In the previous issues, I shared the past and present of the matrix effect of small molecule LC-MS/MS bioanalysis with friends in the circle. This issue returns to the topic of macromolecular bioanalysis.
Ligand Binding Assay (LBA) is the most widely used detection method for macromolecular bioanalysis. LBA is based on the equilibrium reaction between the ligand (that is, the analyte) and the binding molecule, and is generally a multi-step reaction that generates a signal response related to the analyte concentration to achieve quantitative or qualitative analysis.
Since the introduction of the radioimmunoassay (RIA) in 1960, the LBA analysis method has been developed for nearly 60 years, including RIA, enzyme-linked immunoassay (EIA), electrochemiluminescence (ECL), fluorescence Various analysis platforms such as analysis, chemiluminescence and enzyme analysis. Because of its high sensitivity, good specificity and high throughput, LBA is widely used in the field of macromolecular drug analysis, especially PK blood drug concentration, immunogenicity and biomarker analysis. It runs through drug development from early screening to clinical research. Then to the entire process of post-marketing monitoring. Because LBA cannot be pretreated, its matrix effect is more complicated than that of LC-MS/MS. Of course, in addition to the matrix, there are many other key reagents that affect the key properties of LBA. In this issue, I will share with you some thoughts on the selection and application of key reagents for LBA.
What is a key reagent
Critical reagent, literally understood, refers to the reagent components that are essential to the performance of the method in the analysis process. It is an important weapon that determines the success or failure of the analysis. It often has characteristics that are difficult to obtain and difficult to replace. According to the description of the current major regulations, the key reagents for LBA generally include binding proteins, aptamers (oligonucleotides, polypeptide chains), antibodies or markers, enzymes, etc. A common feature of these reagents is that they react with the analyte with high affinity and specificity, and indirectly produce a detectable signal. This signal is also directly related to the concentration of the analyte. It can be quantified through a certain functional relationship or through It is qualitatively compared with the critical threshold. What makes the editor feel entangled is that the following sentence appeared in the 2018 FDA bioanalytical method validation guidelines:
It seems that the reference standard and matrix are also included in the key reagents, but these three items are listed separately in the final record and report form, which makes people feel inconsistent.
There is no doubt that standards are critical analytical materials, and the matrix also has a significant impact on analytical performance at certain times, especially in ADA and biomarker analysis, so the editor speculates that the FDA’s bigwigs actually don’t care how to define key reagents. The focus is to emphasize proper characterization and documentation of these materials. In terms of material management, whether standard products, substrates or key reagents, from source, receipt, storage, use to destruction, all should be strictly controlled and managed. In a more fashionable way, it is the life cycle management of key reagents.
Life cycle management of key reagents
The life cycle management of key reagents runs through all aspects of reagent procurement, storage, change and stability, and there are dual requirements for business implementation and regulatory supervision. In terms of business implementation, key reagents are the basis for ensuring the quality and long-term performance of LBA analysis, and have a decisive impact on the analysis results. When they change, they may significantly change or weaken the performance of the method. In terms of regulatory supervision, all major guidelines set forth requirements for the control of key reagents. When obtaining key reagents, their quality must be properly characterized and recorded. The certification documents should at least include the name, batch number, source, purity, concentration (such as Yes), storage conditions and expiration date/retest date. When the key reagents are changed, the performance of the method must be confirmed through biological analysis to ensure the consistency of the measurement results.
There are two sources of key reagents: internal production or commercial reagents (mass production reagents or special custom reagents). Each has its advantages and disadvantages. In-house production reagents, such as antigens, labeled antibodies, etc., are designed according to the needs of the project, and can be fully characterized and investigated for stability. Customized CoA is provided, and it is also convenient to monitor the differences between batches; the disadvantage is that the resource investment requirements are relatively high. High, such as personnel, facilities and materials, the characterization of such reagents is also a time-consuming and laborious task. Commercial reagents are rich in sources, easy to obtain, and can provide appropriate characterization and CoA, which are more widely used; and generally well-known manufacturers as suppliers can provide strong technical support for end users; the disadvantage is that the information provided by manufacturers may not be sufficient. Usually lack of stability data, characterization may not be enough to meet the demand, it is difficult for users to control the differences between batches.
For the procurement of key reagents, some suggestions that may be useful are as follows:
Recombinant proteins and peptides of the same concentration and different suppliers may have large differences in performance in the analysis system. Therefore, well-known suppliers should be selected, and it is best to prepare a sufficient number of the same batch of key reagents for a research project. If you use such reagents from different sources, you can consider using correction factors to compensate for errors in content and ratio, provided that the correction factors have been established during the method verification process, or experimental data that can support the correction factors have been generated.
Suppliers usually have QC/QA and batch release processes. These processes usually do not need to be audited, but in some cases, users can consider QA audits of suppliers.
Normally, some reagent-related documents (such as documents related to reagent characterization, stability, batch release specifications, etc.) are not provided with the reagents, and users need to make specific requirements to the supplier to obtain these documents. Keeping these documents is also a requirement of the regulatory authorities.
Commercially prepared pre-coated solid-phase carriers, such as ELISA plates, ECL plates and magnetic beads, should be used as key reagents. The user should establish and record internal processes and batch acceptance standards, especially when using detachable slats to ensure that the slabs are evenly coated. When using commercial reagents to construct biomarker detection methods, mixed biological matrices containing endogenous analytes (such as serum/plasma) should be used as QCs to monitor the characteristics of commercial reagent batches.
For internally produced reagents, during the preparation process, it is necessary to ensure that the performance of the facilities and equipment are qualified, to have sufficient training for technical personnel, to operate in strict accordance with the manufacturer’s protocol or standardized procedures, and to record the experimental process truthfully and accurately. After obtaining the reagent, the content, purity and characteristics (such as affinity, activity, specificity) of the reagent should be determined, and the stability of the reagent should be monitored for a long time.
The cycle of clinical trials is generally very long, and it is inevitable that reagents exceed the validity period. After some key reagents (such as some recombinant proteins and labeling reagents) reach the expiration date provided by the manufacturer, their functions may still meet the research needs. If the preset standards are met after the retest, the validity period of these reagents can be extended. Due to space limitations, the next edition will continue to share with you the storage, stability and batch change issues in the life cycle management of LBA key reagents.