Deamidation detection

Isoelectric focusing also finds application in analysing the stability of biopharmaceuticals over the course of their shelf life. Repeat analysis of samples over time will detect deamidation or other degradative processes that alter protein charge characteristics. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Ion-exchange chromatography (both cation and anion) can also be undertaken in HPLC format. Though not as extensively employed as reverse-phase or size-exclusion systems, ion-exchange-based systems are of use in analysing for impurities unrelated to the product, as well as detecting and quantifying deamidated forms. 

The EP limits charged variants of somatropin, e.g., deamidated forms, hy a CZE method that is characterized hy an ammonium phosphate buffer at pH 6.0 as an electrolyte, an uncoated fused silica capillary, and a UV detection at 200 nm (see Eigure 4 for robustness see reference 43). 

Disposition In healthy subjects, renal clearance of eptihbatide accounts for approximately 50% of total body clearance, with the majority of the drug excreted in the urine as eptihbatide, deamidated eptihbatide, and other, more polar metabohtes. No major metabohtes have been detected in human plasma. 

In addition to analyzing a protein for characteristics dictated by its specific nature, routine analytical assays are performed namely, amino acid analyses and amino- and carboxy-terminal sequencing. If necessary, disulfide assignments are made. A search is ordinarily conducted for oxidations and/or deamidations. In the case of recombinant proteins particular attention is paid to detecting signal sequences or proteolytically degraded species. In contrast, deletions and chemical modifications can be a consequence of proteins prepared by chemical synthesis. Lastly, tests are applied to determine if the protein is correctly folded into its native three dimensional structure. In addition, with recombinant proteins, sensitive immunological procedures are performed to ensure that host cell proteins have not been copurified with the protein of interest. 

Electrophoresis methods are among the most common and potent used in the evaluation of protein purity and homogeneity. They are valuable indicators of protein stability because they detect small molecular or chemical changes in the product caused by denaturation, aggregation, oxidation, deamidation, etc. One of the advantages is that they require only microgram amounts of a sample. 

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