2023 White Paper on Recent Issues in Bioanalysis: Deuterated Drugs; LNP; Tumor/FFPE Biopsy; Targeted Proteomics; Small Molecule Covalent Inhibitors; Chiral Bioanalysis; Remote Regulatory Assessments; Sample Reconciliation/Chain of Custody (PART 1A - Recommendations on Mass Spectrometry, Chromatography, Sample Preparation Latest Developments, Challenges, and Solutions and BMV/Regulated Bioanalysis PART 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/IVD/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with this NEW Regulation" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication covers the recommendations on Mass Spectrometry Assays, Regulated Bioanalysis/BMV (Part 1A) and Regulatory Inputs (Part 1B). Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 7 and 8 (2024), respectively.

LC-MS bioanalysis of protein biomarkers and protein therapeutics in formalin-fixed paraffin-embedded tissue specimens.

Formalin-fixed paraffin-embedded (FFPE) is a form of preservation and preparation for biopsy specimens. FFPE tissue specimens are readily available as part of oncology studies because they are often collected for disease diagnosis or confirmation. FFPE tissue specimens could be extremely useful for retrospective studies on protein biomarkers because the samples preserved in FFPE blocks could be stable for decades. However, LC-MS bioanalysis of FFPE tissues poses significant challenges. In this Perspective, we review the benefits and recent developments in LC-MS approach for targeted protein biomarker and protein therapeutic analysis using FFPE tissues and their clinical and translational applications. We believe that LC-MS bioanalysis of protein biomarkers in FFPE tissue specimens represents a great potential for its clinical applications.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 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 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Preclinical Characterization of Linrodostat Mesylate, a Novel, Potent, and Selective Oral Indoleamine 2,3-Dioxygenase 1 Inhibitor.

Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor-binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support clinical development.

High-Throughput Therapeutic Antibody Interference-Free High-Resolution Mass Spectrometry Assay for Monitoring M-Proteins in Multiple Myeloma.

Measurement of monoclonal antibodies (M-proteins) plays an important role in the diagnosis and treatment monitoring of multiple myeloma. Currently available M-protein assays have several limitations, particularly because of their lack of sensitivity and propensity to therapeutic antibody (t-mAb) interference. A previously described mass spectrometry method termed monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) is more sensitive than current clinical tests and can provide a solution for resolving t-mAb interferences. However, the original miRAMM workflow is too complex for the throughput needed to analyze a large number of samples. Here, we describe a high-throughput liquid chromatography-high-resolution mass spectrometry (HT-LC-HRMS) approach that employs a fully automated immunocapture step, significantly improved immunoglobulin recovery, simplified chromatography, and high throughput (HT) data processing. In this HT-LC-HRMS approach, raw spectra of the peaks eluting from the LC column during the predefined time period are automatically deconvoluted without the need to identify and monitor the retention time of each patient-specific M-protein. The deconvoluted peak heights of M-protein and therapeutic antibody light chain are conveniently used for quantitation. With the total LC-HRMS measurement time being only 11.0 min, this method was able to differentiate between the M-protein and elotuzumab mass signatures in 91 out of 92 (98.9%) multiple myeloma serum samples tested. The single interference case was resolved using the mass signature of a heavy chain. In addition to resolving t-mAb interference, the developed assay has a 25-fold improvement in sensitivity over immunofixation electrophoresis and can potentially provide an objective tracking of M-proteins in patients with complete response.

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

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

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

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

Detection of Weak Organic Anion-Transporting Polypeptide 1B Inhibition by Probenecid with Plasma-Based Coproporphyrin in Humans.

Probenecid (PROB) is a clinical probe inhibitor of renal organic anion transporter (OAT) 1 and OAT3 that inhibits in vitro activity of hepatic drug transporters OATP1B1 and OATP1B3. It was hypothesized that PROB could potentially affect the disposition of OATP1B drug substrates. The plasma levels of the OATP1B endogenous biomarker candidates, including coproporphyrin I (CPI), CPIII, hexadecanedioate (HDA), and tetradecanedioate (TDA), were examined in 14 healthy subjects treated with PROB. After oral administration with 1000 mg PROB alone and in combination with furosemide (FSM), AUC (0-24 h) values were 1.39 ± 0.21-fold and 1.57 ± 0.41-fold higher than predose levels for CPI and 1.34 ± 0.16-fold and 1.45 ± 0.57-fold higher for CPIII. Despite increased systemic exposures, no decreases in CPI and CPIII renal clearance were observed (0.97 ± 0.38-fold and 1.16 ± 0.51-fold for CPI, and 1.34 ± 0.53-fold and 1.50 ± 0.69-fold for CPIII, respectively). These results suggest that the increase of CP systemic exposure is caused by OATP1B inhibition. Consistent with this hypothesis, PROB inhibited OATP1B1- and OATP1B3-mediated transport of CPI in a concentration-dependent manner, with IC50 values of 167 ± 42.0 and 76.0 ± 17.2 µM, respectively, in transporter-overexpressing human embryonic kidney cell assay. The inhibition potential was further confirmed by CPI and CPIII hepatocyte uptake experiments. In contrast, administration of PROB alone did not change AUC (0-24 h) of HDA and TDA relative to prestudy levels, although the administration of PROB in combination with FSM increased HDA and TDA levels compared with FSM alone (1.02 ± 0.18-fold and 0.90 ± 0.20-fold vs. 1.71 ± 0.43-fold and 1.62 ± 0.40-fold). Taken together, these findings indicate that PROB displays weak OATP1B inhibitory effects in vivo and that coproporphyrin is a sensitive endogenous probe of OATP1B inhibition. This study provides an explanation for the heretofore unknown mechanism responsible for PROB's interaction with other xenobiotics. SIGNIFICANCE STATEMENT: This study suggested that PROB is a weak clinical inhibitor of OATP1B based on the totality of evidence from the clinical interaction between PROB and CP and the in vitro inhibitory effect of PROB on OATP1B-mediated CP uptake. It demonstrates a new methodology of utilizing endogenous biomarkers to evaluate complex drug-drug interaction, providing explanation for the heretofore unknown mechanism responsible for PROB's inhibition. It provides evidence to strengthen the claim that CP is a sensitive circulating endogenous biomarker of OATP1B inhibition.

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.

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

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

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. 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 bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of Bioanalysis, issues 23 and 24 (2019), respectively.

Perspectives on exploring hybrid LBA/LC-MS approach for clinical immunogenicity testing.

Biological drug products may elicit an antidrug antibody (ADA) response. The current widely used bridging ligand binding assay (LBA) is the gold standard for ADA assessments in drug development, which is a qualitative assay followed by a quasi-quantitative titer analysis but can be prone to interferences from biological matrices, drug targets and circulating drugs. We present our perspectives and findings in exploring a hybrid LBA/LC-MS as an orthogonal bioanalytical tool for clinical immunogenicity assessments. The hybrid LBA/LC-MS is a semiquantitative assay with acceptable specificity, drug tolerance and the capability of multiplexed detection of ADA isotypes. The assay results suggest this technology to be a promising and complementary bioanalytical tool that can provide informative immunogenicity data in drug development.

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

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

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

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

Critical reagent screening and characterization: benefits and approaches for protein biomarker assays by hybrid LC-MS.

In recent years, hybrid ligand-binding assays (LBAs)/LC-MS assays have been increasingly used for quantitation of protein biomarkers in biological matrices. However, unlike in LBAs where the importance of critical reagent screening and characterization is well understood and widely reported, benefits of well-characterized hybrid LC-MS assay reagents are frequently underestimated. Two groups of analyte-specific reagents, binding reagents and assay calibrators, are considered the critical reagents for biomarker assays. In this article, we summarize the similarities and differences of critical reagents used in LBAs and hybrid LC-MS assays, overview the benefits and approaches of critical reagent screening, characterization, antibody conjugation and discuss bioanalytical considerations in hybrid LC-MS assay development for robust measurements of protein biomarkers in biological matrices.

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

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

LC-MS/MS bioanalysis of plasma 1, 14-tetradecanedioic acid and 1, 16-hexadecanedioic acid as candidate biomarkers for organic anion-transporting polypeptide mediated drug-drug interactions.

AIM: A robust LC-MS/MS assay was developed to quantify endogenous 1, 14-tetradecanedioic acid (TDA) and 1, 16-hexadecanedioic acid (HDA) in human plasma as potential biomarkers for evaluating drug-drug interactions mediated by the hepatic drug transporters, organic anion-transporting polypeptides. RESULTS: This assay was validated using fit-for-purpose approach over standard curve range of 2.5-1000 nM for TDA and HDA using analyte-free charcoal-stripped human plasma as the surrogate matrix. Chromatographic separation condition was successfully optimized to separate TDA from an interference peak while maintaining both analytes in neutral forms to minimize carryover issue. CONCLUSION: The described assay is currently applied to a clinical study for evaluating TDA/HDA as potential substitute biomarkers for drug-drug interaction studies.

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

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

UHPLC-MS/MS bioanalysis of human plasma coproporphyrins as potential biomarkers for organic anion-transporting polypeptide-mediated drug interactions.

AIM: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. RESULTS: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. CONCLUSION: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.