Proteins chemical instability

Disruption of the native structure of a protein can also contribute to chemical instability by accelerating the rates of a variety of degradation routes, including deamidation, hydrolysis, oxidation, disulfide exchange, /1-elimination, and racemization. 

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. 

Proteins, peptides, and other polymeric macromolecules display varying degrees of chemical and physical stability. The degree of stability of these macromolecules influence the way they are manufactured, distributed, and administered. Chemical stability refers to how readily the molecule can undergo chemical reactions that modify specific amino-acid residues, the building blocks of the proteins and peptides. Chemical instability mechanisms of proteins and peptides include hydrolysis, deamidation, racemization, beta-elimination, disulfide exchange, and oxidation. Physical stability refers to how readily the molecule loses its tertiary and/or sec-... 

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]. 

Li, S. Shoeneich, C. Borchardt, R.T. Chemical instability of protein pharmaceuticals mechanisms of oxidation and strategies for stabilization. Biotechnol. Bioeng. 1995, 48, 500-590. 

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. 

The stability of biotechnology-produced products, proteins (macromolecules), and peptides is unique when compared with conventional pharmaceuticals (small molecules). Protein degradation by both chemical and physical processes leads to the loss of biological activity, whereas peptides decompose only through chemical instability with loss of efficacy and produce undesirable biological effects. 

Reubsaet JLE et al. Analytical techniques used to study the degradation of proteins and peptides chemical instability. J Pharm Biomed Anal 1988 17 955 - 978. 

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. 

Chemical instability refers to covalent modifications of the molecule that result in a new chemical entity. Some amino acids in the protein are chemically labile and undergo degradation to form a new chemical entity. The chemical reactions that affect the proteins are as follows ... 

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. 

A variety of reactions give rise to the chemical instability of proteins, including hydrolysis, oxidation, racemization, (J-elimination, and disulfide exchange (Fig. 6.26). Each of these changes may cause a loss of biological activity. 

Fig. 6.26. Chemical instability of protein biopharmaceuticals, (a) Hydrolysis, (b) Base-catalyzed racemization. (c) P-Elimination.

Metabolic oxidation reactions may occur to the side chains of sulfor-containing residues, similar to that observed for in vitro chemical instability. Methionine can be oxidized to the sulfoxide, whereas metabolic oxidation of cysteine residues forms a disulfide. Metabolic reductive cleavage of disulfide bridges in proteins may occur, yielding free sulfhydryl groups. 

Li S, Schoneich C, Borchardt RT. Chemical instability of proteins. Pharmaceut News 1995 12-16. 

There also are chemical instability issues associated with insulin. For 40 years, the only rapid-acting form of insulin was a solution of zinc insulin, with pH 2 to 3. If this insulin is stored at 4°C, deamidation of the asparagine at A21 occurs at a rate of 1 to 2% per month. The C-terminal Asn, under acidic conditions, undergoes cyclization to the anhydride, which in turn can react with water, leading to deamidation. The anhydride also can react with the N-terminal Phe of another chain to yield a cross-linked molecule. If stored at 25°C, the inactive deamidated derivative constitutes 90% of the total protein after 6 months (Fig. 32.2) (34). 

Reactive Metabolites or Intermediates Reactive metabolites are chemically unstable and often rapidly bind to proteins or glutathione (GSH). In general, GSH adducts are not present at significant levels in circulation, because they undergo rapid elimination via direct biliary excretion or further metabolism to mercapturic acids that are excreted into urine. Reactive metabolites can be monitored via quantitative analysis of their GSH adducts or mercapturic acids in excreta. However, it is not practical to test the toxicity of reactive metabolites on animals directly since the synthesis and dosing preparation of reactive metabolites are extremely difficult or impossible due to their chemical instability (Davis-Bruno and Atrakchi, 2006). 

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