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

Purpose: 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 [18F]FDG-positron emission tomography (PET) imaging for anticancer therapies in a clinical setting. Methodology/results: 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. In a mouse JIMT-1 tumor model, FDG-monophosphate levels measured by LC-MS/MS correlated with [18F]FDG-PET imaging results. Conclusion: LC-MS/MS analysis of FDG-monophosphate accumulation in tumors is a cost-effective tool to gauge the translational potential of [18F]FDG-PET imaging as a noninvasive biomarker in clinical studies.

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. Implementation of a ligand binding assay was not successful due to extensive bispecific antibody interference. Instead, we explored two types of immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays, one at the protein level and one at the surrogate peptide level. Ultimately, the protein-level IA-LC-MS/MS method was optimized for use in a cynomolgus monkey PK/pharmacodynamic study. In addition, we demonstrated that the method was easily adapted for use with human samples in preparation for translation to the clinic. This work demonstrates the benefit of flexibility and agility in bioanalytical method development in early drug development. Multiplatform suitability assessments enable rapid, resource-sparing identification and qualification of clinically translatable assays. We recommend early adoption of this strategy to provide enough time for critical reagent development and assay validation for analysis of shed targets.

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. The sample matrix for a specific analyte is often chosen based on prior experience or literature searches, without good understanding of the possible effects on analyte quantification. Using a data set of 32 different soluble protein markers measured in matched serum and plasma samples, we examined the differences between serum and plasma and discussed how platelet or immune cell activation can change the quantified concentration of the analyte. We have also reviewed the effect of anticoagulant on analyte quantification. Finally, we provide specific recommendations for biomarker sample matrix selection and propose a systematic and data-driven approach for sample matrix selection. This review is intended to raise awareness of the impact and considerations of sample matrix selection on biomarker quantification.

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. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.

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. 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.

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. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of <11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax >0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.

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. Supreme Court will address how much deference the appellate court should afford to a trial court's claim construction ruling. The effect of this decision will be far-reaching, as how claims are construed can determine whether a patent is infringed or not infringed, valid or invalid.

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. 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.

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. Bioanalytical methods are key to informing PK/PD models and require assessment of both PK and PD end points. Where targets are sequestered in tissues (noncirculating), the ability to quantitatively measure drug or biomarker in tissue compartments becomes particularly important. This perspective provides an overview of contemporary applications of quantitative bioanalysis in tissue compartments as applied to PK and PD assessments associated with novel biotherapeutics. Case studies and key references are provided.

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. Commercially available antibodies were screened to identify optimal combinations that were then used in a multi-factorial design of experiments (DOE) to optimize reaction conditions. Analytical validation included assessments of accuracy and precision (A&P), and dilutional linearity/hook effect, as well as reagent and sample stability. The method is reliable, with total error, a measure of accuracy and precision, less than 30% for all concentrations tested. AFP concentrations were measurable in diseased mice and undetectable in normal mice. Therefore, this novel, low volume AFP immunoassay is suitable for pre-clinical drug development, where its miniaturized format facilitates serial sampling in rodent models of cancer.

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. More recently, LC-MS methods have being adapted to quantitate biologics. LC-MS has attributes that complement the limitations encountered by immunoassays. Whether employing immunoassay or LC-MS methods, compared with traditional chemical-based therapeutics, biological therapeutics present unique analytical challenges to analysts. In this article, we review bioanalytical strategies for supporting biologics and discuss the regulatory and analytical challenges that must be met.

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. Here we extend the common usage of the atomic force microscopy technique to study whether compressive forces applied to bound streptavidin-biotin species can significantly accelerate the rate of dissociation. Presented experimental data indicate that compressive forces can substantially decrease the lifetime of the molecular bond. Surprisingly, the efficiency of accelerating dissociation by compressive forces sometimes exceeds the enhancement of the dissociation rate measured in pulling experiments, indicating that compressive forces applied to the bound species might be efficiently used to control the lifetime of adhesion bonds.

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. The experiment involved the testing of 30 incurred samples at 3 stages with incremental laboratory harmonization in standard/quality controls and assay components: Stage I represented a transfer of a detailed protocol and critical reagents. Stage II, a single source of standards and quality controls were provided to each site. Stage III, standards and quality controls plus a ready-to-use kit were provided. The results indicated that all testing facilities failed agreement testing using the stage I procedure. The introduction of standards from a single source improved the agreement. The modification reduced variation by 33% compared to the stage I approach. There was no additional benefit when a packaged kit was provided. In conclusion, introduction of a single source of standards and quality controls reduced the inter-site component of variation and should allow for combinability of data.

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. Therefore, the kinetic parameters of bond dissociation can be extracted from the dependence of detection probability on probe velocity and the detection threshold. This approach is sensitive to low rupture forces and therefore is complementary to the common most probable force data analysis approach. The developed approach is tested using rupture forces measured with specific bonds between biotin and streptavidin and with nonspecific bonds between linear alkanes in water. Results for the analysis of specific bonds rupture are consistent with the previous measurements, suggesting that rupture forces spanning a wide range of values originate from the same binding potential. Kinetic parameters obtained for linear alkanes are significantly different from previous measurements suggesting possible heterogeneity of the bound state.

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. We determined differences among various manual pipetting techniques by analysts within a laboratory. To reduce variability in pipetting, a flexible modular liquid handling script was created on the Hamilton Microlab Star (HMS) to perform sample dilution, pre-treatment and plate loading. The script is capable of handling variable dilution factors. Additionally, two dilution controls were prepared and tested at concentrations of high and mid quality controls (QC). These same dilution controls were incorporated into both pre-study validation and in-study QCs to monitor dilution processing and assay performance. Variability of manual pipetting among 11 analysts was more negatively biased with increasing dilution. Forward and reverse pipetting delivering different volumes contributed to the discordance. The dilutional bias with manual pipetting was eliminated using the liquid handler. Total error of dilution controls was less than 20%. The in-study pass rate was 100%. Application of liquid handlers minimizes the variability and bias due to manual pipetting differences among analysts. The incorporation of dilution QCs serves a dual purpose to monitor the dilution process of the samples as well as the binding assay performance.

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. We used a statistical design-of-experiments approach to identify angiopoietin interference in bridging immunoassays of anti-AMG 386 antibodies. We also demonstrated that a high-affinity monoclonal antibody, directed against an epitope on angiopoietin that competes with AMG 386 binding, could inhibit the angiopoietin interference while preserving the detection of ADA. This report describes the development and validation of methodologies for evaluating and addressing drug target interference in bioanalytical assays that involve interactions between drug, ADA, immune complexes, and drug target.