N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies

Abstract

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.

FIGURE 1.

Pyroglutamate formation mechanism. The mechanism of pyroglutamate (pyroGlu) formation from Glu or Gln is shown.

FIGURE 2.

mAb1 peptide map. A portion of mAb1 peptide map chromatogram at (A) 214 nm (UV) and (B) mass spectrometry total ion current is shown. Peak 1, HC N-terminal peptide; peak 2, pE modified HC N-terminal peptide.

FIGURE 3.

mAb1 N-terminal pE levels in vivo for the 1000-mg intravenous dosing. A, circles, HC pE levels in subject 1; diamonds, HC pE levels in subject 2; squares, LC pE levels in subject 1; triangles, LC pE levels in subject 2. The trend lines represent the averages from the two subjects. B, comparisons of all the in vivo dosings of mAb1. Two subjects for each dosing are shown. Upper curve, relative HC pE levels. Lower curve, relative LC pE levels in vivo.

FIGURE 4.

Comparison of in vivo and in vitro pE formation rates. N-terminal Glu to pE conversion of mAb1 HC in vivo (circles) and in vitro in PBS at 37 °C at pH 7.4 (squares). The data represent the averages of two data sets for each experiment.

FIGURE 5.

LC N-terminal region homology models of mAb1 and mAb2. Ribbons in green and light blue represent the model structures for mAb1 and mAb2, respectively. Side chain residues for light chain Glu-1, Pro-72 (mAb1), and Asp-72 (mAb2) are depicted as sticks (Kabat numbering). The distance from the Asp-61 delta oxygen and Glu-1 epsilon oxygen on the mAb2 model is given.

FIGURE 6.

Patient exposure profiles based on dosing. The time course profile is based on mAb1 pharmacokinetic profile and the Glu to pE conversion rate for the mAb1 HC. A, 1000-mg intravenous dosing; B, 300-mg intravenous dosing.