Surendra Bansal

Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays

AbstractThe Third AAPS/FDA Bioanalytical Workshop, entitled “Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays” was held on May 1–3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.ConclusionFor quantitative bioanalytical method validation procedure and requirements, there was a relatively good agreement between chromatographic assays and ligand-binding assays. It was realized that the quantitative and qualitative aspects of bioanalytical method validation should be reviewed and applied appropriately.1.Some of the major concerns between the 2 methodologies related to the acceptable total error for precision and accuracy determination and acceptance criteria for an analytical run. The acceptable total error for precision and accuracy for both the methodologies is less than 30. The 4–6–15 rule for accepting an analytical run by a chromatographic method remained acceptable while a 4–6–20 rule was recommended for ligand-binding methodology.2.The 3rd AAPS/FDA Bioanalytical Workshop clarified the issues related to placement of QC samples, determination of matrix effect, stability considerations, use of internal standards, and system suitability tests.3.There was a major concern and issues raised with respect to stability and reproducibility of incurred samples. This should be addressed for all analytical methods employed. It was left to the investigators to use their scientific judgment to address the issue.4.In general, the 3rd AAPS/FDA Bioanalytical Workshop provided a forum to discuss and clarify regulatory concerns regarding bioanalytical method validation issues.

Key elements of bioanalytical method validation for small molecules

Method validation is a process that demonstrates that a method will successfully meet or exceed the minimum standards recommended in the Food and Drug Administration (FDA) guidance for accuracy, precision, selectivity, sensitivity, reproducibility, and stability. This article discusses the validation of bioanalytical methods for small molecules with emphasis on chromatographic techniques. We present current thinking on validation requirements as described in the current FDA Guidance and subsequent 2006 Bioanalytical Methods Validation Workshop white paper.

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns.

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0-3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.

Historical perspective on the development and evolution of bioanalytical guidance and technology.

Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological fluids provide essential regulatory data for bioavailability, bioequivalence, pharmacokinetic and toxicokinetic studies. The quality of these studies is directly related to the underlying bioanalytical data. Data generated by a typical bioanalytical laboratory is submitted to not only the local regulatory agency, but also to multiple regulatory agencies worldwide. Many pharmaceutical companies and CROs are now performing bioanalytical work for global submissions and the regulatory agencies are often reviewing the bioanalytical work performed in other countries. The bioanalytical workplace has become global and therefore needs universal rules for quality and compliance of bioanalysis. This paper provides a historical perspective and insight into the development and evolution of the regulatory guidance for bioanalytical method validation and analysis of samples.

Bioanalysis of alectinib and metabolite M4 in human plasma, cross-validation and impact on PK assessment

BACKGROUND Alectinib is a novel anaplastic lymphoma kinase (ALK) inhibitor for treatment of patients with ALK-positive non-small-cell lung cancer who have progressed on or are intolerant to crizotinib. To support clinical development, concentrations of alectinib and metabolite M4 were determined in plasma from patients and healthy subjects. METHODS LC-MS/MS methods were developed and validated in two different laboratories: Chugai used separate assays for alectinib and M4 in a pivotal Phase I/II study while Roche established a simultaneous assay for both analytes for another pivotal study and all other studies. CONCLUSION Cross-validation assessment revealed a bias between the two bioanalytical laboratories, which was confirmed with the clinical PK data between both pivotal studies using the different bioanalytical methods.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

Regulated Bioanalysis: Documentation and Reports

The early Crystal City Bioanalytical workshops did not discuss the requirements for bioanalytical documentation and reports. The major bioanalytical guidance from FDA and EMA provided only broad outlines for the requirements in bioanalytical documentation and reports. The bioanalytical practitioners were therefore left to decide what to document in the reports and what to maintain in the archives for inspection. This has led to bioanalytical reports of various shapes and sizes. Parallel to the development of the bioanalytical guidance, ICH developed an electronic Common Technical Document (eCTD) guidance for preparing regulatory reports. Recently, FDA issued a mandatory guidance for submission of data and reports electronically for certain submissions. These latter changes have also been influencing how the bioanalytical data and reports would be submitted in the future. This chapter provides an overview of the bioanalytical documentation and reports for bioanalytical studies intended for regulatory submission.