Evaluation of the Conformity of Human Growth Hormone. Concentrations Measured with the DPC Immulite® and the Nichols® Advantage™ Assays

Abstract

Sir, Human growth hormone (hGH) is a polypeptide hormone that is secreted by the anterior pituitary, and consists of a variety of molecular isoforms, the most prevalent in human serum being the 22 kDa (1). These diverse isoforms have different cross reactivity profiles in hGH immuno-assays contributing to the observed discrepancies between these (2). The present study was undertaken in order to evaluate the conformity of two hGH immunochemiluminometric assays (ICMA) (DPC Immulite ® and Nichols ® Advantage™) in serum hGH samples obtained from healthy male subjects exposed to the growth hormone secretagogue (GHS) tabimorelin (NN703) (3). The mechanism of action of GHS seems to rely on several factors, a direct effect on the GH releasing cells in the anterior pituitary, amplification of the effects of GHRH, and functional antagonism of somatostatin (4). The assays studied both utilise a primary monoclonal anti-hGH antibody, and a secondary polyclonal anti-hGH antibody, and GH is sandwiched in between these. The assays are calibrated against the same international standard (WHO 80/505), and the conversion factor in terms of the WHO IRP 80/505 is 1 mg = 2.6 IU.Following approval from the local ethics committee, the blood samples for the comparative study were obtained from 10 healthy male subjects enrolled in a double-blind, randomised, placebo controlled, cross-over study with NN703/placebo administration (3 mg/kg bw) investigating the pharmacokinetics and pharmacodynamics of NN703 administered as capsules. From the total hGH serum profile obtained in this study (0-6 hours post dose, 3 samples/hour analysed by Nichols ® Advantage™), blood samples from 3-6 hours in the periods where the subjects received active treatment (10 samples/subject -in total 100 samples) were chosen for the comparative assay evaluation and subjected to additional analysis by DPC Immulite ® . This time period was chosen in order to have a wide range of hGH levels, with the highest number of samples above the lower limit of quantification (following NN703 administration the median hGH t max was 3.67 hours with a range of 3.33-5.67 hours). The assay procedures were performed in accordance with the manufacturer's instructions. The inter-assay variability was 9.3-9.8% for the DPC Immulite ® , and 3.83-6.94% for the Nichols ® Advantage™ ICMA. The higher variability observed with the DPC Immulite ® , as compared to what has been reported with the United Kingdom National External Quality Assessment Scheme (UKNEQAS), is most likely due to the fact that non-processed pooled serum was used, rather than the processed controls used in the UKNEQAS (5). Of the original 100 samples, 16 samples were excluded from the statistical analysis due to being below the lower limit of quantitation (LLOQ) in one (n=13) or both assays (n=3) (In both assays the LLOQ was determined as the lowest point on the calibration curve, and was found to be 0.15 ng/ml for Nichols ® Advantage™, and 0.05 ng/ml for DPC Immulite ® ). In Figure 1 a Bland-Altman plot of the data is present...