Freeze/thaw of IGG solutions

Area of Science:

• Biopharmaceutical formulation development
• Protein chemistry and stability

Background:

• Monoclonal antibodies (mAbs) like IgG1 are susceptible to physical instability during manufacturing and storage.
• Excipient crystallization, particularly of cryoprotectants such as mannitol and trehalose, can affect protein stability in frozen formulations.
• Understanding the impact of freeze-thaw cycles and pH on mAb aggregation is crucial for developing stable drug products.

Purpose of the Study:

• To investigate the effect of mannitol and trehalose crystallization on IgG1 unfolding in the frozen state.
• To evaluate the influence of repeated freeze-thaw cycles and varying pH conditions on IgG1 aggregation.
• To assess the role of surfactants and cryoprotectant-free formulations in mitigating aggregation.

Main Methods:

• Formulation annealing at -20°C to induce excipient crystallization.
• Repeated freeze-thaw cycles (5 cycles) applied to formulations.
• Characterization using circular dichroism (CD), particle analysis, and size exclusion chromatography (SEC).

Main Results:

• At pH 3, significant insoluble and soluble aggregate formation occurred, which was reduced by surfactant addition.
• Cryoprotectant-free formulations exhibited higher monomer content post-freeze/thaw.
• At pH 5, a single freeze-thaw cycle showed minimal particle increase, but aggregate formation (1-25 µm) was observed across pH 4-7 after 5 cycles.

Conclusions:

• Excipient crystallization during freeze-thaw cycles can induce IgG1 aggregation, particularly at acidic pH.
• Surfactants can mitigate aggregation at low pH, while cryoprotectant-free formulations may offer better monomer retention.
• Formulation pH is a critical factor influencing IgG1 stability under repeated freeze-thaw stress.