Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody

Abstract

The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (approximately 50 kD) ("intact protein"), or corresponding synthetic peptides ("peptide") were stored in Tris buffer at 37 degrees C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N(382) to form the isoaspartate (isoD(382)) and aspartate (D(382)) products in the ratio of approximately 4:1, with a half-life of approximately 3.4 days. The succinimide intermediate (Su(382)) was also detected; deamidation was not observed for the other two sites (N(387) and N(388)) in peptide samples. The intact protein showed a 30-fold slower overall deamidation half-life of approximately 108 days to produce the isoD(382) and D(387) products, together with minor amounts of D(382). Surprisingly, the D(382) and isoD(387) products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N(388). The results indicate that higher order structure influences both the rate of N-deamidation and the product distribution.

Figure 1

Deamidation products in the NNN synthetic peptide. Representative extracted ion chromatogram (EIC) (A) and molecular ion isotope envelopes (B) of IsoD₃₈₂NN (1), NNN (2) and D₃₈₂NN (3) peaks in the EIC for a sample of the NNN synthetic peptide stressed for 90 h at 37°C, pH 7.4. The EIC (A) shows the elution order, with IsoD₃₈₂NN eluting first followed by NNN and D₃₈₂NN. The isotope envelopes (B) show the +1 amu mass increase for the first and third peaks, consistent with deamidated forms of the NNN peptide.

Figure 2

High energy MS1 spectra of peaks 1 (A), 2 (B) and 3 (C) min of Figure 1. The fragmentation pattern, particularly the , , and ions, together with the elution and fragmentation patterns of synthetic peptide standards (see text), identifies the peaks as the IsoD₃₈₂NN, NNN (parent) and D₃₈₂NN forms, respectively. See the electronic version of this article for enlarged figures.

Figure 3

Deamidation products in Fc IgG. Representative extracted ion chromatogram (EIC) (A) and molecular ion isotope envelopes (B) of IsoD₃₈₂NN (1), NNN (2), a doubly deamidated product (3), ND₃₈₇N (4), and D₃₈₂NN (5) peaks in the EIC, for a sample stressed for 28 h at 37°C, pH 7.4 before digestion. The EIC shows the elution order with IsoD₃₈₂NN eluting first followed by NNN, doubly deamidated and D₃₈₂NN peptides. The isotope envelopes show the +1 amu mass increase for the singly deamidated species and +2 amu for the doubly deamidated product.

Figure 4

High energy MS1 spectra of peaks 3 (A) and 4 (B) of Figure 3. The peptides were identified using the y″ ions as the doubly deamidated and the D₃₈₇ peptide, respectively. Other ions were used to confirm the products. See the electronic version of this article for enlarged figures.

Figure 5

Reaction scheme showing the kinetics of deamidation of (A) the NNN synthetic peptide and (B) intact Fc IgG.

Figure 6

Kinetic profile for deamidation of the NNN synthetic peptide stored at 37°C, pH 7.4. The symbols are experimental data points; the lines represent the fit obtained with the kinetic model described in the text. n = 3 ± S.D.

Figure 7

Kinetic profile for deamidation in Fc IgG stored at 37°C, pH 7.4. The symbols are experimental data points; the lines represent the fit obtained with the kinetic model described in the text. n = 3 ± S.D.