Crystallizing amino acids as bulking agents in freeze-drying

Area of Science:

• Pharmaceutical Sciences
• Biotechnology
• Materials Science

Background:

• Bulking agents such as mannitol (Man) and glycine (Gly) necessitate high bulking agent to stabilizer ratios for successful crystallization during freeze-drying.
• Investigating alternative bulking agents is crucial for optimizing lyophilization processes.

Purpose of the Study:

• To explore several amino acids (AA) as potential bulking agents in low amino acid to sucrose (Suc) ratios.
• To evaluate the efficacy of these amino acids in challenging freeze-drying conditions.

Main Methods:

• Fast freeze-drying above the collapse temperature (Tc) of the amorphous phase.
• Characterization of lyophilizates and liquid formulations using differential scanning calorimetry, Karl-Fischer titration, and X-ray powder diffraction.
• Assessment of macroscopic cake appearance, reconstitution times, and monoclonal antibody stability (UV-Vis spectroscopy, light obscuration, size exclusion chromatography).

Main Results:

• Phenylalanine (Phe), leucine (Leu), and isoleucine (Ile) demonstrated crystallization at 5/45 and 10/40 AA/Suc ratios.
• Lower AA/Suc ratios (2.5/47.5) resulted in less pronounced crystallization.
• The presence of monoclonal antibody (2 or 50 mg/mL) did not suppress crystallization peaks in Leu and Ile lyophilizates.
• Mannitol and glycine produced unacceptable cake appearance at low bulking agent to sucrose ratios under fast freeze-drying conditions.
• No protein aggregation was observed post-freeze-drying.

Conclusions:

• Leucine, isoleucine, and phenylalanine are viable alternative bulking agents at lower ratios compared to mannitol or glycine.
• These amino acids offer improved cake characteristics and good protein process stability during freeze-drying.
• Further optimization of reconstitution times may be achieved with surfactant addition.