 2.51E3 2358.9714

z=6

z=5

1310.9833 z=9

800

1000

1200

1400

3000

2948.1959 z=4

1600

1800

2000

2200

2400

2600

2800

3000

m/z

Figure 4. HRMS spectra of BMS-986192 in monkey plasma after immunocapture using (A) 0.1% FA, 0.1% TFA and 5% DMSO in ACN and (B) 0.1% FA and 0.1%TFA in ACN as mobile phase B. Mobile phase A was 0.1% FA and 0.1% TFA in water for both cases.


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120 100

% Remaining

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60

RT

40

On Ice

20 0 0

1

2

4

Time (hr)

Figure 5. Stability of BMS-986192 in monkey plasma at room temperature and on ice.



100

(A1)

S/N100

Peak Height: 3.21E3

0 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

100

S/N 71 (A2)

Peak Height 1.31E3

0 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5

100

S/N 67 (A3)

Peak Height 2.09E3

100

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(B1)

S/N:10988

Peak Height: 4.29E5

: 0 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 100

(B2)

S/N 5815 :

Peak Height: 2.23E5

0 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 S/N 3244 :

100 (B3)

Peak Height: 2.06E5

0 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4

: 0 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4

Time (min)

Time (min)

Figure 6. Chromatograms of processed monkey samples at 10 ng/mL (A1-A3) and 1000 ng/mL (B1B3). Different ions were extracted during the data processing. For A1 and B1, ions 2358.5765, 2358.7754, 2358.9745, 2359.1735, 1965.6350, 1965.8114, 1965.9781, 1966.3110 were extracted and summed; For A2 and B2, 2358.5765, 2358.7754, 2358.9745, 2359.1735 were extracted and summed and for A3 and B3, 1965.6350, 1965.8114, 1965.9781, 1966.3110 were extracted summed similarly. S/N ratio was calculated using Xcalibur software.


Page 25 of 28

100000

10000

Concentration (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LC-MS LBA

1000

100

10

1 0

5

10

15 20 Time (hr)

25

30

Figure 7. Concentration versus time profile of cold [19F]BMS-986192 in monkey plasma measured by LC-MS/MS and LBA assay. Results showed here are an average from three different animals.



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Table 1. Accuracy and Precision Results for BMS-986192 in monkey plasma using A) HRMS and B) MS/MS. The calibration curve range for HRMS is 20 to 1000 ng/ml and 1 to 1000 ng/ml for MSMS assay.

(A) Nominal Conc. (ng/mL) Mean Observed Conc. %Dev

Low (60.00) 58.24 -2.9

GM (200.00 ) 207.05 3.5

Medium (500.00 ) 531.43 6.3

High (800.00 ) 754.46 -5.7

Between Run Precision (%CV) 2.1 Within Run Precision (%CV) 6.3 Total Variation (%CV) 5.3

5.3 6.8 6.5

2.0 2.6 2.8

6.1 6.5 6.8

n Number of Runs

8 2

8 2

8 2

8 2

(B) Nominal Conc. (ng/mL) Mean Observed Conc. %Dev

Low (3.00 ) 2.89 -3.7

GM (30.00 ) 30.01 0.0

Medium (500.00 ) 475.14 -5.0

High (800.00 ) 725.16 -9.4

Between Run Precision (%CV) 9.5 Within Run Precision (%CV) 4.7 Total Variation (%CV) 10.6

1.5 2.7 3.1

0.0 3.3 2.9

3.8 6.6 7.6

n Number of Runs

8 2

8 2

8 2

8 2


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Table 2. Data comparison of the measured concentration of BMS-986192 in a monkey toxicology study using HRMS and LCMS. Sample #

LC-MS/MS (A)

LC-HRMS (B)

% Diff%*

1

8655.71

8994.80

-1.9%

2

562.89

604.64

-3.6%

3

135.7

129.846

2.2%

4

26.35

25.45

1.7%

5

14920.96

15000.96

-0.3%

6

1.34

Strategy for the Quantitation of a Protein Conjugate via Hybrid

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