Multivariate analysis of the sequence dependence of asparagine deamidation rates in peptides.

PURPOSE: To develop a quantitative scheme to describe and predict asparagine deamidation in polypeptides using chemometric models employing reduced physicochemical property scales of amino acids. METHODS: Deamidation rates for 306 pentapeptides, Gly-(n-1)-Asn-(n+1)-Gly, with the residues n-1 and n+1 varying over the naturally occurring amino acids, were obtained from literature. A multivariate regression technique, called projection to latent structures (PLS), was used to establish mathematical relationships between the physicochemical properties and the deamidation half-lives of the amino acid sequences. Three reduced physicochemical property scales, amide hydrogen exchange rates (to describe the relative acidity of the amide protons) and flexibility parameters for the sequences were evaluated for their predictive capacity. RESULTS: The most effective descriptors of the deamidation half-lives were reduced-property parameters for amino acids called zz-scores. The PLS models with the reduced property scales, combined with the hydrogen exchange rates and/or flexibility parameters, explained more than 95% of the sequence-dependent variation in the deamidation half-lives. The amide hydrogen exchange rate (i.e., amide proton acidity), hydrophilicity, polarizability, and size of amino acids in position n+1 were found to be the principal factors governing the rate of deamidation. The effect of amino acids in position n-1 was found to be negligible. CONCLUSIONS: Chemometric analysis employing reduced physicochemical parameters can provide an accurate prediction of chemical instability in peptides and proteins. The relative importance of these various factors could also be determined.

Optimization and applications of CDAP labeling for the assignment of cysteines.

PURPOSE: The aim of the study is to provide a methodology for assigning unpaired cysteine residues in proteins formulated in a variety of different conditions to identify structural heterogeneity as a potential cause for protein degradation. METHODS: 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was employed for cyanylating free cysteines in proteins and peptides. Subsequent basic cleavage of the peptide bond at the N-terminal side of the cyanylated cysteines provided direct information about their location. RESULTS: CDAP was successfully employed to a wide variety of labeling conditions. CDAP was reactive between pH 2.0 and 8.0 with a maximum labeling efficiency at pH 5.0. Its reactivity was not affected by excipients, salt or denaturant. Storing CDAP in an organic solvent increased its intrinsic stability. It was demonstrated that CDAP can be employed as a thiol-directed probe to investigate structural heterogeneity of proteins by examining the accessibility of unpaired cysteine residues. CONCLUSION: CDAP is a unique cysteine-labeling reagent because it is reactive under acidic conditions. This provides an advantage over other sulfhydryl labeling reagents as it avoids potential thiol-disulfide exchange. Optimization of the cyanylation reaction allowed the utilization of CDAP as a thiol-directed probe to investigate accessibility of sulfhydryl groups in proteins under various formulation conditions to monitor structural heterogeneity.

Inverse relationship of protein concentration and aggregation.

PURPOSE: To determine the effect of protein concentration on aggregation induced through quiescent shelf-life incubation or shipping-related agitation. METHODS: All aggregation was measured by size-exclusion high-performance liquid chromatography. Aggregation was induced by time-dependent incubation under stationary conditions or by agitation caused by shaking, vortexing, or vibration using simulated shipping conditions. RESULTS: Protein aggregation is commonly a second- or higher-order process that is expected to increase with higher protein concentration. As expected, for three proteins (PEG-GCSF, PEG-MGDF, and OPG-Fc) that were examined, the aggregation increased with higher protein concentration if incubated in a quiescent shelf-life setting. However, aggregation decreased with higher protein concentration if induced by an air/water interface as a result of agitation. This unexpected result may be explained by the rate-limiting effect on aggregation of the air/water interface and the critical nature of the air/ water interface to protein ratio that is greatest with decreased protein concentration. The non-ionic detergent polysorbate 20 enhanced the aggregation observed in the quiescently incubated sample but abrogated the aggregation induced by the air/water interface. CONCLUSIONS: The effect of protein concentration was opposite for aggregation that resulted from quiescent shelf-life treatment compared to induction by agitation. For motionless shelf-life incubation, increased concentration of protein resulted in more aggregation. However, exposure to agitation resulted in more aggregation with decreased protein concentration. These results highlight an unexpected complexity of protein aggregation reactions.