Glycosylation of Antibody Molecules

Jefferis, Royston; Goodall, Margaret; Tishchenko, V. M.; Nash, P.; Lund, John

doi:10.1007/978-1-4615-1885-3_14

Cited by 3 publications

(2 citation statements)

References 22 publications

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“…This observation is ascribed to the decreased surface exposure of Asn N297 and the attached glycan as it engages in several interactions with the protein backbone and residue side chains at the inner face of the domain [35,36]. Although most plasma IgG molecules possess only the conserved N-glycosylation sequon in the Fc region, approximately 15-25% also express additional glycosylation sites on the variable domain of the Fab region [37,38]. Glycan profiling of Fab-glycosylated serum IgGs has revealed contrasting structural characteristics between Fc-derived and Fab-derived glycans, most notably higher overall heterogeneity and sialylation [39,40].…”

Section: Glycan Profile Analysismentioning

confidence: 99%

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity

et al. 2021

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Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

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Section: Glycan Profile Analysismentioning

confidence: 99%

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity

et al. 2021

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“…Further cross-sub-class variant mAb therapies are in development, with key amino acid substitutions from the IgG2 and IgG4 sub-classes introduced for intentionally suppressed effector functions [4,13]. The removal of the conserved glycosylation site through amino acid substitution of N297 or T299 (aglycosylation) has also demonstrated reduced effector function; however, this happens at the expense of mAb stability and is therefore not a suitable strategy for whole mAb therapeutics [11,14,15,137,138,139,140].…”

Section: Strategic Modulation Of Mab Immune Effector Functionsmentioning

confidence: 99%

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

et al. 2019

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Therapeutic antibody technology heavily dominates the biologics market and continues to present as a significant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions.

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Deglycosylation of anti-ß amyloid antibodies inhibits microglia activation in BV-2 cellular model

Rebe

Solomon

2005

Am J Alzheimers Dis Other Demen

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Immunotherapy has become a strategy for treatment of Alzheimer's disease, by inducing antibody response to amyloid-beta peptide (AbetaP) or by passive administration of anti-AbetaP antibodies. Clearance of amyloid plaques involves interaction of immunoglobulin Fc receptor (FcR)-expressing microglia and antibodyopsonized Abeta deposits, stimulating phagocytosis but may promote neuroinflammation. Carbohydrate moiety of Fc of the immunoglobulin G molecule plays a significant role in modulating binding to FcR and its effector functions. Here, we enzymatically removed Fc glycan from monoclonal antibody 196 raised against AbetaP Antigen binding ability and in vitro stability of deglycosylated antibody were unaffected by deglycosylation. Moreover, the deglycosylated antibody exhibits low affinity to FcR on microglial BV-2 cells and has limited ability to mediate microglial chemotaxis and antibodydependent cytotoxicity compared to native antibody. These data suggest that deglycosylation of anti-Abeta antibodies before in vivo administration might prevent microglial overactivation, thus reducing the risk of neuroinflammatory response during passive immunization.

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Glycosylation of Antibody Molecules

Cited by 3 publications

References 22 publications

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

Deglycosylation of anti-ß amyloid antibodies inhibits microglia activation in BV-2 cellular model

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