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Lecture

Alkaline phosphatase (ALP) activity of the ATDC-5 cell line following exposure to GDF-5 as a basis for its therapeutic or diagnostic application in pre-clinical large animal models

Thursday (09.05.2019)
12:30 - 12:50
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Objectives: Injectable calcium phosphate cements (CPC) represent resorbable, bioactive, osteoconductive alternatives to bioinert polymethylmethacrylate cement for the treatment of osteoporotic vertebral fractures. To improve their regenerative potential, CPC can be loaded with bone specific growth factors, e.g., bone morphogenic proteins such as growth/differentiation factor-5 (GDF-5) 1. The present study assessed the suitability of an in vitro alkaline phosphatase assay (ALP) with the osteogenic indicator cell line ATDC5 for the final choice of a certain GDF-5 protein batch for its therapeutic or diagnostic in vivo application in pre-clinical large animal models.

Methods: Increasing concentrations of different batches of human recombinant GDF-5 were added to culture medium (DMEM/F12 10% FCS, Gold). ATDC-5 indicator cells were then cultured for 3 days in the modified media in 48-well plates at 37°C, 5% CO2 and a density of 4.5 x 103 cells/ well. Osteogenic alkaline phosphatase (ALP) activity in lysed ATDC5 cells was assessed using a p-nitrophenyl phosphate (pNPP) substrate assay, a pNPP standard curve (3.13 to 200 mM), and an ELISA reader at 405 nm. In this system, bioactive GDF-5 induces the expression of the osteoblast-specific marker ALP. The establishment and standardization of the ALP assay is important for the proof of bioactivity and bioequivalence of different batches of GDF-5.

Results: Different batches of GDF-5 showed considerable differences in bioactivity in the ALP induction assay with ATDC5 cells (up to 20%), whereas the same GDF-5 batch in soluble or lyophilized form showed little, if any differences in bioactivity. These results were highly reproducible in repeat assays with the identical lysed cell samples.

Conclusions: In addition to fulfilling standard biochemical quality criteria for therapeutically applied biologicals or biosimilars, the established in vitro ALP bioactivity assay appears suitable for the assessment of the bioactivity of such proteins for therapeutic or, in modified form, diagnostic studies, e.g., concerning the pharmakinetics of GDF-5 following its application in the drug-carrier system of an injectable, resorbable, and bioactive CPC for vertebroplasty and/or kyphoplasty.

1 Bungartz M et al. Spine J. 2017 Nov;17(11):1685-1698

Speaker:
Dr. Martina Stoica
University Hospital Jena
Additional Authors:
  • Francesca Gunnella
    University Hospital Jena
  • Dr. Jörg Bossert
    Friedrich Schiller University Jena
  • Dr. Andreas Borowski
    ImmunoTools GmbH
  • Prof. Dr. Raimund W. Kinne
    University Hospital Jena