Antibody Fc-glycan Analysis
Introduction
Most commercial therapeutic antibodies are of the class IgG and contain an N-glycan in the Fc domain. An important critical quality attribute is the glycosylation profile since it can affect the stability, solubility, pharmacodynamic-, and pharmacokinetic properties. Such posttranslational modifications can occur during the bioprocess development and manufacturing and thereby need to be monitoring closely.
The FabRICATOR (IdeS) enzyme has become a widely accepted analytical tool for the rapid characterization of monoclonal antibodies (mAbs). FabRICATOR digests IgG below the hinge, generating F(ab’)2 and Fc/2 fragments. The single FabRICATOR digestion site and the following accuracy of the mass profile have enabled detection of the Fc glycosylation profile allowing for the monitoring of batch-to-batch consistency and other critical quality attributes.
Fc glycan Analysis using FabRICATOR Digestion
Figure 1. Classical middle down workflow for mAb analysis using LC/MS
A popular method for analyzing Fc glycosylation is hydrophilic interaction liquid chromatography high-performance liquid chromatography (HILIC-HPLC) on released N-glycans labelled with 2-aminobenzamide (2-AB; 1,2). However, this methodology suffers from several time-consuming hands-on steps. Digestion with FabRICATOR (IdeS) is rapid (30 minutes) and due to its specific activity there is no risk of over digestion that otherwise might result in heterogeneity of the antibody sample. It digests human IgG at a specific site below the hinge, generating a homogenous pool of Fc/2 and F(ab’)2 fragments (Fig. 1).
The smaller fragment size of Fc/2 (~25 kDa) compared to the intact heavy chain (HC, ~50 kDa), allows for monoisotopic resolution of the glycan profile when analyzed with ESI-MS. If there is an interest in the Fd’ and LC fragments, the reaction can also be continued with reduction of the antibody fragments for 60 minutes. In an extensive comparison between different methods for Fc glycosylation analysis, the middle-up analysis of Fc/2 glycans using LC-MS was found to be robust, delivering reliable results for the IgG glycoprofile. The relative glycoform distribution has also been shown to correlate well with those of other methods, such as HILIC-UHPLC separation of released and 2-AB labelled glycans and an LC- MS analysis of reduced, intact antibody.
Paired Antibody Glycan Analysis using Intact Fc Fragments
For analysis of intact and paired Fc glycans, bi- or multi-specific antibodies, monovalent binding, higher order structure, disulfide scrambling and antibodies with mutated hinge regions, an alternative method is to use the cysteine protease FabALACTICA (IgdE). It digests the human IgG1 at one specific site above the hinge (...KSCDKT / HTCPPC...) and generates intact Fab and Fc fragments (Fig. 2). The enzyme does not require reducing conditions or co-factors for activity and is active at pHs from 6-8. FabALACTICA is recombinantly expressed in E. coli, contains a His-tag, and the molecular weight is ~70 kDa.
Figure 2. Schematic overview of FabALACTICA digestion of human IgG1.
Antibody Fc glycan Analysis using FabRICATOR
In this application note, the Fc glycosylation profile of the therapeutic antibody trastuzumab (human IgG1) was analyzed. The antibody was digested with FabRICATOR, denatured and reduced, and the resulting fragments Fc/2, Fd’ and light chain (LC) were separated and analyzed by LC-MS. The short digestion time of FabRICATOR in combination with the ~25 kDa Fc/2 fragment, enabled a fast analysis of the glycan profile. The measured monoisotopic masses of Fc/2, LC and Fd’ fragments were compared to theoretical data and the relative distribution of the different Fc glycoforms calculated. The fast and easy workflow presented allows for the determination of the glycan composition, highlighting how the glycan profile can be monitored during process development and help assure batch-to-batch consistency during the production process.
Antibody Fc Glycan Analysis by FabRICATOR and LC-MS
Paired Glycan Analysis using FabALACTICA
In this application note, the glycoforms of different mAbs were analyzed by LC-MS after digestion with FabALACTICA. When analyzing the intact mAb different glycoforms could be identified. However, after FabALACTICA digestion several additional glycan pairs were observed, including those of functional importance. In addition, the method reduced the complexity of the Fab glycosylation to a manageable level.
