Level of Afucosylation
Afucosylation of the IgG N-glycans impacts receptor binding of the antibody and thus the antibody dependent cytotoxicity (ADCC). For this reason, a complete characterization of the degree of antibody afucosylation and total glycosylation is of great importance for all antibody based therapeutics.
Genovis SmartEnzymes have been used to study the degree of afucosylation on therapeutic antibodies in a number of publications.
Quantification of afucosylation and N-glycan site occupancy using GlycINATOR
Lack of core fucose on therapeutic antibodies are associated with increased antibody dependent cytotoxicity (ADCC) and is a desirable property of a drug product. To quantify the level of afucosylation, researchers at Biogen used GlycINATOR to remove the N-linked glycans after the first GlcNAc residue, reduced the inter-chain bindings, and applied liquid chromatography/mass spectrometry analysis (Liu et al. 2016). By using this 1h workflow, the authors obtained precision, sensitivity, robustness and improved throughput. In addition to characterize the afucosylation levels, information on N-glycan site occupancy were obtained. The analysis can be applied to clone selection, process development and other assays where high-throughput and fast results are required.
A similar workflow has been developed by researchers at Covance, where the level of core afucosylation on a therapeutic antibody was studied using GlycINATOR and correlated with an increase in Fc receptor binding affinity and in vitro ADCC activity in a cell based assay (Upton et al. 2016). The scientists also used FabRICATOR to study the total antibody glycan composition of the Fc/2 in a middle-up workflow using LC/MS and obtained robust assessments of the characteristics of the glycans. As IgGZERO does not cleave high-mannose or hybrid type Fc-glycans, the enzyme was used to estimate the mannose content, another important quality attribute of biotherapeutics.
- Afucosylation quantitation using GlycINATOR and LC/MS
- Glycan analysis of Fc/2 using FabRICATOR middle-up analysis
- Estimation of high-mannose content using IgGZERO
Upton, R. et al., 2016. Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action. Analytical Chemistry, pp.acs.analchem.6b02994–7.
Degree of Fucosylation in Clone Selection using IgGZERO
IgGZERO (EndoS) specifically cleaves the N-linked glycan of native IgG after the first GlcNAc, thus leaving a GlcNAc with or without a fucose residue attached. This specific and rapid hydrolysis reduces sample complexity dramatically and allow easy interpretation of MS data. French researchers at Institute Pasteur in collaboration with LFB Biotechnologies have developed a method to study the degree of fucosylation in cell culture supernatant, as part of a clone selection program (Henninot et al. 2015). Briefly, the cell supernatant is concentrated, digested with FabRICATOR and IgGZERO, reduced and analyzed using LC-MS. With this method the fucosylation levels of the antibody could easily be determined and led to a better selection of clone candidates.
- IgGZERO works in cell culture medium
- Rapidly deglycosylates IgG at Fc
- Can be used to study degree of fucosylation
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Fucosylation in Whole Blood
The specificity of the SmartEnzymes allows detailed characterization of antibody attributes even in such a complex medium as serum. To study antibody allelic and glycoform variants, FabRICATOR was employed by researchers at Amgen to generate antibody fragments that were analyzed directly on LC-MS (Goetze et al. 2011). IgGZERO was utilized to cleave off antibody glycans after the first Glc-NAc. The difference of a fucose (-146 Da) allowed detailed characterization of fucosylation degree in the deconvoluted mass spectra. The level of fucosylation was studied between different donors and remarkable differences were found.
- FabRICATOR and IgGZERO can be used in serum
- Fucosylation can be studied in automated analysis
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