Kinetic Bacterial Endotoxin Testing Methods: Specifics of Analytical Conditions Exemplified by Medicinal Products from Different Classes

INTRODUCTION. Kinetic bacterial endotoxin (BE) testing methods based on amoebocyte lysate from the haemolymph of the horseshoe crab can quantify BEs in parenteral medicinal products across a broad range of concentrations. To develop a specific analytical procedure that is based on these methods and can be implemented into pharmaceutical quality control and standardisation, it is required to ascertain the conditions for its use, in particular, to determine the lowest product dilution that would neutralise interfering factors.

AIM. This study aimed to develop an analytical procedure for the quantitative determination of BEs by the kinetic chromogenic method.

MATERIALS AND METHODS. The study examined samples of medicinal products containing active substances from different classes, including a monoclonal antibody (mepolizumab) and a direct-acting anticoagulant (nadroparin). The BE limits for mepolizumab and nadroparin were ≤20 EU/mL and ≤95 EU/mL, respectively. The samples were tested at different dilutions, which did not exceed the maximum valid dilution. The measurements were performed using a BioТek EL×800 photocolourimeter and EndoScan-V software.

RESULTS. The results for mepolizumab samples show that the kinetic chromogenic method can quantify BEs in mepolizumab medicinal products in the dilution range of 1:10 to 1:40. The study detected BEs in quantities of 0.0078–0.224 EU/mL within this dilution range. However, at a dilution ratio of 1:50 or higher, the method is suitable only for qualitative BE tests. For nadroparin medicinal products, the quantitative determination of BEs should start at a dilution of 1:1000 (the study demonstrated no influence of interfering factors at this dilution).

CONCLUSIONS. The authors developed analytical procedures for the determination of BEs in mepolizumab and nadroparin medicinal products by the kinetic chromogenic method (method D). When developing analytical procedures for the quantification of BEs in individual medicinal products, the developer should determine the lowest sample dilution that would neutralise interfering factors. Subsequently, the dilution should be specified in the documentation for the analytical procedure. This approach can enhance assay accuracy and save time and resources in routine testing.

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