Cells Cell-based assays, HPLC

Purity for a small molecule is a relatively simple concept. Normally, an HPLC method is sufficient to measure the content and impurity levels of a small molecule drug. A macromolecule, such as a protein, has a much more complex behavior. Determining protein concentration by UV absorption spectroscopy can give a measure of the total protein in the product, but it will not necessarily differentiate between active protein and inactive protein (i.e., denatured or otherwise degraded). A validated method or methods to determine the biological activity of the molecule is needed. So, whereas protein concentration is usually tested as part of the specifications, it is also normally accompanied by one or more methods that measure or correlate to biological activity. This is the bioassay. These methods can be animal-based or cell-based, protein interaction assays, binding methods such as surface plas-mon resonance or ELISA (enzyme-linked immunosorbent assay) and immunoblot methods. 

MC-LR equivalent concentrations (pgg-1 dry weight) in cyanobacterial cells derived by the colorimetric PP2A inhibition assay, the amperometric PP2A inhibition-based biosensor and HPLC. In parentheses, the RSD values are... 

Based on the concept that the retention time of a polypeptide chain in reverse-phase HPLC increases with the degree of unfolding of the chain 2,4,18,19) we infered that HPLC peaks 1, 2, 3, and 4 corresponded to the isoforms so numbered, respectively, in Fig. 3. In order to determine which of the isoforms is biologically active, the eluted peaks from HPLC were assayed on C6 glioma cells for suppression of proliferation. Table II shows that only... 

In addition to studies on toosendanin, there are a number of other efforts to develop natural products as BoNT inhibitors, most notably from the laboratory of Dr Bavari. Using the peptide-based inhibitor 2-mercapto-3-phenylpropionyl-RATKML developed by Schmidt and Stafford (2002), Burnett et al. (2007) examined the interaction of this molecule with the active site of BoNT/A LC. The investigators then screened small nonpeptide molecules from the National Cancer Institute s Open Repository and tested these for inhibitory activity using an HPLC assay and in chick spinal motor neurons. Several compounds were effective in the HPLC assay (/q = 3-10 JlM), and one, NSC 240898, was found to be effective in producing a concentration-dependent protection of SNAP-25 in cultured spinal cord cells with no evidence of toxicity up to 40 I M. 

Pouget, ).-P., Ravanat, ).-L., Douki, T., Richard, M.-)., and Cadet,). (1999) Measurement of DNA base damage in cells exposed to low doses of y radiation comparison between the HPLC/ECD and the comet assays, frit. 

To determine dose (or assay) the quantity of the compound is assessed in the most specific way by HPLC or specific capillary electrophoresis methods using authenticated reference/working standards for quantitative evaluation are suitable for many products. In the case of viral or cell therapy, total protein determination or number of viable particles or (specific) cells might be appropriate dose definitions. In addition, DNA hybridization assays or the determination of total DNA can be applied. Alternatively, for some products a dose definition may be based on the potency of the applied amount. 

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