Lyophilization, stabilizer crystallization

Lyoprotectants can affect enzyme stability in both stages of lyophilization the freezing and the drying stages. In the freezing stage of lyophilization, ice crystals form and have been shown to be a cause of enzyme denaturation. Studies have shown that when added as a lyoprotectant, the amorphous polyol mannitol stabi-... 

The presence of a solvent, especially water, and/or other additives or impurities, often in nonstoichiometric proportions, may modify the physical properties of a solid, often through impurity defects, through changes in crystal habit (shape) or by lowering the glass transition temperature of an amorphous solid. The effects of water on the solid-state stability of proteins and peptides and the removal of water by lyophilization to produce materials of certain crystallinity are of great practical importance although still imperfectly understood. 

It has been reported that replacing succinate buffer with glycolate buffer improved the stability of lyophilized y-interferon. In this work, it was found that the succinate buffer could crystallize in the frozen state, which limited its ability to maintain the appropriate pH, and therefore led to degradation. On the other hand, use of the glycolate buffer appeared to minimize the freeze-induced pH shifting, and the lyophilized product exhibited superior solid-state stability. 

Many proteins can be stabilized in the lyophilized state if the stabilizer and protein do not phase separate during freezing or the stabilizer does not crystallize out. 

In Example 11-5, freeze crystallization of imipenem. which has lower stability in solution at room temperature, is presented. In this process, the product is rapidly frozen to an amorphous solid state to conserve its chemical purity. The temperature is then raised (still below the fieezing temperature at this stage), and the amoiphous solid converts to a crystalline solid over time. After the completion of the solid-state transition phase, the lyophilization drying cycle is initiated. 

The manufacturing factors that may be affected by the choice of a particular polymorphic form include granulation, milling and compression, stability (particularly for semisolid forms), amount of dose delivered in metered inhalers, crystallization from different solvents at different speeds and temperature, precipitation, concentration or evaporation, crystallization from the melt, grinding and compression, lyophilization, and spray drying. In the manufacturing processing, crystallization is a major problem and it can be avoided by a careful study of polymorphic transition, particularly in supercritical fluids. 

The risk of chemical instability can be assessed from the primary sequence of the protein. The sequence containing labile amino acids such as Asn-Gly and Met would be indicative of potential instability issues. The rate of chemical reactions that alter the primary sequence of the protein is higher in solution conditions and can limit the shelf-life of protein therapeutics. As the mobility of reactants is minimized in the solid state, freeze-drying is often attempted to improve the stability [16]. In such instances, physical instability is a major issue to be dealt with. Freezedrying, also termed as lyophilization, is a dessication process in which the solvent (usually water) is first frozen and then is removed by sublimation in a vacuum [17]. In other words, the protein in solution is frozen, producing discrete ice and solute crystals. The solid ice is sublimed. Controlled heating desorbs any of the tightly bound water. 

---