A Biopharmaceutical Company developed a novel drug delivery platform which increased the absorption and distribution of co-administered biological compounds. The concentration of the biologic could be measured directly in the serum; whereas the efficacy of the biologic and its delivery could be inferred by either the pharmacokinetic profile of the drug being delivered with the biologic, or by measuring the release of a key biomarker directly related to the activity of the biologic.
Two methods of analysis were being utilized to measure the concentration of the key biomarker during clinical studies; however, detection was complicated because the biomarker varied in structure and size. The ELISA based method did not recognize all forms of the biomarker and potentially underestimated the total concentration of the biomarker. The HPLC-fluorescence method required lengthy enzymatic digestion, followed by a derivatization step and subsequent chemical reduction to stabilize the derivative. These procedures were followed by a 45 minute gradient elution which was required to resolve the peak from endogenous interferences with similar digestion products. Additionally, the HPLC-fluorescence method was not sensitive enough to detect endogenous levels of the biomarker.
Our scientists employed a faceted approach to resolve the analytical shortcomings of the two existing methods by developing an LC/MS/MS method.
We carefully optimized the enzymatic digestion step to ensure complete digestion of all forms of the biomarker to a common fragment. We then evaluated several potential MS-compatible derivatives of that molecule, identifying a derivatization reagent with several advantages over the previous fluorescent tag. The derivative chosen was stable, did not require a reduction step, and allowed for sensitive and selective detection in MRM mode. The new derivatization procedure was not affected by moisture, which allowed the digestion to be followed by a simple protein precipitation technique prior to derivatization.
The tag facilitated detection in negative ion mode. This served to mediate the effects of adduct formation, which most of the positive ion-mode tags suffered from. The tag also increased hydrophobicity, allowing a wider range of chromatographic options. Most importantly, a deuterium-labeled form of the tagging reagent was available. This enabled us to create an ideal internal standard by reacting the digested fragment with stable labeled reagent.
All of the facets worked in concert to achieve the accuracy and precision needed for a successful, GLP-compliant validation. During validation, several forms of the biomarker – spanning the range of sizes expected in subjects – were used to make QC samples which were tested with the method. The overall sensitivity of the LC/MS/MS assay allowed determination of basal endogenous levels and the method provided accurate and precise concentration data for all forms of the biomarker evaluated.