Protein instability

Biopharmaceuticals in Plants Toward the Next Century of Medicine 

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There have been many reports from several groups that plant culture medium is not conducive to protein stability, and that the retention of secreted proteins in culture media can be very poor [10, 11, 17, 40, 60, 63-66]. The mechanisms responsible for protein loss from plant culture media are not completely understood however, current indications are that multiple factors may be involved. Processes that have been proposed to affect foreign proteins in plant media include protein degradation due to protease activity [10, 17, 20, 38, 60, 65], protein instability due to defined or... 

As stated previously, the total normal cytoplasmic free copper concentration is less than 10 18 M or less than one copper ion per cell. In thermodynamic terms, almost all hydrated copper ions are immediately and tightly coordinated by amino acids or biopolymers—peptides, proteins, and other species with free sulfur ligands. An excess of copper ions activates metallothionein synthesis for storage or removal of the excess. Copper chaperones mediate transfer of copper ions from extracellular or storage locations to their target proteins. Instability of copper ion concentrations in vivo results in various disease states. Three of these—FALS, Menkes, and Wilson s diseases—are described below. 

Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form. Many protein instabilities result in precipitation of the protein and adsorption to the container. An instability that results in only a modification of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. 

Proteins must be stable during processing, storage, and reconstitution (if lyo-philized). Although liquid formulation is preferred for protein biopharmaceuticals, it may not always be the most stable presentation. The biopharmaceutical protein may need to be lyophilized to maintain stability. Lyophilization involves the removal of water from a frozen substance by sublimation and water vaporization under vacuum.17 But in some cases, this process may itself cause protein instability. 

Pocock, K. F. and Rankine, B. C. (1973). Heat test for detecting protein instability in wine. 

Waters, E. J., Peng, Z., Pocock, K. F., and Williams, P. J. (1995). Proteins in white wine, I Procyanidin occurrence in soluble proteins and insoluble protein hazes and its relationship to protein instability. Aust. ]. Grape Wine Res. 1, 86-93. 

If, after the laboratory tests, a wine is judged to be protein-unstable, a laboratory fining series is run, adding different levels of bentonite to the wine. The series is tested for protein instability with one of the tests previously mentioned, and the level of bentonite needed to prevent haze occurring under the test conditions is selected for use in the cellar. 

Peptide and protein instability in vitro is manifested by the tendency of such molecules to undergo selfassociation in solution, resulting in the formation of multimers and, in the extreme, aggregation and precipitation. For example, insulin at pH 7 exists predominantly as hexameric aggregates, which are too large to be absorbed. 

FIGURE 2 Flow chart of excipients used to prevent/minimize protein instability problems. 

Bilati, U., Allemann, E., and Doelker, E. (2005), Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles, Eur. J. Pharm. Biopharm., 59, 375-388. 

Despite the vast literature on protein instability, the actual protein levels at which wines will remain protein-stable are unknown. Wine proteins are a mixture of probably more than 100 proteins derived from the grape, yeast (Dambrouck et al. 2003), autolyzed yeast (Charpentier et al. 1986) and sometimes Botrytis cinerea when grape berries are not sound (Cilindre et al. 2007). Variety, vintage, maturity of the fruit, pH, and processing methodology affect both the must and the wine protein contents. Yeast proteins, however, have not been shown to play a role in white wine... 

The unusual aspect of wine protein instability is that the proteins responsible for protein haze in the long term are, paradoxically, very stable themselves in the... 

Dawes, H., Boyes, S., Keene, J., Heatherbell, D. (1994). Protein instability of wines Influence of protein isoelectric point. Am. J. Enol. Vitic., 45, 319-326 Derckel, J.P., Legendre, L., Audran, J.C., Haye, B., Lambert, B. (1996). Chitinases of the grapevine (Vitis vinifera L.) five isoforms induced in leaves by salicylic acid are constitutively expressed in other tissues. Plant Sci., 119, 31-37... 

Kwon, S.W. (2004). Profiling of soluble proteins in wine by nano-high-performance liquid chro-matography/tandem mass spectrometry. J. Agric. Food Chem., 52, 7258 - 7263 Lee, T. (1986). Protein instability nature, characterization and removal by bentonite Leiper, K.A., Stewart, G.G., McKeown, I.P., Nock, T, Thompson, M.J. (2005). Optimising beer stabilisation by the selective removal of tannoids and sensitive proteins. J. 1. Brewing, 111, 118-127... 

Sah, H. Protein instability toward organic solvent/water emulsification implications for protein microencapsulation into microspheres. PDA J. Pharm. Sci. Technol. 1999, 53 (1), 3-10. 

Protein instability mechanisms have been reviewed by several investigators.3-13 Chemical reactions such as oxidation, deamidation, proteolysis, racemization, isomerization, disulfide exchange, photolysis, and others will give rise to chemical instability. It is critical that when this happens, the denaturation mechanisms must be identified in order to select appropriate stabilizing excipients. These chemical excipients may be in the form of amino acids, surfactants, polyhydric alcohols, antioxidants, phospholipids, chelating agents, and others. 

White wines are generally treated before bottling, following evaluation of their protein instability (Section 5.5.4). In red wines, treatment generally takes place at the same time as fining. 

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