Journal: Biotechnology progress

Article Title: Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

doi: 10.1002/btpr.3365

Figure Lengend Snippet: FIGURE 1 Schematic of simultaneous analysis of antibody macroheterogeneity and microheterogeneity. Light blue-shaded region indicates involvement of manual steps and light yellow-shaded region indicates automated injection and analysis steps of the LabChip instrument. When the same amount of antibody is analyzed (e.g., 8 μL of 1 mg/mL), the glycoforms can be identified as different peaks, and by comparison to known quantities of glycan standards, macroheterogeneity can be determined by the fluorescence intensity.

Article Snippet: Finally, the sample was reconstituted in 100 μL deionized water and prepared for analysis in a LabChip GXII Touch instrument (Perkin Elmer, Waltham, MA).

Techniques: Injection, Comparison, Glycoproteomics, Fluorescence

Journal: Biotechnology progress

Article Title: Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

doi: 10.1002/btpr.3365

Figure Lengend Snippet: FIGURE 2 Inter-day consistency in the characterization of a monoclonal antibody (mAb) reference standard (NIST mAb). (a) Consistency in the absolute peak area (left axis) of identified glycoforms and relative fraction (right axis) of identified glycoforms, and (b) consistent elution times of all analyzed glycoforms were observed across independent measurements. The absolute area represents the integrated area of respective glycan peaks in relative fluorescence units (RFU) as reported by the LabChip platform. The error bars are representative of the standard error of triplicate samples performed independently on three consecutive days. G0, G0F, G1Fa, G1Fb, and G2F designate galactose (G) addition with either 0, 1, or 2, as noted, while F represent the presence of a fucose residue. G1Fa and G1Fb are the galactosylation isoforms where either branch of the GlcNAc residue is galactosylated. Please refer to Reference [7] for complete glycan notation. Note that, as expected, not all possible glycoforms were detected and analyzed.

Article Snippet: Finally, the sample was reconstituted in 100 μL deionized water and prepared for analysis in a LabChip GXII Touch instrument (Perkin Elmer, Waltham, MA).

Techniques: Glycoproteomics, Fluorescence, Residue

Journal: Biotechnology progress

Article Title: Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

doi: 10.1002/btpr.3365

Figure Lengend Snippet: FIGURE 4 Utilization of the LabChip platform to quantitate antibody deglycosylation. (a) Representative chromatograms of antibody samples of the same concentration (1 mg/mL) but with different degrees of glycosylation. The chromatograms represent intact IgG (green), a 1:1 volumetric mixture of intact and deglycosylated IgG (red), and deglycosylated IgG (blue). G0, G0F, G1Fa, G1Fb, and G2F designate galactose (G) addition with either 0, 1, or 2, as noted, while F represent the presence of a fucose residue. G1Fa and G1Fb are the galactosylation isoforms where either branch of the GlcNAc residues is galactosylated. Please refer to Reference [7] for the complete glycan notation. (b) A linear response of the absolute G0F peak area and the amount of glycosylated IgG (n = 1, R2 = 0.995).

Article Snippet: Finally, the sample was reconstituted in 100 μL deionized water and prepared for analysis in a LabChip GXII Touch instrument (Perkin Elmer, Waltham, MA).

Techniques: Concentration Assay, Glycoproteomics, Residue