title: A high-throughput method for quantification of glycoprotein sialylation.
abstract: Sialic acid can improve qualities of therapeutic glycoproteins such as circulatory half-life, biological activity, and solubility. In production of therapeutic glycoproteins, a high-throughput method is required for process monitoring and optimization to ensure consistent and optimal sialic acid content. Current methods for quantifying sialic acid, however, require chromatographic separation that is time-consuming and cannot rapidly analyze many samples in parallel. Here we present a novel high-throughput method for quantifying glycoprotein sialylation. Using chemical reduction, enzymatic release of sialic acid, and chemical derivatization of the sialic acid, the method can accurately, rapidly (15 min), and specifically analyze many samples in parallel. It requires only 45 μl of sample and has a quantitation limit of 2 μM sialic acid. It has also been validated for monitoring sialylation of recombinant interferon gamma (IFN-γ) produced in Chinese hamster ovary (CHO) cell culture. This method is useful for various applications in upstream and downstream bioprocesses.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Lam Raga A
authors.initials: LR
authors.lastname: Markely
authors.forename: Boon Tee
authors.initials: BT
authors.lastname: Ong
authors.forename: Kong Meng
authors.initials: KM
authors.lastname: Hoi
authors.forename: Gavin
authors.initials: G
authors.lastname: Teo
authors.forename: Michelle Y
authors.initials: MY
authors.lastname: Lu
authors.forename: Daniel I C
authors.initials: DI
authors.lastname: Wang
mesh_terms: D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D007371:Interferon-gamma / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D009439:Neuraminidase / Q000378:metabolism; D009570:Nitriles / Q000737:chemistry; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D006023:Glycoproteins; D009570:Nitriles; D011994:Recombinant Proteins; D007371:Interferon-gamma; C000945:dicyanmethane; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid