Biomarkers have always been one of the hot topics in the field of drug research and development. Especially in the past decade, with the increasing attention to product innovation and research and development efficiency, the application of biomarkers has become more and more medicine. An indispensable part of the R&D strategy of enterprises and biotech companies.
Biomarkers are widely used in different stages of drug development. For example, in the early stage of drug development, biomarkers can be used to screen candidate drugs. By identifying and eliminating candidate drugs that are likely to fail, the cost of drug development can be greatly reduced, and the tragic occurrence of major losses due to drug failure in the later clinical stage can be avoided. In the clinical stage, biomarkers can be used for disease identification and diagnosis, patient screening and treatment effect prediction, monitoring treatment progress and disease progress, and reflecting treatment effects. It is one of the best methods to increase the success rate in drug R&D process, reduce research and development costs, and accelerate the launch of drugs.
All the success of biomarkers applications cannot be achieved without the support of their detection methods. Due to the characteristics of biomarkers themselves, in the field of bioanalysis, the development and verification strategies of their bioanalysis methods are often related to pharmacokinetics (PK). The method of biological analysis is different.
Basic Concepts
Since biomarkers are a relatively broad concept and involve researchers with more disciplinary backgrounds, this often leads to misunderstandings among researchers with different disciplinary backgrounds. So before discussing the biomarker bioanalysis methods, Let’s go over some basic concepts of biomarkers to clarify the scope of this article.
1. What are biomarkers?
In general, a biomarker is an indicator that can be objectively measured and evaluated to indicate normal physiological processes, pathological processes, or pharmacological effects after drug treatment. It can be disease-related or treatment-related. According to the classification of FDA-NIH Biomarker Working Group BEST Resource, it can be divided into:
- diagnostic biomarkers
- monitoring biomarkers
- pharmacodynamic biomarkers
- predictive biomarkers
- prognostic biomarkers
- safety biomarkers
- susceptibility biomarkers
2. Biomarkers in the field of bioanalysis
Biomarkers are also diverse in terms of their detection types, which can be molecular, histological, imaging, physiological characteristics, and so on. In the field of bioanalysis, the detected biomarkers are usually soluble molecular biomarkers in biological fluids, such as soluble IL-6 in serum. The following discussion will only focus on the biomarkers in the field of bioanalysis, and the histological, imaging, physiological and non-fluid molecular biomarkers are beyond the scope of this article.
3. COU and FFP
For the detection of biomarkers, “Context of use (COU)” and “Fit for purpose (FFP)” are two very important concepts.
The FDA’s definition of COU is for a drug development tool, in what environment is it used for drug development and regulatory review. In other words, the COU of a biomarker is a brief description of the specific use of the biomarker in drug development. It includes two parts: the type of biomarker and the use in drug development. For example, HER2 can be used as a predictive biomarker to guide the selection of patients in clinical trials of Herceptin, and specifically select breast cancer patients with HER2 overexpression. A biomarker often has multiple COUs. For example, HER2 can be used as a pharmacodynamic biomarker in combination with pharmacokinetic data to confirm the relationship between the target and the disease and guide the choice of dosage. From the perspective of clinical confirmation of biomarkers, each COU requires a separate confirmation process. The same biomarker is likely to change COU with changes in different stages of drug development. This point cannot be ignored during the development of bioanalytical methods.
On the other hand, FFP means that the verification level of a drug development tool needs to be sufficient to support its COU. FFP is also the basic strategy for the development of current biomarker bioanalysis methods, and this part will be explained in more detail below.
4. Clinical qualification and analytical assay validation of biomarkers
Clinical qualification refers to the process of confirming that a biomarker is related to the biological process or clinical endpoint it represents. The analytical method validation (analytical assay validation) refers to the process of evaluating the characteristic performance parameters of the analytical method to determine whether it can produce reproducible and accurate measurement results. This article aims to discuss the biological analysis methods of biomarkers, so it does not involve the content of clinical confirmation. The “qualification” appearing in the following text refers specifically to the method qualification confirmation in the biological analysis method.
5. Qualification and validation in the field of bioanalysis
For the two words “qualification” and “validation”, researchers from the analytical background, clinical background, and basic research background may have a certain degree of different understanding. In some areas of drug development, there is no strict definition of these two terms and they are often used interchangeably, but for bioanalytical laboratory workers who support drug production and marketing, these two terms are It’s very different.
In the field of bioanalysis, validation usually refers to a documented certification process. This process can ensure to a large extent that a specific process will consistently generate results in compliance with preset regulations and quality standards. Qualification generally refers to the process of proving that a business site, system, or equipment can work correctly and produce expected results in actual work. In general, method qualification (MQ) is a simpler method verification (MV).
Challenges in the development of biomarker bioanalytical methods
In the method development of biomarker bioanalysis, situations and requirements would be different from PK methods, which often brings challenges to method development.
Lack of real standard reference substance
The standard product used in the biomarker method is usually a recombinant protein, which is often different from the endogenous biomarker structurally, physically, and chemically and cannot completely represent the endogenous analyte detected. As a result, coupled with the complexity and diversity of the structure of biomarkers, most of the detection methods of biomarkers are relative quantification, and the accuracy of their detection is essentially relative accuracy, which is very different from the PK method.
Of course, there are also a few biomarkers because their molecular structures are relatively simple, and there are internationally recognized standards (for example, some hormones with a small molecular weight), but these are only a few cases after all.
Non-negligible Biological characteristics
Unlike the PK method, the biological characteristics of biomarkers are an important consideration that cannot be ignored in development. However, analysts often do not pay enough attention to this aspect, resulting in insufficient validity of the final test results. Another situation that may occur is that, due to the complexity of the analyzed biomarkers and the incompletely clear biological characteristics, it is impossible to completely determine whether the test results can really be used to answer the questions they need to answer.
Endogenous Problems
Most biomarkers have a non-negligible endogenous concentration. Blank biological substrates are often difficult to obtain. In the method development stage, it is inevitable to encounter the selection of alternative substrates. This also brought additional challenges to MD processes.
For a small number of PK methods, this problem may also be encountered, such as the difficulty of obtaining biological matrices or high prices, and the drug is an endogenous recombinant protein.
Lack of unified guiding principles
Since the Year of 2000, researchers in the field of bioanalysis have begun to speak up and indicate problems in applying FDA’s guidelines to the development of biomarker bioanalytical methods. A group of related scientists worked on the AAPS (the American Association of Pharmaceutical Scientists) began to set up a discussion group named “Bioanalytical Focus Group” and subsequently published a white paper named “Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement”, which is a pioneering guidance document for biomarkers bioanalytical methods, and more white paper guidance documents followed.
In 2013, the FDA first formally discussed the FFP strategy of biomarkers in the 2013 FDA Draft Guidance for Bioanalytical Method Validation, and then a number of white papers supplemented the FFP strategy to follow up regarding the lack of clarity in the FDA Guidance Part 6 of the specified details. Currently, in the latest version of FDA Guidance, (Bioanalytical Method Validation, Guidance for Industry, 2018), the original description of the FDA on biomarker testing “Biomarkers can be used for a wide variety of purposes during drug development; therefore, an FFP approach should be used when determining the appropriate extent of method validation.“
Although the FDA clearly recommends that the development strategy of biomarker bioanalytical methods is based on the FFP strategy for the purpose of use, it has not made relevant regulations on the specific details. Therefore, for the development of biomarker bioanalytical methods, there are many scientificity and compliance issues are still left to the researchers.
Strategies for the development and validation of bio-analytical methods for biomarkers
A successful biomarker analysis method development verification strategy can ensure the applicability of the biomarker detection data in different drug development stages, and promote drug development in many aspects. As mentioned above, the development and verification of biomarker bioanalytical methods should generally follow the FFP strategy based on its COU. The COU that clarifies the biomarkers can determine the expectations and requirements for the FFP strategy. Before proceeding with the project, the researcher can combine cost requirements and risk-benefit issues to formulate strategies more reasonably.
In the field of bioanalysis, biomarkers are usually detected by the LBA or LC-MS/MS method. Since most of the biomarkers detected in biological matrices are macromolecular proteins or peptides, So in contrast, the LBA method is more widely used. This article will also explain the detection methods of biomarkers based on LBA, but some of the detection principles and strategies can also be referred to when using the LC-MS/MS method.
Clarify the biological characteristics of biomarkers
The first step in developing a bioanalytical method is to clarify what the method is measuring and how the method works. Only when this is clear, can we begin to design experiments to test the analytical method and verify its performance. In the development of biomarker bioanalytical methods, it is necessary to further consider the clinical validity of the test results and whether the results obtained can answer the questions they want to answer. This first requires researchers to have a clear understanding of the biological characteristics of biomarkers, rather than just considering the parameters of the analysis method.
In order to clarify the biological characteristics of biomarkers as much as possible, bioanalytical researchers should collect and learn more relevant data before developing methods, and communicate with the basic drug research and development teams and clinical teams as much as possible. The biological characteristics of endogenous biomarkers include, but are not limited to, post-processing modification, multimerization, the influence of possible binding proteins in the matrix, the expected endogenous level, and its distribution in healthy people and target populations, and so on.
Determine the degree of method verification
In the detection of biomarkers, different COUs determine different requirements for the degree of verification. For example, whether it is in preclinical trials or clinical trials, the data of biomarkers that affect safety need to use fully verified analysis methods Obtained; For the biomarker methods selected to support key phase III clinical trials, the results may be included in the drug labeling instructions, so full verification is also required. For the more exploratory biomarkers (common in non-clinical or early phase I and II clinical trials), it is not required to be fully verified. In many cases, simpler qualification verification or verification is required. Partial verification can be applied.
In the latest version of FDA Guidance, there is also a related description of this point: when the biomarker data is used to support a regulated decision, such as a critical safety or pharmacodynamic decision, it supports the relevant dosage in the product label. When explaining, its analysis method needs to be fully verified. For analysis methods that support early drug development, such as candidate drug selection, go-on-go decision-making, and proof-of-concept (POC), the sponsor can use what it considers appropriate Method validation degree (the original text is: “When biomarker data will be used to support a regulatory decision making, such as the pivotal determination of safety and/or effectiveness or to support dosing instructions in product labeling, the assay should be fully validated. For assays intended to support early drug development (e.g. candidate selection, go-no-go decisions, proof-of-concept), the sponsor should incorporate the extent of method validation they deem appropriate.”)
Regarding the judgment of the specific degree of verification, it is recommended to help the researcher make a decision by answering questions like the following: What is the COU of this method? What is the purpose of the test data? Based on this method, what types of conclusions can be drawn? At a certain research node, is it possible to submit biomarker data to regulatory agencies for review, or just use the data to make internal decisions and better understand the mechanism of action of the drug?
In general, no matter whether the simplest method qualification or complete verification is selected in the end, the purpose is to prove that the analytical method can meet its purpose of use, but there are differences in the degree of verification and the degree of stability of the method parameters. Different pharmaceutical companies also have many differences in the choice of exploratory biomarker analysis method development strategies that are not clearly stated in the regulations. Some companies adopt relatively conservative strategies. For example, they use complete For the validated analysis methods, some companies, out of cost and time cost considerations, use incompletely validated methods for testing in the early and preclinical stages of drug research, and even some early phase I and phase II clinical trials. In fact, as long as the developed biomarker analysis method is FFP, they are good to use regardless of a simple verification or a complete verification. However, many companies may have problems when the method needs to be changed from simple verification to full verification.
The evolution of biomarker bioanalysis methods
Although the development and verification of biomarker bioanalysis methods adopt FFP strategies based on its COU, in real-world situations, there are often some more complicated situations. A situation that often occurs is that, as the drug development process advances, the COU of biomarkers changes, and biomarkers that were originally set as purely exploratory may evolve beyond exploratory uses. For example, the exploratory study of a series of cytokines using multi-component analysis methods in the early clinical stages of drug development may eventually identify a key biomarker to support key clinical decisions.
After the exploratory biomarker COU has evolved, it is necessary to re-examine the degree of verification of the previous method and re-determine the method parameters. At this time, if the analytical methods used in the early stage are not sufficiently rigorous and scientific, there will be certain risks. For example, further method development finds large cross-reactions or discovers that the results of the previous methods have large deviations and data. Draw the wrong conclusion and so on. In order to avoid this situation, it is recommended that when formulating the development and verification strategy of biomarker bioanalysis methods, a continuous overall consideration should be based on the COU that may continue to evolve, and a certain degree of rigor and scientificity should be guaranteed even in the early stage.
Minimum verification experiment
To save time and resources and to maintain appropriate scientific rigor, we recommend that, for purely exploratory biomarker research, carrying out the following key experiments:
- Matrix screening experiment (to determine the biomarkers in the individual matrix Concentration and distribution of substances, evaluate the performance of the standard curve, and initially determine the standard curve points);
- Parallelism experiment (to evaluate the matrix effect of the method, determine the appropriate MRD, evaluate the suitability of the standard and alternative matrix used, and judge the internal measurability of the source substance in the matrix and the preliminary range of its LLOQ. For analytes with a very low endogenous concentration, consider the use of selective experiment provisional);
- QC determination experiments (to determine the quantitative range, the concentration and acceptance range of quality control, precision, and reproducibility).
For substances that are known to be likely to interfere, such as homologs, specific evaluation is recommended (if the kit is used for detection, please refer to the specific evaluation results in the kit first).
Concluding remarks
When developing a bioanalytical method for biomarkers, it is first necessary to understand its biological characteristics and start with its COU to formulate appropriate development and verification strategies.
In the early drug exploratory stage, as long as the testing method can provide reliable data and information, complete verification is not necessarily required. With the continuous advancement of the drug development process, in order to meet the target requirements, corresponding verifications should also be performed simultaneously to ensure the accuracy and reliability of the methods used. In the later stages of drug development, the use of biomarker data to evaluate clinical safety, effectiveness, pharmacodynamics, and surrogate endpoints has increased, and at this time the need for rigorous method validation has also increased accordingly.
In the earliest stage of method feasibility exploration, researchers should strive to obtain information on parallelism, reproducibility, and specificity to ensure that the biomarker test results provide correct answers when answering key hypothetical questions, which can also enable biological The marker method can withstand the test in its subsequent life cycle.
Reference
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