Deamidated, identification

C. M. Hekman, W. S. DeMond, P. J. Kelley, S. F. Mauch, J. D. Williams, Isolation and Identification of Cyclic Imide and Deamidation Products in Heat Stressed Pramlintide Injection Drug Product ,./. Pharm. Biomed. Anal. 1999, 20, 763 - 772. 

Mazzeo MF, De Giulio B, Senger S, Rossi M, Malorni A, Siciliano RA. Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced mass-spectrometric methodologies. Protein Sci 2003 12 2434—2442. 

Absolute identification of the modified amino acids may require more than one enzyme digest to produce different peptides. Some kinds of modifications that are easily identified by MS include phosphorylation of threonine or serine sulfation or phosphorylation of tyrosine deamidation of asparagine or glutamine O-or N-linked glycosylation oxidation of methionine or cysteine and N-terminal modification by formylation or prenylation. Combining enzymatic maps (tryptic mapping) with MS/MS may identify single amino acid variants of the protein that cannot otherwise be seen. 

The forced degradation studies should be part of impurity characterization. When identification of the impurity is not feasible, incorporate the description of unsuccessful experiments (including those conducted in stress-testing studies) in the text of the application. The most frequently encountered protein variants include truncated fragments, deamidated, oxidized, isomerized, aggregated forms, and mismatched disulfide links. 

Isoelectric focusing (lEF) is another routine slab gel technique for the identification of biotechnology products [37]. The isoelectric points (pi) of the produced protein and its variants can be monitored. The presence of several charge variants is a common feature of recombinant antibodies and can be the result of, for example, deamidation or differences in processing of G-terminal lysine residues [38, 39]. The consistency of the charge heterogeneity can be monitored by using lEF. A typical lEF analysis is shown in Fig. 3.21 B. The pi values of the protein isoforms can be identified for aU of the samples. The lEF patterns for antibodies produced in batch, fed-batch and perfusion modes with PER.G6 cells are very similar. 

Rice, M.S. and Dahlquist, F.W. (1991). Sites of deamidation and methylation in Tsr, a bacterial chemotaxis sensory transducer. J. Biol. Chem. 266, 9746-9753. Bidder, l.S. and Dijkstra, B.W. (1999). Identification of the Mg -binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehaloge-nase) superfamily by structural similarity to the response regulator protein CheY. Biochem. J. 339, 223-226. 

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