title: Glycosylation profiling of therapeutic antibodies in serum samples using a microfluidic CD platform and MALDI-MS.
abstract: The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose, and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper, a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic compact-disc (CD) platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified, and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and, simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel, and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.
authors: Department of Analytical Chemistry, Stockholm University, 10691, Stockholm, Sweden.
authors: Tran Thi
authors: TT
authors: Thuy
authors: Gunnar
authors: G
authors: Thorsén
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood* / Q000737:chemistry* / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized / Q000097:blood / Q000737:chemistry / Q000627:therapeutic use; D000941:Antigens / Q000737:chemistry; D000068258:Bevacizumab; D000068818:Cetuximab; D006031:Glycosylation; D006801:Humans; D000069285:Infliximab; D046210:Microfluidic Analytical Techniques; D015394:Molecular Structure; D011134:Polysaccharides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000941:Antigens; D011134:Polysaccharides; D000068258:Bevacizumab; D000069285:Infliximab; D000068818:Cetuximab
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