Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry
ABSTRACTAggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effec...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2024-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2024.2334783 |
| _version_ | 1797238968891211776 |
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| author | Jing Xu John E. Coughlin Malgorzata Szyjka Serene Jabary Sonal Saluja Zoran Sosic Yunqiu Chen Chong-Feng Xu |
| author_facet | Jing Xu John E. Coughlin Malgorzata Szyjka Serene Jabary Sonal Saluja Zoran Sosic Yunqiu Chen Chong-Feng Xu |
| author_sort | Jing Xu |
| collection | DOAJ |
| description | ABSTRACTAggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as “Fab/c”), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments. |
| first_indexed | 2024-04-24T17:44:05Z |
| format | Article |
| id | doaj.art-8a4dd72d29084788975e90ab944ead49 |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-04-24T17:44:05Z |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-8a4dd72d29084788975e90ab944ead492024-03-27T16:47:26ZengTaylor & Francis GroupmAbs1942-08621942-08702024-12-0116110.1080/19420862.2024.2334783Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometryJing Xu0John E. Coughlin1Malgorzata Szyjka2Serene Jabary3Sonal Saluja4Zoran Sosic5Yunqiu Chen6Chong-Feng Xu7Analytical Development, Biogen, Cambridge, MA, USAProcess Biochemistry, Biogen, Cambridge, MA, USAAnalytical Biochemistry, Biogen, Cambridge, MA, USABiologics Product Development, Biogen, Cambridge, MA, USABiologics Product Development, Biogen, Cambridge, MA, USAAnalytical Development, Biogen, Cambridge, MA, USAAnalytical Development, Biogen, Cambridge, MA, USAAnalytical Development, Biogen, Cambridge, MA, USAABSTRACTAggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as “Fab/c”), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.https://www.tandfonline.com/doi/10.1080/19420862.2024.2334783Aggregationaggregation ratefragmentsheterodimersmonoclonal antibodynative mass spectrometry |
| spellingShingle | Jing Xu John E. Coughlin Malgorzata Szyjka Serene Jabary Sonal Saluja Zoran Sosic Yunqiu Chen Chong-Feng Xu Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry mAbs Aggregation aggregation rate fragments heterodimers monoclonal antibody native mass spectrometry |
| title | Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry |
| title_full | Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry |
| title_fullStr | Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry |
| title_full_unstemmed | Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry |
| title_short | Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry |
| title_sort | evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry |
| topic | Aggregation aggregation rate fragments heterodimers monoclonal antibody native mass spectrometry |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2024.2334783 |
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