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Table 2 Standard variables studied under a DoE and QbD approach in the freeze/thaw process

From: Enhancing chemical and physical stability of pharmaceuticals using freeze-thaw method: challenges and opportunities for process optimization through quality by design approach

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]