Proteins in lyophilized solids

Figure 15.2 Typical HX-MS experimental workflow for (a) protein adsorbed on solid surfaces, (b) protein In frozen solution, and (c) protein in lyophilized solid powders. The experimental conditions are shown in the figure...

The degradation of proteins in the solid state occur to a lesser extent and typically via different mechanisms than those that occur in solution [109,110]. Lyophilization is currently the more common technique in the manufacture of dried therapeutic proteins however, there is increasing interest in the use of spraydrying, owing to the unique physical nature of the spray-dried powder and its potential usefulness in protein drug delivery. 

Liu, W.R., Langer, R. Moisture induced aggregation of lyophilized proteins in the solid state. Biotechnol. Bioeng. 37, 177-184,1991... 

Likewise, the strucmre of subtilisin (pH 3.0) suspended in varying ratios of acetonitrile and water demonstrated a-helical content similar to that in the lyophilized powder (Griebenow and Klibanov, 1996). Furthermore, the rate of transesteriflcation reactions of subtilisin (pH 7.8) suspended in DMSO/acetonitrile, formamide/acetonitrile or formamide/dioxane were increased approximately 100-fold over aqueous conditions (Almarsson and Klibanov, 1996). Similar results were obtained for subtilisin (pH 7.8) in a tetrahydrofuran/1-propanol mixture (Affleck et al., 1992). These results can be attributed to the increased structural rigidity of the active conformation of the protein in the solid, and the denaturing characteristics of the solvent at the solvent-particulate interface. Preservation of this molecular memory or molecular imprint of the protein can also be used to stabilize structure and activity (Mishra et al., 1996 Rich and Dordick, 1997 Santos et al., 2001). Subtilisin was lyophilized from crown ethers, resulting in more native like structure, by FTIR, and increased enzyme activity in THF, acetonitrile and dioxane (Santos et al.,2001). 

Figure 204. Effect of added water content on the aggregation of freeze-dried bovine serum albumin stored at 37°C for 24 h. [From W. R. Liu, R. Langer, and A. M. Klibanov, Moisture-induced aggregation of lyophilized proteins in the solid state, Biotechnol. Bioeng. 37,177-184 (1991). Reproduced by permission of Wiley-Liss, Inc., a subsidiary of John Wiley Sons, Inc.]...

HX-MS for proteins in lyophilized powders has developed over the past 5 years. Recent studies suggest that the method can provide detailed information on protein conformation, dynamics, and interactions with excipients in lyophilized solids and that HX with mass spectral peak width analysis can be used to screen protein formulations for the presence of nonnative subpopulations. Though the utility of the method for developing lyophilized protein formulations has not been fully tested, early results promote the wider development and application of the method. 

Over the past 25 years, there has been increasing interest in expanding the use of HX-MS. In this chapter, we have reviewed its development and application for proteins in three different environments proteins adsorbed onto solid surfaces, in frozen solutions, and in lyophilized solids. The results have demonstrated the capability of HX-MS to detect and monitor protein conformation and dynamics with high resolution in these environments that differ from bulk aqueous solution. In addition, HX-MS has provided quantitative and site-specific information, addressing many of the limitations of more established techniques such as FTIR and CD spectroscopy. 

S Yoshioka, Y Aso, S Kojima, S Sakurai, T Fujiwara, H Akutsu. Molecular mobility of protein in lyophilized formulations linked to the molecular mobility of polymer excipients, as determined by high resolution solid-state NMR. Pharm Res 16 1621-1625, 1999. 

In addition to the additives used in a formulation to help stabilize the protein to freezing, the residual moisture content of the lyophilized powder needs to be considered. Not only is moisture capable of affecting the physicochemical stability of the protein itself, equally important is the ability of moisture to affect the Tg of the formulation. Water acts as a plasticizer and depresses the Tg of amorphous solids [124,137,138]. During primary drying, as water is gradually removed from the product, the Tg increases accordingly. The duration and temperature of the secondary drying step of the lyophilization process determines how much moisture remains bound to the powder. Usually lower residual moisture in the finished biopharmaceutical product leads to enhanced stability. Typically, moisture content in lyophilized formulations should not exceed 2% [139]. The optimal moisture level for maximum stability of a particular product must be demonstrated on a case-by-case basis. 

Drug development, proteins in, 20 839 Drug discovery, yeasts in, 26 488 Drug dosage forms, 15 702-718 aerosols, IS 717 biotechnology and, IS 717-718 capsules, 15 708 granules, 15 702-705 liquid, 15 712-713 lyophilization, 15 716 ophthalmic, 15 716 parenteral, IS 713-716 prolonged action/controlled release solid, 15 708-712... 

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. 

Of course, it is common (and often desireable) to have both amorphous and crystalline phases present in a freeze-dried formulation. This is particularly relevant to freeze-dried proteins, where the lyoprotectant is present in the amorphous state, and another component, such as glycine or mannitol, is present as a crystalline solid in order to impart mechanical integrity and pharmaceutical elegance to the lyophilized solid. 

Moreover, simply obtaining a native protein in samples rehydrated immediately after lyophilization is not necessarily indicative of adequate stabilization during freeze-drying or predictive of storage stability. Many proteins unfold during lyophilization but readily refold if rehydrated immediately (cf. [8,11,14]). Without directly examining the structure in the dried solid, it is not possible to know whether an unfolded protein with poor storage stability is present or not. 

For lyophilized formulations, it is often critical to add a surfactant to the formulation to minimize aggregation. Denaturation of the protein may occur during the freezing or dehydration portion of the process. Interactions of the protein at the solid/air, liquid/air, or water/ice interface may result in aggregation, and it is these interactions that the addition of surfactant to the formulation may minimize. ... 

The reaction is performed by dissolving the protein (10 mg/ml) in 0,5 M borate buffer pH 8.8, containing 4 M guanidine hydrochloride. Solid KCNO is added to a final concentration ofO.5 M and the reaction mixture is maintained at 30°C for 24 hr. The mixture is then dialyzed against 0.005 M NH4HCO3, pH 8.2, and the protein derivative lyophilized. 

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