More comprehensive standards for monitoring glycosylation

Nicotiana spp. for the Expression and Purification of Functional IgG3 Antibodies Directed Against the Staphylococcus aureus Alpha Toxin

“…This was attributed to the presence of additional glycosylation sites in the IgG3 heavy chain (Plomp et al, 2015;De Haan et al, 2019), further contributing to the difference in apparent molecular mass compared to IgG1 (Ma et al, 1994). For example, even ER-retained mAbs are modified in tobacco with high-mannose-type N-linked glycans featuring 6-9 mannose residues (Gomord et al, 2004), which have a mass of ∼1.4-1.9 kDa (Guo et al, 2021). Consequently, a mass increase of ∼2 kDa would be anticipated solely based on the additional N-glycosylation site in the IgG3 CH3 region compared to IgG1 (De Haan et al, 2019).…”

USP Reference Standard Monoclonal Antibodies: Tools to Verify Glycan Structure

“…These isomers may coexist, or it is possible that only one of them dominates, as they cannot be distinguished with the current approach. This indicates the limitation of the current approach, although it is widely used currently in industry [ 3 , 8 ]. A combination of this approach with automatic analysis of LC-MSMS data and ion mobility data may help to differentiate the glycan isomers precisely [ 9 ].…”

“…As previously reported [ 8 , 9 ], without validated reference standards, manufacturers cannot create an in-depth glycan profile that includes the minor glycans that may be present or validate the suitability of their equipment for such an analysis. The major glycans present in the USP monoclonal antibodies cover a quantitative range ( Table 5 ) which gives added benefit in their use in evaluating the suitability of an analytical method.…”

“…It is the specificity of binding and mode of action of the monoclonal antibody toward its target that allows these therapeutics to precisely treat the illness. There are numerous structural features that contribute to the specific actions of a given monoclonal antibody, including the glycan structure associated with the monoclonal antibody [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. The glycan structure of a monoclonal antibody is typically heterogeneous with a predominance of a particular structure(s) that can contribute to stability, bioactivity, pharmacokinetics and pharmacodynamics (PK/PD), and immunogenicity of the molecule [ 4 , 5 , 6 , 9 ].…”

“…Analytical characterization of the glycan structure of a monoclonal antibody requires scientifically sound analytical procedures that are accurate, precise, and reproducible [ 1 , 2 , 3 , 7 , 8 ]. To demonstrate that an analytical procedure is scientifically sound often requires characterizing a reference standard in parallel with a test sample to demonstrate that the analytical procedure was performing properly as expected.…”