Fc-Fusion has diverse structures and complex glycosylation modifications. Therefore, in order to reduce the complexity of the sample, different methods are usually used for glycoprotein analysis according to protein size: intact and subunit protein level (Top and Middle-up), glycopeptide (Bottom-up) and free glycan level. Typically, methods used to characterize glycosylation modifications include lectin microarrays, reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), anion exchange chromatography (AEX), porous graphitic carbon (PGC) or Capillary electrophoresis (CE), usually combined with techniques such as matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), amperometric or fluorescence detection (FD), etc.
However, the glycosylation modifications of Fc-Fusion tend to be more complex and diverse than those of common mAb, with multiple N- or O-glycosylation sites, so techniques that are traditionally applied to mAb are not necessarily suitable for analyzing and characterizing Fc-Fusion.
HILIC is the most commonly used technique for glycan analysis in Fc-Fusion process development and quality control, which usually requires pre-separation of free N-glycans from Fc-Fusion and then fluorescence labeling before analysis. The procedure is as follows: first, the N-glycan is separated from the glycoprotein using enzymes such as PNGase F; then the N-glycan is fluorescently labeled by reductive amination with 2-aminobenzamide (2-AB); subsequently, the labeled glycan is separated using a HILIC column and identified according to the gradient of 2-AB-labeled dextran. However, HILIC (2-AB) also has some disadvantages. For example: the sample preparation time is long (∼40 h); it is prone to co-elution. The combination of HILIC-FD detection and MS identification techniques with RapiFluor MS labeling method can overcome these drawbacks, not only by significantly reducing the sample preparation time (∼1 h), but also by significantly improving the sensitivity of fluorescence and mass spectrometry detection. Moreover, the addition of MS detection provides a fully orthogonal identification method for FD, resulting in more reliable characterization and quantification in the case of co-elution.
Sialylation
Similarly, similar to the methods described above, the sialic acid modification of Fc-Fusion can also be characterized using methods to separate and label glycans. In general, N-acetylneuraminic acid and non-human N-glycylneuraminic acid can be used to introduce a negative charge into the target Fc-Fusion molecule and then analyze the sialic acid using AEX chromatography, which separates neutral glycans (non-sialylated) from monosialylated, disialylated and tri-sialylated glycans. It is important to note that these techniques cannot distinguish between different types of sialic acids, e.g. Neu5Ac and Neu5Gc. Due to the immunogenicity of the non-human naturally Neu5Gc, another complementary approach was required to characterize the sialylation of Fc-Fusion. The steps are: first, chemical separation of sialic acid from glycans; then, fluorescent labeling using DMB; and finally, RPLC chromatography and FD detection.
Structures of sialic acids and the diversity of sialylated glycoproteins.
O-glycosylation
Fc-Fusion usually carries multiple O-glycosylation sites in the non-IgG domain, and since it cannot be cleaved with PNGase F, different analytical methods are required for characterization. Typically, O-glycans are isolated by reductive β-elimination and then analyzed by PGC-ESI-MS or MALDI-TOF. Compared with N-glycans, O-glycan core structures are complex and diverse, which makes the characterization of O-glycan structures very challenging. However, since O-glycans can be randomly attached to the hydroxyl groups of amino acids such as serine and threonine, there may be a large number of O-glycosylation sites in the Fc-Fusion molecule. Therefore, obtaining specific information on glycan attachment sites is important for accurate analysis of glycan profiles.
O-glycosylation of immunoglobulin G and Ig-fusion protein
Glycosylation Site Specificity
In general, glycan site-specific characteristics can provide important information on the safety and efficacy of Fc fusion products containing various N- and O-glycosylation sites, and site-specificity of glycosylation is essential for further development of the clinical potential of therapeutic products and for better understanding of CQA.
However, the above-described "separation-and-analyze " glycan analysis method results in the loss of site-specific information that can be obtained with peptide mapping techniques. Specifically: first, glycoproteins are digested using proteases such as trypsin to produce peptides and glycopeptides of about 0.5-5 kDa; then, the obtained peptides are analyzed using methods such as MALDI-MS or ESI-MS to obtain glycan profiles based on accurate mass information. In order to overcome the ion suppression effect of conventional peptides and high-abundance glycopeptides, chromatography such as RPLC, HILIC or electrophoretic separation techniques such as CE are usually used before MS detection to ensure the reliability of low-abundance carbohydrate characterization. In addition, tandem MS analysis of glycopeptides (eg, electrotransfer dissociation) can be used to identify glycan sites and determine site occupancy. Usually, O-glycan sites do not have a sequence similar to that of N-glycans, so O-glycans may be randomly distributed throughout the protein molecule. Therefore, this glycan site specificity and its occupancy information is very valuable for Fc-Fusion containing O-glycans. A distinct advantage of the Middle-up method compared to the Bottom-up method is that it can obtain important site-specific glycan information with fewer sample preparation steps. Furthermore, fully automated analysis of Fc-Fusion glycans at the Middle-up level is possible using the emerging 3D-LC/MS method. Therefore, the Middle-up method provides a very convenient option for routine analysis or rapid batch-to-batch comparison of the glycosylation profiles of therapeutic proteins. Overall, N- and O-glycan characterization techniques for Fc-Fusion are diverse. However, special attention still needs to be paid to the modification sites of polyglycosylation, the site-specific glycosylation characteristics, and the occupancy of N- and O-glycans at the sites. Therefore, it is necessary to combine multiple orthogonal techniques to analyze Fc-Fusion to obtain comprehensive and efficient complex glycosylation information.
Conclusion
The glycosylation of Fc-Fusion has important biological significance, which may change its immune effector function by changing the binding affinity of the target molecule to Fcγ receptors, and may also affect the PK/PD and biosafety of the molecule. Therefore, in-depth understanding and and monitoring of the glycosylation of Fc-Fusion by various characterization techniques are needed.
Since the glycosylation of Fc-Fusion is extremely complex, involving multiple N- and O-glycan sites and a large number of sialic acids, complementary new analytical methods are required for accurate characterization. To cope with the extremely complex features of Fc-Fusion, more robust characterization methods such as multidimensional LC or ion mobility spectroscopy-mass spectrometry (IM-MS) will need to be developed in the future. Furthermore, due to the large amount of sialic acid present in its structure, bio-inert chromatography systems and bio-inert chromatography columns can be employed to limit unnecessary adsorption in glycan analysis. Furthermore, due to the very high number of charge heterogeneities and sialic acids in Fc-Fusion, the usual AEX characterization of Fc-Fusion is often not as powerful as it could be, and MS-compatible analytical steps must be developed in AEX to ensure that a sufficient amount of information can be obtained from such analysis to meet regulatory agency requirements.
References
- Li, F., Ding, J. Sialylation is involved in cell fate decision during development, reprogramming and cancer progression. Protein Cell 10, 550–565 (2019).
- Jonathan Sjögren, Rolf Lood. On enzymatic remodeling of IgG glycosylation; unique tools with broad applications. Glycobiology 30(4) (2019).
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