title: Validation of analytical methods in compliance with good manufacturing practice: a practical approach.
abstract: BACKGROUND
  The quality and safety of cell therapy products must be maintained throughout their production and quality control cycle, ensuring their final use in the patient. We validated the Lymulus Amebocyte Lysate (LAL) test and immunophenotype according to International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, considering accuracy, precision, repeatability, linearity and range.
  
  
  METHODS
  For the endotoxin test we used a kinetic chromogenic LAL test. As this is a limit test for the control of impurities, in compliance with International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, we evaluated the specificity and detection limit.For the immunophenotype test, an identity test, we evaluated specificity through the Fluorescence Minus One method and we repeated all experiments thrice to verify precision. The immunophenotype validation required a performance qualification of the flow cytometer using two types of standard beads which have to be used daily to check cytometer reproducibly set up. The results were compared together.Collected data were statistically analyzed calculating mean, standard deviation and coefficient of variation percentage (CV%).
  
  
  RESULTS
  The LAL test is repeatable and specific. The spike recovery value of each sample was between 0.25 EU/ml and 1 EU/ml with a CV% < 10%. The correlation coefficient (≥ 0.980) and CV% (< 10%) of the standard curve tested in duplicate showed the test's linearity and a minimum detectable concentration value of 0.005 EU/ml.The immunophenotype method performed thrice on our cell therapy products is specific and repeatable as showed by CV% inter -experiment < 10%.
  
  
  CONCLUSIONS
  Our data demonstrated that validated analytical procedures are suitable as quality controls for the batch release of cell therapy products.Our paper could offer an important contribution for the scientific community in the field of CTPs, above all to small Cell Factories such as ours, where it is not always possible to have CFR21 compliant software.
authors: Paediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, City of Science and Health of Turin, Regina Margherita Children's Hospital, P.zza Polonia 94, Turin 10126, Italy. deborah.rustichelli@unito.it
authors: Deborah
authors: D
authors: Rustichelli
authors: Sara
authors: S
authors: Castiglia
authors: Monica
authors: M
authors: Gunetti
authors: Katia
authors: K
authors: Mareschi
authors: Elena
authors: E
authors: Signorino
authors: Michela
authors: M
authors: Muraro
authors: Laura
authors: L
authors: Castello
authors: Fiorella
authors: F
authors: Sanavio
authors: Marco
authors: M
authors: Leone
authors: Ivana
authors: I
authors: Ferrero
authors: Franca
authors: F
authors: Fagioli
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology; D045744:Cell Line, Tumor; D002624:Chemistry, Clinical / Q000379:methods* / Q000592:standards*; D004731:Endotoxins / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D016130:Immunophenotyping; D008033:Limulus Test; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D011786:Quality Control; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D004731:Endotoxins
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