Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis
The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Curr...
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MDPI AG
2021-10-01
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| author | Bastiaan L. Duivelshof Steffy Denorme Koen Sandra Xiaoxiao Liu Alain Beck Matthew A. Lauber Davy Guillarme Valentina D’Atri |
| author_facet | Bastiaan L. Duivelshof Steffy Denorme Koen Sandra Xiaoxiao Liu Alain Beck Matthew A. Lauber Davy Guillarme Valentina D’Atri |
| author_sort | Bastiaan L. Duivelshof |
| collection | DOAJ |
| description | The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the <i>N</i>-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade<sup>®</sup>) was assessed and compared to two of its commercially available biosimilars (Remsima<sup>®</sup> and Inflectra<sup>®</sup>). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method. |
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| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-03-10T05:10:11Z |
| publishDate | 2021-10-01 |
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| record_format | Article |
| series | Pharmaceutics |
| spelling | doaj.art-9ef3402fc0064b6fa7968bd0b3ad24852023-11-23T00:56:56ZengMDPI AGPharmaceutics1999-49232021-10-011311174410.3390/pharmaceutics13111744Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS AnalysisBastiaan L. Duivelshof0Steffy Denorme1Koen Sandra2Xiaoxiao Liu3Alain Beck4Matthew A. Lauber5Davy Guillarme6Valentina D’Atri7Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU—Rue Michel-Servet 1, 1211 Geneva, SwitzerlandResearch Institute for Chromatography (RIC), President Kennedypark 26, 8500 Kortrijk, BelgiumResearch Institute for Chromatography (RIC), President Kennedypark 26, 8500 Kortrijk, BelgiumWaters Corporation, 34 Maple Street, Milford, MA 01757-3696, USAIRPF—Centre d’Immunologie Pierre-Fabre (CIPF), 5 Avenue Napoléon III, 60497 Saint-Julien-en-Genevois, FranceWaters Corporation, 34 Maple Street, Milford, MA 01757-3696, USAInstitute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU—Rue Michel-Servet 1, 1211 Geneva, SwitzerlandInstitute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU—Rue Michel-Servet 1, 1211 Geneva, SwitzerlandThe identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the <i>N</i>-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade<sup>®</sup>) was assessed and compared to two of its commercially available biosimilars (Remsima<sup>®</sup> and Inflectra<sup>®</sup>). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.https://www.mdpi.com/1999-4923/13/11/1744glycan profilingquantitative analysisHILIC-MSprotein subunitsmonoclonal antibodies |
| spellingShingle | Bastiaan L. Duivelshof Steffy Denorme Koen Sandra Xiaoxiao Liu Alain Beck Matthew A. Lauber Davy Guillarme Valentina D’Atri Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis Pharmaceutics glycan profiling quantitative analysis HILIC-MS protein subunits monoclonal antibodies |
| title | Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis |
| title_full | Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis |
| title_fullStr | Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis |
| title_full_unstemmed | Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis |
| title_short | Quantitative <i>N-</i>Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis |
| title_sort | quantitative i n i glycan profiling of therapeutic monoclonal antibodies performed by middle up level hilic hrms analysis |
| topic | glycan profiling quantitative analysis HILIC-MS protein subunits monoclonal antibodies |
| url | https://www.mdpi.com/1999-4923/13/11/1744 |
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