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.

Figure 1

LAL test validation protocol flow-chart. The test was performed three times under the same operating conditions by the QC manager on the same samples (CTPs, CTPs supernatant, pyrogen-free water) to test precision. According to ICH Q2 we evaluated specificity and the detection limit. To evaluate accuracy, the assay includes seeding each sample in duplicate. For linearity, a standard curve with 0.005 endotoxin unit EU/mL was used. The acceptance criteria were: spike recovery between 0.25 EU/ml – 1 EU/ml with a CV% < 10, standard curve with CV < 10% and correlation coefficient ≥ 0.980.

Figure 2

Immunophenotype validation protocol flow-chart. The immunophenotype validation protocol required: a first step which is the titration of each antibody performed by using scalar antibody dilution; a second step, named Performance Qualification (PQ), during which the QC manager used two types of standard beads to check cytometer reproducibly over time. Immunophenotyping analysis is an identity test to evaluate specificity by using FMO method. The test was performed three times to test precision. The acceptance criteria were: inter-experiment CV%  ≤ 10%, BM MSCs positive for CD90, CD73, CD105 and negative for CD45, CD14, CD34, CD19 and HLADR; mDCs positive for CD80, CD86, CD83, CD40, CD11c and HLADR at a high level; CTLs positive for CD3+, CD3 + CD4+, CD3 + CD8+, CD56 + CD3- at a low level and negative for CD19.

Figure 3

LAL test specificity. According to the acceptance criteria, the histogram shows that the mean spike recovery of three replicates for all samples analysed was between 0.25 EU/ml and 1EU/ml (A) with PPC CV% less than 10 (B). Bars are SD. Samples analysed are given in Table 1. Samples: 1 = pyrogen free water (negative control); 2–10 = supernatants with FBS; 11–13 = supernatants with albumin; 14–17 = CTPs; 18–23 = supernatants with HS.

Figure 4

Representative panel of antibody titration. CD90 FITC titration on BM MSCs.

Figure 5

Representative panel of fluorescence minus one method (FMO). Cytofluormetric analysis of BM MSCs using FMO.

Figure 6

Immunophenotype specificity. The histograms show the mean percentage expression of three experiments of each marker for BM MSCs (A), iDCs (B-light grey histograms), mDCs (B-dark grey histograms) (B) and CTLs (C). Bars are SD.