Bioanalysis in drug development

RAPS 2024 Article of the Year  The importance of bioanalytical methods during drug development is well established. These methods are used to determine the concentration of the drug and its metabolites and biomarkers in physiological fluids, such as blood, serum, plasma, urine, and cerebrospinal fluid; tissue, such as skin; and tumor biopsies. Bioanalytical data pertaining to the pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic profile of the drug is crucial in assessing the safety and efficacy of the drug and guiding regulatory decision making. The considerations and challenges associated with developing sensitive and robust bioanalytical methods are discussed in this article, including the choice of sample preparation methods and the advantages and drawbacks of chromatographic methods and ligand binding assays (LBAs). Regulatory guidelines associated with development and validation of phase-appropriate bioanalytical methods are also discussed.
 
Keywords – bioanalytical, assays, nonclinical, clinical, validation, GLP
 

Introduction

Development of new drugs is a lengthy, complex, expensive, and multidisciplinary process. Drug discovery and development takes more than 10 years, with an average cost exceeding $1 billion for each new drug approval for clinical use.¹ Furthermore, 90% of drug development programs fail at the clinical development stage, primarily because of lack of clinical efficacy (40%-50%) and unmanageable toxicity (30%).² Figure 1 shows the process of drug development, duration, and the likelihood of success through each stage.
 
Figure 1. Process of drug development, duration, and likelihood of success through each stage^a
 
 
aIn general, out of 250 drug candidates assessed at drug discovery stage, 10-20 drug candidates proceed to preclinical development, and only 1 drug reaches final approval
after successfully completing clinical evaluation.
 PK, pharmacokinetic 
Adapted from Sun et al.²
 
Strategies should be used to improve each step of drug development and to acquire useful information early in the program. This article describes the role of bioanalytical methods in drug development, including drug discovery and nonclinical and clinical studies. The considerations and challenges associated with developing bioanalytical methods and the validation requirements and parameters are also described. Immunogenicity assays are beyond the scope of this article and are not covered here.
 

Drug development

Drug discovery/initial research

Drug discovery/initial research involves the identification of lead targets or molecules from among a large number of candidate molecules. Subsequently, a selection of these shortlisted molecules is further assessed for their pharmacological and biological activity. Physiochemical properties of the molecule, such as solubility, lipophilicity, and stability, are determined.³ In addition, in silico and in vitro studies are used to determine the absorption, distribution, metabolism, and excretion (ADME) characteristics, such as bio-oral availability, plasma protein binding, metabolic stability, transporter interactions, and plasma-blood distribution. In vivo studies are carried out to characterize the PK properties, including drug exposure and confirmation of in vitro ADME properties in selected animal models.⁴ For these assessments, it is critical to develop sensitive and quantitative bioanalytical methods that can accurately measure the concentration of the drug in physiological fluids.⁵ These methods are also used to determine associated metabolites, proteins, and biomarkers. This in turn helps establish and validate pharmacologically relevant animal models to support drug candidate selection and for subsequent nonclinical studies. At this stage, the bioanalytical assays must be high throughput and need not be validated. However, some parameters, such as selectivity and specificity, range of quantitation, accuracy and precision, and acceptance criteria, are defined to ensure the data generated are fit for purpose.⁶

Nonclinical studies

Nonclinical studies focus on the evaluation of safety and efficacy of drugs in animals. These studies are also known as preclinical studies when performed before conduction of any clinical trials. Extensive nonclinical studies need to be carried out before the drug can be taken in first-in-human (FiH) clinical trials, including pharmacology, PD, PK, and toxicity studies. There are several International Council for Harmonisation (ICH) guidelines available for developing nonclinical safety programs depending on the product type and the therapeutic indication.^4,7-10 Typically, pivotal safety pharmacology, toxicokinetic, genotoxicity, and repeat-dose toxicity studies must adhere to 21 CFR 58 for submission to the US Food and Drug Administration (FDA)⁵ and must be performed in compliance with good laboratory practice (GLP) standards.^4,5
 
The pharmacology studies provide information on the potency of the drug and its PD and PK profile, which in turn helps confirm the drug’s relevance and potential in the human clinical trial.¹¹ The PD studies evaluate the mechanism of action and the therapeutic effects of the drug. Biomarkers as PD endpoints can provide an insight into drug efficacy.⁴ The PK studies provide data on exposure-response curves (ADME properties) in the animal model, which can provide an estimation of the therapeutically relevant and effective therapeutic dose range for humans. Toxicological assessment of the drug helps determine the potential toxic effect of the drug along with any side effects, which helps minimize risk in humans as well as determine the safety biomarkers to be assessed in the clinical trial.^4,11 The toxicity studies also help determine the therapeutic dose range, maximum tolerated dose, and no- observed-adverse-effect level in animals, which predicts the relevant and safe human dose.^4,11
 
Samples are collected and analyzed at various timepoints in the in vivo studies, depending on the drug and therapeutic indication for measuring the drug concentration, metabolites, biomarkers, and proteins/peptides.^11,12 Bioanalytical methods are validated to GLP standards at this stage of development, because the nonclinical data directly affects the FiH clinical study, with specific validations occurring for each species and matrix analysis. Sample analysis must also be conducted within the quality environment.
 

Clinical studies

The drug development program can proceed to FiH clinical trials if the nonclinical studies demonstrate sufficient safety and efficacy of the product. Phase 1 clinical trials are typically conducted in healthy volunteers to determine the PK profile and investigate dose escalation and the toxicity profile of the drug in humans. Approximately 70% of drugs are successful in this phase and move to Phase 2 clinical trials. Phase 2 and 3 trials investigate the efficacy of the drug, dose range, and toxicity profile of the drug in patients.^6,13 Again, bioanalytical methods are required for analyzing samples throughout the clinical studies for determining drug concentration and measuring biomarkers and protein and/or peptides. Bioanalytical methods must be fully validated at this stage of drug development, and while clinical bioanalysis cannot be conducted to GLP, equivalent acceptance criteria are typically used. Regulatory bodies, such as the FDA and European Medicines Agency, are now adopting the ICH M10¹² guidance for harmonized validation, analysis, and reporting for PK methods. For specific bioequivalent and bioavailability studies, the method must also meet the criteria specified in 21 CFR 320 bioequivalence and bioavailability requirements (i.e., 21 CFR 320.29) for submission to FDA.⁵
 
Similar to nonclinical studies, bioanalysis provides invaluable information pertaining to the safety and the efficacy of the drug in humans by investigating PD endpoints, bioavailability, and/or bioequivalence.^12,14 Bioavailability studies determine the rate and extent to which the drug is absorbed in the body and is available at the site of action. Bioequivalence studies determine the bioavailability of the drug compared with pharmaceutically equivalent drugs or alternatives at the same dose. Bioequivalence study is important when formulation changes or manufacturing changes have occurred during the drug development and at postapproval stages.¹⁴
 

Bioanalytical methods

Bioanalytical methods should be sensitive, specific, reproducible, and able to measure drug concentration in the biological matrix with high accuracy.^5,12 Challenges associated with bioanalytical assays include complex composition of biological sample (presence of many interfering substances), low drug concentration in the sample, limited sample volume (limited options for repeat testing), high variability associated with same subjects at different timepoints, and having different subjects within the same group.¹⁵ During method development, it is important to understand and consider the drug’s physiochemical properties, in vitro and in vivo metabolism, and protein binding properties.¹²
 

Detection and quantitation of the drug

Detection and quantitation of the drug in biological samples is typically achieved using chromatographic assays and LBAs.^5,12
 
Chromatographic assays. Although high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UPLC) can be coupled with a range of detectors, including ultraviolet, fluorescence, and charged aerosol detection, among others, the gold standard is now considered to be HPLC or UPLC coupled with liquid chromatography tandem mass spectrometric detection (LC-MS/MS). LC-MS/MS is considered the benchmark analytical methodology for quantifying drugs, peptides, and oligonucleotides in biological samples.¹⁵ A pretreatment step involving cleanup to remove interfering substances and to concentrate the drug to improve detection is usually required because of the complex composition of biological samples. Commonly used pretreatment methods include liquid-liquid extraction, solid-phase extraction, and protein precipitation. Sample cleanup can also be the most time-consuming and labor-intensive step during sample analysis. Important considerations for a sample clean-up method would therefore include: ^15,16
 

• Physicochemical properties of the analyte(s),
• Sample matrix,
• Quantitation range,
• Lower limit of detection required,
• Number of samples to be analyzed,
• Volume of sample available, and
• Analyte stability to extraction/pretreatment.

 
After sample cleanup, the assay’s selectivity is typically further enhanced by the incorporation of a reversed-phase liquid chromatograph column (e.g., C18, phenyl, C18, C8), before MS detection. The advantages of LC-MS/MS include very high sensitivity (the nanomolar or picomolar lower limit of detection and quantitation), high specificity and selectivity, and quantitation range covering four to five orders of magnitude.^17,18 Furthermore, it allows detection of multiple analytes at the same time.¹⁸
 
However, this method also has some drawbacks. The LC-MS/MS method uses an internal standard for accurate detection and quantitation of each analyte. Ideally, the internal standard is a stable isotope-labeled internal standard (SILIS) which is an isotope-labeled analog of the target analyte. The SILIS is physicochemically identical to the analyte and shows potential variation in sample extraction recovery or matrix effects (suppression or enhancement of the analyte’s signal in the MS/MS due to the presence of co-extracted material).^17,18 Unfortunately, the synthesis of the SILIS can be time consuming and expensive. Furthermore, the sample may contain isobaric compounds that co-elute at the same mass-to-charge ratio (m/z, the unit in which a mass spectrometer detects analytes) and that fragment identically to the analyte in the MS/MS. If that is the case, then these contaminations require chromatographic separation before the MS/MS detection – for example, reverse phase separation coupled with ion-exchange chromatography or size-exclusion chromatography.¹⁸
 
Ligand binding assays. An LBA does not directly measure the analyte but indirectly measures the binding interactions between the analyte and the binding target (e.g., antibody, receptor). The enzyme-linked immunosorbent assay (ELISA) is a commonly used platform for LBAs (Figure 2). There are four main types of ELISAs, each differing in how the antigen/antibody is coated to the plate and how the signal is detected: ^19,20
 

• Direct ELISA – The antigen is attached directly onto the wells of the microtiter plate and then incubated with a primary antibody specific for the antigen. The primary antibody is prelabeled with an enzyme for detection.

 

• Indirect ELISA – The antigen is attached onto the wells of the microtiter plate and then incubated with a primary antibody specific for the antigen. A labeled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection.

 

• Sandwich ELISA – This format requires two antibodies specific for different epitopes of the antigen. One of the antibodies (capture antibody) is coated on the plate surface and captures the antigen. The other antibody (detection antibody) facilitates the detection of the antigen.

 

• Competitive ELISA – In this format, a reference antigen or antibody competes with an unlabeled sample antigen or antibody for the same binding site. All the preceding ELISA formats can be adapted for this format. For example, a reference antigen is coated on to the wells of the microtiter plate, and then the sample is pre-incubated with a labeled primary antibody and added to the wells. The sample and reference antigen compete to bind to the labeled antibody. This means the more antigen there is in the sample, the less reference antigen will be detected, thus the lower the detection signal.

 
Figure 2. Types of ELISA assay
 

ELISA, enzyme-linked immunosorbent assay
 Adapted from Promega and Abcam^19,20
 
 
A calibration curve with known concentration of reference antigen is generated to infer the concentration of antigen in the sample. For each type of ELISA, the detection signal can be colorimetric, fluorometric, or chemiluminescent.¹⁹ The Meso Scale Discovery platform is similar to ELISA but uses an alternative detection method of electrochemiluminescence. Other assays have emerged as expansions of LBAs, such as the surface plasmon resonance immunoassay, hybridization assay, immune-PCR assay, and bioassay.²¹ The advantages of the ELISA method are that it has high specificity, sufficient sensitivity, minimal requirements for sample clean-up, high sample throughput, and relatively low sample cost. However, this assay can exhibit high variability, nonlinear calibration curve, narrow dynamic range, and matrix interference issues. Furthermore, the critical reagents required for the assay (e.g., antibodies) need to be custom produced, controlled, and replaced periodically. Any changes in the critical reagent requires assessment to ensure that the assay performance has not been affected.²¹ Critical differences between the chromatographic assays and LBAs are described in Table 1.
 

 

Bioanalytical method development

Bioanalytical method development involves developing and optimizing the procedures and conditions for extracting and detecting the analyte. ICH M10¹² and FDA guidance on bioanalytical method validation⁵ describe the parameters to consider during bioanalytical method development – reference standards, critical reagents, calibration curve, quality control samples, selectivity and specificity, sensitivity, accuracy, precision, recovery, stability of the analyte in the matrix, and minimum required dilution. Typically, quantitation of one analyte is performed using a bioanalytical method; however, measuring more than one analyte is also acceptable. The two analytes may be two different drugs or a parent drug with its metabolite or enantiomers or isomers.¹²
 

Validation of bioanalytical methods

As previously described, fully validated bioanalytical methods are required for analyzing biological samples from the pivotal nonclinical studies and clinical studies.¹² Validation of the bioanalytical methods is executed to demonstrate the reproducibility and reliability of the method. If an assay measures more than one analyte, validation parameters are executed for all analytes of interest.¹² Normally, the validation of the bioanalytical method is executed according to a validation protocol and associated standard operating procedure for the assay. ICH M10¹² and FDA guidance on bioanalytical method validation⁵ describes the parameters evaluated during validation: selectivity, specificity, calibration curve (response function), range (lower limit of quantitation [LLOQ] to upper limit of quantitation [ULOQ]), dilution integrity/linearity, accuracy, and precision. Furthermore, for chromatographic methods, matrix effect, carryover, and reinjection reproducibility are also evaluated. For LBA method validation, dilution linearity is also assessed.¹² Table 2 summarizes the rationale for development and validation parameters.
 

Conclusion

Bioanalytical methods play a crucial role in drug development and in assessing PK, PD, and toxicity data, thereby facilitating evaluation of the drug’s safety and efficacy and supporting regulatory decision making. The challenges associated with developing sensitive and robust bioanalytical methods were discussed in this article, including the choice of sample preparation methods, and advantages and drawbacks of chromatographic and LBA methods. Regulatory guidelines associated with development and validation of bioanalytical methods were discussed.
 
Abbreviations
ADME, absorption, distribution, metabolism, and excretion; ELISA, enzyme-linked immunosorbent assay; FDA, Food and Drug Administration; FiH, first in human; GLP, good laboratory practice; HPLC, high-performance liquid chromatography; LBA, ligand binding assays; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLOQ, lower limit of quantitation; PD, pharmacodynamic; PK, pharmacokinetic; SILIS, stable isotope labeled internal standard; ULOQ, upper limit of quantitation; UPLC, ultra-high-performance liquid chromatography.
 
About the author
Tapasvi Modi, PhD, is a regulatory affairs manager at Parexel International, working as a consultant in chemistry, manufacturing, and controls and nonclinical regulatory submissions for new products. She has 15 years of experience and subject matter expertise in product development (biologics and vaccines) and in development and validation of bioanalytical assays interfacing discovery, nonclinical and clinical studies. She is a Parexel Enterprise RAPS member and can be contacted at Tapasvi.Modi@Parexel.com
 
Acknowledgment The author thanks Graeme Clark for editorial advice and guidance during the preparation of this article.
 
Citation Modi T. Bioanalysis in drug development. Regulatory Focus. Published online 28 August 2023. https://www.raps.org/News-and-Articles/News-Articles/2023/8/Bioanalysis-in-drug-development
 
References
All references last checked and verified on 16 August 2023.
 

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