title: A comparison of three techniques for quantitative carbohydrate analysis used in characterization of therapeutic antibodies.
abstract: A comparison of three techniques for quantitative analysis of galactosylation present on immunoglobulins is described. ESIMS, MALDI-TOF MS, and anion-exchange chromatography with fluorescence detection were evaluated in terms of repeatability, limit of quantitation, selectivity, and linearity. A recombinant monoclonal IgG was enzymatically modified in vitro to produce essentially completely galactosylated and degalactosylated forms of the immunoglobulin. Samples of known galactosylation levels were prepared by mixing the modified forms with the native form of the immunoglobulin. Good repeatability and linearity were demonstrated for all three assays (RSDs<1.0%, correlation coefficients>0.99). Differences in selectivity, sensitivity, and other performance aspects of the three techniques are also discussed in this paper.
authors.affiliation: Analytical Development, Biogen Idec, Inc, Cambridge, MA 02142, USA. joseph.siemiatkoski@biogenidec.com
authors.forename: Joseph
authors.initials: J
authors.lastname: Siemiatkoski
authors.forename: Yelena
authors.initials: Y
authors.lastname: Lyubarskaya
authors.forename: Damian
authors.initials: D
authors.lastname: Houde
authors.forename: Samnang
authors.initials: S
authors.lastname: Tep
authors.forename: Rohin
authors.initials: R
authors.lastname: Mhatre
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005453:Fluorescence; D006031:Glycosylation; D007136:Immunoglobulins / Q000032:analysis*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013296:Streptococcus pneumoniae / Q000201:enzymology
chemical_list: D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D007136:Immunoglobulins; D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase