title: Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH).
abstract: 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.
authors.affiliation: Bioassay-Labor fuer biologische Analytik GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Hohenstein
authors.affiliation: Bioassay-Labor fuer biologische Analytik GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Hebell
authors.affiliation: Minapharm Pharmaceuticals, Mina Street, 3rd Industrial Zone A2, 10th of Ramadan City, Egypt.
authors.forename: Heidi
authors.initials: H
authors.lastname: Zikry
authors.affiliation: Minapharm Pharmaceuticals, Mina Street, 3rd Industrial Zone A2, 10th of Ramadan City, Egypt.
authors.forename: Maria
authors.initials: M
authors.lastname: El Ghazaly
authors.affiliation: Minapharm Pharmaceuticals, Mina Street, 3rd Industrial Zone A2, 10th of Ramadan City, Egypt.
authors.forename: Frank
authors.initials: F
authors.lastname: Mueller
authors.affiliation: Minapharm Pharmaceuticals, Mina Street, 3rd Industrial Zone A2, 10th of Ramadan City, Egypt. Electronic address: jrohde@minapharm.com.
authors.forename: Jan
authors.initials: J
authors.lastname: Rohde
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D010281:Parathyroid Hormone / Q000378:metabolism*; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D057134:Antibodies, Neutralizing; C470125:PTH protein, human; C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D000242:Cyclic AMP
keywords: HEK293; Immunogenicity testing; Parathyroid hormone; Potency assay; TR-FRET