Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors
ABSTRACT This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon‐γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, poten...
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| Format: | Article |
| Language: | English |
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Wiley-VCH
2025-01-01
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| Series: | Engineering in Life Sciences |
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| Online Access: | https://doi.org/10.1002/elsc.202400031 |
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| author | Letha Chemmalil Tanmay Kulkarni Mathura Raman Priya Singh Yueming Qian Chris Chumsae Kyle McHugh Zhuangrong Huang Eric Hodgman Michael C. Borys Julia Ding Gloria Li Anthony Leone |
| author_facet | Letha Chemmalil Tanmay Kulkarni Mathura Raman Priya Singh Yueming Qian Chris Chumsae Kyle McHugh Zhuangrong Huang Eric Hodgman Michael C. Borys Julia Ding Gloria Li Anthony Leone |
| author_sort | Letha Chemmalil |
| collection | DOAJ |
| description | ABSTRACT This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon‐γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, potency, and overall clinical performance due to its influence on protein clearance via hepatic asialoglycoprotein receptors (ASGPR). The ASGPR recognizes and binds to glycoproteins exposed to terminal galactose or N‐acetylgalactosamine residues, leading to receptor‐mediated endocytosis. Recent studies have demonstrated that sialylation of O‐linked glycan plays a role in protecting against macrophage galactose lectin (MGL)‐mediated clearance. In addition to the impact on serum half‐life, sialylation can influence other clinical outcomes, including immunogenicity, potency, and cytotoxicity. Therefore, the level of sialic acid is a critical quality attribute (CQA), and monitoring and regulating sialylation has become a regulatory requirement to ensure desired clinical performance. To achieve consistent levels of sialic acid‐to‐protein ratio, the time of upstream harvest and conductivity of downstream wash buffers must be tightly regulated based on the sialic acid content. Therefore, the utilization of process analytical technology (PAT) tools for generating real‐time or near‐real‐time sialic acid content is a business‐critical requirement. This work demonstrates the utility of an integrated PAT system for near real‐time online sialic acid measurements. The system consists of a micro‐sequential injection analyzer (µSIA) interfaced with SegFlow and an ultra performance liquid chromatography (UPLC). The fully automated architecture exemplifies the execution of online sampling, automatic sample preparation, and subsequent online UPLC analysis. This carefully orchestrated PAT framework effectively supports the requirements of QbD‐driven continuous bioprocessing. |
| format | Article |
| id | doaj-art-a1060ecacf5942e7ad13e4f90f7f926f |
| institution | Kabale University |
| issn | 1618-0240 1618-2863 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley-VCH |
| record_format | Article |
| series | Engineering in Life Sciences |
| spelling | doaj-art-a1060ecacf5942e7ad13e4f90f7f926f2025-01-30T06:40:30ZengWiley-VCHEngineering in Life Sciences1618-02401618-28632025-01-01251n/an/a10.1002/elsc.202400031Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from BioreactorsLetha Chemmalil0Tanmay Kulkarni1Mathura Raman2Priya Singh3Yueming Qian4Chris Chumsae5Kyle McHugh6Zhuangrong Huang7Eric Hodgman8Michael C. Borys9Julia Ding10Gloria Li11Anthony Leone12Biological Process Analytical Group Bristol Myers Squibb Devens Massachusetts USABiological Process Analytical Group Bristol Myers Squibb Devens Massachusetts USABiological Process Analytical Group Bristol Myers Squibb Devens Massachusetts USAAnalytical Science & Technology Bristol Myers Squibb Devens Massachusetts USAHorizon Therapeutics Bristol Myers Squibb Rockville Maryland USAAnalytical Development & Analytical Attribute Science in Biologics Bristol Myers Squibb Devens Massachusetts USATakeda Pharmaceuticals Former BMS Affiliate Lexington Massachusetts USAProcess Development Group Bristol Myers Squibb Devens Massachusetts USAProcess Development Group Bristol Myers Squibb Devens Massachusetts USAProcess Development Group Bristol Myers Squibb Devens Massachusetts USAVertex Pharmaceutical Bristol Myers Squibb Boston Massachusetts USABiological Process Analytical Group Bristol Myers Squibb Devens Massachusetts USAAnalytical Development & Analytical Attribute Science in Biologics Bristol Myers Squibb Devens Massachusetts USAABSTRACT This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon‐γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, potency, and overall clinical performance due to its influence on protein clearance via hepatic asialoglycoprotein receptors (ASGPR). The ASGPR recognizes and binds to glycoproteins exposed to terminal galactose or N‐acetylgalactosamine residues, leading to receptor‐mediated endocytosis. Recent studies have demonstrated that sialylation of O‐linked glycan plays a role in protecting against macrophage galactose lectin (MGL)‐mediated clearance. In addition to the impact on serum half‐life, sialylation can influence other clinical outcomes, including immunogenicity, potency, and cytotoxicity. Therefore, the level of sialic acid is a critical quality attribute (CQA), and monitoring and regulating sialylation has become a regulatory requirement to ensure desired clinical performance. To achieve consistent levels of sialic acid‐to‐protein ratio, the time of upstream harvest and conductivity of downstream wash buffers must be tightly regulated based on the sialic acid content. Therefore, the utilization of process analytical technology (PAT) tools for generating real‐time or near‐real‐time sialic acid content is a business‐critical requirement. This work demonstrates the utility of an integrated PAT system for near real‐time online sialic acid measurements. The system consists of a micro‐sequential injection analyzer (µSIA) interfaced with SegFlow and an ultra performance liquid chromatography (UPLC). The fully automated architecture exemplifies the execution of online sampling, automatic sample preparation, and subsequent online UPLC analysis. This carefully orchestrated PAT framework effectively supports the requirements of QbD‐driven continuous bioprocessing.https://doi.org/10.1002/elsc.202400031µSIAcontinuous bioprocessingFIA labsmonoclonal antibody (mAb)process analytical technology (PAT)quality by design (QbD) |
| spellingShingle | Letha Chemmalil Tanmay Kulkarni Mathura Raman Priya Singh Yueming Qian Chris Chumsae Kyle McHugh Zhuangrong Huang Eric Hodgman Michael C. Borys Julia Ding Gloria Li Anthony Leone Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors Engineering in Life Sciences µSIA continuous bioprocessing FIA labs monoclonal antibody (mAb) process analytical technology (PAT) quality by design (QbD) |
| title | Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors |
| title_full | Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors |
| title_fullStr | Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors |
| title_full_unstemmed | Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors |
| title_short | Integrated SegFlow, µSIA, and UPLC for Online Sialic Acid Quantitation of Glycoproteins Directly from Bioreactors |
| title_sort | integrated segflow µsia and uplc for online sialic acid quantitation of glycoproteins directly from bioreactors |
| topic | µSIA continuous bioprocessing FIA labs monoclonal antibody (mAb) process analytical technology (PAT) quality by design (QbD) |
| url | https://doi.org/10.1002/elsc.202400031 |
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