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Implementation of a Ph. Eur. compliant recombinant method for testing of bacterial endotoxins in sterile pharmaceuticals

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European Statement

Production and Compounding

Author(s)

Randi Hamre Svendsen, Vilde Pettersen, Theo Dogbeten

Why was it done?

Endotoxin testing by the gel-clot method is a limit test and relies on the operator’s subjective evaluation of the results. The procedure itself contains several steps and dilutions, and it is time and resource consuming. The availability of the amoebocyte lysate reagent can also vary since it is harvested from the endangered horseshoe crab. The recombinant factor C method (rFC) is a fluorimetric method based on the gene sequence of the horseshoe crab, providing quantitative results with no interpretation by an operator. The rFC method consists of less handling and is less susceptible to human error.

What was done?

The hospital pharmacy implemented a modern system for testing of bacterial endotoxins as a part of the quality control of raw materials (i.e. water for injection (WFI)) and sterile pharmaceuticals manufactured at the pharmacy, replacing the old gel-clot test.

How was it done?

All sterile pharmaceuticals manufactured at the hospital pharmacy were evaluated for maximum valid dilution (MVD) and endotoxin limit. Firstly, the optimal dilution of the products was established, and then tested with three different batches to ensure valid results regardless of any batch-to-batch variation. WFI was validated undiluted with batches from three separate days. Validation started with the most frequently manufactured products, and subsequently continued over the next year with the rest. To expedite the full validation of some products, expired batches were used simultaneously with at least one recently produced batch due to infrequent production times, otherwise a full transfer of method would not be achieved for up to six years.

What has been achieved?

In total, 25 sterile pharmaceuticals and raw materials were successfully validated for endotoxin testing by rFC during 2021 and 2022. The gel-clot method is no longer in use at the hospital pharmacy, reducing the negative impact on the horseshoe crab population. The rFC method streamlined the testing for endotoxins, reducing the time spent on performing the analysis by 50% with less handling and increased capacity. Results are quantitative and objective, not relying on observations by the operator, thereby improving the quality.

What next?

The rFC method increases both quality and capacity of testing, opening up for expanded testing in pharmacy, and of samples from other departments or hospitals.

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