Minimum Required Dilution (MRD) is used in almost all PK and ADA (including Biomarker and Nab) methods. Usually, all samples in an analysis plate, including standard curve points, quality control samples, and unknown samples, will be diluted multiple folds in the analysis buffer, and proceed with the downstream experiments.
Why setting up MRD?
In bioanalysis, the goal of method development and validation is to serve pre-clinical/clinical sample analysis. Most of the samples are from serum, plasma, and other matrices. Matrices and analysis buffers are essentially different, as the components in matrices are complex, varied, and diverse. These components may interfere with the method, which is what we usually call the matrix effect.
Setting up MRD in PK/ADA methods and diluting the matrices in a certain ratio can remove or reduce the matrix effect so that the method can be more effective without being interfered with by the components of the biological matrix. Therefore, the use of MRD can lead to more stable bioanalysis and less affected by the matrix, and it is a great tool for optimizing results.
How to Set Up MRD
There are no hard restrictions to MRD in PK methods, which means from 1 to 100 or higher. As long as other constraints allow, higher MRD can be applied.
For the ADA method, According to FDA’s guideline, Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection, MRD from 5 to 100 are allowed. The reason to keep the MRD low is that “Higher MRD may result in false-negative responses”.
The Application of MRD in ADA Methods
Threshold factor, method sensitivity, drug tolerance level, and matrix effect are affected by MRD. By adjusting MRD to balance these parameters, the parameters of the method can reach a better state.
In the method development stage, to obtain better sensitivity, background value, and signal window as the evaluation objectives, the types of capture antibody and detection antibody (capture antibody and detection antibody in ADA are generally drugs) are preliminarily determined, and the concentration (at this stage, MRD can also be introduced at the same time for preliminary evaluation), and the various parameters can be balanced by adjusting the MRD.
The influence of MRD on sensitivity
Increasing MRD means reducing the content of the individual matrix and increasing the content of the analysis buffer. The lower the proportion of the matrix, the smaller the difference between different individual matrixes. The screening threshold factor is closer to 1, and the confirmation threshold factor is lower, and vice versa.
When the MRD is large, the value of the tested sample will be infinitely close to the negative control of the analysis batch. At this time, slight signal value fluctuations will affect the negative and positive results of the sample. If the sample is screened as a false positive, the result can be used in the later confirmation experiments and the possibility can be eliminated. But if the sample is screened as false negatives, the result of this inaccurate false negative will be reported in the report. Therefore, the MRD cannot exceed 100 in the ADA experiment.
The influence of MRD on sensitivity
First, in the same method, if assuming two different threshold factors to calculate the sensitivity, then a lower threshold factor would get a better sensitivity.
When the signal values of different individual substrates are relatively consistent (with a few outliers), increase MRD and keep other conditions unchanged, SCPF may decrease. The signal value of the sensitivity curve will also decrease overall. Under two different MRD conditions, the signal of the positive sample with the same concentration will be relatively low when the MRD is larger. But usually, the degree of SCPF reduction is not as much as the signal reduction of positive samples, so the sensitivity becomes poor.
In general, when the signal value between different individual substrates changes significantly, increasing the MRD will therefore reduce the screening threshold factor significantly. At this stage, the reduction of the screening threshold factor dominates and generally makes the sensitivity better.
The influence of MRD on matrix effect
According to the review, MRD and various parameters are closely related, interrelated, and restrict each other. For example, when we want to increase the MRD to remove the matrix effect, we must also consider how the threshold factor will change, whether the sensitivity will decrease or increase, whether the sensitivity is acceptable, whether the drug tolerance level can be appropriately reduced, and so on.
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