2024 White Paper on Recent Issues in Bioanalysis: Three Way-Cross Validation; Urine Clinical Analysis; Automated Methods; Regulatory Queries on Plasma Protein Binding; Automated Biospecimen Management; ELN Migration; Ultra-Sensitivity Mass Spectrometry ( - Recommendations on Advanced Strategies for Mass Spectrometry Assays, Chromatography, Sample Preparation and BMV/Regulated Bioanalysis - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV).

The 18 Workshop on Recent Issues in Bioanalysis (18 WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18 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.

2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs ( - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16 Workshop on Recent Issues in Bioanalysis (16 WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. 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 16th 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.

Acute and Chronic Effects of Rifampin on Letermovir Suggest Transporter Inhibition and Induction Contribute to Letermovir Pharmacokinetics.

Rifampin has acute inhibitory and chronic inductive effects that can cause complex drug-drug interactions. Rifampin inhibits transporters including organic-anion-transporting polypeptide (OATP)1B and P-glycoprotein (P-gp), and induces enzymes and transporters including cytochrome P450 3A, UDP-glucuronosyltransferase (UGT)1A, and P-gp. This study aimed to separate inhibitory and inductive effects of rifampin on letermovir disposition and elimination (indicated for cytomegalovirus prophylaxis in hematopoietic stem cell transplant recipients).

High-throughput LC-MS method to investigate postprandial lipemia: considerations for future precision nutrition research.

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (H-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.

Experimental Medicine Study to Measure Immune Checkpoint Receptors PD-1 and GITR Turnover Rates In Vivo in Humans.

Development of monoclonal antibodies (mAbs) targeting immune-checkpoint receptors (IMRs) for the treatment of cancer is one of the most active areas of investment in the biopharmaceutical industry. A key decision in the clinical development of anti-IMR mAbs is dose selection. Dose selection can be challenging because the traditional oncology paradigm of administering the maximum tolerated dose is not applicable to anti-IMR mAbs.

A Microdose Cocktail to Evaluate Drug Interactions in Patients with Renal Impairment.

Renal impairment (RI) is known to influence the pharmacokinetics of nonrenally eliminated drugs, although the mechanism and clinical impact is poorly understood. We assessed the impact of RI and single dose oral rifampin (RIF) on the pharmacokinetics of CYP3A, OATP1B, P-gp, and BCRP substrates using a microdose cocktail and OATP1B endogenous biomarkers. RI alone had no impact on midazolam (MDZ), maximum plasma concentration (C ), and area under the curve (AUC), but a progressive increase in AUC with RI severity for dabigatran (DABI), and up to ~2-fold higher AUC for pitavastatin (PTV), rosuvastatin (RSV), and atorvastatin (ATV) for all degrees of RI was observed.

Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy.

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic T cell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events.

Effects of CETP inhibition with anacetrapib on metabolism of VLDL-TG and plasma apolipoproteins C-II, C-III, and E.

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE.

Effects of 13-Hour Hyperglucagonemia on Energy Expenditure and Hepatic Glucose Production in Humans.

Glucagon (GCG) acutely stimulates energy expenditure (EE) and hepatic glucose production (HGP) in humans, but whether these effects persist during hyperglucagonemia of longer duration is unclear. Using a prospective, randomized, single-blind, crossover study design, we therefore measured EE and rates of glucose appearance (glucose RA) during three separate infusion protocols in healthy lean males: A) 10-h overnight GCG infusion (6 ng/[kg × min]) followed by 3-h infusion of GCG, octreotide (OCT), and insulin (INS) for basal replacement; B) overnight saline (SAL) infusion followed by GCG/OCT/INS infusion; and C) overnight SAL infusion followed by SAL/OCT/INS infusion. Sleep EE, measured at 6 to 7 h of the overnight infusion, was increased 65-70 kcal/24 h in A compared with B and C.

Achieving efficient digestion faster with Flash Digest: potential alternative to multi-step detergent assisted in-solution digestion in quantitative proteomics experiments.

Rationale: In quantitative analysis of protein biomarkers and therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS), it is a preferred and well-established approach to digest with proteolytic enzymes to produce smaller peptide fragments which are more suitable for LC/MS analysis than the intact protein. In-solution digestion is one widely used method for protein digestion. Proteolytically resistant proteins often require digestion times that extend beyond normal working hours and prohibit same day analysis.

Development and validation of an IA-LC/MS method to quantitate active and total B-type natriuretic peptide in human plasma.

Aim: Patients with elevated levels of B-type natriuretic peptide (BNP) and/or NT-proBNP as measured by clinical tests have an elevated risk of heart failure (HF). Despite utility in large clinical studies, both assays are plagued by large biological variability and specificity issues. To address these concerns and further investigate BNP in the HF setting, we developed an LC/MS assay to characterize the ratio of active to total BNP.

An evaluation of an aptamer for use as an affinity reagent with MS: PCSK9 as an example protein.

Background: For quantitative immunoaffinity IA-LC-MS, the utility of antibodies has been demonstrated many times but the utility of aptamers as affinity reagents is unproven.

Methods: Immunoaffinity reagents including a monoclonal antibody and an aptamer were coupled to magnetic beads and used as part of an enrichment strategy for PCSK9 quantitation in plasma.

Results: With limited method development, we have established a comparison of an anti-PCSK9 aptamer with an anti-PCSK9 monoclonal antibody.

The clinical utility of mass spectrometry based protein assays.

Reports of mass spectrometry based assays for peptides and proteins have become increasingly common in the literature. The growing interest of mass spectrometry for use in clinical laboratories has been primarily driven by the inherent selectivity of the platform relative to more traditional platforms such as immunoassays. However, the adoption of mass spectrometry for peptide and protein analysis in the clinic has been relatively slow compared its adoption in non-clinical laboratories such as in biomarker discovery efforts or within laboratories that support pharmaceutical and academic research.

An LC-MRM method for measuring intestinal triglyceride assembly using an oral stable isotope-labeled fat challenge.

Aim: A traditional oral fatty acid challenge assesses absorption of triacylglycerol (TG) into the periphery through the intestines, but cannot distinguish the composition or source of fatty acid in the TG. Stable isotope-labeled tracers combined with LC-MRM can be used to identify and distinguish TG synthesized with dietary and stored fatty acids.

Results: Concentrations of three abundant TGs (52:2, 54:3 and 54:4) were monitored for incorporation of one or two (2)H11-oleate molecules per TG.

Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test.

Background: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g.

Anacetrapib lowers LDL by increasing ApoB clearance in mildly hypercholesterolemic subjects.

Background: Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib.

Methods: We performed a trial of the effects of anacetrapib on ApoB kinetics.

Applications of low-flow LC-SRM for the analysis of large molecules in pharmaceutical R&D.

Although ligand-binding assays are frequently employed to measure large molecules, the use of LC-SRM assays is increasingly popular due to the inherent selectivity advantage and the ability to operate without exquisitely selective antibodies. Until recently LC-SRM assays have been unable to compete with ligand-binding assays in terms of sensitivity. However, the use of low-flow chromatography prior to mass spectrometry has played a crucial role in increasing the sensitivity of LC-SRM platforms and enabling measurements of large molecules that had previously been unmeasurable.

Development and fit-for-purpose validation of a LC-MS/MS assay for fibrinogen peptide A quantitation in human plasma.

Background: Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of thrombin activity and could potentially be used as a biomarker for anti-thrombotic therapy development. Results/Methodology: A LC-MS/MS assay with a high throughput solid phase extraction procedure was developed and validated to measure FPA in plasma.

Practical immunoaffinity-enrichment LC-MS for measuring protein kinetics of low-abundance proteins.

Background: For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS.

Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.

Rationale: Apolipoprotein(a) is a polymorphic glycoprotein covalently bound to apoB100 in Lp(a) particles and has been described to be both atherogenic and prothrombotic, although its exact mechanism of action is not well defined. Apolipoprotein(a) is routinely measured by immunoassays. Unfortunately, the accuracy of the measurement can be affected by the apolipoprotein(a) size (number of kringles) polymorphism in Lp(a) particles.