Evaluation of a stabilized RSV pre-fusion F mRNA vaccine: Preclinical studies and Phase 1 clinical testing in healthy adults.

Human respiratory syncytial virus (RSV) causes a substantial proportion of respiratory tract infections worldwide. Although RSV reinfections occur throughout life, older adults, particularly those with underlying comorbidities, are at risk for severe complications from RSV. There is no RSV vaccine available to date, and treatment of RSV in adults is largely supportive. A correlate of protection for RSV has not yet been established, but antibodies targeting the pre-fusion conformation of the RSV F glycoprotein play an important role in RSV neutralization. We previously reported a Phase 1 study of an mRNA-based vaccine (V171) expressing a pre-fusion-stabilized RSV F protein (mDS-Cav1) in healthy adults. Here, we evaluated an mRNA-based vaccine (V172) expressing a further stabilized RSV pre-fusion F protein (mVRC1). mVRC1 is a single chain version of RSV F with interprotomer disulfides in addition to the stabilizing mutations present in the mDS-Cav1 antigen. The immunogenicity of the two mRNA-based vaccines encoding mVRC1 (V172) or a sequence-optimized version of mDS-Cav1 to improve transcriptional fidelity (V171.2) were compared in RSV-naïve and RSV-experienced African green monkeys (AGMs). V172 induced higher neutralizing antibody titers than V171.2 and demonstrated protection in the AGM challenge model. We conducted a Phase 1, randomized, placebo-controlled, clinical trial of 25 µg, 100 µg, 200 µg, or 300 µg of V172 in healthy older adults (60-79 years old; N = 112) and 100 µg, 200 µg, or 300 µg of V172 in healthy younger adults (18-49 years old; N = 48). The primary clinical objectives were to evaluate the safety and tolerability of V172, and the secondary objective was to evaluate RSV serum neutralization titers. The most commonly reported solicited adverse events were injection-site pain, injection-site swelling, headache, and tiredness. V172 was generally well tolerated in older and younger adults and increased serum neutralizing antibody titers, pre-fusion F-specific competing antibody titers, and RSV F-specific T-cell responses.

Investigating Performance of the SLIM-Based High Resolution Ion Mobility Platform for Separation of Isomeric Phosphatidylcholine Species.

Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.

Proteomic Discovery and Validation of Novel Fluid Biomarkers for Improved Patient Selection and Prediction of Clinical Outcomes in Alzheimer's Disease Patient Cohorts.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral ß amyloid (Aß) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers.

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.

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.

Correction: Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

[This corrects the article DOI: 10.1371/journal.pbio.2002457.].

Forward and reverse translational approaches to predict efficacy of neutralizing respiratory syncytial virus (RSV) antibody prophylaxis.

BACKGROUND: Neutralizing mAbs can prevent communicable viral diseases. MK-1654 is a respiratory syncytial virus (RSV) F glycoprotein neutralizing monoclonal antibody (mAb) under development to prevent RSV infection in infants. Development and validation of methods to predict efficacious doses of neutralizing antibodies across patient populations exposed to a time-varying force of infection (i.e., seasonal variation) are necessary. METHODS: Five decades of clinical trial literature were leveraged to build a model-based meta-analysis (MBMA) describing the relationship between RSV serum neutralizing activity (SNA) and clinical endpoints. The MBMA was validated by backward translation to animal challenge experiments and forward translation to predict results of a recent RSV mAb trial. MBMA predictions were evaluated against a human trial of 70 participants who received either placebo or one of four dose-levels of MK-1654 and were challenged with RSV [NCT04086472]. The MBMA was used to perform clinical trial simulations and predict efficacy of MK-1654 in the infant target population. FINDINGS: The MBMA established a quantitative relationship between RSV SNA and clinical endpoints. This relationship was quantitatively consistent with animal model challenge experiments and results of a recently published clinical trial. Additionally, SNA elicited by increasing doses of MK-1654 in humans reduced RSV symptomatic infection rates with a quantitative relationship that approximated the MBMA. The MBMA indicated a high probability that a single dose of ≥ 75 mg of MK-1654 will result in prophylactic efficacy (> 75% for 5 months) in infants. INTERPRETATION: An MBMA approach can predict efficacy of neutralizing antibodies against RSV and potentially other respiratory pathogens.

Longitudinal CSF proteomics identifies NPTX2 as a prognostic biomarker of Alzheimer's disease.

INTRODUCTION: Biomarkers that reflect pathologic processes affecting neuronal function during preclinical and early stages of Alzheimer's disease (AD) are needed to aid drug development. METHODS: A targeted, stable isotope, quantitative mass spectrometry-based investigation of longitudinal changes in concentrations of previously identified candidate biomarkers was performed in cerebrospinal fluid (CSF) of Alzheimer's Disease Neuroimaging Initiative participants who were classified as cognitively normal (CN; n = 76) or with mild cognitive impairment (MCI; n = 111) at baseline. RESULTS: Of the candidate biomarkers, the CSF concentration of neuronal pentraxin 2 (NPTX2), a protein involved in synaptic function, exhibited rates of change that were significantly different between three comparison groups (i.e., CN vs. MCI participants; AD pathology positive vs. negative defined by phosphorylated tau181/amyloid beta1-42 ratio; and clinical progressors vs. non-progressors). The rate of change of NPTX2 also significantly correlated with declining cognition. DISCUSSION: CSF NPTX2 concentration is a strong prognostic biomarker candidate of accelerated cognitive decline with potential use as a therapeutic target.

The Discovery of Two Novel Classes of 5,5-Bicyclic Nucleoside-Derived PRMT5 Inhibitors for the Treatment of Cancer.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivo activity, and low human dose projections.

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

A phase 1, randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults.

Respiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.

Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology.

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng µL-1 compared to 0.05 ng µL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.

Mass Spectrometry-Based Biomarkers in Drug Development.

Advances in mass spectrometry, proteomics, protein bioanalytical approaches, and biochemistry have led to a rapid evolution and expansion in the area of mass spectrometry-based biomarker discovery and development. The last decade has also seen significant progress in establishing accepted definitions, guidelines, and criteria for the analytical validation, acceptance and qualification of biomarkers. These advances have coincided with a decreased return on investment for pharmaceutical research and development and an increasing need for better early decision making tools. Empowering development teams with tools to measure a therapeutic interventions impact on disease state and progression, measure target engagement and to confirm predicted pharmacodynamic effects is critical to efficient data-driven decision making. Appropriate implementation of a biomarker or a combination of biomarkers can enhance understanding of a drugs mechanism, facilitate effective translation from the preclinical to clinical space, enable early proof of concept and dose selection, and increases the efficiency of drug development. Here we will provide descriptions of the different classes of biomarkers that have utility in the drug development process as well as review specific, protein-centric, mass spectrometry-based approaches for the discovery of biomarkers and development of targeted assays to measure these markers in a selective and analytically precise manner.

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.

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.

Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.

Automated DBS microsampling, microscale automation and microflow LC-MS for therapeutic protein PK.

AIM: Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS. METHODS & RESULTS: We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold. CONCLUSION: Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.

Development and evaluation of a multiplexed mass spectrometry based assay for measuring candidate peptide biomarkers in Alzheimer's Disease Neuroimaging Initiative (ADNI) CSF.

PURPOSE: We describe the outcome of the Biomarkers Consortium CSF Proteomics Project (where CSF is cerebral spinal fluid), a public-private partnership of government, academia, nonprofit, and industry. The goal of this study was to evaluate a multiplexed MS-based approach for the qualification of candidate Alzheimer's disease (AD) biomarkers using CSF samples from the AD Neuroimaging Initiative. EXPERIMENTAL DESIGN: Reproducibility of sample processing, analytic variability, and ability to detect a variety of analytes of interest were thoroughly investigated. Multiple approaches to statistical analyses assessed whether panel analytes were associated with baseline pathology (mild cognitive impairment (MCI), AD) versus healthy controls or associated with progression for MCI patients, and included (i) univariate association analyses, (ii) univariate prediction models, (iii) exploratory multivariate analyses, and (iv) supervised multivariate analysis. RESULTS: A robust targeted MS-based approach for the qualification of candidate AD biomarkers was developed. The results identified several peptides with potential diagnostic or predictive utility, with the most significant differences observed for the following peptides for differentiating (including peptides from hemoglobin A, hemoglobin B, and superoxide dismutase) or predicting (including peptides from neuronal pentraxin-2, neurosecretory protein VGF (VGF), and secretogranin-2) progression versus nonprogression from MCI to AD. CONCLUSIONS AND CLINICAL RELEVANCE: These data provide potential insights into the biology of CSF in AD and MCI progression and provide a novel tool for AD researchers and clinicians working to improve diagnostic accuracy, evaluation of treatment efficacy, and early diagnosis.

Quantitation of human peptides and proteins via MS: review of analytically validated assays.

Since the development of monoclonal antibodies in the 1970s, antibody-based assays have been used for the quantitation of proteins and peptides and, today, they are the most widely used technology in routine laboratory medicine and bioanalysis. However, in the last couple of decades, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) techniques have been adopted in the quantitation of small molecules, and more recently have made significant contributions in the quantitation of proteins and peptides. In this article, we will review clinical MS-based assays for endogenous peptides, proteins, and therapeutic antibodies, for which validated methods exist. We will also cover the measurement of protein turnover and the unique solutions that MS can offer in this field.

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.