Qualification of a method for the measurement of a potentially therapeutic protein
A recent project completed by Anapharm involved qualifying an analytical method for the measurement of a therapeutic protein in human skin tissue. The protein of interest is one that is expressed ubiquitously in a number of tissues and cell types.
While there are commercially available immunoassay kits to detect and measure this particular protein, the aim here was to develop a fit-for-purpose method, which produced reproducible results within a challenging matrix.
There were a number of factors that had a direct impact on the reproducibility of this assay and were addressed in the following ways:
• With limitations on the availability of the skin sample matrix and the presence of endogenous protein, a surrogate matrix of RIPA buffer was used.
• To maintain sample stability, samples were sliced on dry ice and removed only briefly for weighing.
• Cut samples were homogenised using the Precylls Evolution Homogeniser®, which allowed for the creation of repeatable programs at sub-zero temperatures.
• Additionally, during homogenisation, RIPA buffer, spiked with a protease inhibitor was added to prevent any enzyme degradation.
• To ensure that a similar amount of drug per mg of total protein was generated regardless of the size of sample section taken, a fixed ratio of RIPA buffer was added per mg of skin tissue.
In addition to method parameters normally assessed for a PK assay, such as precision and accuracy, selectivity, parallelism and stability, the qualification of this method also involved the evaluation and optimisation of the homogenization procedure. The use of an automated tissue homogeniser also helped in the generation of a highly reproducible homogenisation process, which then facilitated the completion of the qualification of a fit-for-purpose method.
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