Container–closure integrity was assessed on vials that were pierced, filled, and laser resealed but not capped. The objective
was to assess in one test the integrity at the junction between the stopper and the vial body and also the quality of the
laser resealing. The dye ingress challenge test was used for validation and also for regular performance of batch release
The selected dye test was based on
3.2.9 under the denomination "self-sealing test." The normal procedure consists of immersing vials in methylene blue solution
and challenging them with a –27 kPa vacuum for 10 minutes, followed by a return to atmospheric pressure for 30 minutes. To
be more challenging, three successive cycles were applied, each of them consisting of a vacuum of –30 kPa for 30 minutes,
followed by an overpressure of +15 kPa for 30 minutes. The results showed the absence of dye ingress.
Microbiological tests have been conducted as well. Tests based on immersion in solution with
bacteria showed no contamination of vials filled with Tryptic Soy Broth and laser resealed.
It is crucial to ensure that the endotoxin contamination of vials is acceptable because no depyrogenization process takes
place in the overall vial manufacturing and filling process. To ensure that the level of endotoxin is acceptable, both the
raw material pellets and the vials are tested for endotoxin presence with an acceptable limit of 0.25 EU/mL. The method used
was the Limulus Amebocyte Lysate (LAL) test using Endosafe equipment from Charles River. All batches tested until now showed
low level of endotoxin on raw materials (1 g extracted with 2 mL of WFI, 0.05–0.10 EU/mL for 5 batches of raw materials) and
the level inside the vial was always below the detection limit (<0.05 EU/mL UI for all vial batches). If endotoxin levels
appears to be systematically low thanks to a robust process to avoid contamination, the level of control may be reduced in
the future following a risk analysis.
Because the vials are sterilized by gamma-irradiation, the bioburden should be assessed to ensure that the irradiation dose
is in line with ISO-11137 requirements. Because both vial body and stopper are molded at a temperature in the range of 200
°C and directly assembled in ISO5, the bioburden should be very low. Currently, all the samples tested (10 vials from each
tested batch) showed absence of bioburden. This test should be performed either each batch or every three months according
to ISO-11137 guidelines.