Patrick Bennett

Identifying trends and developing solutions for incurred sample reanalysis failure investigations in a bioanalytical CRO

In 2007, the US FDA recommended that pharmaceutical companies and CROs conduct incurred sample reanalysis (ISR) following the analysis of study samples using validated methods. Between January 2008 and December 2009, over 250 separate analytes were tested for ISR in our laboratory (a bioanalytical CRO). Among these, nine analytes initially failed for ISR. While thorough investigations were conducted to identify the root cause of ISR failure for each study, these investigations were often painfully tedious and very costly, both financially and in terms of project timelines. In this paper, three representative studies are presented to showcase the detailed investigation processes, methodologies and final conclusions of the ISR investigations. Additionally, all nine ISR failures are analyzed to identify trends or common elements of the studies or methods that might help identify potential problems before they occur. Furthermore, suggestions and recommendations to minimize future ISR failures are provided.

Performance assessment of microflow LC combined with high-resolution MS in bioanalysis

BACKGROUND 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. RESULTS 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. DISCUSSION 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.

Method development and validation of six bile acids for regulated bioanalysis: improving selectivity and sensitivity

BACKGROUND Quantification of bile acids using LC-MS has previously been very challenging on triple quadrupole MS systems due to the absence of a primary fragment ion for unconjugated bile acids. RESULTS A LC-high-resolution/accurate mass MS method for the analysis of six bile acids (cholic acid, chenodeoxycholic acid, taurocholic acid, deoxycholic acid, lithocholic acid and ursodeoxycholic acid) was developed and successfully validated. The method includes a single extraction and a single injection with all analytes separated using target-selected ion monitoring (SIM) mode in two periods with a resolution of 70,000 and 140,000, respectively. CONCLUSION This is the first LC-high-resolution/accurate mass assay fully validated to quantify six bile acids for regulated bioanalysis.

Small Molecule Specific Run Acceptance, Specific Assay Operation, and Chromatographic Run Quality Assessment: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Teams

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.

Parallelism experiments to evaluate matrix effects, selectivity and sensitivity in ligand-binding assay method development: pros and cons

Parallelism is an essential experiment characterizing relative accuracy for a ligand-binding assay (LBA). By assessing the effects of dilution on the quantitation of endogenous analyte(s) in matrix, selectivity, matrix effects, minimum required dilution, endogenous levels of healthy and diseased populations and the LLOQ are assessed in a single experiment. This review compares and discusses all available approaches that can be used to assess key assay parameters for pharmacokinetic and biomarker LBAs, as well as the advantages and disadvantages of each approach. This review also summarizes a systematic approach that can apply to guide endogenous LBA method development and optimization with a suggested way to interpret parallelism data.

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this years closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industrys best practices and the conclusions from the discussion of these topics is included in this meeting report.

Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance

Flow cytometry is increasingly becoming an important technology for biomarkers used in drug discovery and development. Within clinical development flow cytometry is used for the determination of PD biomarkers, disease or efficacy biomarkers or patient stratification biomarkers. Significant differences exist between flow cytometry methodology and other widely used technologies measuring soluble biomarkers including ligand binding and mass spectrometry. These differences include the very heavy reliance on aspects of sample processing techniques as well as sample stabilization to ensure viable samples. These differences also require exploration of new approaches and wider discussion regarding method validation requirements. This paper provides a review of the current challenges, solutions, regulatory environment and recommendations for the application of flow cytometry to measure biomarkers in clinical development.

Meeting the challenges of bioanalytical outsourcing: understanding the hidden cost of price pressure

Historically, it is important for any company to continually reduce the cost of developing their products. These pressures exist in both small start-up pharmaceutical companies, as well as the largest companies. These efforts include reducing outsourcing costs for drug development. The focus of this editorial is to describe the impact this pressure is having on the relationship between pharmaceutical companies and the bioanalytical CROs that support them.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canadas Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

Logistical and Operational Practice in the Regulated Bioanalysis Laboratory

This chapter provides information on the internal and external logistics and practices required to operate a regulated bioanalytical laboratory. Despite commonality afforded by health authority guidances , the organizational structure of laboratories conducting regulated bioanalysis varies across the industry. For example, an internal bioanalytical laboratory operating within a pharmaceutical company is likely to be structured and operate significantly differently from a contract research organization (CRO) laboratory focused on the same discipline. From personal experiences, we can attest to the potential benefits for different operational structures, tools, and practices depending on the size and geographical footprint of a bioanalytical organization. Particularly, as a bioanalytical laboratory grows, the need to adapt to the scale of data handling, information management, and associated communications requires operational structures to evolve accordingly. These and other variables discussed in this chapter demonstrate the operational and logistical differences between different types of bioanalytical laboratories. Despite the structural differences, there are also some common logistical and operational requirements for all regulated bioanalytical laboratories including: (1) Information Technology (IT) systems that provide security, data management, and automation, (2) Standard Operating Procedures (SOPs) and policies that drive both regulated and business activities, (3) metric tracking that assists in both business operations and scientific operations, and (4) document and sample lifecycle management.