Analysis of Afucosylation Levels
Introduction
Afucosylation of the N-glycans found on IgGs can impact the receptor binding of the antibody and thus the antibody dependent cytotoxicity (ADCC). For this reason, complete characterization of the degree of antibody afucosylation and total glycosylation of antibody-based therapeutics is of great importance. SmartEnzymes are a popular tool for the studying the degree of afucosylation on therapeutic antibodies, highlighted by the publications below.
Quantification of Afucosylation and N-glycan Site Occupancy
Figure 1. GlycINATOR workflow
To quantify the level of afucosylation researchers Liu et al. at Biogen developed a quick, one-hour workflow achieving high precision, sensitivity, robustness and improved throughput. The N-linked glycans were removed after the first GlcNAc residue using GlycINATOR. Reduction of the inter-chain disulfide bridges was then performed prior to LC-MS analysis. In addition to the level of afucosylation, the of the afucosylation characterization also provided information on the N-glycan site occupancy. This approach can be applied to clone selection, process development and other assays where high-throughput and fast analysis is required.
Figure 2. IgGZERO workflow
A similar workflow was developed by researchers Upton et al. at Covance, where the level of core afucosylation on a therapeutic antibody was studied using GlycINATOR. The results correlated with an increase in Fc receptor binding affinity and in vitro ADCC activity in a cell-based assay. 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 a robust assessment of the characteristics of the glycans. Compared to GlycINATOR, IgGZERO does not cleave high-mannose or hybrid type Fc-glycans so the enzyme was used to estimate the mannose content, another important quality attribute of biotherapeutics.
Degree of Fucosylation in Clone Selection using IgGZERO
Absence of a core fucose on the N-glycan of a therapeutic antibody is associated with an increased ADCC, a desirable property of a drug product.
IgGZERO (EndoS) specifically hydrolyses the N-linked glycan on native IgG after the first GlcNAc, thus leaving the 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 took advantage of this and developed a technique for studying 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 then analyzed using LC-MS. This allows for the easy determination of the fucosylation levels of the antibody and enhanced selection of clone candidates.
Fucosylation in Whole Blood
The specificity of the SmartEnzymes allows for the detailed characterization of antibody attributes even in complex medium such as serum. Hereditary, allelic gene variations and glycoform variants were studied by researchers at Amgen. They employed FabRICATOR to generate antibody fragments that were analyzed directly on LC-MS (Goetze et al. 2011). IgGZERO was utilized to hydrolyze the antibody glycans after the core Glc-NAc. The difference in mass of the fucose (-146 Da) allowed detailed characterization of the degree of fucosylation in the deconvoluted mass spectra. The level of fucosylation was compared in different donors and remarkable differences were observed.
Figure 3. FabRICATOR workflow