Cassette assay

Sample pooling is also used in a process called cassette assay in which samples are pooled from multiple (typically 5 or 6) dosing experiments.24-83 87-88 Hsieh et al.89 showed that one could pool the plasma from six NCEs into one sample per time point to reduce sample assay time. Kuo et al.88 used a similar sample pooling approach for NCEs dosed into rats. The advantage is that cassette assay requires fewer samples. Two disadvantages are the need to dilute samples and the difficult set-up. 

One recent example of a high-throughput assay for metabolic stability was reported by Fonsi et al. (2008). In this report, the authors described the use of a robotic platform to prepare the compounds for a microsomal stability assay. The authors selected five time points (20, 30, 45, 60, and 90 min) so the intrinsic clearance (CL nt) could be calculated with an Excel spreadsheet. The assay was performed with a triple quadrupole tandem mass spectrometer (MS/MS) system. The system included software tools for automated MS/MS method development—an important requirement for any MS/MS system that will be used for high-throughput assays for microsomal stability assays. The authors also made use of a cassette assay system where samples were pooled after the incubation step was completed. Up to four analytes were pooled and were assayed in one HPLC—MS/MS procedure with a generic chromatographic gradient system. 

There are a number of solutions that have been proposed to address the limitations on throughput in PK assays, including cassette dosing [37], where typically five compounds are dosed in a mixture, pooling of plasma samples from multiple animals receiving a specific dose, or the cassette-accelerated rapid rat screen where the processing of samples is streamlined [38]. 

Typically, fewer than five compounds are cassette-dosed to animals with one of the compounds as the benchmark. The advantage of cassette-dosing is quite obvious it can process more compounds at a time and reduce the number of animals needed for an assay. However, the PK exposure of testing compounds may be compromised due to the potential of systemic drug-drug interactions. ... 

Figure 1.4. NCE/NME progression scheme showing the various discovery stage liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS) assays used for selecting NME/NCE to advance into development. (Reprinted with permission from Korfmacher, 2005.) (CARRS, Cassette accelerated rapid rat screening IV, Intravenous administration PO, Oral administration NCE, New chemical entity)...

Smalley, J., Kadiyala, P., Xin, B., Balimane, P., and Olah, T. (2006). Development of an online extraction turbulent-flow chromatography tandem mass spectrometry method for cassette analysis of Caco-2 cell based bi-directional assay samples. J. Chromatogr. B Ana. Technol. Biomed. Life Sci. 830 270-277. 

The success of transfection, either stable or transient, depends on several factors that must be taken into account and can be summarized as follows (i) the transfectability and physiology of the cell line (ii) the characteristic of the genetic marker in the expression vector (iii) the type of expression desired (iv) the size of the expression cassette and the quality of the DNA to be introduced (v) the compatibility of the transfection method and/or reagents with the cell line (vi) the type of assay to be used for detection of... 

An interesting and more recent expression system for the development of cell-based assays is based on BacMams. These recombinant baculoviruses containing mammalian cell-active expression cassettes seem to be an efficient strategy to speed up assay development. These viruses are produced in insect cells and transiently express but do not replicate in transduced mammalian cells. The expression level can be well controlled by titrating the amount of virus. Highly reproducible transient expression levels, which are a prerequisite to use transient transfection for HTS, might thus be an attractive alternative for some approaches. 

Huang R, Qian M, Chen S, Lodenquai P, Zeng H, Wu J-T. Effective strategies for the development of specific, sensitive and rapid multiple-component assays for cassette dosing pharmacokinetic screening. Int. J. Mass Spect. 2004 238 131-137. 

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