Bioanalysis is an important part of the drug development process, including pharmacokinetics (PK) and biomarker analysis, which runs through the entire process of drug development from early screening to clinical research to post-marketing monitoring. Pharmacokinetics is usually an important end point of clinical research. Non-clinical and clinical pharmacokinetics not only guide the dosage selection of clinical trials, but also data on drug absorption, distribution, metabolism and excretion are also important considerations in the drug development process.
Biomarkers are indicators for objectively evaluating physiological conditions and judging the occurrence, development and prognosis of diseases. They can reflect the characteristic changes that can be measured in the interaction of the organism in the environment. As an auxiliary means, biomarkers have a variety of predictable applications in different stages of drug development. However, biological analysis still faces many challenges:
In the case of low-dose administration, effective pharmacokinetic studies may require an ultra-sensitive detection platform to quantify the drug concentration in biological samples.
Many proteins in the body are not fully expressed due to low expression levels, especially some protein biomarkers that have been proven to have clinical significance.
Some biological samples (such as cerebrospinal fluid) are difficult to collect, and the concentration of related biomarkers in blood or other matrices is much lower than that of cerebrospinal fluid, or even lower than the detection limit of traditional techniques, so it is difficult to use conventional detection methods to detect.
For biological drugs, in addition to high analytical sensitivity requirements, it is also necessary to increase throughput, reduce sample size requirements and reduce matrix interference .
Therefore, it is necessary to use high-quality, high-sensitivity trace detection technology to obtain reliable data and promote the development of drugs.
Ligand binding assays (Ligand binding assays, LBAs) are a commonly used method in the detection of protein analytes, and enzyme-linked immunosorbent assays (ELISAs) have become the use of ligand binding assays for protein analysis in a variety of biological matrices The preferred method of processing objects, its operational advantages and disadvantages are also widely known.
In recent years, the development of LBA detection platforms has become more diversified, which is largely due to the advancement of new technologies, from electrochemiluminescence platforms to new generation platforms, including Gyrolab™ , Erenna® (Singulex) , Simoa (Quanterix) and Immuno-PCR .
Compared with the traditional immunoassay platform, the new technology has obvious technical advantages, has the potential to solve the biological analysis challenges faced in the drug development process, and achieves a larger dynamic range. Based on the introduction of the bead method, coupled with the standardization of single-molecule detection and the ability to amplify the detection signal, the sensitivity of many immunoassays has been improved, even by several orders of magnitude in some cases.
Sensitivity is the most critical part of a bioanalytical method, which represents the ability of the method to detect analytes in a concentration range related to drug efficacy, mode of action and safety . However, most methods to improve the sensitivity of ultra-sensitive technology platforms focus on the amplification of the detection signal. For example, Immuno-PCR relies on the immune PCR platform to amplify the detection signal to detect a single DNA molecule. Although Immuno-PCR has significantly higher sensitivity than ELISA and other techniques, there is still high background interference, and this interference will be further amplified in the operation steps . Other technology platforms, including MSD, DELFIA and AlphaLISA, are signal amplification based on enzymatic reactions, which are detected by fluorescence, electrochemiluminescence and other modes. Analytical sensitivity largely depends on the use of reagents with high affinity, selectivity and specificity for its target. By optimizing analysis conditions, such as buffer, reagent concentration and incubation conditions, the sensitivity is gradually improved. But this will be limited by the catalytic efficiency of the enzyme, the binding ability of the carrier molecule, and the non-selective amplification of binding and non-binding substances . Therefore, signal amplification may not increase the sensitivity to the level required for certain analytes, and there are few ultra-sensitive detections that can reach the sub-picogram or femtogram per milliliter level. The single molecule count can be used to achieve hypersensitivity and protein quantification at the level of nanograms per milliliter . Currently, the detection platforms using single molecule counting include Quanterix’s Simoa™, Singulex’s SMC™ and Chimera’s Imperacer®.
The three platforms have their own merits, and each platform shows its unique advantages when applied to biological analysis. However, when choosing a certain technology platform, we must not only thoroughly understand the advantages and disadvantages of emerging single molecule detection technologies, but also thoroughly understand the costs and risks associated with the application. The choice of platform should be driven by project requirements and the intended use of the generated data. This article mainly focuses on Simoa, and introduces the application of this trace detection technology in biological analysis.