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Bioanalytical method validation is the backbone of effective drug discovery and development. Its pivotal role ensures the generation of reliable and reproducible data from diverse species, leading to safe and effective therapeutics. As critical as this process is, scientists face many challenges, particularly regarding validation across different species in preclinical and clinical studies.
Accommodating species differences requires an understanding of species-specific metabolic and physiologic characteristics. Therefore, customizing a validating method may involve modifying sampling procedures, adjusting analytical parameters, or incorporating species-specific biomarkers. Whether method developers and sponsors do this work in-house or call on a laboratory partner for help, successful validation is when the method consistently delivers accurate and precise results within the required acceptance criteria and timeline.
To that end, we will explore challenges faced during method validation and examine some unexpected events that can occur during the process. We will frame this discussion through the lens of gaining regulatory compliance. The goal is to shed some light on this complex and nuanced stage of drug discovery and development.
Bioanalytical method validation challenges
Analytical methods are crucial in measuring analyte concentration or evaluating immunogenicity response. Validating each method for each specified species is required by regulators but can be especially challenging when trying to achieve precision, accuracy and specificity.
During a clinical study, a method might have been previously validated in a preclinical study, such as those involving rodent and/or non-human primates (NHPs). However, as the method progresses to a clinical stage, it becomes necessary to avoid using detection antibodies that were used previously, as their specificity cannot be confirmed. This change demands a different validation timeline and approach, extending the overall method development time.
A further challenge arises from target interferences. Many drugs, especially those based on monoclonal antibodies, are designed to target specific receptors related to a disease in tissue or on the cell surface. These circulating target levels in biological matrices such as blood can differ significantly between animal models and humans, leading to variations in assay specificity. Furthermore, the absence of a disease profile for certain animal models means matrix effect/interference is not evaluated in most preclinical studies. But when the assay is moved to a clinical study, the variations on the assay specificity regarding target is critical to ensure the method’s specificity and the data accuracy for clinical study.
The question of accessing different species also introduces challenges in the revalidation process. According to guidelines, each species-specific validation method must be validated for the specific matrix, which is usually related to the species’ biological matrices and the potential interferences.
In preclinical studies, the number of animals used is usually limited, and the validation focuses on pharmacokinetic/pharmacodynamic (PK/PD) and ADA immunogenicity assays. However, when validating a method for clinical studies, the specificity must relate to a larger, population-based context. For example, an initial study might be performed using a healthy donor matrix. But as the assay moves to patients, the method needs to be partially or even fully revalidated, depending on the extent of changes. This transition presents yet another layer of complexity, as the method must adapt to the different matrices between healthy donors and patients.
Variance in metabolic rates, protein binding and genetics remain the fundamental challenges in revalidating bioanalytical methods across species. Missteps can lead to inaccurate data interpretation, potential misjudging of a drug’s efficacy or safety, and ultimately result in failed clinical trials or adverse patient effects.
Handling unexpected events during revalidation
During any validation process, unexpected events arise, requiring adjustments and shifts in methodology. Before the validation of a method, a significant amount of development work occurs, during which many of the critical parameters are tested. These parameters often include accuracy, precision, target interference direction and preliminary stability for PK assays. For ADA, this might involve estimating cut points and selectivity.
While most of these parameters can be confirmed during development before moving into validation, some cannot be evaluated at this stage. An example of this would be the selectivity of an ADA/PK assay in disease serum. This is mainly due to the limited availability of the disease matrix. Consequently, a typical ADA/PK assay might be unable to test selectivity even at the validation stage. Moreover, in-study cut point flexibility is needed to check clinical studies for ADA assays. In-study cut points may also be evaluated towards the clinical population matrix, when the method is validated using different population matrices (i.e., from a healthy donor).
To address this issue, scientists usually use pre-dose study samples to evaluate selectivity. As a result, some work that should ideally be assessed or confirmed during the validation process must be shifted to the study sample analysis stage. This adjustment is necessary due to the limited availability of the disease matrix. Therefore, dealing with these unexpected events requires a certain level of flexibility and adaptability during the validation process.
Unexpected events can also significantly impact development timelines and costs. Lab testing partners often receive requests from clients whose timeline is a significant concern. Therefore, completing the method revalidation within a reasonable period can significantly benefit clients, their clinical studies, and future projects. If the process needs to be shifted to a new species, and there are no existing data or results from previous testing, method development must start from scratch. This means creating a new assay, which entails evaluating fundamental parameters such as sensitivity and the concentration of critical reagents. This validation process applies to both PK assays and ADA assays.
The time spent on method validation and development for one species would have to be reinvested for the new species. This inevitably extends the project timeline, incurring additional costs and requiring more resources. However, if previous experience or data can be applied to the new process, it can significantly shorten discovery and development timelines.
It is common for clients to plan for long-term success from the early stages of their preclinical studies, sometimes even while planning their toxicology studies and clinical studies. Thus, an efficient and cost-effective revalidation process is crucial to a project’s long-term success.
Gaining regulatory compliance during revalidation
Regulatory bodies play pivotal roles in ensuring the reliability and accuracy of bioanalytical method validation. They provide guidelines, including parameters for method validation like precision, accuracy and specificity. Their primary concern is ensuring that any method used in drug discovery and development is scientifically sound and reproducible. That said, some bioanalytical techniques are too new or too complex to have clear guidance.
Global regulatory bodies have diverse expectations, and achieving compliance can be multifaceted. A typical starting point is participating in meetings and reading published papers from the American Association of Pharmaceutical Scientists (AAPS) and Workshops on Recent Issues in Bioanalysis (WRIB). These resources offer opportunities to learn about cutting-edge developments, raise concerns with regulators or provide feedback on guidance documents on bioanalysis. This industry collaboration is especially valuable in cases where regulators gather input for drafting guidelines for specific processes that lack official guidance—qPCR and flow cytometry assays are two examples of this.
Maintaining robust documentation and record-keeping systems is another vital aspect of gaining regulatory compliance. Regular internal quality control (QC) checks and/or quality assurance (QA) inspections can also ensure compliance with the current regulations. Performing these checks and inspections routinely—as opposed to only when preparing for regulatory inspections—can help developers and sponsors prepare thorough documentation and reduce timeline delays.
Investing in relationships with laboratory partners
Laboratories with experience in method transfer can help shorten drug development timelines when transitioning to a new species. But while method transfer from one lab to another is common, it’s often not straightforward. Transitioning from one analytical team to another can foster miscommunication and even loss of information. Sticking with the same lab partner from drug discovery to development has these additional advantages:
Continuity: Using the same lab provides continuity as it would be familiar with the project from the outset. These partners would undoubtedly understand the compound, the validation and analytical methods used, and any specific challenges associated with the project. This in-depth knowledge would likely lead to more efficient troubleshooting and problem-solving as the project advances.
Streamlined communication: Working with a single lab partner reduces the risk of miscommunication or information loss when transitioning between different labs. The same team would have been part of the process, fostering better understanding and reducing the time required for catch-up or context-setting.
Data consistency: Data consistency is critical in all stages of drug development. The same lab, employing consistent methodologies, would likely produce uniform data sets, simplifying data interpretation and potentially leading to more accurate conclusions.
Time & cost efficiency: Onboarding a new lab partner requires bringing them up to speed, aligning them with project specifics and ensuring regulatory compliance—all of which takes time and diminishes resources. In addition, long-term collaborations often enable cost-effective packages, further adding to cost efficiencies.
Regulatory compliance: Using a single lab from the preclinical to clinical stages would clarify the regulatory considerations specific to the project. It would also help organize the data and documentation generated at each stage, ensuring smooth transitions between stages.
Trust and relationship building: Over time, a strong partnership can develop, leading to better collaboration, communication and problem-solving.
An often-overlooked advantage of working with a comprehensive lab partner from preclinical testing to clinical trials is that it eliminates the need for sponsors to invest in costly technology and train staff to use it. Assay development can be lengthy and can change significantly during each development stage. Building these in-house capabilities for limited use cases can waste time and resources.
A final word on bioanalytical methods
Scientists sometimes struggle to perfectly replicate bioanalytical methods across species, even with complete and fully validated protocols. Challenges arise from species-specific metabolic and physiological differences, necessitating different protocols for large and small molecules. Not to mention, unexpected events often demand flexibility and timely adjustments.
Teaming up with a lab partner with a proven track record, sufficient technical capabilities, and a robust quality assurance system can mitigate these challenges and ultimately create better therapeutics. The bottom line is that the potential for efficient drug development cannot compromise the quality and reliability of the testing process.
Jinping Lai, Ph.D, joined WuXi AppTec, the Laboratory Testing Division (NEJ) in July 2022 as Technical Director for Global Large Molecule Bioanalysis. Currently he is working as Director, Large Molecule Bioanalysis. Dr. Lai earned his Ph.D in Analytical Chemistry. His academic research was focused on Nano medicine and biomaterials, led to over 30 peer reviewed publications and 2 US patents. Beyond these, Dr. Lai has earned extensive experience in GLP/GCP/GCLP regulated preclinical/clinical bioanalysis of large molecule pharmacokinetics (PK), immunogenicity and PD biomarkers for various drug modalities, from method development, validation to study sample analysis.
Yasuhiro (Jim) Yamashita, Ph.D., joined WuXi AppTec Laboratory Testing Division (NEJ) as a Senior Director of Small Molecule Bioanalytical Services (BAS) in July 2021. He is the head of the bioanalysis group in support of regulated bioanalytical analysis using LC-MS/MS. Dr. Yamashita has more than 21 years of experience in regulated bioanalysis, primarily using LC-MS/MS to quantify various types of drugs (e.g., small molecule, oligonucleotide, peptide, antibody) under GxP. Prior to joining WuXi AppTec, Dr. Yamashita worked for regulated bioanalytical departments at multiple CROs in the United States and Japan and has extensive knowledge of regulations from both countries. He received his Ph.D. in Biotechnology and Applied Microbiology from Kansai University, Osaka, Japan.