Document Detail


A high-throughput method for quantification of glycoprotein sialylation.
MedLine Citation:
PMID:  20692221     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Lam Raga A Markely; Boon Tee Ong; Kong Meng Hoi; Gavin Teo; Michelle Y Lu; Daniel I C Wang
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-08-06
Journal Detail:
Title:  Analytical biochemistry     Volume:  407     ISSN:  1096-0309     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2010 Dec 
Date Detail:
Created Date:  2010-09-22     Completed Date:  2011-01-18     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  128-33     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier Inc. All rights reserved.
Affiliation:
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cricetinae
Cricetulus
Glycoproteins / chemistry*
Glycosylation
Interferon-gamma, Recombinant / chemistry*,  isolation & purification,  metabolism
N-Acetylneuraminic Acid / analysis*
Neuraminidase / metabolism
Nitriles / chemistry
Oxidation-Reduction
Spectrometry, Fluorescence / methods*
Chemical
Reg. No./Substance:
0/Glycoproteins; 0/Interferon-gamma, Recombinant; 0/Nitriles; 109-77-3/dicyanmethane; 131-48-6/N-Acetylneuraminic Acid; EC 3.2.1.18/Neuraminidase

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