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.

The homogeneous, gas-phase pyrolytic thermal degradation of 2-chlorophenol was studied in a 1 cm i.d. fused silica flow reactor at a concentration of 90 ppm, with a reaction time of 2.0 s, and over a temperature range of 300-1000 °C. Observed products included dibenzo-p-dioxin (DD), 1-monochlorodibenzo-p-dioxin (1-MCDD), dibenzofuran (DF), naphthalene, chloronaphthalene, phenol, chlorobenzene, and benzene. These results differed significantly from other reported lower temperature studies with reaction times of 10-100 s. At temperatures lower than 700 °C, formation of the 2-chlorophenoxyl radical, which decomposes through CO elimination to form a chlorocyclopentadienyl radical, forms naphthalene and 2-chloronaphthalene through radical recombination/rearrangement reactions. The formation of DD and 1-MCDD are attributed to radical-radical reactions involving the 2-chlorophenoxyl radical with the carbon-chlorine-centered radical mesomer and the carbon-hydrogen-centered radical mesomer of 2-chlorophenoxyl radical, respectively. Unlike previously reported studies with 2-chlorophenol, 4,6dichlorodibenzofuran, which is purported to be due to radicalradical recombination pathways, was not detected. This is attributed to pyrolytic conditions of our experiments and the resulting shorter reaction times and higher temperatures that favor reaction intermediates that form DD and 1-MCDD.

The homogeneous, gas-phase oxidative thermal degradation of 2-chlorophenol was studied in a 1 cm i.d., fused silica flow reactor at a concentration of 88 ppm, reaction time of 2.0 s, over a temperature range of 300 to 1000 °C. Observed products in order of yield were as follows: 4,6dichlorodibenzofuran (4,6-DCDF) > dibenzo-p-dioxin (DD) > 1-monochlorodibenzo-p-dioxin (1-MCDD), 4-chlorodibenzofuran (4-MCDF), dibenzofuran (DF), naphthalene, chloronaphthalene, 2,4-dichlorophenol, 2,6-dichlorophenol, phenol, chlorobenzene, and benzene. In contrast to pyrolysis, 4,6-DCDF is the major product rather than DD, and 1-MCDD and naphthalene are formed at temperatures as low as 400 °C. Under oxidative conditions, •OH and Cl• are the major carriers, which favors 4,6-DCDF formation over DD or 1-MCDD through abstraction of H• through diketo-and ether-intermediates. It is proposed that below 500 °C, unimolecular tautomerization/HCl elimination and CO elimination/isomerization reactions result in the formation of 1-MCDD and naphthalene, respectively.

Brominated hydrocarbons are the most commonly used flame retardants. Materials containing brominated hydrocarbons are frequently disposed in municipal and hazardous waste incinerators as well as being subjected to thermal reaction in accidental fires. This results in the potential for formation of brominated dioxins and other hazardous combustion byproducts. In contrast to chlorinated hydrocarbons, the reactions of brominated hydrocarbons have been studied only minimally. As a model brominated hydrocarbon that may form brominated dioxins, we studied the homogeneous, gas-phase pyrolytic thermal degradation of 2-bromophenol in a 1-cm i.d., fused-silica flow reactor at a concentration of 90 ppm, with a reaction time of 2.0 s, and over a temperature range of 300 to 1000 degrees C. Observed products included dibenzo-p-dioxin (DD), 1-monobromodibenzo-p-dioxin (1-MBDD), 4-monobromodibenzofuran (4-MBDF), dibenzofuran (DF), naphthalene, bromonaphthalene, 2,4- and 2,6-dibromophenol, phenol, bromobenzene, and benzene. These results are compared and contrasted with previous results reported for 2-chlorophenol. At temperatures lower than 700 degrees C, formation of 2-bromophenoxyl radical, which decomposes through CO elimination to form a bromocyclopentadienyl radical, forms naphthalene and 2-bromonaphthalene through radical recombination/rearrangement reactions. However, unlike the results for 2-chlorophenol, where naphthalene is the major product, DD becomes the major product for the pyrolysis of 2-bromophenol. The formation of DD and 1-MBDD are attributed to radical-radical reactions involving 2-bromophenoxyl radical with the carbon- (bromine) centered radical and the carbon- (hydrogen) centered radical mesomers of 2-bromophenoxyl radical, respectively. The potential product, 4,6-dibromodibenzofuran (4,6-DBDF) for which the analogous product, 4,6-dichlorodibenzofuran (4,6 DCDF), was observed in the oxidation of 2-chlorophenol, was not detected. This is attributed to the pyrolytic conditions of our experiments (e.g., shorter reaction times and higher temperatures) that favor reaction intermediates that form DD and 1-MBDD.

The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called “bottom-up” approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced – the “Sequence Validation Percentage.” Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only −2 Da in the natalizumab Fd domain, were corrected as a result of this work.