Freeze-drying approach to enhance antibody stability
Abstract
Preparations containing antibodies are more easily formulated in solution, but their stability is often impaired, concerning the fact that aqueous solutions are prone to numerous physical and chemical changes, such as denaturation, aggregation, adsorption, hydrolysis, deamidation, nonreducible cross-linking, formation/exchange of disulfide bonds, isomerization, fragmentation, Maillard-reaction, etc. Freeze-drying is often used process in biopharmaceutical production, because the chemical and physical degradation rate can be significantly reduced in dry state. This process enables preservation of the desired characteristics of the product for a longer period of time, thus increasing its shelf life. Part of the challenges associated with the first phase of the freeze-drying process can be addressed with its’ corresponding design, i.e. defining the freezing rate, but also addition of bulking crystalline agents as mannitol, glycine or disaccharides (sucrose, trechalose), that can provide fine, microporous structure. Recent research had shown that slow freezing, which includes gradual cooling of the sample to a given temperature with a certain rate, and fast freezing, including direct introduction of the sample to a previously cooled freeze-dryer, are the most favored.
Our experience was in designing a freeze-drying process in order to formulate freeze-dried kit, containing conjugated Anti-CD-20 antibody, ready-to label with radioactive isotopes. Using freeze-dryer able to controll process parameters, (Labconco Free Zone Stoppering Tray Dryer, USA), two types of freeze-drying protocols were applied, a protocol with direct freezing to the initial temperature of -40 °C and a protocol with gradual freezing from 5 °C to -40 °C, at a rate of 0,40 °C/min. Appearance after reconstitution of the finished product and presence of aggregates were evaluated, as well as the IR and Raman spectra.
Results have shown that the protocol using gradual freezing from 5 °C to -40 °C, at a rate of 0,40 °C/min was more successful in maintaining the stability of the antibody during freeze-drying, giving clear solution after reconstitution, no presence of aggregates and IR and Raman spectra most similar to the spectra of the native conjugated form.
The research performed, enabled formulation of ready-to label anti-CD-20 antibody, intended for preclinical research for Non-Hodgkin’s lymphoma therapy.
