Proteins physical instability

All proteins and peptides display chemical and physical instability that affects the way they are distributed and cleared in the body and their delivery to the site of action. Physical and chemical instability is affected by primary sequences and secondary and tertiary structures and the degree of glyco-sylation of protein. Chemical degradation of proteins and peptides involves deamidation, racemization, hydrolysis, oxidation, beta elimination, and disulfide exchange. Physical degradation of proteins involves denaturation and aggregation. 

Figure 5.10. Possible mechanisms of chemical and physical instability that influence biological activities of protein pharmaceuticals. (Adapted from Manning et al. [18])...

Degradation pathways for proteins can be separated into two distinct classes chemical and physical. Chemical instability is any process which involves modification of the protein by bond formation or cleavage. Physical instability refers to changes in the protein structure through denatur-ation, adsorption to surfaces, aggregation, and precipitation [15]. 

During stability studies, the concentration of the protein is monitored to ensure that there is no loss due to physical instability of the protein. Most typical routes of chemical degradation do not result in a change of absorbance. A change in color of the solution may occur as a result of degradation of some excipients, (notably His) or in the presence of a reducing sugar. 

Physical instability is a phenomenon which is rarely encountered with small organic molecules but arises in peptides and proteins because of the many conditions under which... 

Reubsaet JL, Beijnen JH, Bult A, et al. Analytical techniques used to study the degradation of proteins and peptides physical instability. J Pharm Biomed Anal 1998 17(6-7) 979-984. 

As a result of the complex structure of the proteins, formulation of protein therapeutics pose unique difficulties as it is susceptible to physical and chemical instabilities. The complexity develops from the hierarchical nature of its structure primary, secondary, tertiary, and quaternary structures. Primary structure is the amino acid sequence of the polypeptide chains secondary structure refers to local-ordered conformation tertiary structure deals with the spatial arrangement of secondary structural elements (often referred as global fold) and the quaternary structure is the spatial arrangement of subunits. In general, chemical instability is related to primary structure of the protein, whereas physical instability is associated with the global fold or 3D structure of the molecule. The common problems encountered for protein products are listed in Table 6.2-1. 

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. 

Severai detaiied reviews on the stabiiity of proteins and protein pharmaceuticais written for pharmaceutical scientisls are available (43,45,46,47,48,49,50). A brief overview of these resources foiiows and provides additionai information. The instability of proteins, including protein pharmaceuticals, can be separated into two distinct ciasses. Chemicai instabiiity resuits from bond formation or cleavage yielding a modification of the protein and a new chemical entity. Physical instability involves a change to the secondary or higher-order structure of the protein rather than a covalent bond-breaking modification. 

Generally not encountered in most small organic molecules, physical instability is a consequence of the polymeric nature of proteins. Proteins adopt secondary,... 

Delivery of large-molecular-weight, biotechnology-produced drugs info the body is difficult because of the poor absorption of these compounds, the acid lability of peptide bonds, and the rapid enzymatic degradation of these drugs in the body. In addition, protein pharmaceuticals are susceptible to physical instability, complex feedback control mechanisms, and peculiar dose-response relationships. 

The third advantage, the chemical stability of DNA aptamers, can solve the main problem of protein-based biosensors. The chemical and physical instability of protein-based biosensors is always claimed in practical use, and this limits the range of biosensor application. However, DNA is chemically stable. It is stable within the pH range 2 to 12 and is thermally renaturable Even if it is denatured at 100°C, it is refolded at room temperature. Even RNA aptamers can gain stability upon 1 modification therefore, aptamers have the potential to enhance the applicability of biosensors in practical contexts. Additionally, aptamers can be immobilized onto substrates using DNA microarray fabrication technology, and aptamer microarrays can be created. 

They show some physical instability and early burst releases of encapsulated drugs as proteins or water-insoluble drugs as ibuprofen. This phenomenon could be due to the location of a part of the associated drug at the outer surface of the particles or by the presence of surfactants at the interface. 

Multiple emulsions are unique in that a true liquid phase is maintained separate from an external aqueous phase. This may be especially important for bioactive molecules that cannot be appropriately stabilized in the solid state. In addition, the separation of aqueous phases enables highly specialized environments, conducive to protein activity, to be prepared. The physical instability of conventional systems remains a major factor limiting their wider application. Attempts to improve the physical stability of the aqueous dispersions through interfacial complexation and the use of microemulsions are improving the short-term stability. As an alternative approach, solid-state emulsions attempt to store the multiple emulsion as a solid. Although solid-state emulsions appear to have the potential to be useful protein delivery systems, a substantial experimental data base has yet to be generated. 

Physical properties of the protein structure should be considered in designing strategies to achieve stable formulations because they can often yield clues about which solution environment would be appropriate for stabilization. For example, the insulin molecule is known to self-associate via a nonspecific hydrophobic mechanism66 Stabilizers tested include phenol derivatives, nonionic and ionic surfactants, polypropylene glycol, glycerol, and carbohydrates. The choice of using stabilizers that are amphiphilic in nature to minimize interactions where protein hydrophobic surfaces instigate the instability is founded upon the hydro-phobic effect.19 It has already been mentioned that hydrophobic surfaces prefer... 

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