Bioanalytical Scientists Research Articles

How the bioanalytical scientist plays a key role in interdisciplinary project teams in the development of biotherapeutics - a reflection of the European Bioanalysis Forum.

The bioanalytical scientist plays a key role in the project team for the drug development of biotherapeutics from the discovery to the marketing phase. Information from the project team members is required for assay development and sample analysis during the discovery, preclinical and clinical phases of the project and input is needed from the bioanalytical scientist to help data interpretation. The European Bioanalysis Forum target team 20 discussed many of the gaps in information and communication between the bioanalytical scientist and project team members as a base for providing a perspective on the bioanalytical scientist's role and interactions within the project team.

Integrating internal and external bioanalytical support to deliver a diversified pharmaceutical portfolio.

The portfolios of pharmaceutical companies have diversified substantially over recent years in recognition that monotherapies and/or small molecules are less suitable for modulating many complex disease etiologies. Furthermore, there has been increased pressure on drug-development budgets over this same period. This has placed new challenges in the path of bioanalytical scientists, both within the industry and with contract research organizations (CROs). Large pharmaceutical, biotechnology and small-medium healthcare enterprises have had to make important decisions on what internal capabilities they wish to retain and where CROs offers a significant strategic benefit to their business model. Our journey has involved asking where we believe an internal bioanalytical facility offers the greatest benefit to progressing drug candidates through the drug-development cycle and where externalization can help free up internal resources, adding flexibility to our organization in order to deal with the inevitable peaks and troughs in workload.

Resolution of matrix interference: quantitative and quasi-quantitative ligand-binding assays case studies.

Matrix effects pose a constant challenge in developing robust ligand-binding assays to be validated for use in nonclinical and clinical study support. When notable matrix effects of any kind are present, it can render an otherwise sound method ineffective. We present two case studies detailing the mitigation of observed matrix effects. A dimeric protein was removed from unknown samples in an anti-therapeutic antibody assay through protein extraction. Nonspecific matrix effects in a quantitative ligand-binding assays were mitigated through development of a specialized buffer. The protein extraction method reproducibly reduced the artificially high responses of naïve samples, enabling the accurate detection of anti-therapeutic antibodies. Design of experiments was used to evaluate and select the optimal components and associated concentrations in order to reduce the observed matrix effect to acceptable limits. Our results suggest there are multiple techniques available for the bioanalytical scientist to mitigate both matrix effects in ligand-binding assays.

Performance Assessment of Microflow LC Combined with High-Resolution MS in Bioanalysis

There continues to be consistent pressure for bioanalytical scientists to achieve lower limits of quantitation. The reasons range from smaller sample volumes available for analysis, to more potent analytes and the growth of biologics in drug development. This has led scientists to investigate alternative LC techniques, including microflow and nanoflow. These techniques have been shown to increase sensitivity of electrospray methods and reduce ionization matrix effects. Because high-resolution MS has significant benefits for the analysis of biologics, this type of mass spectrometer is becoming increasingly important in bioanalysis. For microflow analysis, a new ion source and significant extra sample preparation or chromatographic separation are not required. However, increased sensitivity and reduced matrix effects were consistently demonstrated when compared with UHPLC flow rates. The extent of matrix effects observed were compound dependent. This paper presents the utility of combining high-resolution/accurate mass with microflow LC from a quantitative standpoint. This includes evaluating the typical quantitative parameters of sensitivity, linearity/dynamic range, precision and accuracy. It also includes the evaluation of changes in signal suppression using microflow LC and microspray ionization. The benefits and disadvantages of using the combination of these two technologies for quantitative bioanalysis are also discussed.

Managing Scientific, Technical and Regulatory Innovation in Regulated Bioanalysis: A Discussion Paper from The European Bioanalysis Forum

On 12-13 June 2012, the European Bioanalysis Forum hosted its third Focus Meeting in Brussels (Belgium). At the meeting, a panel discussion was held on the hurdles that the bioanalytical community encounters when adopting new technologies or managing regulated bioanalysis expectations around emerging technologies. Over the last few years, the industry has seen many new technologies maturing. As they became available, the bioanalytical scientist has observed that implementing these technologies in the regulated environment has become increasingly challenging. For one, scientific developments and regulatory expectations may not go hand in hand. At the same time, the pharmaceutical industry has become increasingly risk averse in their response to these real or perceived higher expectations in regulated bioanalysis. As a downstream consequence, the potential result of overinterpretation of guidance or occasional widespread and premature implementation of responses to health authority inspections, industry may be contributing significantly to raising the bar on some processes related to day-to-day practices in the bioanalytical laboratory. Last but not least, with the community being satisfied with the performance of the current tools, potential complacency can be observed in the regulated bioanalytical community because existing technologies, such as LC-MS/MS and ligand-binding assays, have served and still are serving them extremely well. Hence, the question 'what's next after LC-MS/MS or ELISA?' is not resonating with many scientists as pertinently compared with 'What's next after RIA, GC or LC-UV?', which was the key question in the 1990s, certainly in the context of an increasing effort needed to validate these new tools. With this article, the European Bioanalysis Forum aims to stimulate an open dialogue between all stakeholders in regulated bioanalysis to positively influence how we balance science, process and regulations in day-to-day work. This discussion should facilitate the evaluation and the subsequent implementation of innovative techniques for the benefit of the patient, while stimulating our community to raise the bar on added-value science, but at the same time removing the bar on processes with limited or no added value.

Free And Total Biotherapeutic Evaluation in Chromatographic Assays: Interference From Targets And Immunogenicity

Measurement of drug concentrations is critical during drug development, supporting evaluation of safety and efficacy in the context of pharmacokinetics. Protein-based therapeutics have been historically measured by immunoassay methods. Technological advances provide new opportunities to measure these biotherapeutics using previously incompatible chromatographic techniques, such as MS. These advances are breaking down the barriers between 'large-molecule' and 'small-molecule' bioanalysis, and pushing scientists outside their comfort zones. One challenge in measuring biotherapeutic concentration is potential impact from other matrix components, such as therapeutic target or antidrug antibodies. Depending on the specific assay development objective, target interference could be either desired (favoring free measurement) or undesired (favoring total measurement). Orthogonal techniques provide additional tools to meet this challenge. The goal of this review is to introduce both small- and large-molecule bioanalytical scientists to the opportunities and challenges to consider while evaluating orthogonal methods for biotherapeutic bioanalysis.

Submission of scientifically sound and ethical manuscripts to peer‐reviewed journals – a reviewer's personal perspective on bioanalytical publications

In the pharmaceutical industry, bioanalysis is very dynamic and is probably one of the few fields of research covering the entire drug discovery, development and post-marketing process. Important decisions on drug safety can partially rely on bioanalytical data, which therefore can be subject to regulatory scrutiny. Bioanalytical scientists have historically contributed significant numbers of scientific manuscripts in many peer-reviewed analytical journals. All of these journals provide some high-level instructions, but they also leave sufficient flexibility for reviewers to perform independent critique and offer recommendations for each submitted manuscript. Reviewers play a pivotal role in the process of bioanalytical publication to ensure the publication of high-quality manuscripts in a timely fashion. Their efforts usually lead to improved manuscripts. However, it has to be a joint effort among authors, reviewers and editors to promote scientifically sound and ethically fair bioanalytical publications. Most of the submitted manuscripts were well written with only minor or moderate revisions required for further improvement. Nevertheless, there were small numbers of submitted manuscripts that did not meet the requirements for publications because of scientific or ethical deficiencies, which are discussed in this Letter to the Editor.

Mars: Bringing The Automation of Small-Molecule Bioanalytical Sample Preparations to A New Frontier

In recent years, there has been a growing interest in automating small-molecule bioanalytical sample preparations specifically using the Hamilton MicroLab(®) STAR liquid-handling platform. In the most extensive work reported thus far, multiple small-molecule sample preparation assay types (protein precipitation extraction, SPE and liquid-liquid extraction) have been integrated into a suite that is composed of graphical user interfaces and Hamilton scripts. Using that suite, bioanalytical scientists have been able to automate various sample preparation methods to a great extent. However, there are still areas that could benefit from further automation, specifically, the full integration of analytical standard and QC sample preparation with study sample extraction in one continuous run, real-time 2D barcode scanning on the Hamilton deck and direct Laboratory Information Management System database connectivity. We developed a new small-molecule sample-preparation automation system that improves in all of the aforementioned areas. The improved system presented herein further streamlines the bioanalytical workflow, simplifies batch run design, reduces analyst intervention and eliminates sample-handling error.

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.

Using High-Resolution Quadrupole Tof Technology in Dmpk Analyses

Recent advances in quadrupole TOF (Q-TOF) MS have some bioanalytical scientists referring to a 'paradigm shift' in their field. They are speaking of a potential move away from workflows based upon triple-quadrupole MS. Gone would be the optimizing of numerous parameters in selected-reaction monitoring (SRM) experiments, replaced with more generic workflows provided by Q-TOF instruments with high data acquisition rates, excellent mass accuracy (≤5 ppm) and high resolving power (≥30,000). Such a move could pay real dividends for high-throughput workflows, especially in drug metabolism and pharmacokinetics analyses where quantitation and qualification studies could actually be merged. But, are modern Q-TOF-MS instruments, touted as high-resolution MS, ready for this? If not, how close is it? This article will examine these questions by reviewing recent advances in Q-TOF technology and some fascinating orthogonal technology (such as ion mobility) that modern Q-TOFs employ for even greater analytical power.

Ligand binding assays in the 21st Century laboratory: platforms.

The 21st Century Bioanalytical Laboratory Platforms initiative in the BIOTEC section of the American Association of Pharmaceutical Scientists (AAPS) began with a pre-conference workshop held in Seattle, WA in June of 2009. This workshop brought together members of the pharmaceutical and biotechnology industries, with instrument and reagent manufacturers to discuss the current and potential future state of the bioanalytical laboratories supporting biologics development. At the conclusion of the workshop, four sub-teams were formed to further develop the ideas and concepts raised during the 2-day workshop. The sub-teams are reagents, automation, e-solutions, and platforms. This paper discusses the critical attributes of a research and development ligand binding assay (LBA) platform and the desired characteristics new platforms should strive to offer in the future. This paper is not intended to be a review and comparison of the current platforms on the market, as this has been done and published elsewhere (1–9). The platforms team consists of a balanced cross-section of the industry with representatives from pharmaceutical, biotechnology, contract research organizations, and instrument manufacturers. The Platforms team have collaborated to discuss and arrive at a consensus regarding the most useful characteristics of a bioanalytical platform for biologics. We present here the results of these discussions. A platform is the technology employed in an analytical method to transduce a biochemical event into a measureable output or signal. This signal allows the bioanalytical scientist to accurately and reproducibly make measurements to analyze different aspects of a specific biologic target (therapeutic, biomarker, and anti-drug antibody) such as its pharmacokinetics, immunogenicity, potency, or effect of biomarkers. An instrument is the tool utilized minimally to measure a platform’s output and convert the resultant signal into interpretable information the analytical scientist can use but can incorporate other aspects such as liquid handling. Many platforms employ optical signals including the absorbance of light through a medium (10) or the emission of fluorescence (10) or luminescence (11). A variety of light detectors are used to measure these optical signals including photo diodes, charge-coupled device cameras, and photo-multiplier tubes (Table I). Sections in this paper provide details on the desirable analytical characteristics, multiplexing, platform flexibility and throughput, desirable instrument characteristics, and finally, life cycle management of the ideal LBA platform. Table I Commonly Used Platforms The analytical characteristics of today’s ligand binding assays are primarily influenced by three major factors—the quality of the reagents, assay format, and the choice of the analytical platform. This paper describes only those aspects derived from the analytical platform.

The Application of SIMCA P+ in Shotgun Metabolomics Analysis of ZICⓇHILIC-MS Spectra of Human Urine - Experience with the Shimadzu IT-T of and Profiling Solutions Data Extraction Software

The search for bio markers of disease have moved from metabolites to proteins and to genes and back again as technology which was developed. Bio-analytical scientists have been trained for decades to methodically isolate, identify and measure specific molecules. Rapid separations coupled to mind blowing rapid mass spectral characterization and generating analytical profiles within minutes which contain incomprehensible amounts of data. As the old saying goes we “can’t see the wood for the trees”. The realization is that in this data rich age we no longer have to methodically isolate characterize and measure specific molecules. What is important is to identify which of the 100’s or 1000’s of resolved and measured “unknown” molecules is associated with the pathology for which we are interested. The goal is markers of clinical relevance and statistical examination of patterns of association is the new mantra of the biomedical analyst - so called shotgun analysis. However, as any clinical biomedical scientist will tell you; a biomarker has to be robust, easily, rapidly and cheaply measured if it is to be adopted in a hospital laboratory. Any shotgun analysis has to recognize these constraints and not just identify statistical outliers that are seen only sometimes in a few samples and not consistently in the majority of the pathology in question. We have applied the SIMCA P+ plus software in shotgun metabolomic analysis of pregnancy urine.

A Practical Approach to Reduce Interference Due to In-Source Collision-Induced Dissociation of Acylglucuronides in LC–MS/MS

In LC-MS/MS, glucuronide conjugated metabolites may convert back to the parent drug due to in-source collision-induced dissociation (CID). During the bioanalysis of naproxen, it was noticed that naproxen acylglucuronide exhibited intense in-source CID to the naproxen [M+H](+) ion under positive ESI. However, no in-source CID of the acylglucuronide to the naproxen [M+NH(4)](+) adduct was observed. Furthermore, absolutely no in-source CID was detected under negative ESI. This phenomenon was not only observed for naproxen acylglucuronide but for eight other acylglucuronides compounds. We have shown that monitoring the parent drug [M-H](-) or [M+NH(4)](+) whenever possible could be an easy approach used by bioanalytical scientists to minimize the impact of in-source CID of acylglucuronides to the parent drug.

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid–liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.

Bioanalytical Solutions to Acetonitrile Shortages

The acetonitrile shortage during 2008 to 2009 challenged bioanalytical scientists due to the ubiquitous role that acetonitrile plays in sample preparation and analysis. Replacement, reduction and reuse of acetonitrile were the core tenants behind each approach used to tackle the shortage. Sample preparation of biological matrices can be accomplished by protein precipitation using a variety of solvents; methanol is usually the best substitute for acetonitrile. The potential liabilities in using methanol can be handled with appropriate modifications. Often methanol is superior to acetonitrile for both protein precipitation and chromatography if phospholipid interference is a problem. Solvent consumption can be minimized by reducing column dimensions and particle size. Separations can be achieved at greatly reduced run times using sub-2-μm and fused-core particle columns. Emerging technologies, such as desorption ESI, direct analysis in real time and laser diode thermal desorption, eliminate the need for chromatography and achieve significant solvent and time savings. Acetonitrile recyclers can purify HPLC waste for reuse.

Validated quantitation method for a peptide in rat serum using liquid chromatography/high‐field asymmetric waveform ion mobility spectrometry

The analysis of peptides presents serious challenges for bioanalytical scientists including low total ion current and non-selective fragmentation during tandem mass spectrometry (MS/MS). During method validation of a peptide in rat serum matrix some interferences could not be easily removed and thus prevented accurate and precise measurement. These problems associated with peptide quantitation were resolved by using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). This selectivity-enhancing technique filters out matrix interferences, and the resulting pseudo-selected reaction monitoring (pseudo-SRM) chromatograms were nearly free from interferences. Control blank matrix samples contained an acceptable level of interference (only 7% signal as compared to the lower level of quantitation). Chromatographic peaks were easily, accurately and precisely integrated resulting in a validated liquid chromatography (LC)/FAIMS-MS/MS method for the analysis of a peptide drug in rat serum according to United States Food and Drug Administration (US FDA) bioanalytical guidelines. These results confirm that new selectivity-enhancing technologies aid the pharmaceutical industry in reliably producing acceptable pharmacokinetic data.

A novel approach for in‐process monitoring and managing cross‐contamination in a high‐throughput high‐performance liquid chromatography assay with tandem mass spectrometric detection

Cross-contamination among wells of a high-throughput, high-density assay is a risk that cannot be detected or controlled by the performance of calibration standards and quality control samples. In the current practice, carryover and cross-contamination is detected only when analytes are detected in blank, zero, placebo, pre-dose samples, in a low standard or low quality control sample. There is no mechanism that allows bioanalytical scientists to determine if cross-contamination has occurred among other samples. As a result, erroneous results can be released to clients even though a batch meets the acceptance criteria. We tested a new approach that quantifies the cross-contamination of each sample and allows the scientist to make quality decisions with documentation. The approach will also detect carryover in over 90% of the wells. Briefly, two additional analytes were added as contamination markers. The markers were added to a multi-well plate alternatively creating a pattern of a checkerboard. The spiked multi-well plate was then used to perform the assay. If both markers were detected in a well, the sample was considered contaminated. The amount of the unexpected marker detected in a well measures the degree of contamination and may be used to make deactivation decisions. Depending on the relative impact of the contamination, a scientist can choose to tolerate the bias, reject the sample, reject the batch or raise the lower limit of quantitation for the batch. A guideline for rejection decisions is presented for discussion.