title: A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation.
abstract: The serum half-life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high-throughput method for semi-quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry.
authors.affiliation: Cell Culture Development-High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Lam Raga Anggara
authors.initials: LR
authors.lastname: Markely
authors.affiliation: Cell Culture Development-High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Lila
authors.initials: L
authors.lastname: Cheung
authors.affiliation: Cell Culture Development-High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Young Jun
authors.initials: YJ
authors.lastname: Choi
authors.affiliation: Cell Culture Development-High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ryll
authors.affiliation: Cell Culture Development-High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Scott
authors.initials: S
authors.lastname: Estes
authors.affiliation: Cell Culture Development-High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Shashi
authors.initials: S
authors.lastname: Prajapati
authors.affiliation: Analytical Development - High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Iva
authors.initials: I
authors.lastname: Turyan
authors.affiliation: Analytical Development - High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Ruth
authors.initials: R
authors.lastname: Frenkel
authors.affiliation: Analytical Development - High-Throughput Analytical Group, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Sosic
authors.affiliation: Cell Culture Development, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: James
authors.initials: J
authors.lastname: Lambropoulos
authors.affiliation: Cell Culture Development, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Lia
authors.initials: L
authors.lastname: Tescione
authors.affiliation: Cell Culture Development, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ryll
authors.affiliation: Biomolecular and Small Molecule Science, Biogen, 125 Broadway, Cambridge, MA, 02142.
authors.forename: Melissa
authors.initials: M
authors.lastname: Berman
mesh_terms: D018929:Cell Culture Techniques; D002853:Chromatography, Liquid; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*
chemical_list: D006023:Glycoproteins; D019158:N-Acetylneuraminic Acid
keywords: carbohydrate; cell culture; glycoprotein; process monitoring; sialic acid