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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A systematic assessment of structural heterogeneity and IgG/IgE-binding of ovalbumin
Ovalbumin (OVA), one of the major allergens in hen egg, exhibits extensive structural heterogeneity due to a range of post-translational modifications (PTMs). However, analyzing the structural heterogeneity of native OVA is challenging, and the relationship between heterogeneity and IgG/IgE-binding of OVA remains unclear. In this work, ion exchange chromatography (IXC) with salt gradient elution and on-line detection by native electrospray ionization mass spectrometry (ESI MS) was used to assess the structural heterogeneity of OVA, while inhibition-ELISA was used to assess the IgG/IgE binding characteristics of OVA. Over 130 different OVA proteoforms (including glycan-free species and 32 pairs of isobaric species) were identified. Proteoforms with acetylation, phosphorylation, oxidation and succinimide modifications had reduced IgG/IgE binding capacities, whereas those with few structural modifications had higher IgG/IgE binding capacities. OVA isoforms with a sialic acid-containing glycan modification had the highest IgG/IgE binding capacity. Our results demonstrate that on-line native IXC/MS with salt gradient elution can be used for rapid assessment of the structural heterogeneity of proteins. An improved understanding of the relationship between IgG/IgE binding capacity and OVA structure provides a basis for developing biotechnology or food processing methods for reducing protein allergenicity reduction.
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- Award ID(s):
- 1709552
- NSF-PAR ID:
- 10333287
- Date Published:
- Journal Name:
- Food & Function
- Volume:
- 12
- Issue:
- 17
- ISSN:
- 2042-6496
- Page Range / eLocation ID:
- 8130 to 8140
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks. The MP-11 structural heterogeneity was studied as a function of the starting pH ( e.g. , pH 3.1–6.1) and concentration ( e.g. , 1–50 μM) conditions and adduct coordination. Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) showed the MP-11 structural dependence of the charge state distribution and molecular ion forms with the starting pH conditions. The singly charged ( e.g. , [M] + , [M − 2H + NH 4 ] + , [M − H + Na] + and [M − H + K] + ) and doubly charged ( e.g. , [M + H] 2+ , [M − H + NH 4 ] 2+ , [M + Na] 2+ and [M + K] 2+ ) molecular ion forms were observed for all solvent conditions, although the structural heterogeneity ( e.g. , number of mobility bands) significantly varied with the pH value and ion form. The MP-11 dimer formation as a model for heme-protein protein interactions showed that dimer formation is favored toward more neutral pH and favored when assisted by salt bridges ( e.g. , NH 4 + , Na + and K + vs. H + ). Inspection of the dimer mobility profiles (2+ and 3+ charge states) showed a high degree of structural heterogeneity as a function of the solution pH and ion form; the observation of common mobility bands suggest that the different salt bridges can stabilize similar structural motifs. In addition, the salt bridge influence on the MP-11 dimer formations was measured using collision induced dissociation and showed a strong dependence with the type of salt bridge ( i.e. , a CE 50 of 10.0, 11.5, 11.8 and 13.0 eV was observed for [2M + H] 3+ , [2M − H + NH 4 ] 3+ , [2M + Na] 3+ and [2M + K] 3+ , respectively). Measurements of the dimer equilibrium constant showed that the salt bridge interactions increase the binding strength of the dimeric species.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d0fo02980g
