Antipeptide Immunocapture with In-Sample Calibration Curve Strategy for Sensitive and Robust LC-MS/MS Bioanalysis of Clinical Protein Biomarkers in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Despite huge promises, bioanalysis of protein biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for clinical applications is still very challenging. Here, we describe a sensitive and robust LC-MS/MS assay to quantify clinical protein biomarkers in FFPE tumor sections using automated antipeptide antibody immunocapture followed by in-sample calibration curve (ISCC) strategy with multiple isotopologue reaction monitoring (MIRM) technique. ISCC approach with MIRM of stable isotopically labeled (SIL) peptides eliminated the need for authentic matrices for external calibration curves, overcame the matrix effects, and validated the quantification range in each individual sample. Specifically, after deparaffinization, rehydration, antigen retrieval, and homogenization, the protein analytes in FFPE tumor tissues were spiked with a known concentration of one SIL peptide for each analyte, followed by trypsin digestion and antipeptide immunocapture enrichment prior to MIRM-ISCC-based LC-MS/MS analysis. This approach has been successfully used for sensitive quantification of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in 15 representative FFPE tumor samples from lung, colorectal, and head and neck cancer patients. Except for one sample, PD-L1 and PD-1 in all samples were quantifiable using this assay with concentrations of 27.85-798.43 (amol/µg protein) for PD-L1 and 16.96-129.89 (amol/µg protein) for PD-1. These results were generally in agreement with the immunohistochemistry (IHC) data but with some exceptions. This approach demonstrated the feasibility to quantify low abundant protein biomarkers in FFPE tissues with improved sensitivity, specificity, and robustness and showed great potential as an orthogonal analytical approach to IHC for clinical applications.

In-Sample Calibration Curve Using Multiple Isotopologue Reaction Monitoring of a Stable Isotopically Labeled Analyte for Instant LC-MS/MS Bioanalysis and Quantitative Proteomics.

A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM-ISCC-LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid-liquid extraction. The potential applications of the MIRM-ISCC-LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM-ISCC-LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.

Eliminating Preparation of Multisample External Calibration Curves and Dilution of Study Samples Using the Multiple Isotopologue Reaction Monitoring (MIRM) Technique in Quantitative LC-MS/MS Bioanalysis.

Preparation of multisample external calibration curves and dilution of study samples are critical steps in bioanalytical sample processing for quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics, and biomarkers, but they can be time-consuming and prone to error. It is highly desired to simplify or eliminate these two steps in order to improve the assay throughput and robustness. While multisample external calibration curve preparation using authentic matrices can be eliminated with a previously reported in-sample calibration curve (ISCC) approach using multiple isotopologue reaction monitoring (MIRM) of a stable isotopically labeled (SIL) analyte, dilution of study samples is still inevitable due to limited LC-MS/MS assay ranges. In this work, a one-sample multipoint external calibration curve and isotope sample dilution, both using MIRM of an analyte, for quantitative LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking a known amount of an analyte into one blank authentic matrix sample, a one-sample multipoint external calibration curve in an authentic matrix can be established on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the MS/MS responses in the corresponding MIRM channels. This one-sample multipoint external calibration curve can be used in the same way as the traditional multisample external calibration curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance in each MIRM channel can be calculated and measured accurately, isotope sample dilution can be achieved by simply monitoring one or a few of the MIRM channels of the analyte in addition to the most abundant MIRM channel for study samples. While the most abundant MIRM channel (isotopic abundance of 100%) is used for the quantitation of samples having concentrations within the assay calibration curve range, less abundant MIRM channels (isotopic abundance of IA%) can be used for the quantitation of samples having concentrations beyond the assay upper limit of quantitation (ULOQ), resulting in isotope dilution factors (IDF) of 100%/IA%. The approaches of one-sample multipoint external calibration curve and isotope sample dilution were evaluated and demonstrated in this work with an example of the quantitation of daclatasvir in human plasma extracted with liquid-liquid extraction. Using these approaches together with the MIRM-ISCC methodology, accurate and reliable LC-MS/MS bioanalysis can be achieved without the need of preparation of multisample external calibration curve and dilution of study samples.

Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance.

A simple procedure for selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays is reported. The correct weighting factor is determined by the relationship between the standard deviation of instrument responses (σ) and the concentrations (x). The weighting factor of 1, 1/x, or 1/x(2) should be selected if, over the entire concentration range, σ is a constant, σ(2) is proportional to x, or σ is proportional to x, respectively. For the first time, we demonstrated with detailed scientific reasoning, solid historical data, and convincing justification that 1/x(2) should always be used as the weighting factor for all bioanalytical LC-MS/MS assays. The impacts of using incorrect weighting factors on curve stability, data quality, and assay performance were thoroughly investigated. It was found that the most stable curve could be obtained when the correct weighting factor was used, whereas other curves using incorrect weighting factors were unstable. It was also found that there was a very insignificant impact on the concentrations reported with calibration curves using incorrect weighting factors as the concentrations were always reported with the passing curves which actually overlapped with or were very close to the curves using the correct weighting factor. However, the use of incorrect weighting factors did impact the assay performance significantly. Finally, the difference between the weighting factors of 1/x(2) and 1/y(2) was discussed. All of the findings can be generalized and applied into other quantitative analysis techniques using calibration curves with weighted least-squares regression algorithm.

A contrast in safety, pharmacokinetics and pharmacodynamics across age groups after a single 50 mg oral dose of the γ-secretase inhibitor avagacestat.

AIM: To evaluate the single dose pharmacokinetics, pharmacodynamics, and preliminary tolerability of the γ-secretase inhibitor BMS-708163 (avagacestat) in young and elderly men and women. METHODS: All subjects received double-blinded administration of a single 50 mg dose of avagacestat in capsule form or matching placebo. Main evaluations included pharmacokinetics, safety, plasma amyloid-ß (Aß)(1-40) concentratios and exploration of Notch biomarkers. RESULTS: Avagacestat 50 mg capsule was well tolerated and rapidly absorbed among young and elderly subjects, with a median t(max) between 1 and 2 h post dose and an average half-life between 41 and 71 h. In general, subjects aged 75 years or more had higher AUC(0,∞) values than those aged less than 75 years. An exploratory analysis of Aß(1-40) serum concentrations showed a pattern of decreasing concentrations over the first 4-6 h followed by a rise above baseline that was maintained until the end of the assessment period. Adverse events were generally mild, occurring more frequently in elderly subjects, with no observed difference between subjects receiving avagacestat and placebo. No dose limiting gastrointestinal effects of avagacestat were observed and exploratory biomarkers of Notch inhibition did not change significantly. CONCLUSIONS: The favourable safety profile and pharmacokinetic effects of avagacestat in this study support its continued development, especially in the target population of elderly subjects with mild cognitive impairment or Alzheimer's disease.

Practical and efficient strategy for evaluating oral absolute bioavailability with an intravenous microdose of a stable isotopically-labeled drug using a selected reaction monitoring mass spectrometry assay.

A strategy of using selected reaction monitoring (SRM) mass spectrometry for evaluating oral absolute bioavailability with concurrent intravenous (i.v.) microdosing a stable isotopically labeled (SIL) drug was developed and validated. First, the isotopic contribution to SRM (ICSRM) of the proposed SIL drug and SIL internal standard (IS) was theoretically calculated to guide their chemical synthesis. Second, the lack of an isotope effect on drug exposure was evaluated in a monkey study by i.v. dosing a mixture of the SIL and the unlabeled drugs. Third, after the SIL drug (100 µg) was concurrently i.v. dosed to humans, at T(max) of an oral therapeutic dose of the unlabeled drug, both drugs in plasma specimens were simultaneously quantified by a sensitive and accurate SRM assay. This strategy significantly improves bioanalytical data quality and saves time, costs, and resources by avoiding a traditional absolute bioavailability study or the newer approach of microdoses of a radio-microtracer measured by accelerator mass spectrometry.

Calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry assays and its application in supporting microdose absolute bioavailability studies.

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

Development of an intrabiliary MR imaging-monitored local agent delivery technique: a feasibility study in pigs.

PURPOSE: To investigate the feasibility of using magnetic resonance (MR) imaging to monitor intrabiliary delivery of motexafin gadolinium (MGd) into pig common bile duct (CBD) walls. MATERIALS AND METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. Initially, human cholangiocarcinoma cells were treated with various concentrations of MGd, a compound serving as a T1-weighted MR imaging contrast agent, chemotherapy drug, and cell marker. These cells were then examined by means of confocal microscopy to confirm the intracellular uptake of MGd. In addition, an MGd/trypan blue mixture was locally infused into CBD walls of six cadaveric pigs using a microporous balloon catheter. CBDs of six pigs were infused with saline to serve as controls. Ex vivo T1-weighted MR imaging of these CBDs was performed. For in vivo technical validation, the microporous balloon catheter was placed in the CBD by means of a transcholecytic access to deliver MGd/trypan blue into CBD walls of six living pigs. T1-weighted images were obtained with both a surface coil and an intrabiliary MR imaging guidewire, and contrast-to-noise ratios of CBD walls before and after MGd/trypan blue infusions were compared in the two groups by means of paired t test, with subsequent histologic analysis to confirm the penetration and distribution of the MGd/trypan blue agent into CBD walls. RESULTS: In vitro experiments confirmed uptake of MGd by human cholangiocarcinoma cells. The ex vivo experiments demonstrated the penetration of MGd/trypan blue into the CBD walls. The in vivo experiment confirmed the uptake of MGd/trypan blue, showing an increased contrast-to-noise ratio for the CBD after administration of the mixture, compared with images obtained prior to MGd/trypan blue administration (11.6 ± 4.2 [standard deviation] vs 5.7 ± 2.8; P = .04). Histologic results depicted the blue dye stains and red fluorescence of MGd in CBD walls, confirming the imaging findings. CONCLUSION: It is feasible to use MR imaging to monitor the penetration of locally delivered MGd into pig CBD walls.

Biomarker Assay Validation by Mass Spectrometry.

Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.

MGAT2 inhibitor decreases liver fibrosis and inflammation in murine NASH models and reduces body weight in human adults with obesity.

Monoacylglycerol acyltransferase 2 (MGAT2) is an important enzyme highly expressed in the human small intestine and liver for the regulation of triglyceride absorption and homeostasis. We report that treatment with BMS-963272, a potent and selective MGAT2 inhibitor, decreased inflammation and fibrosis in CDAHFD and STAM, two murine nonalcoholic steatohepatitis (NASH) models. In high-fat-diet-treated cynomolgus monkeys, in contrast to a selective diacylglycerol acyltransferase 1 (DGAT1) inhibitor, BMS-963272 did not cause diarrhea. In a Phase 1 multiple-dose trial of healthy human adults with obesity (NCT04116632), BMS-963272 was safe and well tolerated with no treatment discontinuations due to adverse events. Consistent with the findings in rodent models, BMS-963272 elevated plasma long-chain dicarboxylic acid, indicating robust pharmacodynamic biomarker modulation; increased gut hormones GLP-1 and PYY; and decreased body weight in human subjects. These data suggest MGAT2 inhibition is a promising therapeutic opportunity for NASH, a disease with high unmet medical needs.

Ultrasound guidance for the creation of a small animal model of aortic injury.

PURPOSE: To develop a small animal model of controlled aortic intimal injury with ultrasound (US) imaging guidance. MATERIALS AND METHODS: Via carotid artery cutdown, a custom-made microcatheter/angled metal device system was advanced to damage the intima of the ascending aortas of 20 Sprague-Dawley rats and 10 JCR atherosclerotic rats. This minimally invasive endovascular procedure was monitored by a clinical US imaging system. Injured aortas were harvested for histologic confirmation via a grading system: grade I, intima injury; grade II, injury to media; and grade III, injury through the entire aortic wall. Neointimal reactions at the injury site were compared by calculating the ratio of intimal to medial thickness among different animal groups at various survival times (week 1, weeks 2-3, and weeks 4-7). RESULTS: Clear visualization of the architecture of the heart and great vessels and the exact location of the angled metal device by US imaging ensured consistent intimal damage of the aorta. Histopathologic analysis confirmed that most of the aortic injures were classified as grade I. There was no significant difference between the two rat groups. Analysis of pathophysiologic reactions at the injury sites revealed increased thickening of neointimal hyperplasia as animal survival times extended from week 1 to weeks 4-7 after the aortic interventions. CONCLUSIONS: This study demonstrates the feasibility of clinical US imaging to precisely guide the creation of controlled aortic intimal injury in rats, which may become a useful tool to facilitate research involving the prevention and treatment of atherosclerotic cardiovascular disease.

Effect of mobile phase pH, aqueous-organic ratio, and buffer concentration on electrospray ionization tandem mass spectrometric fragmentation patterns: implications in liquid chromatography/tandem mass spectrometric bioanalysis.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on selected reaction monitoring (SRM) is the standard methodology in quantitative analysis of administered xenobiotics in biological samples. Utilizing two SRM channels during positive electrospray ionization (ESI) LC/MS/MS method development for a drug compound containing two basic functional groups, we found that the response ratio (SRM1/SRM2) obtained using an acidic mobile phase was dramatically different from that obtained using a basic mobile phase. This observation is different from the well-established phenomenon of mobile phase affecting the [M+H](+) response, which is directly related to the amount of the [M+H](+) ions produced during the ionization. Results from follow-up work reported herein revealed that the MS/MS fragmentation patterns of four drug or drug-like compounds are affected not only by the pH, but also by the aqueous-organic ratio of the mobile phase and the buffer concentration at a given apparent pH. The observed phenomenon can be explained by invoking that a mixture of [M+H](+) ions of the same m/z value for the analyte is produced that is composed of two or more species which differ only in the site of the proton attachment, which in turn affects their MS/MS fragmentation pattern. The ratio of the different protonated species changes depending on the pH, aqueous-organic ratio, or ionic strength of the mobile phase used. The awareness of the mobile phase dependency of the MS/MS fragmentation pattern of precursor ions of identical m/z value will influence LC/MS/MS-based bioanalytical method development strategies. Specifically, we are recommending that multiple SRM transitions be monitored during mobile phase screening, with the MS/MS parameters used for each SRM optimized for the composition of the mobile phase (pH, organic percentage, and ionic strength) in which the analyte elutes.

Application of in-sample calibration curve methodology for regulated bioanalysis: Critical considerations in method development, validation and sample analysis.

Traditionally, for a liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC-MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification. This methodology has great potential in many applications, for example biomarker measurement, quantitative proteomics and clinical diagnosis. Here, we assessed the feasibility of applying this ISCC-LC-MS/MS methodology in regulated bioanalysis using BMS-984478, a drug candidate, as the model compound. We also proposed method validation procedures/processes for this new approach for industry peers' consideration and feedback. A LC-MS/MS method using the ISCC strategy was successfully developed and validated for the quantitative analysis of BMS-984478 in human plasma over the range of 1.33-993.42 ng/mL. The validated ISCC-LC-MS/MS method was compared with a previously validated method using the conventional external calibration curve approach, and the two methods showed equivalent performance. Critical considerations and practical approaches in method development, validation and sample analysis were also discussed. Our work demonstrated that the ISCC-LC-MS/MS methodology is a promising approach for regulated LC-MS/MS bioanalysis. ISCC-LC-MS/MS methodology has its unique advantages and has great potential to be widely applied for various quantitative applications, and may even change the landscape of quantitative analysis.

Fit-for-purpose protein biomarker assay validation strategies using hybrid immunocapture-liquid chromatography-tandem-mass spectrometry platform: Quantitative analysis of total soluble cluster of differentiation 73.

In recent years, biomarkers have played more extensive roles as indicators of disease progression, safety, and drug efficacy. Targeted quantitative analysis of biomarkers including drug targets have become increasingly important to drive critical decision-making in various drug development stages, as well as to improve the success rates of clinical trials. There are many analytical challenges when developing and validating the bioanalytical methods associated with the measurement of an endogenous protein biomarker, especially when using LC-MS based analysis. Moreover, the current regulatory guidelines for assay development and validation using LC-MS platform mainly focuse on regulated bioanalysis for therapeutic drugs. In this manuscript, we use total soluble CD73 (sCD73) as an example to present a "fit-for-purpose" assay using a hybrid immunocapture-LC-MS/MS assay platform. A non-competing antibody (to the therapeutic drug) was used to isolate and enrich the total sCD73 from biological matrix. The enriched sample was digested after immunocapture and a surrogate peptide was monitored for quantification. The assay showed good accuracy, precision, specificity and sensitivity with the LLOQ of 1.00 ng/mL, and was applied in a clinical study to measure the total sCD73 as a potential pharmacodynamic (PD) marker. Some recommendations and considerations for "fit-for-purpose" validation of this assay, and hybrid LC-MS assays in general, for the quantitative analysis of an endogenous protein biomarkers is also discussed.

Modeling and Simulation of the Pharmacokinetics and Target Engagement of an Antagonist Monoclonal Antibody to Interferon-γ-Induced Protein 10, BMS-986184, in Healthy Participants to Guide Therapeutic Dosing.

BMS-986184 is a human, second-generation, anti-interferon-γ-induced protein 10 (IP-10) monoclonal antibody. In this study the pharmacokinetics and target engagement (TE) of BMS-986184 in healthy participants were characterized using population-based target-mediated drug disposition (TMDD) modeling and data from a first-in-human study (NCT02864264). The results of the first-in-human study and the model generated were used to conduct stochastic simulations of a virtual population of healthy participants to predict pharmacokinetic exposures and TE responses for different dosage regimens. A 2-compartment, 2-target, TMDD structural model, assuming quasi-steady-state and stimulated production on treatment, was developed by simultaneous fitting of the total drug, serum-free IP-10, and serum total IP-10 concentration data, with the second unobservable target contribution to drug elimination described by the Michaelis-Menten elimination term. Model evaluation confirmed agreement between model predictions and observed data. Simulation of a virtual population of healthy individuals demonstrated that steady state was reached at the eighth dosing interval, and that around 150 mg subcutaneously every other week could be a suitable target dosage regimen for future clinical trials. Integrated modeling strategies such as this can be used to help guide rational clinical trial development of drugs with TMDD, leading to improved dose selection and greater patient benefits.

Critical considerations for immunocapture enrichment LC-MS bioanalysis of protein therapeutics and biomarkers.

In recent years, immunocapture enrichment coupled with LC-MS technology has seen more applications for the measurement of low abundant protein therapeutics and biomarkers in biological matrices. In this article, several critical considerations for the application of immunocapture enrichment to LC-MS bioanalysis of protein therapeutics and biomarkers, including reagent selection, reagent characterization, designing of capture format, etc. are discussed. All these considerations are critical in developing reliable and robust bioanalytical assays with high assay specificity and sensitivity. Successful examples using the immunocapture LC-MS approach in the quantification of biotherapeutic and low abundant protein biomarkers will also be discussed.

Immunoaffinity LC-MS/MS for quantitative determination of a free and total protein target as a target engagement biomarker.

AIM: IP-10 is a protein target for the treatment of Crohn's disease. Inhibition of IP-10 by anti-IP-10 mAbs neutralizes its various biological activities. The measurement of free IP-10 suppression as a target engagement biomarker is required for the assessment of drug effect on the target. RESULTS: The development of highly sensitive immunoaffinity-LC-MS/MS assays for quantifying free and total IP-10 in cynomolgus monkey serum is reported for the first time. This paper details strategies for maximizing assay sensitivity by selecting digestion routes, and optimizing immunocapture to achieve full recovery and minimal matrix effect. For the free IP-10 assay, bioanalytical strategies have been established to minimize drug/ligand dissociation. CONCLUSION: The assays have been implemented for target engagement measurement, pharmacokinetic-pharmacodynamic correlation, and human dose projections.

Role of regional absorption and gastrointestinal motility on variability in oral absorption of a model drug.

Variability in oral absorption in pre-clinical species makes human dose projection challenging. In this study, we investigated the mechanistic basis of variability in oral absorption of a model hydrophobic compound with pH-dependent solubility, BMS-955829, after oral dosing in rats, dogs, and cynomolgus monkeys. The contribution of regional absorption to pharmacokinetic variability was assessed in ported monkeys by direct intraduodenal and intraileal administration. The effect of BMS-955829 on gastric emptying and intestinal motility was investigated by radiography after co-administration of barium. BMS-955829 exhibited species dependent oral bioavailability, with high variability in monkeys. During regional absorption studies, highest rate of drug absorption was observed after direct intraduodenal administration. Radiography studies indicated that BMS-955829 slowed gastric emptying and intestinal motility. The effect of rate and site of drug release on oral exposure was studied using different drug product formulations. Reducing the rate of drug release reduced oral exposure variability without compromising exposure in cynomolgus monkeys. This effect was likely mediated by avoidance of rapid initial absorption and drug effect on gastric emptying and intestinal transit within the biorelevant timeframe. Thus, drug release rate can modulate the effect of physiological factors on variability in the oral absorption of sensitive compounds.

Automated Tecan programming for bioanalytical sample preparation with EZTecan.

This paper reports the first successful automated Tecan (Tecan US, Research Triangle Park, NC) programming for bioanalytical sample analysis with an in-house developed software, called EZTecan. Based on the sample run list in the Watson Laboratory Information Management System (InnaPhase Corp., Philadelphia, PA), EZTecan can generate a Tecan program for bioanalytical sample preparation, including sample transfer, sample dilution, extraction, and reconstitution. Many built-in functions for EZTecan ensure the flexibility, quality, and efficiency of the liquid handling. It takes about 90 min to prepare two 96-well plates from sample transfer to reconstitution. Analytical results shown good inter- and intra-assay accuracy and precision, which meet the +/-15% Good Laboratory Performance acceptance criteria. With EZTecan, bioanalysts can prepare samples without writing a single line of Tecan program.