title: A practical approach for the validation of sterility, endotoxin and potency testing of bone marrow mononucleated cells used in cardiac regeneration in compliance with good manufacturing practice.
abstract: BACKGROUND
  Main scope of the EU and FDA regulations is to establish a classification criterion for advanced therapy medicinal products (ATMP). Regulations require that ATMPs must be prepared under good manufacturing practice (GMP). We have validated a commercial system for the determination of bacterial endotoxins in compliance with EU Pharmacopoeia 2.6.14, the sterility testing in compliance with EU Pharmacopoeia 2.6.1 and a potency assay in an ATMP constituted of mononucleated cells used in cardiac regeneration.
  
  
  METHODS
  For the potency assay, cells were placed in the upper part of a modified Boyden chamber containing Endocult Basal Medium with supplements and transmigrated cells were scored. The invasion index was expressed as the ratio between the numbers of invading cells relative to cell migration through a control insert membrane. For endotoxins, we used a commercially available system based on the kinetic chromogenic LAL-test. Validation of sterility was performed by direct inoculation of TSB and FTM media with the cell product following Eu Ph 2.6.1 guideline.
  
  
  CONCLUSIONS
  The calculated MVD and endotoxin limit were 780x and 39 EU/ml respectively. The 1:10 and 1:100 dilutions were selected for the validation. For sterility, all the FTM cultures were positive after 3 days. For TSB cultures, Mycetes and B. subtilis were positive after 5 and 3 days respectively. The detection limit was 1-10 colonies. A total of four invasion assay were performed: the calculated invasion index was 28.89 +/- 16.82% (mean +/- SD).
  
  
  CONCLUSIONS
  We have validated a strategy for endotoxin, sterility and potency testing in an ATMP used in cardiac regeneration. Unlike pharmaceutical products, many stem-cell-based products may originate in hospitals where personnel are unfamiliar with the applicable regulations. As new ATMPs are developed, the regulatory framework is likely to evolve. Meanwhile, existing regulations provide an appropriate structure for ensuring the safety and efficacy of the next generation of ATMPs. Personnel must be adequately trained on relevant methods and their application to stem-cell-based products.
authors.affiliation: The Cell Therapy Unit, Cardiocentro Ticino, Via Tesserete 48, CH-6900 Lugano, Switzerland. sabrina.soncin@cardiocentro.org
authors.forename: Sabrina
authors.initials: S
authors.lastname: Soncin
authors.forename: Viviana
authors.initials: V
authors.lastname: Lo Cicero
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Astori
authors.forename: Gianni
authors.initials: G
authors.lastname: Soldati
authors.forename: Mauro
authors.initials: M
authors.lastname: Gola
authors.forename: Daniel
authors.initials: D
authors.lastname: Sürder
authors.forename: Tiziano
authors.initials: T
authors.lastname: Moccetti
mesh_terms: D001681:Biological Assay / Q000379:methods; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology; D004731:Endotoxins / Q000378:metabolism*; D005062:European Union; D006321:Heart / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000502:physiology*; D008420:Manufactured Materials / Q000382:microbiology / Q000592:standards; D009206:Myocardium / Q000166:cytology*; D010602:Pharmacopoeias as Topic / Q000592:standards; D012038:Regeneration / Q000502:physiology*; D015203:Reproducibility of Results; D013242:Sterilization / Q000379:methods / Q000592:standards; D014481:United States
chemical_list: D004731:Endotoxins