Lyophilisation of bacterial test strains in a manifold-type apparatus: Effects of freezing and drying parameters, ampoule fill volume, and cotton filter density

Scientific relevance. Lyophilisation is the preferred method at the National Collection of Pathogenic Microorganisms (NCPM) of the Scientific Centre for Expert Evaluation of Medicinal Products of the Ministry of Health of the Russian Federation. Lyophilisation is used to provide for high standards of test-strain deposition, storage, and transportation and to ensure that test strains maintain their properties. Successful lyophilisation requires conducting experiments to establish the key parameters and critical conditions of the process. Aim. The study aimed to evaluate the effects that the speed and time of freezing, the time of drying, the fill volume of ampoules, and the density of cotton filters have on the quality of NCPM indicator microorganisms lyophilised in a manifold-type apparatus. Materials and methods. Pseudomonas aeruginosa NCTC 12924, Staphylococcus aureus NCTC 10788, and Salmonella Abony NCTC 6017 were freeze-dried using a manifold-type apparatus (M. S. R. 18, Usifroid). The authors used a low-temperature freezer at –70±2 °C for slow freezing and a mixture of dry ice and alcohol for quick freezing. The statistical analysis was performed using Microsoft Excel and Statistica 10. Results. The minimum time needed for freezing the samples in a low-temperature freezer at –70±2 °C was 4 hours. Further storage at this temperature for up to 1 month was shown possible without compromising the quality of the final product. The time needed for freezing the samples in a mixture of dry ice and alcohol was under 1 minute. No differences in quality parameters were observed between the lyophilised samples frozen slowly or quickly, except for the cake appearance. Quick freezing resulted in cakes that were non-uniform, crumbled, and pulled away from the ampoule walls, which is considered undesirable. The primary drying stage for ampoules with a fill volume of 0.2 mL took 6–8 hours. The secondary drying stage of 11, 18, 35, and 59 hours resulted in comparable lyophilisate quality: the authors observed no statistically significant differences in viable cell counts (CFU/mL) at the end of lyophilisation and at the end of stress testing. The residual moisture content after 59-hour secondary drying was less than 2%. The cotton filter density had a critical influence on the lyophilisate quality. Therefore, the authors recommend using cotton filters weighing 50 mg or less. Conclusions. The authors analysed the main stages of the lyophilisation process used for NCPM test strains and considered the effects that the speed and time of freezing, the time of drying, the fill volume of ampoules, and the density of cotton filters have on the quality of the final lyophilised product. The NCPM has implemented the results of this study in its work.