Identifying beta-glucans and peptidoglycans in medicinal products

INTRODUCTION. Beta-glucans and peptidoglycans are cell wall components of bacteria and fungi that are potential sources of pyrogenic contamination of parenteral medicines. Such impurities can cause adverse immune reactions. Therefore, recently, certain attention has been paid to identification of beta-glucans and peptidoglycans in medicines. Despite the lack of a harmonised detection method for beta-glucans and/or peptidoglycans, pharmaceutical industry uses several methods for identifi­ cation and quantitation of these impurities.

AIM. This study aimed to assess applicability of the existing detection methods for beta-glucans and / or peptidglycans in the medicinal products.

MATERIALS AND METHODS. Applicability of detection was examined for beta-glucans and peptidoglycans using amoebocyte lysate and silkworm larvae plasma as reagents. Beta-glucans were detected in Bupivacaine, the product that was previously found to have random glucan impurities. In the drug tests, three types of amoebocyte lysate reagents of different compositions were used reacting to 1) bacterial endotoxins and beta-glucans (lysate with factors C and G); 2) only bacterial endotoxins (lysate with factor C); 3) only beta-glucans (lysate with factor G). Peptidoglycans in Icodextrin were assessed using a reagent from the silkworm larvae plasma. For qualitative analysis, colour of the test solutions was visually assessed after heating them in a dry-air block heater. Kinetic photocolorimetric method was used for quantitation; the primary data were processed using R software.

RESULTS. As a result of two tests with amoebocyte lysate reagents (factors C and G) and (factor C), beta-glucans were detected in Bupivacaine. Chromogenic kinetic method using amoebocyte lysate (factor G) quantified the impurity, which exceeded 2,000 pcg/mL. For Icodextrin, peptidoglycan reference content (not more than 200 pcg/ml) was not exceeded.

CONCLUSIONS. Identification methods for beta-glucans and/or peptidoglycans using amoebocyte lysate and silkworm plasma are applicable for identifying these impurities in the medicinal products. While choosing a study method, product composition and analytical purpose are to be considered.

1. Ławniczek-Wałczyk A, Górny RL. Endotoxins and β-glucans as markers of microbiological contamination – characteristics, detection, and environmental exposure. Ann Agric Environ Med. 2010;17(2):193–208. PMID: 21186760.

2. Barton C, Vigor K, Scott R, Jones P, Lentfer H, Bax HJ, Josephs DH, Karagiannis SN, Spicer JF. Beta-glucan contamination of pharmaceutical products: How much should we accept? Cancer Immunol Immunother. 2016;65(11):1289-301.

3. https://doi.org/10.1007/s00262-016-1875-9

4. Johnson DW, Krediet RT. New peritoneal dialysis solutions and solutions on the horizon. In: Khanna R, Krediet RT, eds. Nolph and Gokal’s Textbook of peritoneal dialysis. Springer, Cham; 2023. P. 393–415. https://doi.org/10.1007/978-3-030-62087-5_11

5. Martis L, Patel M, Giertych JA, et al. Methods and compositions for detection of microbial contaminants in peritoneal dialysis solution. Patent of the USA No. US7618392B2; 2004.

6. Martis L, Patel M, Giertych J, et al. Aseptic peritonitis due to peptidoglycan contamination of pharmacopoeia standard dialysis solution. Lancet. 2005;365(9459):588–94. https://doi.org/10.1016/S0140-6736(05)17908-2

7. Neun BW, Cedrone E, Potter TM, et al. Detection of beta-glucan contamination in nanotechnology-based formulations. Molecules. 2020;25(15):3367. https://doi.org/10.3390/molecules25153367

8. Shapovalova OV, Neugodova NP, Sapozhnikova GA, et al. Detection of bacterial endotoxins in drugs: disturbing factors and their elimination. Pharmacy. 2017;(6):9–14 (In Russ.). EDN: ZGZTFT

9. Song J, Kim S, Park J, et al. Comparison of two β-D-glucan assays for detecting invasive fungal diseases in immunocompromised patients. Diagn Microbiol Infect Dis. 2021;101(1):115415. https://doi.org/10.1016/j.diagmicrobio.2021.115415

10. Kumar M, Mugunthan M. β-d-Glucan and Aspergillus Galactomannan assays in the diagnosis of invasive fungal infections. Med J Armed Forces India. 2019;75(4):357–60. https://doi.org/10.1016/j.mjafi.2017.10.005

11. Burgmaier L, Illes B, Leiss M, et al. Effects of different container types on (1→3)-β-D-glucan recovery. Molecules. 2023;28(19):6931. https://doi.org/10.3390/molecules28196931