Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH).

Parathyroid hormone (PTH) is the primary regulator of serum calcium homeostasis and plays a major role in bone metabolism. Its actions are mediated via the PTH1 receptor (PTH1R) resulting in adenylate cyclase activation and consequently production of cyclic adenosine mono-phosphate (cAMP). The latter stimulates cellular metabolic pathways. This study describes the development, validation and applications of a novel cell-based potency assay for PTH using HEK293 cells over-expressing PTH1R. PTH concentration-dependent cAMP formation in these cells was quantitatively analyzed employing time-resolved fluorescence technology (TR-FRET). The optimized assay was precise, reproducible and exhibited a high sensitivity to PTH with a limit of quantification in the low picogram range. The potencies of differently manufactured PTH1-34 peptides, as well as a full-length variant (PTH1-84), were all accurately measured. Since PTH activity is inhibited by neutralizing antibodies against PTH, the assay was adapted to detect and measure neutralizing antibodies in human serum. Thus, applications of this novel cell-based PTH potency assay were extended to immunogenicity testing of PTH preparations in non-clinical and clinical settings.

Quantification of a pegylated interferon-alpha2a product by a customised and validated reverse phase-high performance liquid chromatography method.

There is increasing development of pegylated proteins as clinical products for therapeutic interventions in human disease. Quantification of such products relies on appropriately calibrated traditional methods, including reverse phase-high performance liquid chromatography (RP-HPLC). However, currently available pharmacopoeia calibrants, e.g., chemical reference substances (CRS), are highly purified non-pegylated proteins of known concentration. These are uncertain to be suitable for calibration purposes where the precise quantification of the mass of pegylated proteins, often heterogeneous with respect to polyethylene glycol (PEG) chain size, structure, attachment sites and isomer numbers and proportions, is required. In this study, a customised RP-HPLC method was developed and validated for the analysis of a pegylated IFN-α2a product having a linear 20kDa PEG chain (PEG20-IFN-α2a; Reiferon Retard(®)). Since the PEG20 moiety generated no signal at the detection wavelength of 210nm, the concentration of the base IFN-α2a molecules in PEG-IFN-α2a could be determined. By calculating the UV absorbance at 210nm of peak areas in their respective chromatographic profiles, a high correlation (r(2) ≥ 0.995) of PEG20-IFN-α2a concentrations with equal concentrations of the CRS of IFN-α2a, or of a well-characterised PEG20-IFN-α2a internal reference substance (IRS) was found. This finding confirmed the suitability of this CRS as a primary calibrant for mass determinations of PEG20-IFN-α2a by the customised RP-HPLC method. Application of this method to the quantitative analysis of 10 batches of Reiferon Retard(®) yielded accurate and consistent results, indicating its utility for mass determinations of current and future Reiferon Retard(®) batches.