Introduction . In the lyophilization technology, the freezing stage plays a key role in determining the time of primary sublimation, secondary desorption, and often the appearance of the lyophilisate. The duration of lyophilization is related to the composition and freezing conditions through the parameter resistance to mass transfer, which depends on the size of ice crystals.Objective: to select temperature regimes of thermal cycling (“annealing”) to obtain the most vapor-permeable sublimation layer, calculate the value of resistance to mass transfer of water and select the most optimal lyophilization mode for preparing lyophilisate GK-2 for preparing solutions for injections.Materials and methods . Substance: GK-2 (hexamethyleneamide bis-(N-monosuccinyl-L-glutamyl-L-lysine)) (V.V. Zakusov Research Institute of Pharmacology, Russia); excipients intended for parenteral use: lyoprotector – sucrose (CompriSugar®) (CristalUnion, France), cryoprotector – polyethylene glycol 4000 (PEG, macrogol) (Polyglykol® 4000, Panreac, Spain). Edwards EF-6 Lyophilic Dryer (Italy); microscope Nikon Eclipse E200; digital camera Nikon DS-Ri2; confocal microscope LEXT OLS 4100. Various freezing, lyophilization, direct optical microscopy in a cold chamber, laser microscopy and mathematical formulas were used to determine the values of resistance to mass transfer of water vapor from model lyophilisates.Results . This article discusses the possibilities of optimizing the freezing stage and, accordingly, the entire lyophilization cycle by introducing an intermediate thermal cycling stage, provides calculations of the mass transfer coefficient of the composition under different freezing conditions and ice crystal sizes.Conclusion . A simplified equation for calculating the coefficient of resistance to mass transfer for model solutions of GK-2 based on the correlation dependence was determined. The selection of freezing and “annealing” modes was carried out on the basis of the most important indicators, such as productivity and time of lyophilization.