Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection
The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxi...
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| Format: | Article |
| Language: | English |
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MDPI AG
2013-03-01
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| Series: | Pharmaceuticals |
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| Online Access: | http://www.mdpi.com/1424-8247/6/3/393 |
| _version_ | 1819007754405150720 |
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| author | Yang Wang Melissa Hamm Richard R. Rustandi |
| author_facet | Yang Wang Melissa Hamm Richard R. Rustandi |
| author_sort | Yang Wang |
| collection | DOAJ |
| description | The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis. |
| first_indexed | 2024-12-21T00:29:36Z |
| format | Article |
| id | doaj.art-c0a71f3f58154af3bb57859ed6460a42 |
| institution | Directory Open Access Journal |
| issn | 1424-8247 |
| language | English |
| last_indexed | 2024-12-21T00:29:36Z |
| publishDate | 2013-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceuticals |
| spelling | doaj.art-c0a71f3f58154af3bb57859ed6460a422022-12-21T19:21:55ZengMDPI AGPharmaceuticals1424-82472013-03-016339340610.3390/ph6030393Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence DetectionYang WangMelissa HammRichard R. RustandiThe N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis.http://www.mdpi.com/1424-8247/6/3/393CE-LIFAPTSHILIC-HPLCglycansmonoclonal antibody NS0capillary electrophoresis |
| spellingShingle | Yang Wang Melissa Hamm Richard R. Rustandi Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection Pharmaceuticals CE-LIF APTS HILIC-HPLC glycans monoclonal antibody NS0 capillary electrophoresis |
| title | Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection |
| title_full | Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection |
| title_fullStr | Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection |
| title_full_unstemmed | Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection |
| title_short | Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection |
| title_sort | characterization of n linked glycosylation in a monoclonal antibody produced in ns0 cells using capillary electrophoresis with laser induced fluorescence detection |
| topic | CE-LIF APTS HILIC-HPLC glycans monoclonal antibody NS0 capillary electrophoresis |
| url | http://www.mdpi.com/1424-8247/6/3/393 |
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