Bioburden determination for sterile process validation in the context of drug shortage during the COVID-19 pandemic

6 October 2021

A. Cèbe1, A. Venet1, B. Dessane1, 2, V. Servant1, J. Héloury1, S. Crauste-Manciet1, 2
1 Pharmaceutical Technology Department, Bordeaux University Hospital, France
2 ARNA Laboratory ChemBioPharm U1212 INSERM - UMR 5320 CNRS, Bordeaux University, France

In the context of essential drug shortages during the Covid-19 pandemic, we were involved in the network driven by ANSM1 for studying feasibility of hospital pharmacy production of 10mg/mL rocuronium bromide. One of the main differences in comparison to our usual process was the use of non sterile raw material. In this context we had to implement a bioburden evaluation to estimate the microbiological load prior sterilization.

Materials and methods
The preparation process was based first on mixing the drug and excipients in sterile pyrogen-free container to produce a bulk solution. The bulk solution was then double filtered through 0.22µm filters and transferred in sterile pyrogen-free vials in a grade A unidirectional airflow surrounded with a grade B environment. To simplify the validation the first filtration was not considered as sterilization step. Bioburden was determined by membrane filtration method as described in European Pharmacopeia (EP). Samples were collected before and after the first filtration of the rocuronium solution at D0, D7, and D14. To ensure the test sensitivity, a volume of 2% of the bulk or 100mL whichever is the greater was taken. These samples were filtered onto the 0.45µm PVDF membrane (Sartorius). Three canisters were used for each test: 1 for bacteria, 1 for fungi and 1 for negative control. The membranes were transferred to agar plates: TSB for bacteria and negative control incubated at 32.5°C for 5 days and Sabouraud for fungi incubated at 22.5°C for 7 days. Prior the test, suitability in the presence of product was conducted in triplicate with the 5 EP aerobic strains.

Results – Discussion
Suitability was validated with the 5 strains. Before first filtration, 6 CFU were found on each of the TSB and Sabouraud agar plates, identified as Penicillium and Staphylococcus cohnii. After first filtration, no colony was found at D0, D7, and D14. Despite the storage of the bulk solution, microbial load stayed below the limit of 10 CFU/100mL which is the limit given by EMA guidelines2 for sterilization by filtration method. These results are encouraging for the transposition to other drugs unable to be heat sterilized (i.e. cisatracurium).

Bioburden determination both validate the use of nonsterile raw material and the possibility to postpone the repartition and sterilization of the bulk stored in pyrogen-free sterile container. These results open the option to deliver the bulk to another site of repartition in case of major national shortage.

1 ANSM : French National Agency for Medicines and Health Products Safety
2 Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container (EMA/CHMP/CVMP/QWP/850374/2015) 6 March 2019, European Medicine Agency (EMA)

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