Influence of glycosylation pattern on the molecular properties of monoclonal antibodies

Figures & data

Figure 1. (A) Crystal structure of the Fc region of an IgG1 antibody with glycan attached (PDB: 1H3X). (B) Glycoform patterns of G0, afucosylated, and high mannose forms. Symbols: blue squares, N-acetylglucosamine (GlcNAc); green circles, mannose; red triangles, fucose.

Table 1. Mass of mAb heavy chains

	Glycan variants
Mass (Da)	G0	Afucosylated	High mannose	Deglycosylated
Theoretical	51004.38	50858.24	51424.96	49560.06
Experimental	51004.00	50858.00	51425.00	49560.00

Figure 2. FTIR spectra of glycoforms. Insert: calculated second derivatives of the amide I region. Spectra line colors in both figures: black, control; red, high mannose form; blue, deglycosylated form; magenta, G0 form; cyan, afucosylated form.

Figure 3. Trp emission spectra of glycoforms. Filled markers: black squares, control; red circles, high mannose form; blue triangles, deglycosylated form. Open markers: magenta squares, G0 form; cyan diamonds, afucosylated form.

Figure 4. Thermal unfolding of glycoforms from DSC measurements. Thermogram line colors: black, control; red, high mannose form; blue, deglycosylated form; magenta, G0 form; cyan, afucosylated form.

Figure 5. Thermal unfolding of control (filled squares), high mannose (open squares), and deglycosylated (filled triangles) forms from fluorescence measurements. Ratios of fluorescence intensities at 325 and 375 nm are plotted vs. temperature.

Figure 6. Thermal unfolding of control (solid line), high mannose (dashed line), and deglycosylated (dotted line) forms from DSC measurements. Thermograms: (A) at pH 4.0, (B) at pH 5.5, (C) at pH 6.5, (D) at pH 7.0, (E) at pH 7.5.

Table 2. Thermal transition temperatures (°C) measured by DSC at various pH conditions

	pH 4.0			pH 5.5			pH 6.5			pH 7.0			pH 7.5
Glycoform	Tm1	Tm2	Tm3	Tm1	Tm2	Tm3	Tm1	Tm2	Tm3	Tm1	Tm2	Tm3	Tm1	Tm2	Tm3
Control	54.7	71.0	NA	69.1	77.7	NA	NA	77.0	82.3	NA	76.9	82.6	NA	76.6	82.6
High mannose	47.3	71.3	NA	64.2	77.9	NA	67.7	77.2	82.7	68.2	76.9	82.4	68.3	76.7	82.4
Deglycosylated	45.0	71.1	NA	61.4	77.5	NA	65.5	76.5	82.5	66.1	76.4	82.9	65.8	76.0	82.8

Tm, transition temperature; NA, not applicable.

Figure 7. Protease digestion of glycoforms monitored by SEC. Digestion profiles: (A) HNE, (B) papain, (C) pepsin. Line colors in all three figures: black, control; red, high mannose form; blue, deglycosylated form; magenta, G0 form; cyan, afucosylated form.

Figure 8. Stability profiles of glycoforms measured by SEC. Profiles after three-month storage at 30 °C: (A) HMWS and (B) LMWS. Profiles after three-month storage at 40 °C: (C) HMWS and (D) LMWS. Filled markers in all figures: black squares, control; red diamonds, high mannose form; blue triangles, deglycosylated form. Open markers in all figures: magenta squares, G0 form; cyan triangles, afucosylated form.