Development of a protective lyophilisation medium and conditions to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin

Liquid erythrocyte diagnostic preparations have a practical disadvantage; i.e., long-distance transportation involving possible non-compliance with cold-chain requirements may result in a complete loss of biological activity. A lyophilisation technology is necessary to ensure that the preparations retain their original properties for a long time. The aim of the work was to develop a protective medium and conditions for lyophilisation to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin. Materials and methods: Gelatin, thiourea, trehalose, sucrose, dextran, and Tween 80 were used as excipients for protective media. The authors used nine strains of homologous and heterologous microorganisms of different genera and species to control the lyophilised diagnostic preparation sensitivity and specificity. Evaluation of the main stability-related quality attributes (appearance of the dried preparation, loss on drying, solubility, appearance after reconstitution, appearance after settling, sensitivity, specificity) considered the temperatures specific to the climatic zones where the in vitro diagnostics is intended to be marketed and used. Results: The authors developed protective stabilising media with different compositions, used them in freeze-drying of the preparation and carried out control testing. The most promising was the lyophilisation medium containing a smaller amount of ingredients —6% of dextran, 0.06% of Tween 80 and up to 0.01% of sodium azide—as it was the simplest one to prepare and ensured complete preservation of the quality attributes. The authors carried out practical evaluation of lyophilisation procedures, and the 12–14-hour procedure proved to be the most cost-effective. Conclusions: The results of long-term, or real time, and accelerated stability testing of the lyophilised diagnostic preparation demonstrated the possibility of two-year storage at a labelled temperature of 2–8 °C, as well as at elevated and low temperatures of 30±2 °С and –18 °С, respectively. The tests showed no negative effects of the temperatures on the controlled quality attributes.

1. Iraklionova NS, Sysuev EB, Mas ES. Natural foci of dangerous and especially dangerous pathogens of infectious diseases. Tularemia. Uspekhi sovremennogo estestvoznaniya = Advances in Current Natural Sciences. 2013;9:118–9 (In Russ.)

2. Kudryavtseva TYu, Popov VP, Mokrievich AN, Kulikalova ES, Kholin AV, Mazepa AV, et al. Epizootiological and epidemiological situation on tularemia in Russia in 2020, the forecast for 2021. Problemy osobo opasnykh infektsii = Problems of Particularly Dangerous Infections. 2021;(1):32–42 (In Russ.) https://doi.org/10.21055/0370-1069-2021-1-32-42

3. Startseva OL, Kurcheva SA. Shelf life prediction of diagnostic fluorescent tularemia dry immunoglobulins. Zdorov`e naseleniya i sreda obitaniya – ZNiSO = Public Health and Life Environment – PH&LE. 2019;(2):56–60 (In Russ.)

4. Tyumenceva IS, Afanasev EN, Alieva EV, Kurcheva SA, Garkusha YY. Antigens and antisera of F. tularensis: on the issue of tularemia immunodiagnosis. Meditsinskiy vestnik Severnogo Kavkaza = Medical News of North Caucasus. 2012;1:49–52 (In Russ.)

5. Zharnikova IV, Zhdanova EV, Zharnikova TV, Startseva OL, Kurcheva SA, Geogjayan AS, et al. Comparative characteristics of biotechnology for the production of erythrocyte and latex diagnosticums to identify the causative agent of tularemia. Vestnik biotekhnologii i fiziko-khimicheskoy biologii im. Yu.A. Ovchinnikova = Yu.A. Ovchinnikov Bulletin of Biotechnology and Physical and Chemical Biology. 2019;15(4):27–31 (In Russ.)

6. Zharnikova IV, Efremenko VI, Zharnikova TV, Kurcheva SA, Kalnoj SM, Efremenko DV, et al. Serological methods for detection of the causative agent of tularemia and their evaluation. Zhurnal mikrobiologii, epidemiologii i immunobiologii = Journal of Microbiology, Epidemiology and Immunobiology. 2019;(4):32–8 (In Russ.) https://doi.org/10.36233/0372-9311-2019-4-32-38

7. Koshkidko AG, Kurcheva SA, Zharnikova IV, Starceva OL. Development of a protective drying medium to stabilize erythrocyte antigenic tularemia diagnosticum. Vestnik biotekhnologii i fiziko-khimicheskoy biologii im. Yu.A. Ovchinnikova = Yu.A. Ovchinnikov Bulletin of Biotechnology and Physical and Chemical Biology. 2020;16(4):12–6 (In Russ.)

8. Zhuchenko MA, Pashkova MA, Potapenko OV. Freeze-drying of biopreparations in dual-chamber syringes. Biotekhnologiya = Russian Journal of Biotechnology. 2015;(4):79–84 (In Russ.)

9. Mustafina EN, Mustafin TR, Sadykov NS, Yusupov SA. Ways of storage Clostridium botulinum. Uchenye zapiski KGAVM im. N.E. Baumana = Scientific Notes Kazan Bauman State Academy of Veterinary Medicine. 2017;229(1):27–30 (In Russ.)

10. Jena S, Krishna Kumar N, Aksan A, Suryanarayanan R. Stability of lyophilized albumin formulations: role of excipient crystallinity and molecular mobility. Int J Pharm. 2019;569:118568. https://doi.org/10.1016/j.ijpharm.2019.118568

11. Piszkiewicz S, Pielak GJ. Protecting enzymes from stress-induced inactivation. Biochemistry. 2019; 58(37):3825–33. https://doi.org/10.1021/acs.biochem.9b00675

12. Manohar P, Ramesh N, Improved lyophilization conditions for long-term storage of bacteriophages. Sci Rep. 2019;9(1):15242. https://doi.org/10.1038/s41598-019-51742-4

13. Zharnikova IV, Koshkidko AG, Kurcheva SA, Zhdanova EV, Startseva OL, Tyumentseva IS, et al. Method of preparation of erythrocyte immunoglobulin tularemia diagnosticum. Patent of the Russian Federation № 2747420; 2021 (In Russ.)

14. Chang BS, Kendrick BS, Carpenter JF. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci. 1996;85(12):1325–30. https://doi.org/10.1021/js960080y

15. Crowe JH, Carpenter JF, Crowe LM, Anchordoguy TJ. Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology. 1990;27:219–31. https://doi.org/10.1016/0011-2240(90)90023-W

16. Prestrelski SJ, Arakawa T, Carpenter JF. Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization: II. Structural studies using infrared spectroscopy. Arch Biochem Biophys. 1993;303(2):465–73. https://doi.org/10.1006/abbi.1993.1310

17. Carpenter JF, Prestrelski SJ, Arakawa T. Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization: I. Enzyme activity and calorimetric studies. Arch Biochem Biophys. 1993;303(2):456–64. https://doi.org/10.1006/abbi.1993.1309

18. Crowe LM, Reid DS, Crowe JH. Is trehalose special for preserving dry biomaterials? Biophys J. 1996;71(4):2087–93. https://doi.org/10.1016/S0006-3495(96)79407-9

19. Izutsu K, Yoshioka S, Kojima S. Increased stabilizing effects of amphiphilic excipients on freeze-drying of lactate dehydrogenase (LDH) by dispersion into sugar matrices. Pharm Res. 1995;12(6):838–43. https://doi.org/10.1023/A:1016252802413

20. Han Y, Jin BS, Lee SB, Sohn Y, Joung JW, Lee JH. Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage. Arch Pharm Res. 2007;30(9):1124–31. https://doi.org/10.1007/BF02980247

21. Bolje A, Gobec S. Analytical techniques for structural characterization of proteins in solid pharmaceutical forms: an overview. Pharmaceutics. 2021;13(4):534. https://doi.org/10.3390/pharmaceutics13040534

22. Gracheva IV, Osin AV. Mechanisms of damaging bacteria during lyophilization and protective activity of shielding media. Problemy osobo opasnykh infektsii = Problems of Particularly Dangerous Infections. 2016;(3):5–12 (In Russ.) https://doi.org/10.21055/0370-1069-2016-3-5-12

23. Ohapkina VYU. Methods of microbe cultures maintenance. Part 2. Liophylisation. Teoreticheskaya i prikladnaya ekologiya = Theoretical and Applied Ecology. 2009;(4):21–32 (In Russ.)