Characterization of Monoclonal Antibody Glycosylation: Comparison of Expression Systems and Identification of Terminal α-Linked Galactose

Sheeley, Douglas M.; Merrill, Barbara M.; Taylor, Lester C. E.

doi:10.1006/abio.1997.2036

Cited by 148 publications

(98 citation statements)

References 25 publications

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“…Further analysis of this ion from each sample (MS 3 , Figure 6A and B) clearly shows their nonidentity and strongly suggests them to be mixtures, along with the two putative structures. In the commercial IgG sample ( Figure 6A), one can see the major B 3 ion structure (Hex-HexNAc-Hex) as indicated by the prominent peak at m/z 486, and in the corresponding IgG sample ( Figure 6B Determination of the Hex-Hex moiety linkage from the Hex-Hex-HexNAc antenna (D, Figure 6) was pursued to corroborate this effort with earlier reported enzymatic studies 16 (Gal-α(1-3)-Gal). Due to limitations in sensitivity, obtaining an MS 5 spectrum of the m/z 445 ion (B 2 ) from the MS 4 spectrum of the m/z 690 precursor, as shown in Figure 4, was not possible.…”

Section: Resultssupporting

confidence: 66%

“…An identical ion has been observed in the MS/MS spectrum from a different recombinant IgG glycan, and this was further characterized by specific glycosidase digestion. 16 To determine the structure of this fragment, collision analysis was run on the m/z 690 ion (MS 4 , Figure 4, bottom left) and the fragments were found to be consistent with a Hex-Hex-HexNAc linear structure. The MS 4 spectrum of its putative complement, m/z 1333, is also shown, (MS 4 , Figure 4, bottom right).…”

Section: Resultsmentioning

confidence: 99%

“…6 In both cases, however, the product structures remain to be characterized. A large body of work has shown that spectral comparisons of isomers at different stages of sequential molecular fragmentation (MS n ) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] are clearly different, although in one case, reproducibility has been reported to be "vague". 25 In our experience, spectral reproducibility has been excellent, 26 and this very feature has provided an opportunity to build a searchable fragment library 27 and derive glycan structure with supporting tools.…”

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confidence: 99%

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Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

et al. 2007

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Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (MS n ) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following MS 2 and required multiple (MS n>2 ) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation "scars" throughout the MS n tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the MS n data sets.A comprehensive carbohydrate sequence is particularly challenging when considering the constitutional and stereoisomers that are abundant in glycan arrays. Constitutional isomers, also referred to as structural isomers, are defined as an identical atomic composition arranged in a different structure. Stereoisomers are exemplified with those frequently encountered in the hexoses, mannose, galactose, and glucose, while the two G1 glycans found in IgG are representative of simple structural isomers. In this latter case, an identical monomer may be linked on either of two antennae providing a different topology. In another nomenclature clarification, the term isomer is often confused with isobar. which relates to a different composition of atoms occurring at a nominal (unit) mass resolution. 1 At the MS profile stage, isobars are not usually a significant problem in carbohydrate structural characterization, but structural isomers and stereoisomers are. During fragmentation, however, isobaric fragments may be formed, but identification will require higher mass resolving power and mass accuracy.

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Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

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Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

et al. 2007

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“…For example, the terminal galactose-α-1,3-galactose (α-Gal) antigen can react with circulating anti α-Gal antibodies, which are present in most individuals, and induce an anaphylactic response [26] . It is now understood that murine cell lines, such as NS0 or SP2/0, contain the necessary biosynthetic machinery to produce proteins containing α-Gal epitopes [27][28][29] . However, it is generally accepted that CHO cells lack the biosynthetic machinery to synthesize glycoproteins with α-Gal antigens [30] .…”

Section: Wwwchinapharcom Yi Ch Et Almentioning

confidence: 99%

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

Ruan

Wang

et al. 2014

Acta Pharmacol Sin

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Aim: To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. Methods: The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. Results: The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. Conclusion: The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

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“…Published reports for structural elucidation generally involve an enzymatic digestion or a chemical deglycosylation step to obtain either glycopeptides or released glycans for subsequent analysis using HPLC coupled with ESI-Q-TOF MS, ESI-ion trap MS [30 -32], ESI triple quadrupole MS [33], or linear ion trap Fourier transform MS (LTQ-FTMS) [34]. Nevertheless, these methods are time consuming, requiring high-resolution separation of the peptides and glycans on the HPLC system, and/or long separation time.…”

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confidence: 99%

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab

Damen

Chen

Chakraborty

et al. 2009

J. Am. Soc. Mass Spectrom.

126

102

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Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied. (J Am Soc Mass

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Characterization of Monoclonal Antibody Glycosylation: Comparison of Expression Systems and Identification of Terminal α-Linked Galactose

Cited by 148 publications

References 25 publications

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab

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