Lyophilized state, proteins stabilized Lyophilizer

Pikal, M.J. Mechanisms of protein stabilization during freeze drying and storage the relative importance of thermodynamic stabilization and glassy state relaxation dynamics. In Freeze Drying/Lyophilization of Pharmaceutical... 

Solid state forms of macromolecules are a common method for preparing protein pharmaceuticals, due to stability advantages. Lyophilization is often utilized for proteins and peptides because it limits the moisture content and increases protein stability. Proteins are routinely lyophilized from aqueous solutions, but lyophilization from co-solvent conditions have also been documented (Santos et al., 2001). 

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. 

KLH also should not be frozen. Freeze-thaw effects cause extensive denaturation and result in considerable amounts of insolubles. Commercial preparations of KLH are typically freeze-dried solids that no longer fully dissolve in aqueous buffers and do not display the protein s typical blue color due to loss of chelated copper. The partial denatured state of these products often makes conjugation reactions difficult. Pierce Chemical is the only commercial source of KLH that includes special (proprietary) stabilizers to provide the protein in a lyophilized form that is almost completely soluble upon reconstitution and with its blue copper-binding characteristics still intact. Reconstitution of the Pierce product with water yields a buffered solution ready for conjugation reactions. 

The effect of moisture on the stability of proteins has been reviewed in the literature [13]. The conventional view that the residual amount of water in the lyophilized plug should be minimized to as low a level as possible has been challenged by many in the field. It is believed that protein molecules need at least a monolayer of water molecules to maintain their native structure in the dried state... 

Frokjaer, S. Otsen, D.E. Protein dmg stability a formulation challenge. Nat. Rev. Drug Discov. 2005,4 (4), 298-306. Molowa, D.T. Mazanet The state of biopharmaceutical manufacturing. Biotechnol. Annu. Rev. 2003,9(1), 285-302. Teixeira, J. Lyophilization growing with biotechnology. Gen. Engin. News 2005, 25 (16), 52-54. 

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. 

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. 

In general therefore, proteins are more stable in the solid state. For example, lyophilized (freeze dried, powdered) ribonuclease in the dry state or suspended in anhydrous solvents exhibits appreciable stability even in temperatures well above 100°C whereas in an aqueous solution undergoes rapid inactivation. Even mild humidity will lead to the aggregation and inactivation of lyophilized tissue-type plasminogen activator (tPA) and growth hormone. 

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