Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
All IgG mAbs  contain the Asn297-X-Ser/Thr-Y sequon (exact sequence of amino acids) in the constant CH2 domain, which is the prerequisite for N-glycosylation, where X is any amino acid besides proline [42, 43]. This sequon is common to the IgG CH2 domain but is also observed in the variable domains of the Fab region in around 20% of IgGs . As a consequence, the most commonly encountered PTMs in IgG mAbs are N-linked glycans, although the glycosylation on the two HC within a single IgG is not necessarily identical and in some cases they are not glycosylated at all .
The degree of glycosylation can have a high impact upon the efficacy, toxicology, and structure of the mAb therapeutic, most commonly by altering the binding affinity to the target receptor [45, 46]. The main glycoforms observed in IgGs expressed in CHO cells are G0 and its fucosylated counterpart, G0F, along with G1F, G2F, and the high-mannose glycan Man5 [21, 47]. Each of the glycans named possesses the same trimannosyl core; G denotes the number of galactose units connected to the two additional GlcNAc units, and for high- mannose species the number denotes the total number of mannose moieties connected to the chitobiose core (GlcNAc-GlcNAc). F denotes the presence of core fucosylation (see Figure 10.2). It has been reported on several occasions, for example, that the absence of the fucose residue at the core of the ^-glycans
Figure 10.2 Common N-glycans present in CHO produced IgGs. N-glycans are posttranslational modifications that add to the Asn-X-Ser/Thr sequons of IgG heavy chains. Variations of the aforementioned also occur. G and the sequential number indicate the number of galactose units attached to the GlcNAc residues. F denotes the presence of fucose within the chitobiose core (GlcNAc-GlcNAc).
of the Fc constant region (referred to as Fc glycan core afucosylation) has a large impact upon binding to protein receptors [43, 44, 48-51]. In trastuzumab (Herceptin®, Genentech) that specifically targets the human epidermal growth factor receptor type 2 (HER2) that is overexpressed in 20-30% of breast carcinomas , a high level of afucosylation increases the antibody-dependent cell-mediated cytotoxicity (ADCC) of the mAb. The increase in binding affinity of the therapeutic protein toward the Fcylll receptor (FcyRIII) on the effector function immune system cells is achieved by the reduction in steric hindrance surrounding the N-glycosylated CH2 domain of the Fc region . With the binding of FcyR, macrophages and natural killer (NK) cells are recruited, hence stimulating the required immune response and activating the ADCC pathway [39, 54].
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