| Author | Search for: Rambotham, Anna1; Search for: Raymond, Celine1; Search for: Durocher, Yves1; Search for: Kelly, John1; Search for: Tikhomirov, Ilia |
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| Affiliation | - National Research Council of Canada. Human Health Therapeutics
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| Format | Text, Article |
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| Conference | Bioprocessing Summit, August 18-22 2014, Boston, USA |
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| Abstract | Monoclonal antibodies (mAbs) and related products such as antibody-drug conjugates (ADCs) are the fastest evolving calss of therapeutic agents today. They are also amongst the most difficult to analyze effectively which can pose significant challenges during the the development process and when attempting to demonstrate regulatory compliance. Increasingly, the power of mass spectrometry (MS) is being exploited to create novel and effective solutions for characterizing biotherapeutics. "Middle-up" LC-MS is a simple technique that is effective for characterizing glycosylation and for mapping other modifications and sequence abnormalities to specific regions of the antibody. Antibodies are selectively cleaved into a handful of large polypeptides which are then analyzed by LC-MS. "Middle-up" LC-MS provided an elegant and rapid solution for separately profiling Fc and Fab N-glycosylation on anti-EGFR antibodies. Furthermore, the resulting glycan profiles are less likely to be biased by the presence of certain sugars such as sialic acid and are more representative of their true abundances. "Middle-up" LC-MS analysis has also been effective for monitoring the results of efforts to remodel Fc glycosylation through glycoengineering. An example will be presented here for Trastuzumab. LC-MS is also an effective method for measuring the drug-antibody ration (DAR) of ADCs provided that the additional molecular diversity due to PTMs can be eliminated. We developed a procedure that simplifies the molecular profiles of antibodies containing multiple sites of glycosylation using conditions that are compatible with LC-MS. The antibody is treated with a combination of exo- and endoglycosidases as well as a carboxypeptidase to remove C-terminal lysines. In this manner the complex mass profile of the antibody collapses into one major peak. Employing this approach for ADCs derived from these antibodies will enable their DAR to be determined. |
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| Publication date | 2014 |
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| Note | poster |
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| Language | English |
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| NRC number | NRC-HHT-53259 |
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| NPARC number | 21277510 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 27fc4e03-4514-4e72-ac5f-5fba9a269bce |
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| Record created | 2016-03-10 |
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| Record modified | 2020-10-14 |
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