Regulatory changes are reshaping toxicology, with precision now at the forefront. We explore how accurate target analysis is transforming drug development, particularly for biotherapeutics.
Scientists in lab coat working with test tubes
In recent years, regulatory focus has shifted from sensitivity to accuracy, marking a pivotal change in how we approach toxicological assessments. While sensitivity remains essential, our growing understanding of toxicology highlights the critical importance of precise target analysis. This shift in focus is especially critical in toxicology, where accurate target analysis plays a vital role in identifying toxic effects and ensuring patient safety, particularly as the field transitions from traditional drugs to the promising realm of biotherapeutics, especially for rare diseases. Despite these advancements, analysing biologically derived targets remains a formidable challenge due to interference from endogenous molecules. This issue poses a significant hurdle for drug developers, with no universal protocol currently in place to address these complexities.
In this article, we share our hard-earned insights into overcoming these critical challenges. As a contract research organisation, we have supported numerous clients in overcoming matrix interference, particularly small and emerging biotherapeutics developers who may lack the in-house expertise to navigate such complex problems. Matrix interference in toxicology can lead to misinterpretation of data, potentially resulting in unsafe drug dosages or overlooked toxic effects. In this context, collaboration is essential to achieving the sensitivity and accuracy required by regulatory bodies. Accurate toxicology data is crucial for regulatory approval. Our approach, grounded in Good Laboratory Practice (GLP) and honed through thousands of studies, ensures that even the most subtle toxicological indicators are reliably detected. By employing advanced analytical techniques like high-resolution mass spectrometry (HRMS) and carefully calibrating systems with optimal internal standards (IS), we effectively mitigate matrix effects and provide reliable preclinical toxicology data. This approach not only boosts our clients’ ability to achieve high accuracy and meet regulatory requirements but also supports the seamless transition of innovative therapies from the lab to the clinic with confidence.
Starting with the right internal standard
Selecting the appropriate internal standard (IS) is vital in toxicology, as inaccuracies could obscure toxic metabolites and lead to flawed safety evaluations. This decision is pivotal in crafting biologically relevant calibration curves. Analysts need to thoroughly understand the analyte, matrix, and any inactive ingredients to choose an IS that won’t interfere with the analysis. Purity and radioactive enrichment are key factors in ensuring the IS aligns with product specifications.
Special attention is required when dealing with antibody-based therapeutics due to their high susceptibility to biotransformation. For these studies, a comprehensive approach to drug metabolism and pharmacokinetics (DMPK), along with immunogenicity is essential, drawing on expertise from multiple disciplines. Once the IS has been selected, a quantitation range can be set using reference solutions.
Defining the quantitation range
An accurate calibration range is essential in toxicology to ensure precise detection of toxic substances, preventing both false positives and false negatives. To determine the quantitation range for bioanalytical methods, you must first set this range based on the expected analyte concentrations in a study. This step is critical for ensuring that the method is both suitable and capable of delivering high-quality data.
Once the quantitation range is defined, precise quantification within this range relies on using reference solutions. These solutions must contain analytes and internal standards at known concentrations. The lower limit of quantification (LLOQ) should correspond to the lowest calibration standard concentration, ensuring complete recovery. For studies with narrow calibration ranges, additional quality controls (QCs) are included to maintain accuracy. If peak plasma concentrations (Cmax) significantly differ from expected QC levels, adjustments to calibration curves and the addition of extra QCs are necessary.
When to recalibrate in toxicology studies
Proper recalibration in toxicology studies is crucial to prevent skewed data, which could compromise the assessment of a drug’s safety profile. To ensure high interassay precision, calibration standards must be accurate within 15 percent of nominal values, with the lower limit of quantification (LLOQ) within 20 percent. Preparing calibration curves (CC) and quality control (QC) samples requires meticulous attention, particularly when dealing with endogenous compounds that can interfere with the analyte. The challenge lies in ensuring that calibrators and QCs are prepared in blank matrices that are completely free from interference or matrix effects, as even trace amounts of endogenous molecules can distort results and lead to inaccurate toxicological assessments.
Common solutions to this challenge include chemical precipitation and liquid-liquid extraction. However, these methods can be labour-intensive and may not always be effective, making them less practical for routine toxicological studies. At Sannova, we have developed a method whereby a charcoal matrix is used to eliminate endogenous compounds before analysis. Charcoal effectively adsorbs a wide range of interfering substances, ensuring a cleaner sample for analysis. However, this method isn’t universally applicable, as some matrices may not be compatible with charcoal treatment due to their specific composition or the nature of the analytes involved. The choice of method depends on the matrix and analyte characteristics, emphasising the need for flexible solutions in toxicology.
Interassay precision
In toxicology, maintaining high interassay precision is key to ensuring that toxicological data is consistent and reliable across different study phases. Choosing the right IS and establishing a quantitation range that ensures linearity and optimised calibration curves are vital steps in achieving reliable analytical results. Additionally, incurred sample reanalysis (ISR) plays a crucial role in validating the precision and reliability of reported analyte concentrations, which is particularly important for toxicological assessments where consistent data is critical for identifying potential safety risks. Given that ISR is a mandatory requirement for studies submitted in New Drug Applications (NDAs), Biologics License Applications (BLAs), or Abbreviated New Drug Applications (ANDAs), it ensures that toxicology data meets the stringent criteria necessary for regulatory approval, further underscoring the importance of accuracy in toxicological evaluations.
Leveraging efficient techniques to overcome signal suppression
Overcoming signal suppression is crucial in toxicology to ensure the accurate detection and quantification of low-abundance toxic compounds. While much of what we’ve discussed applies to traditional MS, HRMS stands out for its exceptional sensitivity and accuracy, especially when analysing complex matrices. Unlike traditional mass spectrometry methods, HRMS offers superior ionization and fragmentation efficiency, which effectively mitigates signal suppression issues common in intricate sample environments. This heightened resolution ensures remarkable precision for both untargeted and targeted analyses, resulting in more reliable and detailed outcomes.
HRMS excels in providing quantitative insights, which is particularly valuable for understanding complex biological processes. This is especially important in areas such as drug transporter inhibition, where precise time profiles are crucial. HRMS also helps differentiate and quantify low-abundance analytes, even in the presence of endogenous compounds. In addition to tackling the complexities of endogenous molecule measurement, HRMS is also effective in evaluating excipients, potential drug metabolites, and drug-drug interactions. This highlights its vital role in modern biotherapeutic research. As biotherapeutic development continues to grow, regulatory bodies are increasingly endorsing advanced mass spectrometry techniques.
A shifting analytical landscape
The regulatory shift from sensitivity to accuracy marks a transformative change in toxicological assessments, emphasising the need for precise target analysis as the field moves from traditional drugs to biotherapeutics, particularly for rare diseases. An approach grounded in GLP, coupled with advanced analytical techniques, is essential for effectively mitigating matrix effects. Such an approach ensures reliable preclinical toxicology data and supports the transition of innovative therapies from the lab to clinical trials. For small- and medium-sized pharmaceutical companies, outsourcing represents a strategic leap forward, offering a gateway to unparalleled expertise and cutting-edge analytical technologies. This approach not only accelerates the regulatory review process for preclinical submissions but also positions companies to navigate the complex landscape with greater agility and precision.
BHUPESH-D-SOMPURA
About the author
Bhupesh D. Sompura, Team Lead of Method Development at Sannova Analytical
Bhupesh D. Sompura, Method Development Team Lead at Sannova Analytical, brings over 18 years of experience in the Bioanalytical Laboratory Department. He has a strong background in LC-MS/MS analysis and specialises in developing high-quality, robust, and high-throughput bioanalytical methods. Bhupesh excels in method validation following protocols, GLP, and SOPs, with extensive expertise in calibrating analytical instruments such as HPLC, LC/MS/MS, ICP-MS, HRMS, and GC/MS.