Product | Factors (CPPs) – tested levels | Response Variables (CQA) | Observations/ Main findings | Reference |
---|---|---|---|---|
L-lactic dehydrogenase (LDH) in a 700-mL pilot-scale | • Freezing time (1–12 h) • Thawing time (1–12 h) • Holding time (0–11 h) • Set temperature (–10ºC, –24ºC, –38ºC) • Fill volume (250 mL, 475 mL, 700 mL) • Recirculation of the protein solution during thawing (Yes or No) | • Specific activity (%) • Protein concentration (%) • Aggregate number (104/mL) • Aggregate size (μmECD) | Application of A- and D-optimal experimental design. The freezing temperature was the most critical process parameter of LDH stability | [129] |
Lyophilized LDH from rabbit muscle | Freeze temperature (− 40 ºC) | • LDH activity (%) • Protein concentration (%) • Aggregation (detection) | The used model helps predict temperature development and the spatial geometry of macroscopic cryoconcentration during a freezing process | [131] |
Monoclonal antibody (mAb1) | • Start temperature (5, 10, 20, and 30 ºC • HTF temperature (-20, -30, -40, and -50 ºC) • Freeze-thaw cycles • Presence of cryoprotectants and surfactants | • Soluble aggregates (%) • Turbidity (NTU) • Polydispersity index by DLS (%) • Particles > 1 μm by LO (particles/mL) | Application of a Full Factorial Design. Intermediate cooling and freezing times favor quality attributes, where cryoprotectants and non-ionic surfactants in formulations reduce the effect of the freeze-thaw process on stability | [136] |
Myoglobin and LHD | • Four different freeze-thaw modalities • Presence of surfactants | • Enzymatic activity (%) • changes in the conformation (UV absorbance) | Factorial design with three-way ANOVA. Model outputs suggested that a low cooling rate during freezing is beneficial for proteins prone to unfold at the ice surface In contrast, a high freezing rate improves the recovery of extremely unstable molecules in bulk | [130] |
Interferon (IFN), two monoclonal antibodies (MAbs), and an Fc-fusion protein | • Freezing rate (2–10 h) • Thawing rate (2–10 h) | Aggregates content | Application of a 2k Factorial Design: two-factors, two-levels face-centered composite surface response. Once the design space was created, it was possible to define the most appropriate conditions of freeze-thaw to get protein stability | [137] |
Liquid Drug Nanosuspensions (Itraconazole) | • Freezing rate = − 1 (0.2◦C/min), + 1 (4◦C/min) • Steric stabilizer concentration = − 1 (20 mg/mL), + 1 (33 mg/mL) • Cryoprotectant concentration = − 1 (25 mg/mL), + 1 (50 mg/mL) | • Particle size (nm) • Nanoparticle stability | 23 for a complete factorial design. The study revealed the combination of “steric stabilizer concentration” and “cryoprotectant concentration” must be carefully chosen to impart nanoparticle stability during freezing | [132] |
self-microemulsifying astaxanthin | Freeze‑thaw cycles (1–3) | • Droplet size (nm) • PDI • Zeta potential (mV) • Active ingredient content (%) | Optimized formulation by mixture design. Results showed stability properties when the freeze-thaw study was conducted | [133] |