Characterizing the effect of multiple Fc glycan attributes on the effector functions and FcγRIIIa receptor binding activity of an IgG1 antibody

Abstract

N-linked Fc glycosylation of IgG1 monoclonal antibody therapeutics can directly influence their mechanism of action by impacting IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, identification and detailed characterization of Fc glycan critical quality attributes (CQAs) provides important information for process design and control. A two-step approach was used to identify and characterize the Fc glycan CQAs for an IgG1 Mab with effector function. First, single factor experiments were performed to identify glycan critical quality attributes that influence ADCC and CDC activities. Next, a full-factorial design of experiment (DOE) to characterize the possible interactions and relative effect of these three glycan species on ADCC, CDC, and FcγRIIIa binding was employed. Additionally, the DOE data were used to develop models to predict ADCC, CDC, and FcγRIIIa binding of a given configuration of the three glycan species for this IgG1 molecule. The results demonstrate that for ADCC, afuco mono/bi has the largest effect, followed by HM and β-gal, while FcγRIIIa binding is affected by afuco mono/bi and β-gal. CDC, in contrast, is affected by β-gal only. This type of glycan characterization and modeling can provide valuable information for development, manufacturing support and process improvements for IgG products that require effector function for efficacy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1181-1192, 2016.

Comprehensive manipulation of glycosylation profiles across development scales

“…A broad body of literature shows that different glycoforms can mediate cellular effector functions, like ADCC 3-6 (reviewed in refs. 1,7, and 8), and CDC 3,6,35 (reviewed in refs. 10,11).…”

“…The presence or absence of fucose on the first core GlucNAc dramatically impacts antibody-dependent cell-mediated cytotoxicity (ADCC). 1,[3][4][5][6][7][8] Similarly, the level of terminal galactosylation affects complement-dependent cytotoxicity (CDC). 3,6,[9][10][11] Antibodies with high mannose-containing glycosylation structures (Man5 and higher) are cleared from the bloodstream at a faster rate than those with mature, complex type oligosaccharides, which has direct implications for drug efficacy.…”

“…1,[3][4][5][6][7][8] Similarly, the level of terminal galactosylation affects complement-dependent cytotoxicity (CDC). 3,6,[9][10][11] Antibodies with high mannose-containing glycosylation structures (Man5 and higher) are cleared from the bloodstream at a faster rate than those with mature, complex type oligosaccharides, which has direct implications for drug efficacy. 7,10,[12][13][14] Therefore, the product quality profile should be defined and maintained early on.…”

Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans

“…Thus, whether the bisecting GlcNAc directly affects IgG functionality has still not been adequately addressed. The function of the rest of the glycan features is less clear14163233, although a recent study indicates an additional role of galactose for FcγRIIIa binding343536.…”

Impact of IgG Fc‐Oligosaccharides on Recombinant Monoclonal Antibody Structure, Stability, Safety, and Efficacy

“…The impact of removal of galactose is highly dependent on the IgG subclass, receptor class, and the specific cell types or other factors used for the assays . There are several studies indicating that removal of terminal galactose has no impact on Fcγ receptor bindings or ADCC . Also, several studies have demonstrated that removal of terminal galactose results in a slight decrease in antibody binding to Fcγ receptors .…”