Stability of Rituximab in Freeze-Dried Formulations Containing Trehalose or Melibiose Under Different Relative Humidity Atmospheres

The objective of the study was to compare the effectiveness of trehalose with that of melibiose in protecting a monoclonal antibody (rituximab) from aggregation, fragmentation, and secondary structure alterations during processing and subsequent storage. Because reducing disaccharides such as melibiose participate in Maillard reaction with proteins, especially in the presence of water, the lyophilizates were stored under different relative humidity (RH 5%, 11%, and 23%) atmospheres. Freeze drying was shown to cause clear alterations in rituximab secondary structure, an increase in noncovalent protein aggregation, and in some cases fragmentation. However, these changes were less pronounced in the formulation containing melibiose. Storing the lyophilizates under low RH (5%) proved to be most harmful to the stability of rituximab, intensifying secondary structure alterations and increasing protein aggregate content. Again, these changes were less aggravated in the formulation containing melibiose. Surprisingly, the concentration of aggregates larger than 1 μm decreased in some cases during storage at RH 11% and 23%. There was no indication that storage even under the highest RH (23%) would have caused significant amounts of Maillard reaction end products to be formed during 3 months of storage.