The Formidable Challenge of Controlling High Mannose-Type N-Glycans in Therapeutic mAbs

Mastrangeli, Renato; Audino, Maria Concetta; Palinsky, Wolf; Broly, Hervé; Bierau, Horst

doi:10.1016/j.tibtech.2020.05.009

Cited by 38 publications

(41 citation statements)

References 97 publications

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“…Endogenous human IgG1 contains <1% of high mannose glycans ( Mastrangeli et al, 2020 ). In contrast, recombinant therapeutic mAbs include ∼10% high mannose glycans ( Goetze et al, 2011 ).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, recombinant therapeutic mAbs include ∼10% high mannose glycans ( Goetze et al, 2011 ). Thus, there is interest in limiting excessive high mannose glycan formation in IgGs during commercial manufacturing ( Mastrangeli et al, 2020 ). Current trends in biopharmaceutical manufacturing such as process intensification, development of fully humanized mAbs, growth in biosimilar products, development of high concentration mAb formulations, and the emergence of next-generation biopharmaceuticals such as anti-drug conjugates and bispecific antibodies, necessitates the control of high mannose content as a critical aspect in the process development ( Berkel et al, 2009 ; Shi and Goudar, 2014 ; Feinberg et al, 2017 ; Sumit et al, 2019 ; Talbot et al, 2019 ; Mastrangeli et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

“…The most abundant high-mannose structure observed was the M5 form, along with M3, M4, M6, M7, and M9 forms that were present in relatively very low abundance as compared to the M5 form. The occurrence of Man 5 glycans could potentially be attributed either by the action of mannosidases in the Golgi complex or from an incomplete/truncated biosynthesis of lipid linked oligosaccharides (LLO) in the endoplasmic reticulum (ER) ( Breitling and Aebi, 2013 ; Mastrangeli et al, 2020 ). The M6, M7 glycans also could be an outcome of inefficient glycan processing in the Golgi complex, although they could also originate from incomplete LLO biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Singh

Lee

2022

Front. Bioeng. Biotechnol.

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Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Singh

Lee

2022

Front. Bioeng. Biotechnol.

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“…Therapeutic IgGs containing high mannose-type N-glycans are known to be selectively cleared by a mannose receptor-mediated mechanism 16 17 23 24 . Since high mannose-type glycans on Fc region can have a great impact on therapeutic mAb pharmacokinetics 25 , monitoring and controlling high mannose-type N-glycan levels in manufacturing processes is important. Finally, the principal component analysis (PCA) of SFC-Erexim dataset clearly illustrated glycomic landscape-dependent classification of therapeutic mAbs ( Fig.…”

Section: Main Textmentioning

confidence: 99%

Fast and ultra-sensitive glycoform analysis by supercritical fluid chromatography-tandem mass spectrometry

Haga

Yamada

Fujii

et al. 2021

Preprint

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Therapeutic monoclonal antibodies (mAbs) are currently the largest and fastest growing category of biopharmaceuticals. Glycosylation of mAbs has a significant impact on their effector functions, as well as on their safety and pharmacokinetics. Heterogeneity of glycoforms is affected by various factors such as the producing cells and cell culture process. Therefore, accurate glycoform characterization is essential for drug design, process optimization, manufacturing, and quality control of therapeutic mAbs. In this study, we developed a fast, quantitative, and highly sensitive analytical platform for mAb glycan profiling by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). An 8-minute analysis of bevacizumab, nivolumab, ramucirumab, rituximab, and trastuzumab by SFC-MS detected a total of 102 glycoforms, with a detection limit of 5 attomole. The dynamic range of glycan abundance was over 6 orders of magnitude for bevacizumab analysis by SFC-MS, compared to 3 orders of magnitude for conventional fluorescence HPLC analysis. This method revealed the glycan profile characteristics and lot-to-lot heterogeneity of various therapeutic mAbs. We were also able to detect a series of structural variations in pharmacologically important glycan structures. SFC-MS-based glycoform profiling method will provide an ideal platform for in-depth analysis of precise glycan structure and abundance.

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“…IgG monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders including cancer, autoimmunity, neurodegenerative and infectious diseases. IgG activities, as mediated by their effector functions, depend on the chemistry of their core N-glycans linked to Asn297 (24)(25)(26)(27)(28). Several studies have J o u r n a l P r e -p r o o f demonstrated that the carbohydrate composition of this N-glycan critically affects the interaction with the Fc-receptor and the complement system, mediating the effector functions of IgGs (29,30).…”

Section: Introductionmentioning

confidence: 99%

GH18 endo-β-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans

Trastoy

et al. 2021

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The Formidable Challenge of Controlling High Mannose-Type N-Glycans in Therapeutic mAbs

Cited by 38 publications

References 97 publications

Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Fast and ultra-sensitive glycoform analysis by supercritical fluid chromatography-tandem mass spectrometry

GH18 endo-β-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans

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