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

Abstract

Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247-253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C(H)2 and C(H)3 domains.

Figure 1

Stacked box plot of the measured hydrogen exchange for GSK-mAb. The sequence of GSK-mAb is shown with stacked boxes indicating exchange. The length of box indicates the peptide for which hydrogen exchange was measured. Each box in the stacks of seven boxes represents a different time of exchange, starting with 30 s for the top box and increasing to 30000s for the bottom box. The colour of the boxes represents the amount of exchange that was measured, as has been indicated. The domains of the protein are indicated above the sequence, as is the site of glycan attachment.

Figure 2

Comparison of the hydrogen exchange measured for the peptide FLFPPKPKDTL (HC[242–252]) in native and oxidised samples of GSK-mAb. Plotted values are the average of 4 measurements, with the error bars showing 2 standard deviations of the measurement.

Figure 3

Crystal structure of Fc region of an IgG (PDB entry 1HZH). Glycans are coloured green, residues coloured pink are peptides that exchanged faster in oxidised sample than in native sample of GSK-mAb. Residues coloured red are from peptides that exchanged much faster in the oxidised sample. Residue coloured dark blue is Met253. Residue coloured light blue is Met430. Sulfur atoms of methionine residues are coloured yellow. Residues 247–252 have been coloured a darker shade of red.

Figure 4

Hydrogen exchange measured for Met253 in native and oxidised samples of GSK-mAb. Standard deviations shown were calculated from a combination of those from the measurements used to calculate exchange of Met253.

Figure 5

Comparison of the measured hydrogen exchange for peptides FLFPPKPKDTLM and the same peptide containing oxidised methionine FLFPPKPKDTLMox (HC[242–253]) in native and oxidised samples of GSK-mAb. Plotted values are the average of 4 measurements, with the error bars showing 2 standard deviations of the measurement.

Figure 6

CGE-LIF analysis of APTS labelled GSK-mAb glycans from the oxidised and unoxidised samples released using the PNGase F digestion protocol. Capillary temperature was maintained at 20°C.

Figure 7

Positive ion MALDI mass spectra of glycans from oxidised and unoxidised GSK-mAb samples released using the PNGase F digestion protocol.