Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC-UV-MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC-UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/j.jchromb.2007.09.030 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Multivariate data analysis of growth medium trends affecting antibody glycosylation.
Biotechnol Prog
January 2020
U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Product Quality, Office of Biotechnology Products, Division of Biotechnology Review and Research II, Silver Spring, Maryland.
Use of multivariate data analysis for the manufacturing of biologics has been increasing due to more widespread use of data-generating process analytical technologies (PAT) promoted by the US FDA. To generate a large dataset on which to apply these principles, we used an in-house model CHO DG44 cell line cultured in automated micro bioreactors alongside PAT with four commercial growth media focusing on antibody quality through N-glycosylation profiles. Using univariate analyses, we determined that different media resulted in diverse amounts of terminal galactosylation, high mannose glycoforms, and aglycosylation.
View Article and Find Full Text PDFEpitope located N-glycans impair the MHC-I epitope generation and presentation.
Electrophoresis
June 2016
Institute of Biochemistry, Romanian Academy, Bucharest, Romania.
The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma.
View Article and Find Full Text PDFMass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates.
Bioconjug Chem
February 2014
Rinat-Pfizer Inc. , 230 East Grand Avenue, South San Francisco, California 94080, United States.
Antibody drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical conjugation methods that yield heterogeneous mixtures of varying number of drugs attached at different positions.
View Article and Find Full Text PDFGlycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture.
J Biosci Bioeng
May 2014
Institute of Technology and Science, The University of Tokushima, Minamijosanjima-cho 2-1, Tokushima 770-8506, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.
N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody.
View Article and Find Full Text PDFApi m 10, a genuine A. mellifera venom allergen, is clinically relevant but underrepresented in therapeutic extracts.
Allergy
October 2011
Institute of Biochemistry and Molecular Biology, University of Hamburg, Germany.
Background: Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!