Clinical Bioanalysis Research Articles

Incurred sample reanalysis in AstraZeneca small molecule portfolio - what have we learned and where do we go next?

In this paper, experiences and learnings are shared from the 10-year application of incurred sample reanalysis (ISR) in support of the AstraZeneca small molecule portfolio. The conclusions from including ISR in every clinical bioanalysis study for a period of 5 years, generating ISR data from 550 studies, are shared. Our preclinical ISR approach is described and data generated using capillary microsampling demonstrate confidence in its routine application. The data demonstrate that ISR failures are very rare and the assessment can and should therefore be limited. Dialogue between the bioanalytical teams internally, as well as with the partner contract research organizations, is however critical for a successful bioanalytical method validation and to avoid any ISR failures.

Development and validation of generic heterogeneous fluoroimmunoassay for bioanalysis of bevacizumab and cetuximab monoclonal antibodies used for cancer immunotherapy

This study describes, for the first time, the development and validation of a highly selective and sensitive heterogeneous fluoroimmunoassay (FIA) for the bioanalysis of two monoclonal antibodies (mAbs) used for cancer immunotherapy: bevacizumab (BEV) and cetuximab (CET). The assay combines reliable non-competitive binding of BEV and CET to their specific cell receptor proteins (human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), respectively) with the highly specific fluorescence activity of the fluorescein isothiocyanate labeled anti-human IgG (FITC-IgG) used as label. The limits of detection were 14.14 and 1.27 × 103 ng mL−1 for BEV and CET, respectively. The accuracy and precision of the assay were demonstrated. The assay is simple, convenient, and requires very small volume (~ 5 µL) of plasma sample for analysis. The assay can offer high throughput analysis in clinical settings when modern microplates of multiplies of 96 (up to 6144-wells) are used and/or integrated as a part of automated robotic system. The proposed assay can be used for routine clinical bioanalysis of mAbs with potential application in pharmacokinetics, pharmacodynamics and therapeutic drug monitoring (TDM).

AstraZeneca and Covance Laboratories Clinical Bioanalysis Alliance: an evolutionary outsourcing model.

The AstraZeneca and Covance Laboratories Clinical Bioanalysis Alliance (CBioA) was launched in 2011 after a period of global economic recession. In this challenging environment, AstraZeneca elected to move to a full and centralized outsourcing model that could optimize the number of people supporting bioanalytical work and reduce the analytical cost. This paper describes the key aspects of CBioA, the innovative operational model implemented, and our ways of ensuring this was much more than simply a cost reduction exercise. As we have recently passed the first 5-year cycle, this paper also summarizes some of the concluding benefits, wins and lessons learned, and how we now plan to extend and develop the relationship even further moving into a new clinical laboratory partnership.

Bilirubin enzyme biosensor: potentiality and recent advances towards clinical bioanalysis.

Bilirubin detection plays a major role in healthcare. Its high concentration in human serum is lethal and must be determined accurately. Clinically, it is vital for assessing patients with deleterious health conditions such as jaundice or icterus, hepatitis, mental disorders, cerebral palsy and brain damage especially in the case of neonates. In evaluating the drawbacks regarding the conventional methodology of bilirubin detection, there is need for a superior analytical tool. Bilirubin oxidase (BOx)-based sensors have been designed for the ultrasensitive analysis of bilirubin and quality deliverance of treatment and this review highlights the different mechanisms of bilirubin detection using different modified electrodes. Further, it also addresses the exploitation of highly attractive electrocatalytic properties of elite nanoparticles such as gold and zirconia- coated silica nanoparticles in enhancing the reproducibility and specificity of bilirubin biosensors.

Programa Estudiantes Mentores, "Una experiencia valiosa en la FAREM-Carazo"

El programa de alumnos mentores se inició en el 2016, en el primer semestre con estudiantes de primer ingreso y con las asignaturas básicas como son: Lengua y Literatura, Introducción a la Física, Introducción a la Química y Matemática General, como iniciativa de la dirección de docencia de grado en conjunto con los departamentos docentes de la UNAN-Managua.Uno de los problemas más comunes con los que se encuentra un alumno de primer ingreso en el entorno universitario es su profundo desconocimiento del mismo, sobre todo si tenemos en cuenta el cambio radical en cuanto a metodología de trabajo y estudio con el que se afronta, lo que dificulta aún más su integración en la vida universitaria.Una de las medidas que se toman en UNAN-Managua, específicamente en la Facultad Regional de Carazo, es la implementación del programa alumnos mentores, quienes con su disponibilidad han permitido que el proyecto en este primer semestre del 2016 haya sido todo un éxito mejorando la calidad en el estudiantado y por ende, el rendimiento académico en la facultad.En este artículo se describe en detalle la experiencia llevada a cabo en la FAREM-Carazo, sobre la puesta en marcha del programa de mentoría. En el Proyecto Mentor los estudiantes de más de un año académico y bajo la supervisión de los profesores, ayudan y orientan a los estudiantes con algunos problemas académicos para que puedan superarlo y la experiencia muestra que se ha logrado el objetivo de mejorar la calidad y el rendimiento académico.Cabe recalcar, que el Departamento de Ciencias de la Educación y Humanidades fue el que estuvo al frente de este programa ejecutado por el equipo coordinador y dirigido por la Unidad Metodológica. La mayoría de los mentores fueron los alumnos del mismo departamento quienes se están formando para ser docentes, con excepción de dos estudiantes del Departamento de Ciencia Tecnología y Salud quienes asumieron el reto y lo cumplieron a cabalidad (un alumno de segundo año de Ingeniería Industrial que trabajó con la asignatura Introducción a la física y una alumna de tercer año de Bioanálisis Clínico que trabajó con Introducción a la Química).

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4°C and used within 10days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

A novel pretreatment method combining sealing technique with direct injection technique applied for improving biosafety.

People today have a stronger interest in the risk of biosafety in clinical bioanalysis. A safe, simple, effective method of preparation is needed urgently. To improve biosafety of clinical analysis, we used antiviral drugs of adefovir and tenofovir as model drugs and developed a safe pretreatment method combining sealing technique with direct injection technique. The inter- and intraday precision (RSD %) of the method were <4%, and the extraction recoveries ranged from 99.4 to 100.7%. Meanwhile, the results showed that standard solution could be used to prepare calibration curve instead of spiking plasma, acquiring more accuracy result. Compared with traditional methods, the novel method not only improved biosecurity of the pretreatment method significantly, but also achieved several advantages including higher precision, favorable sensitivity and satisfactory recovery. With these highly practical and desirable characteristics, the novel method may become a feasible platform in bioanalysis.

Highly sensitive protein detection based on DNAzyme cycling activated surface assembly of peptide decorated nanoparticles

In this work, a new method for the sensitive detection of proteins has been developed by combining the highly efficient DNAzyme cycling processing of DNA with nanoparticles which have an affinity for DNA. Specifically, DNAzymes are designed to be activated after recognition between the target protein and its aptamer, pre-hybridized with a DNAzyme sequence. In the meantime, DNAzyme substrates are pre-modified on an electrode surface so as to expose their 5′ phosphate end to conjugate with zirconia nanoparticles (ZrNPs), which have strong phosphoric affinity. The nanoparticles are also decorated with a large amount of signal molecule, peptide GHK. Consequently, a greatly amplified electrochemical signal can be achieved for the assay of the target protein. In addition to high sensitivity and simplicity, this strategy is also versatile, because it can easily be adapted to assay a large spectrum of targets simply by changing the sequence of the aptamer. Thus it holds great potential for the development of ultrasensitive biosensors in clinical bioanalysis.

Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes.

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

Biomarker discovery in mass spectrometry-based urinary proteomics.

Urinary proteomics has become one of the most attractive topics in disease biomarker discovery. MS-based proteomic analysis has advanced continuously and emerged as a prominent tool in the field of clinical bioanalysis. However, only few protein biomarkers have made their way to validation and clinical practice. Biomarker discovery is challenged by many clinical and analytical factors including, but not limited to, the complexity of urine and the wide dynamic range of endogenous proteins in the sample. This article highlights promising technologies and strategies in the MS-based biomarker discovery process, including study design, sample preparation, protein quantification, instrumental platforms, and bioinformatics. Different proteomics approaches are discussed, and progresses in maximizing urinary proteome coverage and standardization are emphasized in this review. MS-based urinary proteomics has great potential in the development of noninvasive diagnostic assays in the future, which will require collaborative efforts between analytical scientists, systems biologists, and clinicians.

An interlaboratory transfer of a multi-analyte assay between continents.

Alex has worked at GlaxoSmithKline for the past 15years and currently works within the bioanalytical and toxicokinetic group in the United Kingdom. Alex's role in previous years has been the in-house support of preclinical and clinical bioanalysis, from method development through to sample analysis activities as well as acting as PI for GLP bioanalysis and toxicokinetics. For the past two years, Alex has applied this analytical and regulatory experience to focus on the outsourcing of preclinical bioanalysis, toxicokinetics and clinical bioanalysis, working closely with multiple bioanalytical and in-life CRO partners worldwide. Alex works to support DMPK and Safety Assessment outsourcing activities for GSK across multiple therapeutic areas, from the first GLP study through to late stage clinical PK studies. Transfer and cross-validation of an existing analytical assay between a laboratory providing current analytical support, and a laboratory needed for new or additional support, can present the bioanalyst with numerous challenges. These challenges can be technical or logistical in nature and may prove to be significant when transferring an assay between laboratories in different continents. Part of GlaxoSmithKline's strategy to improve confidence in providing quality data, is to cross-validate between laboratories. If the cross-validation fails predefined acceptance criteria, then a subsequent investigation would follow. This may also prove to be challenging. The importance of thorough planning and good communication throughout assay transfer, cross-validation and any subsequent investigations is illustrated in this case study.

The quest for assay robustness across the life cycle of a bioanalytical method.

White has been an analytical chemist for 28 years, with a range of knowledge and experience across multiple disciplines including pharmaceuticals, foodstuffs, environmental and forensic analysis. Steve works within the regulated bioanalytical group at GSK supporting both preclinical and clinical bioanalysis. His responsibilities have included leadership of bioanalytical, dose formulation and sample management teams as well as assisting with the set up of the Anti-Doping Testing Laboratory for the London 2012 Olympics. In his current role, Steve is part of the UK bioanalytical leadership team with a focus on regulatory & compliance issues. Steve is the small molecule rep for GSK within the European Bioanalysis Forum (EBF), a member of the EBF Steering Committee and also contributed to the Global Bioanalysis Consortium S2 team. The successful validation or qualification of an assay plus incurred sample reproducibility gives the bioanalyst confidence of assay suitability. However, these elements alone do not give the full picture of how reliable an assay may be during routine production use for analysis of unknown study samples. The robustness of an assay can be understood by applying key performance indicators to monitor assay performance pre- and post-transfer between laboratories, plus a cross-validation step during assay transfer. The measurement of assay robustness (beyond the context of cross-validation) should not be considered as yet another regulatory layer, but more as a means for the bioanalyst to better understand an individual assay within a large portfolio, when used for its intended purpose.

The 2nd China Bioanalysis Forum Annual Conference: clinical bioanalysis and large molecule bioanalysis.

Beijing, China, 7-8 June 2014. The second China Bioanalysis Forum (CBF) conference was successfully held in Beijing, China, in June 2014 and covered the theme of clinical bioanalysis and large molecule bioanalysis. Compared with the first conference in 2013, this conference was attended by scientists from a greater number of scientific disciplines and geographic areas. This report summarizes the major topics in the conference as well as the future plan of the CBF.

A community of practice approach to bioanalysis delivery: the role of the Preclinical Bioanalysis and Toxicokinetics department at AstraZeneca.

Retaining regulated bioanalysis within AstraZeneca (AZ) is important for the effective delivery of nonclinical and clinical bioanalysis. The focus for the in-house team is the delivery of high-throughput bioanalysis and toxicokinetics support for investigational toxicology; however, by retaining GLP compliance, AZ will have an in-house understanding of every candidate drug that is progressed into development by supporting the first rodent GLP study for each project. The majority of nonclinical bioanalysis and all of the clinical bioanalysis are delivered by two external partnership models. This article describes the key benefits of retaining GLP compliance, the partnership interactions, discusses the management of non-GLP support in a GLP environment and how, having practical experience of regulatory facing method development, validation and study execution adds value to the AZ portfolio delivery.

In vivo and ex vivo SPME: a low invasive sampling and sample preparation tool in clinical bioanalysis.

Solid phase microextraction (SPME) is well-established technology in bioanalysis. Current review discusses the features of SPME, which determine the non- or low-invasiveness of the method in biomedical analysis. In the first section we analyze the factors, which have significant influence on the SPME sampling device performance in the view of sampling safety and efficiency. In the later sections applicability of various SPME approaches for analysis of easily accessible samples routinely used for analysis (e.g., urine, blood) as well as limited availability samples (tissues) is discussed. Moreover, the examples of sampling alternative matrices such as hair, saliva, sweat or breath are presented. The advantages and limitation of the technology in the view of future development of SPME are also reviewed.

Evaluation of Dried Blood Spots for The Quantification of Therapeutic Monoclonal Antibodies and Detection of Anti-Drug Antibodies

Recently, the potential of dried blood spots (DBS) for small-molecule bioanalysis by LC-MS has been explored. The goal of this investigation was to evaluate the use of DBS for the quantification of biologics, where bioanalysis is with immunoassay. Therapeutic monoclonal antibodies were successfully eluted from DBS and detected by immunoassays, and the procedure could be validated in alignment with current guidelines. Accuracy, precision, selectivity and dilution linearity were all within the acceptance criteria currently used for the validation of binding assays with serum samples. Serum and DBS samples obtained in parallel during a PK research study in rats were analyzed for drug and anti-drug antibodies using AlphaLISA(®) technology. Drug concentrations in both sample types showed a strong correlation, and there was very good alignment in detection of immunogenicity positive animals. Using two examples, we have demonstrated that therapeutic monoclonal antibodies can be accurately quantified in DBS, and since anti-drug antibodies could also be successfully detected, there is scope for application of DBS to preclinical and clinical bioanalysis of monoclonal antibody drugs and anti-drug antibodies.

Conference Report: Bioanalysis Highlights From The 2012 American Association of Pharmaceutical Scientists National Biotechnology Conference

The American Association of Pharmaceutical Scientists (AAPS) is an international forum for the exchange of knowledge among scientists to enhance their contributions to drug development. The annual National Biotechnology Conference, organized by the AAPS on 21-23 May 2012 in San Diego, CA, USA, brings together experts from various disciplines representing private industry, academia and governing institutions dedicated toward advancing the scientific and technological progress related to discovery, development and manufacture of medical biotechnology products. Over 300 scientific poster presentations and approximately 50 oral presentation and discussion sessions examined a breadth of topics pertaining to biotechnology drug development, such as the advancement of vaccines and biosimilars, emerging and innovative technologies, nonclinical and clinical bioanalysis, and regulatory updates. This conference report highlights the existing challenges with ligand-binding assays, emerging challenges, innovative integration of various technology platforms and applicable regulatory considerations as they relate to immunogenicity and pharmacokinetic bioanalytical assessments.

Development of A Bioanalytical Method For Quantification of A 15-Mer Oligonucleotide At Sub-Ng/Ml Concentrations Using Lc–MS/MS

LC-MS/MS provides a powerful technique for the selective quantification of therapeutic oligonucleotides; however, the LOQ (typically >1 ng/ml) may be higher than desirable for clinical bioanalysis. A method has been developed to allow quantification of a 15-mer unmodified DNA oligonucleotide in human plasma using SPE and UHPLC with MS/MS detection. The LOQ of this assay was 0.05 nM (∼250 pg/ml). This method was then further developed by the inclusion of online SPE to increase loading and apply additional sample cleanup. This allowed for improved assay precision at lower concentrations and increased signal, thus allowing the method to be validated over the range of 10-4000 pM (approximately 50-20,000 pg/ml). The method is accurate, precise and selective and it provides proof-of-concept for sub-ng/ml, high-throughput quantification of oligonucleotides using online SPE coupled to ion-pair, reversed-phase LC-MS/MS.

NEW TRENDS IN FLUORESCENCE IMMUNOCHROMATOGRAPHY

During the last decade, immunochromatographic techniques, in particular fluorescence labeled immunochromatography, have become more popular for the determination of low concentrations of analytes. It offers several potential advantages including highly sensitive detection and wide applications in clinical chemistry, bioanalysis, and environmental analysis. Currently, fluorescence labeled immunochromatography is not the exclusive preserve for a few specialists who are well experienced with antigen–antibody analysis, since the recent developments in fluorescence labeled immunochromatography have become widespread, from simple screening application to quantitative analysis of analytes. In this review, we assembled recent advances in the development and applications of fluorescence labeled immunochromatography techniques, considering their potential benefits in the future.

Conducting Remote Bioanalytical Data Monitoring and Review Based on Scientific Quality Objectives

For bioanalytical laboratories that follow GLP regulations and generate data for new drug filing, ensuring quality standards set by regulatory guidance is a fundamental expectation. Numerous guidelines and White Papers have been published by regulatory agencies, professional working groups and field experts in the past two decades, and have significantly improved the standards of good practices for bioanalysis. From a sponsor's perspective, continuous quality monitoring of the data generated by CRO laboratories, identifying adverse trends and taking corrective and preventative actions against issues encountered, are critical aspects of effective bioanalytical outsourcing management. This is especially important for clinical bioanalysis, where one validated assay is applied for analyzing a large number of samples of diverse demographics and disease states. This perspective article presents thoughts toward remote data monitoring and its merits for scientific quality oversight, and introduces a novel Bioanalytical Data Review software that was custom-developed and platform-neural, to conduct remote data monitoring on raw or processed LC-MS/MS data from CROs. Flexible, adaptive and user-customizable queries are applied for conducting project-, batch- and sample-level data review based on scientific quality performance factors commonly assessed for good bioanalytical practice.