Bioanalytical Challenges Research Articles

The 3rd China Bioanalysis Forum annual meeting.

The third China Bioanalysis Forum conference was successfully held in Shanghai in June 2015. This year's conference focused on biologics, biomarkers and regulated bioanalytical challenges. More than 150 delegates from pharmaceutical/biotech industry, CRO, clinical research centers, research institutes and regulatory agencies attended. This report summarizes the major discussion topics at the conference.

Bioanalytical approaches for characterizing catabolism of antibody-drug conjugates.

The in vivo stability and catabolism of antibody-drug conjugates (ADCs) directly impact their PK, efficacy and safety, and metabolites of the cytotoxic or small molecule drug component of an ADC can further complicate these factors. This perspective highlights the importance of understanding ADC catabolism and the associated bioanalytical challenges. We evaluated different bioanalytical approaches to qualitatively and quantitatively characterize ADC catabolites. Here we review and discuss the rationale and experimental strategies used to design bioanalytical assays for characterization of ADC catabolism and supporting ADME studies during ADC clinical development. This review covers both large and small molecule approaches, and uses examples from Kadcyla® (T-DM1) and a THIOMAB™ antibody-drug conjugate to illustrate the process.

Antibody-drug conjugates nonclinical support: from early to late nonclinical bioanalysis using ligand-binding assays.

The objective of antibody-drug conjugate (ADC) bioanalysis at different stages of drug development may vary and so are the associated bioanalytical challenges. While at early drug discovery stage involving candidate selection, optimization and preliminary nonclinical assessments, the goal of ADC bioanalysis is to provide PK, toxicity and efficacy data that assists in the design and selection of potential drug candidates, the late nonclinical and clinical drug development stage typically involves regulated ADC bioanalysis that delivers TK data to define and understand pharmacological and toxicological properties of the lead ADC candidate. Bioanalytical strategies and considerations involved in developing successful ligand binding assays for ADC characterization from early discovery to late nonclinical stages of drug development are presented here.

A multiplexed hybrid LC-MS/MS pharmacokinetic assay to measure two co-administered monoclonal antibodies in a clinical study.

Combination therapies with monoclonal antibodies (mAbs) enhance therapeutic activity and may circumvent drug resistance. However, these studies present bioanalytical challenges for ligand-binding assays (LBAs). Recent MS-based protein quantification offers an alternative for bioanalysis. A hybrid LC-MS/MS assay was developed to simultaneously measure human serum concentrations of two mAbs. Anti-idiotypic reagents that did not work in LBAs were successfully used for mAb affinity capture enrichment. Stable isotope-labeled peptide internal standards were employed. The mAb quantification involved measuring a signature CDR peptide derived from each mAb as a surrogate. Selected clinical samples were successfully analyzed. The multiplexed LC-MS/MS method provided a powerful quantitative tool for clinical PK assessment of co-administered mAbs without the requirement for stringent affinity capture reagents.

Practical considerations for the pharmacokinetic and immunogenic assessment of antibody-drug conjugates.

Currently, the most bioanalytically challenging drugs are antibody-drug conjugates (ADCs), constructs comprising a monoclonal antibody and a cytotoxic drug connected by a linker. The bioanalytical challenges arise from the heterogeneous nature of ADCs and their complex in vivo behavior, resulting in a high number of analytes to be measured. Measuring the concentration of biologics in blood/plasma/serum is a necessity to properly assess their pharmacokinetic (PK)/pharmacodynamic behaviors in vivo. An additional bioanalytical challenge is to monitor the stability of the ADCs, as cytotoxic drugs released from the ADC in blood circulation may pose a potential safety risk because of their high cytotoxic potency. The nature of ADCs does not only complicate bioanalysis, but also immunogenicity assessment. Questions, such as 'Which part of the ADCs is the anti-drug antibodies directed against?' may arise, and their answer normally includes several immunogenicity risk assessment strategies. This review will focus on the bioanalytical challenges of ADCs, current approaches involving ligand-binding assays (LBAs), liquid chromatography and mass spectrometry platforms, and recommendations on which approach to use for which stage of drug development, and will close with immunogenicity assessment. In order to appropriately tackle the bioanalytical and immunogenic challenges of ADCs and consider every angle, the authors of this review have expertise in ligand binding and liquid chromatography-mass spectrometry.

Bioanalytical Challenges of Biosimilars

Biologics such as monoclonal antibodies and recombinant proteins represent a significant portion of the pharmaceutical market. With many of the first generation biologics' patents expiring, an increasing number of biosimilars will be submitted for approval in the near future. The successful development of a biosimilar requires the demonstration of biosimilarity in terms of efficacy, safety and purity to an innovator-approved product. While regulatory frameworks have been established for the approval of biosimilars in several countries, there is not an established guidance for bioanalytical testing of biosimilars. Although there are regulatory guidances and White Papers on testing requirements for biologics in general, there is a need to address the bioanalytical challenges and solutions that apply specifically to the analysis of biosimilars in biological samples. This paper will focus on components of the PK and immunogenicity assays that are critical to biosimilar drug development.

Analytical approaches for quantification of a Nrf2 pathway activator: overcoming bioanalytical challenges to support a toxicity study

Activation of the Nrf2 stress pathway is known to play an important role in the defense mechanism against electrophilic and oxidative damage to biological macromolecules (DNA, lipids, and proteins). Chemical inducers of Nrf2 such as sulforaphane, dimethyl fumarate (Tecfidera®), CDDO-Me (bardoxolone-methyl), and 3-(dimethylamino)-4-((3-isothiocyanatopropyl)(methyl)amino)cyclobut-3-ene-1,2-dione (a synthetic sulforaphane analogue; will be referred to as ) have the ability to react with Keap1 cysteine residues, leading to activation of the Antioxidant Response Element (ARE). Due to their electrophilic nature and poor matrix stability, these compounds represent great challenges when developing bioanalytical methods to evaluate in vivo exposure. like SFN reacts rapidly with glutathione (GSH) and nucleophilic groups in proteins to form covalent adducts. In this work, three procedures were developed to estimate the exposure of in a non-GLP 7 day safety study in rats: (1) protein precipitation of blood samples with methanol containing the free thiol trapping reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) to measure GSH- and N-acetylcysteine conjugated metabolites of ; (2) an Edman degradation procedure to cleave and analyze N-terminal adducts of at the valine moiety; and (3) treatment with ammonium hydroxide to measure circulating free- and all sulfhydryl bound .

Current Status and Bioanalytical Challenges in The Detection of Unknown Anabolic Androgenic Steroids in Doping Control Analysis

Androgenic anabolic steroids (AAS) are prohibited in sports due to their anabolic effects. Doping control laboratories usually face the screening of AAS misuse by target methods based on MS detection. Although these methods allow for the sensitive and specific detection of targeted compounds and metabolites, the rest remain undetectable. This fact opens a door for cheaters, since different AAS can be synthesized in order to evade doping control tests. This situation was evidenced in 2003 with the discovery of the designer steroid tetrahydrogestrinone. One decade after this discovery, the detection of unknown AAS still remains one of the main analytical challenges in the doping control field. In this manuscript, the current situation in the detection of unknown AAS is reviewed. Although important steps have been made in order to minimize this analytical problem and different analytical strategies have been proposed, there are still some drawbacks related to each approach.

The analytical challenges of anti-doping analysis

The development of new anti-doping analyses presents a fascinating and ever-changing set of bioanalytical challenges to those who endeavor to devise tests to detect prohibited substances used by athletes. Perhaps unlike any other field of analysis, anti-doping analysis is one with a constantly moving target; as new analyses are developed, new doping techniquesmove to the forefront. This “arms race” can be seen in both the evolution of doping methods, from synthetic steroids to recombinant human proteins, and the associated tests, from gas chromatography to specialized immunoassays. The challenges associated with anti-doping analysis are growing ever more complex, as more and more compounds become available, which not only mimic the function of endogenous compounds in terms of function, but also become almost indistinguishable in their chemistry. The development of new analytical methods to identify and quantify the presence of such compounds, likely at very low concentrations, requires great analytical skill and knowledge from both chemical and biochemical analysis perspectives. Additionally, there is the continuous challenge of identifying the next dopant and developing a test for it before it can become widely used. Fortunately, there are a growing number of scientists dedicating their analytical efforts to tackling these challenges and bringing a range of new tools and techniques to bear in the process. This topical collection is focused on the recent advances that have been made in anti-doping analysis. To a certain extent, the focus of this collection has been directed towards the use of novel or emerging techniques and methods for antidoping analysis. Although it is without question that mass spectrometry, coupled with chromatographic separation, is the dominant analytical technique in the field, it is becoming somewhat limited in its applicability. The next generations of doping agents, those that are recombinant forms of endogenous compounds, will require new tools for their identification. In this collection, the reader will find critical reviews and new research articles by research groups dedicated to developing some of these new tools, such as the detection of gene transfer vectors, a lateral flow assay for erythropoietin, and a DNAbased approach to identifying blood transfusions. The role of mass spectrometry, of course, cannot be neglected, particularly with advances in its coupling with novel sampling approaches, such as solid-phase microextraction, and its use in the detection of the latest small-molecule doping agents. Although urine may be the simplest and commonest fluid sampled for antidoping tests, the analysis of blood samples is growing. There are inherent challenges with this matrix which are not present with urine samples; however, these are being overcome by many research groups to yield new modes of dopant analysis. Additionally, this collection contains a research article on the identification of doping agents in equine athletes, lest we forget that humans are not the only athletes who may be doped. As the guest editor of this special collection, I hope that as the reader of these contributions you are inspired to consider how your own research may assist in the ongoing challenges of anti-doping analysis. The challenges in anti-doping analysis are significant and continue to evolve; therefore, those at the forefront of analytical and bioanalytical research need to work together to devise the next generation of tests to overcome these challenges.

Bioanalysis of Antibody–Drug Conjugates: American Association of Pharmaceutical Scientists Antibody–Drug Conjugate Working Group Position Paper

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

Conference Report: Drug Metabolism Discussion Group Short Meeting: Microsampling – The Next Big Thing

On behalf of the Drug Metabolism Discussion Group, Regulatory Bioanalysis AstraZeneca (UK) recently organized and hosted an extremely successful Drug Metabolism Discussion Group Short Meeting on 'microsampling--the next big thing'. This attracted over 140 delegates and a strong line up of presenters of respected scientists within the field. This meeting focused on the impact of taking a reduced sample (5-20 µl) from an animal, or later in the clinic, particularly neonates. The agenda covered the spectrum of microsampling, from capillary plasma microsampling, as championed by Ove Jonsson and Kristian Königsson, through to dried blood spots. The day was split up in to three sections, the morning concentrating on the sampling aspects from animals. A highlight of the first section was the 'poster blitz' where four poster presenters gave a quick overview of their work. This introduced the poster session and created a good atmosphere for general debate between the delegates. The mid-session saw the bioanalytical challenges discussed from the discovery to the preclinical stage. To encourage interaction between the presenters and the audience, a panel discussion was used that led to interesting insights into study design from toxicological and bioanalytical viewpoints. The final session was left to clinical aspects of microsampling and a particularly interesting presentation from Hitesh Pandya from the Pediatric Respiratory Medicine Department (University of Leicester, Leicester, UK). An eloquent and hard-hitting presentation put into perspective the importance of advancements in this field that enables sample to be taken in a noninvasive manner. The meeting was well received with excellent feedback from all concerned.

Implementation of A Universal Analytical Method in Early-Stage Development of Human Antibody Therapeutics: Application to Pharmacokinetic Assessment For Candidate Selection

Effective bioanalytical support for pharmacokinetic assessment of therapeutics in early development remains challenging. Concurrent evaluation of multiple candidates per program is typical, requiring efficient characterization in various preclinical species; an ambitious effort often complicated by assay reagent unavailability and limited sample volume. Accordingly, a universal anti-human Fc assay for human monoclonal antibody and derived therapeutics was developed using a microfluidics platform to address these bioanalytical challenges. The universal assay with standardized format was qualified for quantitation of human IgG Fc-containing biotherapeutics in matrices from commonly used preclinical species. Results from this assay compared well with those from traditional colorimetric immunoassays. Furthermore, result comparison between the universal and target-specific assays provided additional information on the effect of antidrug antibodies and in vivo drug catabolism. This assay has wide applicability as a default bioanalytical approach in therapeutic candidate selection and preliminary pharmacokinetics evaluation during early-stage therapeutic development.

Bioanalysis Young Investigator: Dajana Vuckovic

Dajana currently has 21 peer-reviewed articles published or accepted for publication in top analytical chemistry journals including Analytical Chemistry and Angewandte Chemie International Edition. She has co-authored eight book chapters, and made 22 presentations at international conferences such as Pittcon and ASMS conferences. Dajana's commitment to excellence is also illustrated by the awards she has won to date, including a Postdoctoral Fellow Award offered by the Natural Science and Engineering Research Council of Canada (2010-2012) and the Douglas E Ryan Analytical Chemistry Student Award of the Canadian Society for Chemistry (2010). During her PhD in my group, Dajana excelled in very diverse areas of interest including the development of targeted LC-MS assays for drugs and metabolites, application of in vivo solid-phase microextraction (SPME) in mice and LC-MS metabolomics for the first time, and evaluation of a high-throughput 96-well plate automated SPME system. Since her graduation from my group she has further extended her bioanalytical expertise to proteomics. I think Dajana's excellent research productivity, accomplishments to date, willingness to address complex bioanalytical challenges and extensive expertise in both small molecule and protein bioanalysis make her an excellent candidate for the Bioanalysis Young Investigator Award.

Conference Report: ‘Large Meets Small‘: Connecting The Bioanalytical Community Around Peptide And Protein Bioanalysis With Lc–Ms(/Ms)

This conference report provides an overview of the discussions at the 2nd European Bioanalysis Forum (EBF) Focus Meeting 'Large Meets Small' held on 20 and 21 June 2011 in Brussels. The meeting discussed scientific progress in the bioanalysis of peptides and proteins with MS-based techniques. Bioanalytical experts in ligand-binding assays (LBAs) and MS from industry and academia presented at the meeting or joined the discussion. The conference hosted sessions on technology developments, validation requirements, cutting edge (bio)analytical approaches for both proteins and peptides and discussions on the analytical challenge presented by the metabolism of peptides or proteins. The engagement of the scientists as well as the bioanalytical challenges identified were real: by shifting peptide or protein analysis from the LBAs laboratory into the LC-MS laboratory, the bioanalytical scientist is moving into partially uncharted territory. The conference delegates strongly shared the feeling that success in overcoming the challenges of peptide and protein bioanalysis will require further integration of the expertise of LBAs and LC-MS/MS experts.

Bioanalytical strategies for developing highly sensitive liquid chromatography/tandem mass spectrometry based methods for the peptide GLP‐1 agonists in support of discovery PK/PD studies

Highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods have been developed and implemented for the quantitative determination of a number of peptides under evaluation in our Glucagon-Like Peptide-1 (GLP-1) discovery program for the treatment of diabetes. These peptides are GLP-1 receptor agonists. Due to the high potency, low dose, and low exposure of these peptides, LC/MS/MS-based methods with Lower Limits of Quantitation (LLOQs) (low picomolar range) were required to support discovery pharmacokinetic/ pharmacodynamic (PK/PD) studies. Compared with small molecules, many of these peptides posed significant bioanalytical challenges in the development of highly sensitive methods because of their parent signal splitting as a result of the formation of multiply charged states, the unfavorable fragmentation patterns for Selected Reaction Monitoring (SRM) transitions due to the generation of a large number of small mass product ions with relative low intensities, and adsorption issues observed during sample preparation. This paper details the strategies developed to maximize the sensitivity and improve LLOQs from aspects of mass spectrometry, chromatography, and sample preparation. A LLOQ of 10 picomolar was achieved for all of the investigated peptides using 100 μL of mouse plasma. This is a 100-fold improvement on LLOQs over generic LC/MS/MS-based methods when the same sample volume and the same mass spectrometer platform were used. The methods have been implemented in the support of discovery PK/PD studies.

EBF Recommendation on the Validation of Bioanalytical Methods for Dried Blood Spots

Over the last few years bioanalysts, pharmacokineticists and clinical investigators have rediscovered the technique of dried blood spots. The revival has provided pharmaceutical R&D a wealth of opportunities to optimize the drug-discovery and development process with respect to animal and patient ethics, new scientific insights and costs savings. On the bioanalytical front, multiple experiments have been performed and a lot of experience has been gained. Nevertheless, the technique still has a number of bioanalytical challenges. The European Bioanalysis Forum discussed the advantages and hurdles of the technique and summarized their current thinking in a recommendation on the validation of bioanalytical methods for dried blood spots, which can be used as a cornerstone for further discussions and experiments.

Book Review: Drug Development Perspectives: Considerations, Challenges and Strategies

Book Review: Drug Development Perspectives: Considerations, Challenges and Strategies

Conference Report: from Challenges to Solutions

The European Bioanalysis Forum is a bioanalytical nonprofit organization comprised of European pharmaceutical companies (27 members to date) and currently expanding to include CROs as well. The European Bioanalysis Forum provides a broad European bioanalytical network for the discussion of scientific, technological and regulatory topics of bioanalytical interest. The 3rd Annual Open Symposium was again much anticipated after the two previous successful meetings. The symposium included sessions on thinking outside the 'commodity' box, bioanalytical challenges with blood, global harmonization, assay platforms, dried blood spots, immunogenicity, matrix effects, anomalous results, biomarkers and two plenary technology sessions hosted by the Platinum sponsors. Experts and key opinion leaders were invited as guest speakers. A total of 424 delegates registered from 113 companies representing a large percentage of the European bioanalytical community. In addition to 48 oral presentations, 88 posters were presented and there was a vendor exposition of 40 companies.

Increased rheumatoid factor interference observed during immunogenicity assessment of an Fc-engineered therapeutic antibody

Protein therapeutics may elicit an anti-therapeutic antibody (ATA) response in patients. This response depends on a number of factors including patient population, disease state, route of delivery or characteristics specific to the product. Therapeutics for immunological indications often target relatively young and healthy patients with hyperactive immune systems who have periodic flares and remissions. The hyperactive immune system of these patients can add several levels of bioanalytical complexity due to the presence of cross reactive molecules such as autoantibodies. In addition, the long-term chronic dosing regimen often necessary in this patient population can increase their risks of immunogenicity against the therapeutic and lead to safety concerns. Therefore, development of a sensitive and drug-tolerant ATA method is important. Bridging ATA assays are usually very sensitive and drug-tolerant methods for immunogenicity assessment; however these methods are particularly vulnerable to any factor that is able to bridge the conjugated therapeutics used as reagents and can generate false positive signal. Although there are many potential interfering factors in serum, rheumatoid factors (RFs), autoantibodies associated with rheumatoid arthritis (RA), are of particular concern in this type of assay. MTRX1011A is a non-depleting anti-CD4 monoclonal antibody therapeutic that was clinically tested in RA patients. This paper will discuss the bioanalytical challenges encountered during development of a clinical ATA assay for MTRX1011A. These challenges highlight interference due to patient disease state, in this case presence of RF in RA patients, as well as specific molecule-related interference caused by an engineered mutation in the Fc region of MTRX1011A designed to enhance its binding to the neonatal Fc receptor (FcRn). We will discuss the characterization work used to identify the cross-reactive epitope and our strategy to overcome this interference during development of an effective ATA assay to support clinical evaluation of MTRX1011A.

Identifying and overcoming bioanalytical challenges associated with chlorine-containing dehydrogenation metabolites

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is a widely utilized analytical tool for quantifying small molecules in complex biological matrices. In certain situations the mass-selection capabilities of the tandem mass spectrometer may be insufficient to discriminate between the analyte of interest and its metabolites, particularly those metabolites that are isobaric with the analyte. One scenario by which isobaric interference may occur is the metabolism of a chlorine- or bromine-containing small molecule to a metabolite with the concomitant loss of 2 Da. This report describes the detection and characterization of two distinct dehydrogenation [M-2] metabolites during LC/MS/MS quantification of a chlorinated small molecule in rat plasma samples derived from a toxicokinetic study. The potential isotope-related impact of these metabolites on quantification of the parent compound was assessed. Several alternate precursor ion and product ion combinations were evaluated and shown to minimize the quantitative impact of the interfering metabolites without having to rely on their stringent chromatographic resolution from the parent compound. These results indicate that when quantifying chlorine- or bromine-containing small molecules from in vivo samples or in vitro metabolic incubations: (1) efforts to detect potential dehydrogenation metabolites should be undertaken and (2) if such metabolites are detected, the judicious choice of alternate multiple-reaction monitoring (MRM) transitions can limit their impact on quantification of the parent molecule without the need for robust chromatographic resolution.