Qualification of the aseptic process for preparing 10%-5% glycerol-fructose bags

Introduction
Colorectal polyps, although mostly benign, can develop into colorectal cancer, requiring endoscopic resection. In our center, a 5% fructose and 10% glycerol solution, more viscous than the previously used 4% Gélofusine^®, is employed. Given its use as a submucosal injectable preparation, the solution must adhere to strict sterility standards. This study aims to validate the aseptic process of the automated preparation of sterile glycerol-fructose solution bags, manufactured from non-sterile raw materials, through a Media Fill Test (MFT) in accordance with french Good Manufacturing Practices (GMP).

Materials and methods
Following PIC/S (Pharmaceutical Inspection Co-operation Scheme) guidelines, three simulations were conducted by different operators using tryptone soy casein broth (Merck Millipore). The MFT included weighing fructose in the preparatory area, dissolving it with 0.9% sodium chloride (NaCl) and adding glycerol under a laminar flow cabinet, then sterilizing filtration (Sterivex^®, Merck Millipore), dilution with 0.9% injectable NaCl in bags and distribution into 250 mL bags (Baxter) and 5 mL syringes in an isolator using metering pump. Challenging conditions included lack of weekly cleaning and unchanged sterile gloves after tubing replacement. Samples were incubated for 14 days at room temperature (20-25°C). Environmental monitoring was done on air, gloves, and surfaces. Previously, a fertility test, following European Pharmacopoeia 2.6.1 guidelines was performed on the same batch of broth to ensure filtration did not affect the medium’s fertility. Three microbial strains were used: A. brasiliensis, B. subtilis, and C. albicans (Biomerieux).

Results
No microbial growth was observed in bags or syringes across all three batches after 14 days. Growth was detected after 4-5 days in inoculated filtered and non-filtered broth bags. No growth was seen in non-inoculated bags, whether filtered or non-filtered. Environmental monitoring over the 7-day incubation period showed no colony formation on the agar plates.

Discussion and conclusion
The microbial growth observed in the inoculated bags confirms that the sterilizing filtration process did not compromise the fertility of the broth. Despite the use of non-sterile raw materials and the multiple preparation steps involved, the absence of bacterial growth in the MFT bags and syringes across all three batches produced, as well as the absence of colonies on the agar plates used for environmental control, confirm the validation of the MFT. These results demonstrate that the process adheres to the stringent standards of sterility according to the french GMP.

Key words: Media fill test – Glycerol-fructose bags – Aseptic processing – Sterility testing – Injectable preparations.

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