The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies
Monoclonal antibodies (mAbs) feature a conserved N-linked glycosylation site in the CH2 domain, which exhibits heterogeneities in both occupancy and glycan structures. Previous studies have suggested that the unoccupied (nonglycosylated) variant exhibits decreased thermal stability, potentially impa...
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| Format: | Article |
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Taylor & Francis Group
2025-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2025.2543768 |
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| author | Gao-Yuan Liu Jenny Kim Kim Shuli Tang Yuetian Yan Mandi Hopkins Dalia Laredo Teng-Chieh Yang James Mutino Douglas E. Kamen Kenneth S. Graham Mohammed Shameem Shunhai Wang Ning Li |
| author_facet | Gao-Yuan Liu Jenny Kim Kim Shuli Tang Yuetian Yan Mandi Hopkins Dalia Laredo Teng-Chieh Yang James Mutino Douglas E. Kamen Kenneth S. Graham Mohammed Shameem Shunhai Wang Ning Li |
| author_sort | Gao-Yuan Liu |
| collection | DOAJ |
| description | Monoclonal antibodies (mAbs) feature a conserved N-linked glycosylation site in the CH2 domain, which exhibits heterogeneities in both occupancy and glycan structures. Previous studies have suggested that the unoccupied (nonglycosylated) variant exhibits decreased thermal stability, potentially impacting the overall stability of mAb products. This hypothesis, however, has remained largely unconfirmed, due to the low abundance of nonglycosylated variants in typical mAb products and the lack of effective analytical tools for detailed characterization of large aggregates with glycoform-specific information. Here, we used a postcolumn denaturation-assisted size exclusion chromatography mass spectrometry technique (SEC-PCD-MS) to reevaluate the effects of the nonglycosylated mAb variant on the thermal stability of mAb drugs during forced degradation studies. Our findings confirmed the compromised thermal stability of the nonglycosylated variant and its increased propensity to form large aggregates at elevated temperatures relevant to mAb-forced degradation studies. We also showed that this thermal stress-induced, nonglycosylation-mediated aggregation pathway could be widely observed in a diverse group of mAb molecules with varying properties. This study offers valuable insights into the rationale of selecting the appropriate temperature for mAb-forced degradation studies and highlights key considerations for data interpretation. |
| format | Article |
| id | doaj-art-aee4e133da484a488ac862ce77e8e954 |
| institution | Kabale University |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj-art-aee4e133da484a488ac862ce77e8e9542025-08-20T03:40:33ZengTaylor & Francis GroupmAbs1942-08621942-08702025-12-0117110.1080/19420862.2025.2543768The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studiesGao-Yuan Liu0Jenny Kim Kim1Shuli Tang2Yuetian Yan3Mandi Hopkins4Dalia Laredo5Teng-Chieh Yang6James Mutino7Douglas E. Kamen8Kenneth S. Graham9Mohammed Shameem10Shunhai Wang11Ning Li12Analytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAFormulation Development Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAAnalytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USAMonoclonal antibodies (mAbs) feature a conserved N-linked glycosylation site in the CH2 domain, which exhibits heterogeneities in both occupancy and glycan structures. Previous studies have suggested that the unoccupied (nonglycosylated) variant exhibits decreased thermal stability, potentially impacting the overall stability of mAb products. This hypothesis, however, has remained largely unconfirmed, due to the low abundance of nonglycosylated variants in typical mAb products and the lack of effective analytical tools for detailed characterization of large aggregates with glycoform-specific information. Here, we used a postcolumn denaturation-assisted size exclusion chromatography mass spectrometry technique (SEC-PCD-MS) to reevaluate the effects of the nonglycosylated mAb variant on the thermal stability of mAb drugs during forced degradation studies. Our findings confirmed the compromised thermal stability of the nonglycosylated variant and its increased propensity to form large aggregates at elevated temperatures relevant to mAb-forced degradation studies. We also showed that this thermal stress-induced, nonglycosylation-mediated aggregation pathway could be widely observed in a diverse group of mAb molecules with varying properties. This study offers valuable insights into the rationale of selecting the appropriate temperature for mAb-forced degradation studies and highlights key considerations for data interpretation.https://www.tandfonline.com/doi/10.1080/19420862.2025.2543768monoclonal antibodiesnonglycosylated mAbthermal stabilityAggregatesSEC-PCD-MS |
| spellingShingle | Gao-Yuan Liu Jenny Kim Kim Shuli Tang Yuetian Yan Mandi Hopkins Dalia Laredo Teng-Chieh Yang James Mutino Douglas E. Kamen Kenneth S. Graham Mohammed Shameem Shunhai Wang Ning Li The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies mAbs monoclonal antibodies nonglycosylated mAb thermal stability Aggregates SEC-PCD-MS |
| title | The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies |
| title_full | The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies |
| title_fullStr | The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies |
| title_full_unstemmed | The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies |
| title_short | The nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies |
| title_sort | nonglycosylated variant in therapeutic monoclonal antibodies preferentially forms large aggregates under typical thermal stresses used in forced degradation studies |
| topic | monoclonal antibodies nonglycosylated mAb thermal stability Aggregates SEC-PCD-MS |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2025.2543768 |
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