Quantification of recombinant human parathyroid hormone (rhPTH(1-84)) in human plasma by immunoassay: commercial kit evaluation and validation to support pharmacokinetic studies.

Immunoassays utilizing commercial kits designed for diagnostic use can be adapted and validated to meet Good Laboratory Practice (GLP) requirements to support pharmacokinetic (PK) studies. We illustrate in this paper a systematic approach for commercial kit evaluation and GLP-compliant method validation to establish selectivity, sensitivity, linearity, accuracy, precision and stability. Immunoassay kits for human parathyroid hormone (hPTH) quantification from three different vendors were assessed in a side-by-side comparison for their suitability for the PK analysis of recombinant humanPTH (rhPTH) in EDTA plasma. Two immunoradiometric (IRMA) assay kits and one immunoluminometric assay (ILMA) kit were evaluated. Since PTH is present as an endogenous component of human plasma, QC preparation in the biological matrix was handled differently than for a xenobiotic drug compound. The endogenous concentration of PTH was determined in plasma samples from 32 individual lots using the three kits. The lots with the lowest endogenous concentrations of PTH were selected, pooled to form the low QC and spiked with rhPTH to prepare the mid and high QCs. Four evaluation batches were run with each of the three commercial kits to evaluate reference standard linearity, and QC accuracy and precision. Selectivity against PTH peptide fragments PTH(7-84) and PTH(3-84) were assessed by cross-reactivity and accurate spike-recovery to the QC samples at two concentrations. One of the kits was chosen for full method validation because it had the lowest cross-reactivity against hPTH fragments (3-84) and (7-84), a wider dynamic range and the least total error. The accuracy and precision from six validation batches of the QCs were <or=8.1% C.V. and <or=7.9% R.E., respectively. Method robustness was shown by acceptable QC performance using a different batch of capture antibodies, through 12 batches of iodinated detection antibodies, and by the use of four analysts over 2 years of patient sample analysis from clinical trials.