Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques

Abstract

The European Medicines Agency received recently the first marketing authorization application for a biosimilar monoclonal antibody (mAb) and adopted the final guidelines on biosimilar mAbs and Fc-fusion proteins. The agency requires high similarity between biosimilar and reference products for approval. Specifically, the amino acid sequences must be identical. The glycosylation pattern of the antibody is also often considered to be a very important quality attribute due to its strong effect on quality, safety, immunogenicity, pharmacokinetics and potency. Here, we describe a case study of cetuximab, which has been marketed since 2004. Biosimilar versions of the product are now in the pipelines of numerous therapeutic antibody biosimilar developers. We applied a combination of intact, middle-down, middle-up and bottom-up electrospray ionization and matrix assisted laser desorption ionization mass spectrometry techniques to characterize the amino acid sequence and major post-translational modifications of the marketed cetuximab product, with special emphasis on glycosylation. Our results revealed a sequence error in the reported sequence of the light chain in databases and in publications, thus highlighting the potency of mass spectrometry to establish correct antibody sequences. We were also able to achieve a comprehensive identification of cetuximab's glycoforms and glycosylation profile assessment on both Fab and Fc domains. Taken together, the reported approaches and data form a solid framework for the comparability of antibodies and their biosimilar candidates that could be further applied to routine structural assessments of these and other antibody-based products.

Keywords: biosimilar; cetuximab; glycosylation; mass spectrometry; sequence.

Figure 1. Deconvoluted ESI-Q-TOF mass spectrum of intact cetuximab. The calculated MW for the G0F/G0F; G2FGal2/G2FGal2 isoform with pyroglutamic acid formation at the N-termini of the heavy chains is 152235 Da, which is 116 Da lower than the experimental MW at 152351 Da.

Figure 2. Sequences of the light and heavy chains of cetuximab as reported in the IMGT and in the literature.^, The N-glycosylation sites are indicated in bold character.

Figure 3. Total ion chromatogram of IdeS cleaved and reduced cetuximab separating the three major subunits. For each of the Fc/2 and Fd subunits, two peaks were observed. Fc/2 shows lysine clipping at the heavy chain’s C-terminus, Fd exhibits glycosylation heterogeneity (presence of sialic acid).

Figure 4. Deconvoluted spectra (red) of the main isoforms of the subunits after IdeS digestion and reduction, (A) Fc/2, (B) LC and (C) Fd. The monoisotopic MWs were calculated from the baseline resolved isotopic peak patterns using the SNAP peak picking algorithm. The isotopic patterns calculated from the sequence of the antibody as specified in the IMGT database were also displayed for the three subunits (blue). The position of the average mass is marked with an arrow. For Fc/2, the isotopic pattern was calculated taking into account lysine clipping at the C-terminus and G0F glycosylation. For the Fd subunit N-terminal pyroglutamic acid formation and G2FGal2 glycosylation was assumed. While the agreement between the measured and expected monoisotopic mass is well below 1 ppm for the Fc/2 and Fd fragments [-1.5 mDa for Fc/2 (A) and 0.4 mDa for Fd (C)], a significant MW difference of 58.006 Da was observed for the LC (B) clearly indicating a sequence or structural variation.

Figure 5. LC-MALDI-ISD middle-down spectrum of cetuximab’s light chain after chromatographic separation, using sDHB as matrix. The N-terminal sequence of the first 86 residues matches the spectrum precisely (top) while the C-terminal sequence displays a +58 Da offset. After assuming an A to E exchange (+58 Da), the C-terminal sequence is in accordance with the ISD-spectrum.

Figure 6. LC-MALDI-MS/MS mass spectrum of the gluC C-terminal peptide of cetuximab’s light chain confirming E213 instead of A.

Figure 7. LC-ESI-MS spectra of (A) Fc/2 subunit and (B) Fd subunit. Only 19 out of 20 are displayed, the lowest abundant species is out of the mass range shown. The peaks were annotated with the correspondent glycoform.

Figure 8.(A) Relative abundance of glycoforms attached to the N299 glycosylation site (Fc) as derived from middle-up glyco-profiling (Left). Eleven glycoforms were assigned to this glycosylation site. (B) Relative abundance of glycoforms of glycosylation site N88 (Fd) as derived from middle-up glyco-profiling (Right). Twenty glycoforms could be assigned to this glycosylation site.