Shifting Sands: From Sensitivity to Accuracy in Bioanalysis

Introduction

In recent years, regulatory focus has notably shifted from sensitivity to accuracy, underscoring a pivotal change in how we approach toxicological assessments. While sensitivity remains a critical requirement, our advancing understanding of toxicology has highlighted the crucial need for precise target analysis. This shift in focus is particularly crucial in toxicology, where the accuracy of target analysis directly impacts the identification of toxic effects and ensures patient safety, especially as the field moves away from traditional drugs and embraces the exciting potential of biotherapeutics, particularly for rare diseases.

Despite these advancements, analyzing biologically derived targets remains a formidable challenge due to the interference from endogenous molecules. This persistent issue poses a significant hurdle for drug developers, with no universal protocol currently in place to address these complexities. In this article, we offer our hard-won insights into navigating these critical challenges. As a contract research organization, we’ve assisted countless clients in overcoming matrix interference, particularly for small and emerging biotherapeutics developers who may lack the in-house expertise to tackle such intricate problems. Matrix interference in toxicology can lead to misinterpretation of toxicological data, potentially resulting in unsafe drug dosages or overlooked toxic effects. Here, collaboration becomes key to achieving the level of sensitivity and accuracy demanded by regulatory bodies.

Accurate toxicology data is essential for regulatory approval. Our approach, rooted in Good Laboratory Practice (GLP) and refined through extensive experience from thousands of studies, ensures even the most subtle toxicological indicators are reliably detected. By employing advanced analytical techniques like high-resolution mass spectrometry (HRMS) and meticulously calibrating systems with optimal internal standards (IS), we effectively mitigate matrix effects and deliver reliable preclinical toxicology data. This framework not only enhances our client’s ability to achieve high levels of accuracy, helping to meet regulatory requirements but also propels 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 here 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) and immunogenicity is essential, drawing on expertise from multiple disciplines. Once the IS is selected, a quantitation range can be set using reference solutions.

Defining the Quantitation Range

An accurate calibration range is critical in toxicology to ensure that the detection of toxic substances is precise, preventing both false positives and 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

Proper recalibration in toxicology studies is essential to avoid skewed data, which could compromise the assessment of a drug’s safety profile. To ensure high inter-assay precision, calibration standards must be accurate within 15% of nominal values, with the LLOQ within 20%. 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 truly free from interference or matrix effects, as even trace amounts of endogenous molecules can distort results, leading to inaccurate toxicological assessments.

Common solutions to this challenge include chemical precipitation and liquid-liquid extraction. However, these methods can be labor-intensive and are not always 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; 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, highlighting the need for flexible solutions in toxicology.

Inter-assay Precision

In toxicology, maintaining high inter-assay 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 optimized 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 critical in toxicology to ensure that low-abundance toxic compounds are accurately detected and quantified. While much of what we’ve discussed is relevant to traditional MS, high-resolution mass spectrometry (HRMS) distinguishes itself with exceptional sensitivity and accuracy, particularly in analyzing 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 especially valuable for understanding complex biological processes such as drug transporter inhibition, where precise time profiles are crucial, and for differentiating and quantifying low-abundance analytes in the presence of endogenous compounds.

In addition to tackling the complexities of endogenous molecule measurement, HRMS is also adept at evaluating excipients, potential drug metabolites, and drug-drug interactions. This underscores its vital role in modern biotherapeutic research. Reflecting its advantages and the increasing focus on biotherapeutics development, regulatory bodies are progressively endorsing advanced mass spectrometry techniques.

A Shifting Analytical Landscape

The regulatory shift from sensitivity to accuracy marks a transformative change in toxicological assessments, emphasizing the need for precise target analysis as the field advances from traditional drugs to biotherapeutics, particularly for rare diseases. An approach grounded in GLP and leveraging advanced analytical techniques is the key to 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.

Author Details 

Shukla Antriksh Pushpakbhai - Team Lead, Sannova Analytical

Shukla Antriksh Pushpakbhai, a Team Lead at Sannova Analytical, started his career as a research scientist and has about two decades of experience in bioanalytical method development and scientific review of study and validation results. Antriksh is a subject matter expert in instruments like LC/MS/MS and HPLCs, performing IQ, OQ, and PQs. He has also trained and mentored various junior scientists, contributing significantly to their professional growth and the success of bioanalytical research projects.

Publication Details 

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