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Bacille Calmette-Guerin (BCG) is a potential tool in the control of Mycobacterium bovis in European badgers (Meles meles). A five year Test and Vaccinate or Remove (TVR) research intervention project commenced in 2014 using two BCG strains (BCG Copenhagen 1331 (Years 1-3/ BadgerBCG) and BCG Sofia SL2222 (Years 4-5). Badgers were recaptured around 9 weeks after the Year 5 vaccination and then again a year later. The Dual-Path Platform (DPP) Vet TB assay was used to detect serological evidence of M. bovis infection. Of the 48 badgers, 47 had increased Line 1 readings (MPB83 antigen) between the Year 5 vaccination and subsequent recapture. The number of BCG Sofia vaccinations influenced whether a badger tested positive to the recapture DPP VetTB assay Line 1 (p 0.001) while the number of BadgerBCG vaccinations did not significantly affect recapture Line 1 results (p = 0.59). Line 1 relative light units (RLU) were more pronounced in tests run with sera than whole blood. The results from an in_house MPB83 ELISA results indicated that the WB DPP VetTB assay may not detect lower MPB83 IgG levels as well as the serum DPP VetTB assay. Changes in interferon gamma assay (IFN-γ) results were seen in 2019 with significantly increased CFP-10 and PPDB readings. Unlike BadgerBCG, BCG Sofia induces an immune response to MPB83 (the immune dominant antigen in M. bovis badger infection) that then affects the use of immunodiagnostic tests. The use of the DPP VetTB assay in recaptured BCG Sofia vaccinated badgers within the same trapping season is precluded and caution should be used in badgers vaccinated with BCG Sofia in previous years. The results suggest that the DPP VetTB assay can be used with confidence in badgers vaccinated with BadgerBCG as a single or repeated doses.

The COVID-19 pandemic has strained the biological matrix supply chain. An upsurge in demand driven by numerous COVID-19 therapeutic and vaccine development programs to combat the pandemic, along with logistical challenges sourcing and transporting matrix, has led to increased lead times for multiple matrices. Biological matrix shortages can potentially cause significant delays in drug development programs across the pharmaceutical and biotechnology industry. Given the current circumstances, discussion is warranted around what will likely be increased use of surrogate matrices in support of pharmacokinetic (PK), immunogenicity, and biomarker assays for regulatory filings. Regulatory authorities permit the use of surrogate matrix in bioanalytical methods in instances where matrix is rare or difficult to obtain, as long as the surrogate is appropriately selected and scientifically justified. Herein, the scientific justification and possible regulatory implications of employing surrogate matrix in PK, immunogenicity, and biomarker assays are discussed. In addition, the unique challenges that cell and gene therapy (CGT) and other innovative therapeutic modalities place on matrix supply chains are outlined. Matrix suppliers and contract research organizations (CROs) are actively implementing mitigation strategies to alleviate the current strain on the matrix supply chain and better prepare the industry for any future unexpected strains. To maintain ethical standards, these mitigation strategies include projecting matrix needs with suppliers at least 6 months in advance and writing or updating study protocols to allow for additional matrix draws from study subjects and/or re-purposing of subject matrix from one drug development program to another.

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.

In this chapter, we describe the importance of bioanalytical methods to drug development. These assays, measuring the active drug and metabolites, provide critical information on the exposure of the drug that is used to correlate with safety and efficacy data to demonstrate the appropriate selection of the dosage strength and frequency. Technologies discussed include those supporting small molecule drugs (chromatographic assays (LC-MS/MS)), biologics (immunoassays), and gene therapies (polymerase chain reaction assays (PCR)). This chapter reviews the predominant technologies and critical features in their use to provide reliable data, the characteristics of the assays that must be demonstrated during validation, and case studies highlighting many of these aspects.

Aim: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7β-hydroxy-DHEA, cortisone and 6β-hydroxycortisone as potential biomarkers to predict CYP3A activity. Results: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7β-hydroxy-DHEA. Conclusion: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug–drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

With the growing focus on translational research and the use of biomarkers to drive drug development and approvals, biomarkers have become a significant area of research within the pharmaceutical industry. However, until the US Food and Drug Administration’s (FDA) 2013 draft guidance on bioanalytical method validation included consideration of biomarker assays using LC-MS and LBA, those assays were created, validated, and used without standards of performance. This lack of expectations resulted in the FDA receiving data from assays of varying quality in support of efficacy and safety claims. The AAPS Crystal City VI (CC VI) Workshop in 2015 was held as the first forum for industry-FDA discussion around the general issues of biomarker measurements (e.g., endogenous levels) and specific technology strengths and weaknesses. The 2-day workshop served to develop a common understanding among the industrial scientific community of the issues around biomarkers, informed the FDA of the current state of the science, and will serve as a basis for further dialogue as experience with biomarkers expands with both groups.

BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid-liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC-MS/MS assay was validated over a concentration range of 0.200-200ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2h on ice. The analytes were also stable in human plasma for 25h at room temperature, 34days at -20°C and -70°C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study.

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO–Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives’ sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Background: The bioanalytical strategy for antibody–drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. Methods: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. Results & conclusion: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure–response relationship.

Herein we report a rapid, accurate and robust UHPLC–MS/MS assay for the quantitation of BMS-911453, a Janus kinase 2 inhibitor under clinical development for the treatment of myeloproliferative disorders, in human plasma. A systematic method development approach was used to optimize the mass spectrometry, chromatography, and sample extraction conditions, and to minimize potential bioanalytical risks. The validated method utilizes stable-isotope labeled 13 C 4 -BMS-911543 as the internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved within 2 min on a Zorbax Extend-C18 column with an isocratic elution. BMS-911543 and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The assay range was from 1 to 500 ng/mL, and the standard curve was fitted with 1/ x 2 weighted linear regression. The intra-assay precision was within 5.0% CV and the inter-assay precision was within 2.6% CV. The inter-assay mean accuracy, expressed as percents of theoretical, was between 99.8% and 102.3%. The assay has high recovery (∼80%) and minimal matrix effect (0.95–1.00). BMS-911543 was stable in human plasma for at least 24 h at room temperature, 90 days at −20 °C, and following three freeze–thaw cycles. The validated method was successfully applied to sample analysis in clinical studies.

Aims: A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. Materials & methods: An average blood volume of 10.6 μl was absorbed by the samplers across the different HCTs investigated (20–65%). Results: No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. Conclusion: The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

Apixaban (Eliquis™) was developed by Bristol-Myers Squibb (BMS) and Pfizer to use as an antithrombotic/anticoagulant agent and has been recently approved for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. A clinical study of apixaban, sponsored by BMS and Pfizer, included a pilot exploratory portion to evaluate the potential for future drug concentration monitoring using dried blood spot (DBS) sample collection. For DBS sample collection, a fixed blood volume was dispensed onto a DBS card by either regular volumetric pipette (venous blood collection) or capillary dispenser (finger prick blood collection). A 96-well semi-automated liquid-liquid extraction sample preparation procedure was developed to provide clean extracts for UHPLC-MS/MS quantitation. Assays using both partial-spot center punch and whole spot punch were developed and validated. The linear dynamic ranges for all the analyses were from 0.5 to 500 ng/mL. The coefficient of determination (r(2)) values was >0.9944 for all the validation runs. For the center punch approach, the intra-assay precision (%CV) was within 4.4% and inter-assay precision was within 2.6%. The assay accuracy, expressed as %Dev., was within ± 5.4% of the nominal concentrations. One accuracy and precision run was performed using the whole spot approach, the intra-assay precision (%CV) was within 7.1% and the accuracy was within ± 8.0% of the nominal concentrations. In contrast to the center punch approach, the whole spot approach eliminated the effect of hematocrit and high lipids on the analysis of apixaban in human DBS when an accurate sample blood volume was collected on DBS cards.

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL with 50 μL of plasma was obtained, which reflects a 5-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for selective reaction monitoring (SRM) detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin's major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to contract research organizations (CROs), validated, and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga or any combination therapy containing dapagliflozin.

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing.

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion. The Workshop was built around four thematic sessions (Common Topics, Chromatographic, Ligand-Binding Assays, and Biomarkers) and a final session with international regulators, concluding with a review of the outcomes and recommendations from the thematic sessions. This Workshop report summarizes the outcomes and includes topics of agreement, those where the FDA will consider the Industry's perspective, and those where the workshop provided a first open dialogue. This article will be available to the bioanalytical community at http://www.aaps.org/BMV13 .

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event – A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies’ Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.

Bioanalytical data submitted to global health authorities in support of drug approval applications is expected to meet certain regulatory standards and requirements. Bioanalytical assay validation and the conduct of study sample analysis is addressed with a multitude of regulatory guidance documents. This chapter provides an overview of the regulations from a historical perspective to current day developments. Originally written around bioanalysis to support pharmacokinetic studies, bioanalysis regulatory language is now being applied to endogenous biomarker assays and evaluating immunogenicity of large molecule therapeutics. Quality management and documentation are also discussed in this chapter along with the process of how modern bioanalysis regulations are continuing to develop.

Background: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum. Results & discussion: Taking advantage of a uniquely acid-labile aspartyl–prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200–50000 ng/ml. The assay accuracy and precision were between 92.8–99.8% and 3.9–14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE. Conclusion: A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.

A sensitive, accurate and rugged UHPLC–MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid–liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100 ng/mL was fitted to a 1/x2 weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (∼90%) and matrix effect was minimal (1.02–1.05). EpoD was stable in stabilized rat plasma for at least 30 h at room temperature, 180 days at −20 °C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.

Aim: To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC–MS/MS assay was developed for the first in-human multiple ascending dose study. Materials & methods: The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC–MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28. Results & conclusion: The fully validated LC–MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.

The last 10 years have witnessed robust debate within the bioanalytical community and regulatory authorities on the topic of metabolite monitoring and safety assessment. Of particular interest to regulated bioanalytical laboratories was the acceptance by the US FDA and other major regulatory bodies of a tiered approach to bioanalytical assay validation. The tiered approach defines a sliding scale of regulatory rigor for the evaluation of significant human metabolites that encompasses a range of assessments from semi-quantitative assays to fully validated assays, all of which can be used in support of regulatory submissions. This article describes the utilization of a tiered approach at Bristol-Myers Squibb and the decision trees guiding the selection of the appropriate level of assay qualification. Case studies illustrate how decisions are made, how different scientific situations influence the assay choice, and what criteria may be set to continue or discontinue metabolite monitoring in later drug development.

RATIONALE Sensitivity is one major challenge limiting the application of liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for bioanalysis of proteins. A novel selective peptide derivatization (SPD) strategy was proposed to improve assay sensitivity. The main concept of the SPD strategy is to selectively derivatize surrogate peptides of the target protein in the digests, while not derivatizing the abundant background peptides, thereby improving the separation of target peptides during sample extraction and chromatography, and increasing the sensitivity. Additional benefits may help improve sensitivity include (1) increased ionization efficiency; (2) improved fragmentation pattern; and (3) increased sample extraction recovery of target peptides. METHODS Feasibility assessment of the SPD strategy was conducted using BMS-986012, a monoclonal antibody, as the model protein, and with malondialdehyde (MDA) to selectively derivatize the arginine-containing surrogate peptide SLIY in tryptic-digested monkey serum samples. RESULTS The decreased polarity and basicity of the MDA-derivatized peptide SLIY (MDA-SLIY) helped improve its separation from the background peptides during solid-phase extraction (SPE) and chromatography. The recovery of MDA-SLIY was 36.1–44.2%, which was ~3-fold higher than the recovery of peptide SLIY (11.9–16.1%). There was no significant ion suppression for MDA-SLIY. Overall, SPD improved the sensitivity ~5-fold. SPD methodology was then successfully applied to the development of a sensitive LC/MS/MS assay for BMS-986012 in monkey serum. CONCLUSIONS This work demonstrates the feasibility of the SPD strategy for sensitivity enhancement. SPD can provide a simple, cost-efficient, and antibody-free sample preparation approach to improve sensitivity. Copyright © 2014 John Wiley & Sons, Ltd.

Two rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of propylparaben, its major metabolite, p-hydroxybenzoic acid (pHBA), and their sulfate conjugates have been developed and validated in citric acid-treated rat plasma. To prevent propylparaben being hydrolyzed to pHBA ex vivo, rat plasma was first treated with citric acid; then collected and processed at a reduced temperature (ice bath). Stable isotope labeled internal standards, d4-propylparaben, (13)C6-pHBA, and the d4-labeled internal standards of their sulfate conjugates were used in the methods. The analytes were extracted from the matrix using protein precipitation, followed by chromatographic separation on a Waters ACQUITY UPLC HSS T3 column. Quantification using negative ion electrospray was performed on a Sciex API 4000 mass spectrometer. The analytical ranges were established from 2.00 to 200 ng/mL for propylparaben, 50.0-5000 ng/mL for pHBA, 50.0-10,000 ng/mL for the sulfate conjugate of propylparaben (SPP) and 200-40,000 ng/mL for the sulfate conjugate of pHBA (SHBA). Inter- and intra-run precision for the quality control samples were less than 5.3% and 4.4% for all analytes; and the overall accuracy was within ±5.7% of the nominal values. The validated bioanalytical methods demonstrated excellent sensitivity, specificity, accuracy and precision and were successfully applied to a rat toxicology study under the regulations of Good Laboratory Practices (GLP). Strategies have been developed and applied toward overcoming the challenges related to analyte stability, and environmental and endogenous background.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These ‘hot’ topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.

Background: Increasing assay sensitivity is critical for promoting the application of LC–MS/MS quantitative bioanalysis of therapeutic proteins. A sample processing method that can selectively remove the abundant background peptides in the serum tryptic digests and retain the target peptides can greatly improve the assay sensitivity. Results: Mixed-mode strong-cation exchange SPE was systematically investigated as an orthogonal sample separation technique to reversed-phase UHPLC for the analysis of a test monoclonal antibody, BMS-986012, in monkey serum after pellet digestion. Strong cation exchange SPE efficiently removed most of the background peptides and reduced the matrix effect and background level in the monitored mass transition channels. As a result, improved sensitivity was observed for the surrogate peptides VVSV and SLIY. Conclusion: This orthogonal approach provides a simple and easy-to-develop sample preparation method that can selectively remove most background peptides and extract the target peptides, therefore, improving the LC–MS/MS assay sensitivity.

An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 × 100 mm, 1.7 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. Four peptides, a unique peptide for each mAb and two confirmatory peptides from different antibody domains, were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. The LC-MS/MS assay showed lower limit of quantitation (LLOQ) at 5 μg/mL for mAb-A and 25 μg/mL for mAb-B. The intra- and interassay precision (%CV) was within 10.0% and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4% for all the peptides. Other validation parameters, including sensitivity, selectivity, dilution linearity, processing recovery and matrix effect, autosampler carryover, run size, stability, and data reproducibility, were all evaluated. The confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. The robustness of the LC-MS/MS assay and the data agreement with the ligand binding data demonstrated that LC-MS/MS is a reliable and complementary approach for the quantitation of coadministered antibody drugs.

In recent years, there has been increasing interest in using LC-MS/MS to measure protein drugs (biotherapeutics) in plasma/serum to support pharmacokinetic/toxicokinetic (PK/TK) studies. A number of strategies have been proposed to quantify different types of protein drug candidates in biological matrices. However, the application of LC-MS/MS in biotherapeutics is still very limited in regulated bioanalysis due to practical challenges. In this manuscript, we propose a "data-dependent" approach for the LC-MS/MS support of protein drug candidates, focusing on operational aspects. We have developed and validated a fast, simple and reliable LC-MS/MS method to quantify a protein drug candidate in mouse serum. This method can fit into our current workflow for routine small molecule LC-MS/MS assays with an overall sample preparation time of less than two hours. The method has been demonstrated to be accurate, precise and rugged, and it has been successfully applied to analyze samples from toxicology studies. The results were verified using a ligand binding assay and tested by standard-addition sample reanalysis. In addition, general recommendations for developing and validating an LC-MS/MS assay based on surrogate peptide(s) of protein drug candidates are also proposed for regulated bioanalysis.

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC–MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D 9 -asunaprevir as the internal standard. The samples were extracted by liquid–liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000 ng/mL for asunaprevir, was fitted to a 1/ x 2 weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species.

Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC-MS/MS analysis. Presented here are three LC-MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.

Aim To determine the absolute oral bioavailability (Fp.o.) of saxagliptin and dapagliflozin using simultaneous intravenous 14C‐microdose/therapeutic oral dosing (i.v.micro + oraltherap).

The paradigm shift of globalized research and conducting clinical studies at different geographic locations worldwide to access broader patient populations has resulted in increased need of correlating bioanalytical results generated in multiple laboratories, often across national borders. Cross-validations of bioanalytical methods are often implemented to assure the equivalency of the bioanalytical results is demonstrated. Regulatory agencies, such as the US FDA and European Medicines Agency, have included the requirement of cross-validations in their respective bioanalytical validation guidance and guidelines. While those documents provide high-level expectations, the detailed implementation is at the discretion of each individual organization. At Bristol-Myers Squibb, we practice a fit-for-purpose approach for conducting cross-validations for small-molecule bioanalytical methods using LC–MS/MS. A step-by-step proposal on the overall strategy, procedures and technical details for conducting a successful cross-validation is presented herein. A case study utilizing the proposed cross-validation approach to rule out method variability as the potential cause for high variance observed in PK studies is also presented.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event – A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the 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. This white paper is published in 3 parts due to length. This part (Part 1) discusses the recommendations for small molecules, peptides and small molecule biomarkers by LCMS. Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in the Bioanalysis journal, issue 23.

Background: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations. Conclusion: A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 μL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 μL of [ 13 C 2 , D 4 ]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 μL of 100 mM NH 4 OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 μL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity ® UPLC BEH C18 Column (2.1 × 50 mm, 1.7 μm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL −1 for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC–MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.

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.

Biological sample dilution is a rate-limiting step in bioanalytical sample preparation when the concentrations of samples are beyond standard curve ranges, especially when multiple dilution factors are needed in an analytical run. We have developed and validated a Microsoft Excel-based robotic sample preparation program (RSPP) that automatically transforms Watson worklist sample information (identification, sequence and dilution factor) to comma-separated value (CSV) files. The Freedom EVO liquid handler software imports and transforms the CSV files to executable worklists (.gwl files), allowing the robot to perform sample dilutions at variable dilution factors. The dynamic dilution range is 1- to 1000-fold and divided into three dilution steps: 1- to 10-, 11- to 100-, and 101- to 1000-fold. The whole process, including pipetting samples, diluting samples, and adding internal standard(s), is accomplished within 1 h for two racks of samples (96 samples/rack). This platform also supports online sample extraction (liquid-liquid extraction, solid-phase extraction, protein precipitation, etc.) using 96 multichannel arms. This fully automated and validated sample dilution and preparation process has been applied to several drug development programs. The results demonstrate that application of the RSPP for fully automated sample processing is efficient and rugged. The RSPP not only saved more than 50% of the time in sample pipetting and dilution but also reduced human errors. The generated bioanalytical data are accurate and precise; therefore, this application can be used in regulated bioanalysis.

Rationale Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. Methods Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. Results In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). Conclusions The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1-50 ng/mL for saxagliptin and 0.2-100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis(®) dC18 column (50 mm × 2.1 mm, 5 μm) for LC-MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at -20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.