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.

Please find below the poster for your ready reference:


02/12/2021 14:33

Challenges in the Detection of Metabolic Biomarkers Using a Multi Plex Assay

A 5 Plex method for the detection of metabolic biomarkers in human plasma was developed using the U-PLEX technology on the MSD platform.

The complexities surrounding this method were the establishment of endogenous QCs for each chosen biomarker and evaluating the potential use and need for buffer QCs vs matrix QCs where applicable. Additional challenges were faced while trying to ascertain a single minimum required dilution to enable accurate quantification of all biomarkers simultaneously, encompassing the different detection ranges and limits for each biomarker.

02/12/2021 10:27

Considerations to properly assess drug stability within biological samples

Assessing drug stability during method development and validation is of paramount importance. The concentration of the target analyte must remain unaffected throughout the lifecycle of the samples to ensure the reliability of the assay data. However, a decrease in analyte concentration in a biological fluid is not always due to a lack of stability.

14/06/2021 15:46

Challenges in development of robust analytical methods for the quantitation of low molecular weight compounds by LC-MS/MS

Method development can be considered one of the most challenging task in a Bioanalytical laboratory. When working with LC-MS/MS and low molecular weight drugs, low degree of ionization, poor fragmentation, higher presence of isobaric compounds or analyte loss due to their high volatility can get in the way of a successful method development.