A quantitative LC-MS/MS approach for monitoring 2'-fluoro-2'-deoxy-D-glucose uptake in tumor tissue.

Develop a quantitative LC-MS/MS method for FDG, FDG-monophosphate, glucose and glucose-monophosphate in mouse tumor models to assist in validating the use of [F]FDG-positron emission tomography (PET) imaging for anticancer therapies in a clinical setting. Analytes were isolated from tumors by protein precipitation and detected on a Sciex API-5500 mass spectrometer. Improved assay robustness and selectivity were achieved through chromatographic separation of FDG-monophosphate from glucose-monophosphate, selection of a unique ion transition and incorporation of stable isotope labeled internal standards.

A Flexible Multiplatform Bioanalytical Strategy for Measurement of Total Circulating Shed Target Receptors: Application to Soluble B Cell Maturation Antigen Levels in the Presence of a Bispecific Antibody Drug.

B cell maturation antigen (BCMA) is a membrane-bound receptor that is overexpressed on multiple myeloma cells and can be targeted with biotherapeutics. Soluble shed forms of membrane-associated receptors in circulation can act as a drug sink, especially when it is present in high molar ratio compared to drug concentration, potentially derailing the intended pharmacological mechanism and impacting pharmacokinetic (PK) measurements and efficacious dose predictions. In this study, we present a bioanalytical strategy for assessing dynamic levels of total soluble BCMA before and during treatment with a bispecific antibody targeting BCMA and CD3.

Considerations for Soluble Protein Biomarker Blood Sample Matrix Selection.

Blood-based soluble protein biomarkers provide invaluable clinical information about patients and are used as diagnostic, prognostic, and pharmacodynamic markers. The most commonly used blood sample matrices are serum and different types of plasma. In drug development research, the impact of sample matrix selection on successful protein biomarker quantification is sometimes overlooked.

Characterization of the Selective Indoleamine 2,3-Dioxygenase-1 (IDO1) Catalytic Inhibitor EOS200271/PF-06840003 Supports IDO1 as a Critical Resistance Mechanism to PD-(L)1 Blockade Therapy.

Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment.

Workshop Report: Crystal City VI-Bioanalytical Method Validation for Biomarkers.

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.

Feasibility of Singlet Analysis for Ligand Binding Assays: a Retrospective Examination of Data Generated Using the Gyrolab Platform.

There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible.

Teva Pharmaceuticals v. Sandoz: availability of generic glatiramer acetate and the impact to patent litigation claim construction.

In the upcoming case of Teva Pharmaceuticals v. Sandoz, the U.S.

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

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.

Tissue bioanalysis of biotherapeutics and drug targets to support PK/PD.

Contemporary drug discovery leverages quantitative modeling and simulation with increasing emphasis, both to gain deeper knowledge of drug targets and mechanisms as well as improve predictions between preclinical models and clinical applications, such as first-in-human dose projections. Proliferation of novel biotherapeutic modalities increases the need for applied PK/PD modeling as a quantitative tool to advance new therapies. Of particular relevance is the understanding of exposure, target binding and associated pharmacology at the target site of interest.

Development and validation of an alpha fetoprotein immunoassay using Gyros technology.

Circulating alpha fetoprotein (AFP) is a diagnostic and prognostic biomarker for hepatocellular carcinoma (HCC) with potential utility as a pharmacodynamic endpoint in rodent tumor models. This application is limited, however, by low sample volumes, highlighting the need for sensitive, sample-sparing biomarker assay methods. In order to improve the utility of AFP as an oncology biomarker, we developed a method for AFP using the Gyrolab™, an automated microimmunoassay platform.

Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs.

Regulatory recommendations for providing bioanalytical support for biological therapeutics have co-evolved with the increasing success of these unique pharmaceuticals. Immunoassays have been used to quantify biological macromolecules for more than 50 years. These assays rely on the use of antigen-specific antibodies.

Mechanical distortion of protein receptor decreases the lifetime of a receptor-ligand bond.

Substantial experimental evidence indicates that the mechanical force applied to pull apart non-covalent molecular bonds (such as receptor-ligand pairs) can significantly decrease the bond lifetime. This evidence is often generated in single-molecule experiments that are designed to specifically test effects of pulling forces. However, the effect of compressive forces on the lifetime of receptor-ligand bonds remains largely unexplored.

Kinetic parameters from detection probability in single molecule force spectroscopy.

The detection probability of rupture events in AFM force spectroscopy measurements presents a viable alternative to standard methods for extracting kinetic parameters of dissociation. The detection probability has a maximum as a function of the probe velocity where (1) the probability to form a molecular bond is independent of the probe velocity and (2) the detection of rupture events is limited by noise and performed with a constant density of data points per distance of the probe displacement. This newly developed model indicates that the optimal detection velocity is independent of dissociation rate and depends on the distance to the barrier kinetic parameter.

Strategies to minimize variability and bias associated with manual pipetting in ligand binding assays to assure data quality of protein therapeutic quantification.

Bioanalytical laboratories require accurate and precise pipetting to assure reproducible and accurate results for reliable data. Two areas where pipetting differences among analysts lead to poor reproducibility are long term stability testing and sample dilution. The purpose of this paper is to illustrate the problems with manual pipetting, describe an automation strategy to mitigate risks associated with manual pipetting, and provide recommendations on a control strategy that properly monitors samples requiring dilutions.

A strategy for improving comparability across sites for ligand binding assays measuring therapeutic proteins.

Outsourcing and multi-site testing has increased for ligand binding assays supporting protein therapeutic measurement. It is common to combine and compare data across studies with data from multiple bioanalytical sites. We designed a prospective study to determine the benefits of increasing control over the transfer process to improve ruggedness.

Identification and inhibition of drug target interference in immunogenicity assays.

A well-designed anti-drug antibody (ADA) immunoassay is critical for appropriately monitoring the immunogenicity profile of a therapeutic protein during its development. AMG 386 is a peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of angiopoietins with the Tie2 receptor. In bridging immunoassays for ADA, interference by the drug target, present in the assay sample, can result in false positive antibody detection.

Antenatal diagnosis and perinatal management of extremely giant fetal cervicopharyngeal teratoma resulting in impossible fetal intubation: a case report.

We report the case of an extremely large giant neck teratoma diagnosed on routine sonogram and confirmed by 3-D and 4-D sonography and MRI in a 19-year-old primigravida at 18 weeks. Rapid growth, polyhydramnios, premature contractions and premature rupture of membranes necessitated delivery at 28 weeks. Under general anesthesia, with a multidisciplinary team attendant, efforts by the otolaryngologist to establish an airway during an EXIT (ex utero intrapartum treatment) procedure failed as did subsequent attempts by the neonatologist, leading to an early neonatal death.

Association kinetics from single molecule force spectroscopy measurements.

Single molecule force spectroscopy is often used to study the dissociation of single molecules by applying mechanical force to the intermolecular bond. These measurements provide the kinetic parameters of dissociation. We present what to our knowledge is a new atomic force microscopy-based approach to obtain the activation energy of the association reaction and approximate grafting density of reactive receptors using the dependence of the probability to form molecular bonds on probe velocity when one of the interacting molecules is tethered by a flexible polymeric linker to the atomic force microscopy probe.

Application of multi-factorial design of experiments to successfully optimize immunoassays for robust measurements of therapeutic proteins.

Developing a process that generates robust immunoassays that can be used to support studies with tight timelines is a common challenge for bioanalytical laboratories. Design of experiments (DOEs) is a tool that has been used by many industries for the purpose of optimizing processes. The approach is capable of identifying critical factors and their interactions with a minimal number of experiments.

Effects of multiple-bond ruptures on kinetic parameters extracted from force spectroscopy measurements: revisiting biotin-streptavidin interactions.

Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe.

Pairwise interactions between linear alkanes in water measured by AFM force spectroscopy.

Pairwise interactions between n-alkanes from decane to octadecane in water have been studied by single-molecule force spectroscopy. The interacting molecules are covalently tethered to the glass substrate and to the probe of an atomic force microscope by water-soluble linkers to facilitate single-molecule detection. However, the measured distribution of rupture forces deviates significantly from the distribution predicted by theoretical models for rupture of individual bonds.