High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry.

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered "undruggable". The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC-MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein-ligand adducts. The screening results displayed a high degree of fidelity, with a Z' factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC-MS showcased the AEMS platform's low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

Automated Bioanalytical Workflow for Ligand Binding-Based Pharmacokinetic Assay Development.

The growth of therapeutic monoclonal antibodies (mAbs) continues to accelerate due to their success as treatments for many diseases. As new therapeutics are developed, it is increasingly important to have robust bioanalytical methods to measure the pharmacokinetics (PK) of circulating therapeutic mAbs in serum. Ligand-binding assays such as enzyme-linked immunosorbent assays (ELISAs) with anti-idiotypic antibodies (anti-IDs) targeting the variable regions of the therapeutic antibody are sensitive and specific bioanalytical methods to measure levels of therapeutic antibodies in a biological matrix. However, soluble circulating drug mAb targets can interfere with the anti-IDs binding to the therapeutic mAb, thereby resulting in an underestimation of total drug concentration. Therefore, in addition to a high binding affinity for the mAb, the selection of anti-IDs and the assay format that are not impacted by soluble antigens and have low matrix interference is essential for developing a robust PK assay. Standardized automated approaches to screen and select optimal reagents and assay formats are critical to increase efficiency, quality, and PK assay robustness. However, there does not exist an integrated screening and analysis platform to develop robust PK assays across multiple formats. We have developed an automated workflow and scoring platform with multiple bioanalytical assay parameters that allow for ranking of candidate anti-IDs. A primary automated indirect electrochemiluminescence (ECL) was utilized to shortlist the anti-IDs that were selected for labeling and screening in pairs. A secondary screen using an ECL sandwich assay with labeled-anti-ID pairings was used to test multiple PK assay formats to identify the best anti-ID pairing/PK assay format. We developed an automated assay using fixed plate maps combined with a human-guided graphical user interface-based scoring system and compared it to a data-dependent scoring system using Gaussian mixture models for automated scoring and selection. Our approach allowed for screening of anti-IDs and identification of the most robust PK assay format with significantly reduced time and resources compared with traditional approaches. We believe that such standardized, automated, and integrated platforms that accelerate the development of PK assays will become increasingly important for supporting future human clinical trials.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

Direct Analysis from Phase-Separated Liquid Samples using ADE-OPI-MS: Applicability to High-Throughput Screening for Inhibitors of Diacylglycerol Acyltransferase 2.

The primary goal of high-throughput screening (HTS) is to rapidly survey a broad collection of compounds, numbering from tens of thousands to millions of members, and identify those that modulate the activity of a therapeutic target of interest. For nearly two decades, mass spectrometry has been used as a label-free, direct-detection method for HTS and is widely acknowledged as being less susceptible to interferences than traditional optical techniques. Despite these advantages, the throughput of conventional MS-based platforms like RapidFire or parallel LC-MS, which typically acquire data at speeds of 6-30 s/sample, can still be limiting for large HTS campaigns. To overcome this bottleneck, the field has recently turned to chromatography-free approaches including MALDI-TOF-MS and acoustic droplet ejection-MS, both of which are capable of throughputs of 1 sample/second or faster. In keeping with these advances, we report here on our own characterization of an acoustic droplet ejection, open port interface (ADE-OPI)-MS system as a platform for HTS using the membrane-associated, lipid metabolizing enzyme diacylglycerol acyltransferase 2 (DGAT2) as a model system. We demonstrate for the first time that the platform is capable of ejecting droplets from phase-separated samples, allowing direct coupling of liquid-liquid extraction with OPI-MS analysis. By applying the platform to screen a 6400-member library, we further demonstrate that the ADE-OPI-MS assay is suitable for HTS and also performs comparably to LC-MS, but with an efficiency gain of >20-fold.

Quantitation of a Therapeutic Antibody in Serum Using Intact Sequential Affinity Capture, Trypsin Digestion, and LC-MS/MS.

Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.

Generic automated method for liquid chromatography-multiple reaction monitoring mass spectrometry based monoclonal antibody quantitation for preclinical pharmacokinetic studies.

Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy, and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.

Quantitative LC-MS/MS. 1. Impact of Points across a Peak on the Accuracy and Precision of Peak Area Measurements.

The number of points across a chromatographic peak has long been recognized as a key determinant of the accuracy and precision of the measured peak area. In LC-MS-based quantitation experiments in drug discovery and development, the "rule-of-thumb" has been to use 15 or more points. This "rule" is based on the literature describing chromatographic methods where the goal was to achieve the lowest possible imprecision in the measurements, especially when unknown analytes are being detected. Restricting methods to the requirement of at least 15 points across a peak can be detrimental to the development methods that fully optimize the signal-to-noise ratio for the assay using longer dwell times and/or transition summing. This study aims to show that 7 points across the peak for peaks that are 9 s or less wide provide more than sufficient accuracy and precision for drug quantitation studies. Data from simulated Gaussian curves using a sampling interval of 7 points across the peak gave peak area calculations within 1% of the expected total peak area using the Trapezoidal and Riemann rules and 0.6% for the Simpson rule. Low and high concentration samples (n = 5) were assayed using three different LC methods on three different days on two different instruments (API5000 and API5500). The difference in peak area (%ΔPA) and relative standard deviation of the peak areas (%RSD) was less than ∼5%. No significant difference was observed from the data that were obtained from different sampling intervals, different peak widths, different days, different peak sizes, and different instruments. Three core analytical runs were performed on three different days. In each core run, the lower limit of quantitation (LLOQ, n = 5), low quality control (LQC, n = 5), middle quality control (MQC, n = 5), and high-quality control samples (HQC, n = 5) were processed and run simultaneously with a standard curve. The range of the intra- and interday accuracy and precision for 3 core runs was 98.0-105% and 0.9-3.0% for 7 data points and 97.5-105% and 0.8-4.3% for 17 data points, respectively. No significant difference was observed for the different sampling intervals. The results show a sampling interval of 7 points for peaks up to 9 s wide is sufficient to define a peak accurately and precisely for drug quantitation studies in drug discovery and development.

Ultrahigh-Throughput Intact Protein Analysis with Acoustic Ejection Mass Spectrometry.

The need for high-throughput intact protein analysis has been rising as drug discovery increasingly requires the analysis of large sets of covalent modifiers and protein therapeutics. Liquid chromatography-mass spectrometry (LC-MS) is the primary analytical tool used to date to characterize proteins within the biopharmaceutical industry. However, the speed of LC-MS prevents the analysis of large-scale sample sets (>1000 within a day). Acoustic ejection mass spectrometry (AEMS) has recently been established as an electrospray ionization (ESI)-MS based platform with both fast analytical throughput and high data quality. Since its introduction, this technology has been applied in numerous fields with a primary focus on small-molecule analysis in high-throughput drug discovery and development. Here we explore the application of AEMS to high-throughput intact protein analysis for proteins ranging in molecular weight from 17 to 150 kDa on a prototype high-resolution quadrupole time-of-flight (HR QTOF) based AEMS system. Data quality obtained on this platform is comparable to LC-MS, while the analysis speed is significantly improved to one-second-per-sample. This ultrahigh-throughput intact protein analysis platform has the potential to be used broadly in drug discovery.

Intact Mass Quantitation of Therapeutic Antibodies for Pharmacokinetic Studies Using Immuno-Purification.

The quantitation of therapeutic antibodies by mass spectrometry often utilizes a surrogate peptide approach following enzymatic digestion of the antibody. Although this approach has been widely adopted, it is labor intensive with limited throughput in most instances. In addition, this approach can pose challenges when attempting to infer details such as quantity and modification state of the intact analyte. Recent enhancements in instrumentation and sample preparation have enabled quantitation through mass spectrometry detection of the intact protein circumnavigating many limitations of the surrogate peptide approach. Presented here is a method for quantitative analysis of therapeutic monoclonal antibodies (mAb) at the fully intact level in a complex pharmacokinetic study. This methodology yielded sensitivity down to 0.1µg/mL from 30µL of a biological sample volume to be utilized across multiple preclinical species without the need for pooling.

A Model-Based Approach to Bridging Plasma and Dried Blood Spot Concentration Data for Phase 3 Verubecestat Trials.

In-clinic venous dried blood spot (DBS) pharmacokinetic (PK) sampling was incorporated into two phase 3 studies of verubecestat for Alzheimer's disease (EPOCH [NCT01739348] and APECS [NCT01953601]), as a potential alternative to plasma PK sampling. Initially, plasma and DBS PK samples were collected concurrently to better understand the DBS-plasma verubecestat concentration relationship, with the intention of discontinuing DBS or plasma sampling following interim analysis. Following initial analyses and comparison of results with prespecified selection criteria, plasma PK sampling was discontinued; however, a stability issue resulting in generally lower DBS verubecestat concentrations with longer collection-to-assay times was subsequently discovered (associated with non-compliance in DBS sample handling), prompting reintroduction of plasma sampling. To enable inclusion of DBS data in population PK analyses, a conversion algorithm for calculating plasma-equivalent concentrations (accounting for DBS sample instability) was developed using paired (time-matched) plasma and DBS data from the EPOCH study. Verubecestat population PK models developed from pooled phase 1/1b and EPOCH data using either (1) plasma-only data or (2) plasma and plasma-equivalent concentrations (calculated from non-paired DBS samples) yielded similar results. The algorithm robustness was demonstrated using DBS data from paired samples from the APECS study and comparison between plasma and plasma-equivalent concentrations. The population PK model was updated with APECS data (both plasma and, if no plasma sample available, plasma equivalents). The results demonstrated similar PK in the two phase 3 populations and exposures consistent with expectations from phase 1 data. This case study illustrates challenges with employing new sampling techniques in large, global trials and describes lessons learned.

An Investigation of Instability in Dried Blood Spot Samples for Pharmacokinetic Sampling in Phase 3 Trials of Verubecestat.

In-clinic dried blood spot (DBS) pharmacokinetic (PK) sampling was incorporated into two phase 3 studies of verubecestat for Alzheimer's disease (EPOCH [NCT01739348] and APECS [NCT01953601]), as a potential alternative to plasma PK sampling for improved logistical feasibility and decreased blood volume burden. However, an interim PK analysis revealed verubecestat concentrations in DBS samples declined with time to assay in both trials. An investigation revealed wide variation in implementation practices for DBS sample handling procedures resulting in insufficient desiccation which caused verubecestat instability. High-resolution mass spectrometry evaluations of stressed and aged verubecestat DBS samples revealed the presence of two hydrolysis degradants. To minimize instability, new DBS handling procedures were implemented that provided additional desiccant and minimized the time to analysis. Both verubecestat hydrolysis products were previously discovered and synthesized during active pharmaceutical ingredient stability characterization. A liquid chromatography-mass spectrometry assay to quantitate the dominant verubecestat degradant in DBS samples was developed and validated. The application of this method to stressed and aged verubecestat DBS samples confirmed that degradant concentrations accounted for the observed decreases in the verubecestat concentration. Furthermore, after increasing desiccant amounts, degradant concentrations accounted for approximately 7% of the verubecestat concentration in DBS clinical samples, indicating that issues with sample handling were minimized with new storage and shipping conditions. This case study illustrates the challenges with employing new sampling techniques in large, global trials, and the importance of anticipating and mitigating implementation risks.

Volumetric absorptive microsampling (VAMS®) in therapeutic protein quantification by LC-MS/MS: Investigation of anticoagulant impact on assay performance and recommendations for best practices in method development.

Microsampling techniques have been employed as an alternative to traditional serum/plasma sampling because of their inherently proven and desirable advantages across the pharmaceutical industry. These include reduced animal usage in pre-clinical studies, as well as, permitting the collection of samples that would otherwise be inaccessible in clinical studies. The application of volumetric absorptive microsampling (VAMS®) technology, a second-generation dried microsampling method, coupled with LC-MS, has been extensively explored for small molecule drugs at various drug development stages. However, the potential of using VAMS technology and LC-MS analysis for biological therapeutic development has yet to be well-established. In this work, we describe the method development, validation, and a proof-of-concept non-human primate study of a LC-MS/MS method for VAMS utilized to obtain pharmacokinetic (PK) data for a therapeutic monoclonal antibody. A good correlation between VAMS data and data from conventional serum samples was established in rhesus monkeys and indicated the possibility of using of this novel sampling technology in clinical studies. However, during the initial clinical study, a significant difference in internal standard (IS) response between the patient fingerstick samples and the standard/QC samples was observed, which posed a question on the accuracy of the clinical results. A comprehensive investigation confirmed that the EDTA anticoagulant used in the standard/QC samples was the root cause of the observed anomalous IS responses. Special considerations and corresponding best practices during method development and validation are proposed to ensure early detection of potential issues and appropriate implementation of VAMS technology in clinical studies in the future.

Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective.

Recent advancements in immunocapture methods and mass spectrometer technology have enabled intact protein mass spectrometry to be applied for the characterization of antibodies and other large biotherapeutics from in-life studies. Protein molecules have not been traditionally studied by intact mass or screened for catabolites in the same manner as small molecules, but the landscape has changed. Researchers have presented methods that can be applied to the drug discovery and development stages, and others are exploring the possibilities of the new approaches. However, a wide variety of options for assay development exists without clear recommendation on best practice, and data processing workflows may have limitations depending on the vendor. In this perspective, we share experiences and recommendations for current and future application of mass spectrometry for biotherapeutic molecule monitoring from preclinical and clinical studies.

Digitally Enabled, Patient-Centric Clinical Trials: Shifting the Drug Development Paradigm.

The rapidly advancing field of digital health technologies provides a great opportunity to radically transform the way clinical trials are conducted and to shift the clinical trial paradigm from a site-centric to a patient-centric model. Merck's (Kenilworth, NJ) digitally enabled clinical trial initiative is focused on introduction of digital technologies into the clinical trial paradigm to reduce patient burden, improve drug adherence, provide a means of more closely engaging with the patient, and enable higher quality, faster, and more frequent data collection. This paper will describe the following four key areas of focus from Merck's digitally enabled clinical trials initiative, along with corresponding enabling technologies: (i) use of technologies that can monitor and improve drug adherence (smart dosing), (ii) collection of pharmacokinetic (PK), pharmacodynamic (PD), and biomarker samples in an outpatient setting (patient-centric sampling), (iii) use of digital devices to collect and measure physiological and behavioral data (digital biomarkers), and (iv) use of data platforms that integrate digital data streams, visualize data in real-time, and provide a means of greater patient engagement during the trial (digital platform). Furthermore, this paper will discuss the synergistic power in implementation of these approaches jointly within a trial to enable better understanding of adherence, safety, efficacy, PK, PD, and corresponding exposure-response relationships of investigational therapies as well as reduced patient burden for clinical trial participation. Obstacle and challenges to adoption and full realization of the vision of patient-centric, digitally enabled trials will also be discussed.

The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor.

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.

IQ consortium perspective: complementary LBA and LC-MS in protein therapeutics bioanalysis and biotransformation assessment.

Increasingly diverse large molecule modalities have driven the need for complex bioanalysis and biotransformation assessment involving both traditional ligand-binding assays (LBA) and more recent hybrid immunoaffinity LC-MS platforms. Given the scientific expertise in LBA and LC-MS typically resides in different functions within the industry, this has presented operational challenges for an integrated approach for bioanalysis and biotransformation assessment. Encouragingly, over time, the industry has recognized the complementary value of the two platforms. This has not been an easy transition as organizational structures vary widely within the industry. However, there are tremendous benefits in adopting fully integrated strategies for biopharma. This IQ consortium paper presents current perspectives across the biopharma industry. It highlights the technical and operational challenges in current large molecule bioanalysis, the value of collaborations across LBA and LC-MS, and scientific expertise for fully integrated strategies for bioanalysis and biotransformation.

Development of an automated and High throughput UHPLC/MS based workflow for cleaning verification of potent compounds in the pharmaceutical manufacturing environment.

Cleaning verification (CV) is a critical step in the pharmaceutical manufacturing process to eliminate or reduce unacceptable contamination of a product as a result of insufficiently cleaned equipment surfaces. The main concern is cross contamination with active pharmaceutical ingredients (APIs) from previous runs that may impact patient safety. Current conventional approaches involve rather tedious sample preparation and analytical methods with relative lengthy analysis time. Potent APIs possessing low acceptable daily intake (ADI) values require analytical methods for CV with very low detection limits to confirm that these APIs are below their acceptance limits prior to the next manufacturing process. In this work, a novel end to end CV workflow was developed, which includes the automated sample and calibration solution preparation as well as high throughput analysis by ultra-high-performance liquid chromatography (UHPLC) coupled with single quadrupole mass spectrometry in multiple injection chromatography and selected ion monitoring mode (MIC-MS-SIM). The method was validated using ten model compounds. Acceptable specificity, linearity (R2 > 0.997) and single digit ng/mL LOQ and LOD were achieved for all model compounds. This approach was also successfully applied to the analysis of 22 internal CV samples from an internal program.

High-sensitivity nanoLC-MS/MS analysis of urinary desmosine and isodesmosine.

Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium-based cross-linker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D(4)-DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 microL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 +/- 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 +/- 3.1 ng/mg crea), (c) healthy smokers (13.2 +/- 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 +/- 2.9 ng/mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.

Direct quantitation of therapeutic antibodies for pharmacokinetic studies using immuno-purification and intact mass analysis.

Aim: The quantitation of therapeutic antibodies by MS often utilizes a surrogate peptide approach. Recent enhancements in instrumentation and sample preparation have enabled quantitation by detection of the intact molecule using MS. Methods & Results: A comparison of three methods for quantitative analysis of therapeutic monoclonal antibodies including analysis after deglycosylation, after hinge digestion and at the fully intact antibody level is reported. The optimized methodology provided sensitivity down to 0.1 µg/ml and a lower limit of quantitation of 0.5 ug/ml from a 30 µl sample volume. Conclusion: Application of this approach to a pharmacokinetic study compared with a conventional surrogate peptide and a ligand-binding assays provided consistent data with direct detection of the dosed molecule.

Leveraging Digital Health Technologies and Outpatient Sampling in Clinical Drug Development: A Phase I Exploratory Study.

Merck & Co, Inc (Kenilworth, NJ) is investing in approaches to enrich clinical trial data and augment decision making through use of digital health technologies, outpatient sampling, and real-time data access. As part of this strategy, a phase I study was conducted to explore a few technologies of interest. In this fixed-sequence two-period trial, 16 healthy subjects were administered 50-mg once-daily sitagliptin packaged in a bottle that electronically captured the date and time study medication was dispensed (period 1) and in a traditional pharmacy bottle (period 2). Dried blood spot samples were collected for sitagliptin concentration analysis on select study days, both in clinic and at home, with collection time recorded using an electronic diary in period 1 and by clinic staff in period 2. Study results demonstrated the feasibility and subject acceptance of collecting digital adherence data and outpatient dried blood spot samples in clinical trials and highlighted areas for future improvements.