Thermodynamic and fluorescence analyses to determine mechanisms of IgG1 stabilization and destabilization by arginine.

PURPOSE: To investigate mechanisms governing the stabilization and destabilization of immunoglobulin (IgG1) by arginine (Arg). METHODS: The effects of Arg on the aggregation/degradation, thermodynamic stability, hydrophobicity, and aromatic residues of IgG1 were respectively investigated by size-exclusion chromatography, differential scanning calorimetry, probe fluorescence, and intrinsic fluorescence. RESULTS: Arg monohydrochloride (Arg-HCl) suppressed IgG1 aggregation at near-neutral pH, but facilitated aggregation and degradation at acidic pH or at high storage temperature. Equimolar mixtures of Arg and aspartic acid (Asp) or glutamic acid (Glu) suppressed aggregation without facilitating degradation even at high temperature. Arg-HCl decreased the thermodynamic stability of IgG1 by enthalpic loss, which was counteracted by using Asp or Glu as a counterion for Arg. The suppression of aggregation by Arg-HCl was well correlated with the decrease in hydrophobicity of IgG1. The intrinsic fluorescence of IgG1 was unaffected by Arg-HCl. CONCLUSIONS: Suppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.

Effect of buffer species on the unfolding and the aggregation of humanized IgG.

The aggregation propensity of humanized antibody after heat treatment is evaluated in the presence of six buffer species. The comparison under equivalent pH showed high aggregation propensity on phosphate and citrate buffer. In contrast, 2-(N-Morpholino) ethane sulfonate (MES), 3-(N-Morpholino) propane sulfonate (MOPS), acetate and imidazole buffer showed lower aggregation propensity than the above two buffers. Meanwhile, unfolding temperature evaluated by differential scanning calorimetry measurement was not altered among these buffer species. The light scattering analysis suggested that heat-denatured intermediate was aggregated slightly on MES and acetate buffer. Therefore, it was found that the different aggregation propensity among buffer species was caused from the aggregation propensity of heat-denatured intermediate rather than the unfolding temperature. Furthermore, it was revealed that the aggregation dependency on buffer species is accounted for by the specific molecular interaction between buffer and IgG, rather than the ionic strength. On the contrary, on the analyses of unfolding and aggregation propensity by molecular dissection of IgG into Fab and Fc fragments, aggregation propensity of Fc fragment on MES, acetate and phosphate buffer was almost the same as whole IgG. From the above results, it was suggested that the specific interaction between buffer molecule and Fc domain of IgG was involved in the aggregation propensity of heat-denatured IgG.

A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N.

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.

Effect of the conformational stability of the CH2 domain on the aggregation and peptide cleavage of a humanized IgG.

To examine the effect of the conformational stability of the CH2 domain on aggregation and peptide cleavage of a humanized IgG1, we carried out size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of incubated sample solutions. By comparing the residual percentage of monomer after incubation at 60 and 80°C at various pH levels, we found that aggregation and peptide cleavage of the humanized IgG1 occurred during long incubation at 60°C under acidic conditions. Next, we confirmed cleavage of the Asp272-Pro273 peptide bond in the CH2 domain. Comparison of the cleavage rates of the IgG1 monomer and a peptide containing the same Asp-Pro sequence revealed that the conformational stability of the CH2 domain retards cleavage of the Asp272-Pro273 peptide bond at 60°C and pH 4.0. The finding of aggregation and peptide cleavage of the humanized IgG1 after long incubation at 60°C under acidic conditions was supported by another finding: there were lower unfolding temperatures of the CH2 domain at pH 4.0 and 5.0. We conclude that the conformational stability of the CH2 domain is closely related to aggregation and peptide cleavage of the humanized IgG1 under acidic conditions. We also found that the 2-[N-morpholino] ethane sulfonate buffer inhibits aggregation of the IgG1 at pH 4.0-5.0 and 7.0-8.0.