Protein aggregation and precipitation

An increase in temperature generally causes an increase in the solubility of solutes. This general rule is followed by most proteins up to about 40°C. Above this temperature, however, many proteins aggregate and precipitate from solution. If the protein of interest is heat stable and still water soluble above 40°C, a major step in protein purification can be achieved because most other proteins precipitate at these temperatures and can be removed by centrifugation. 

Immunogenicity may be affected by the route of administration. Extravascular injection has been shown to stimulate antibody formation more than IV application, but this is most likely due to the increased immunogenicity of protein aggregates and precipitates formed at the injection site [44]. A recent study investigated the effect of the route of administration of INF-P preparations on inducing anti-INF-P antibodies in multiple sclerosis patients. The results indicate that IM injections appear less immunogenic compared to SC injections, resulting in both a lower serum level of anti-INF-P antibodies as well as a delay in their appearance [45]. 

Aromatic Amino Acids (Histidine, Tryptophan and Tyrosine). Histidine residues are highly susceptible to oxidation, as shown for human growth hormone [65] and relaxin [66]. The resulting degradation products are aspartic acid, asparagines, and 2-oxo-histidine [65, 67]. Metal catalyzed oxidation of histidine may alter the secondary/tertiary structures of proteins. As has been demonstrated, oxidation of the human relaxin histidine which exists in an extended loop that joins two a-helices, alters the protein conformation, resulting in pH-dependent protein aggregation and precipitation [66, 68]. 

Extravascular injection is known to stimulate antibody formation more than intravenous application, most likely due to the increased immunogenicity of protein aggregates and precipitates formed... 

The shaking of protein solutions may lead to aggregation and precipitation as a result of several mechanisms, such as air oxidation, denaturation at the interface, adsorption to the vessel, or mechanical stress. These possibilities were systematically examined for solutions of human fibroblast interferon [50]. In this example, mechanical stress was identified as the causative factor in the inactivation. The proposed mechanism of inactivation by mechanical stress was through orientation of the asymmetrical protein in the... 

It is necessary to adjust the Ca, Mg, phosphate, and citrate content of the concentrate to control aggregation and precipitation of the proteins and minerals during sterilization. By controlling protein aggregation, this adjustment provides optimum viscosity to stabilize the protein, mineral, and milk fat emulsion systems during prolonged storage of the sterile product. Some milk concentrates are stabilized by addition of Ca and Mg salts, whereas others are stabilized by addition of phosphate or citrate salts (Parry, 1974). Chemical compounds approved for addition to evaporated milk include calcium chloride, sodium citrate, and disodium phosphate (CFR 1982). 

Finally, changes in pH and temperature have been used effectively to promote selective protein precipitation. A change in the pH of the solution alters the ionic state of a protein and may even bring some proteins to a state of charge neutrality. Charged protein molecules tend to repel each other and remain in solution however, neutral protein molecules do not repel each other, so they tend to aggregate and precipitate from solution. A protein is least soluble in aqueous solution when it has no net charge, that is, when it is isoelectric. This characteristic can be used in protein purification, since different proteins usually have different isoelectric pH values. 

Integral proteins are dissolved into the lipid bilayer of the membrane through interactions of the hydrophobic amino acid side chains and fatty acyl groups of phospholipids. In order to remove integral membrane proteins, the membrane must be disrupted by addition of detergents or other chaotropic reagents to solubilize the protein and to prevent aggregation and precipitation of the hydrophobic proteins upon their removal from the membrane. 

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]. 

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. 

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