Structure and Stability Changes of Human IgG1 Fc as a Consequence of Methionine Oxidation

Abstract

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

“…D. Liu et al reported analysis by DSC of mutants of Human IgG1 Fc that lacked either methionine residue analysis of Human IgG1 Fc demonstrated that oxidation of either methionine resulted in structural changes to the C H 2 and C H 3 domains, the results described here add further weight to this conclusion. 17 It may Figure 7. Positive ion MALDI mass spectra of glycans from oxidised and unoxidised GSK-mAb samples released using the PNGase F digestion protocol.…”

“…[17][18][19] Liu et al analysed oxidised recombinant human IgG1 Fc that had been expressed in E. coli using a variety of biophysical and biochemical techniques. 17 Analysis by CD and NMR of an isotopically labelled Fc fragment showed that methionine oxidation resulted in detectable secondary and tertiary structural changes in the interface between the C H 2 and C H 3 domains. Differential scanning calorimetry (DSC) was used to measure the melting temperature (T m ) of the C H 2 and C H 3 domains.…”

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

“…Such analysis will provide the ability to assess the structural impact of a range of parameters, including post-translational modifications such as methionine oxidation. 52 In the case study of the small protein therapeutic filgrastim, resonance assignments allowed a detailed mapping of the structural effects for different formulation conditions. 53 For mAb therapeutics, the isolated Fab and Fc are known to retain a similar HOS to that observed for these fragments in the intact NISTmAb.…”

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

“…This increase is predictive of decreased long-term stability, as oxidation can trigger further degradation events such as aggregation. 28,32 Thus, MS emerged as a critical stability-indicating assay for the evaluation of oxidative degradation pathways.…”