Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Magnelli, Paula; Bielik, Alicia; Guthrie, Ellen P.

doi:10.3791/3749

Cited by 23 publications

(19 citation statements)

References 26 publications

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“…The electrophoretic mobility of the IgG1 Fc glycoforms shown in Figure 3 was also consistent with slower migration for the larger branched N-linked glycoproteins with HM-Fc migrating slowest, Man5-Fc migrating intermediately, and the GlcNAc-Fc and N297Q-Fc forms migrating fastest. 76 In addition, the non-reduced SDS-PAGE indicated complete intermolecular disulfide bond formation corresponding to 100% dimerized form for all glycoforms. As illustrated in Figure 4, the SEC results indicate the IgG1 Fc glycoforms are mostly monomeric (96–99%) with low levels of aggregates present across the IgG1 Fc samples (1–3 %).…”

Section: Discussionmentioning

confidence: 91%

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

Okbazghi

White

et al. 2016

Journal of Pharmaceutical Sciences

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Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated, high mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, SEC, and cIEF confirmed the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods utilizing biolayer interferometry (BLI), one with protein G immobilized IgG1 Fc and the other with streptavidin immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The HM-Fc and Man5-Fc were highly similar to one another with high affinity for FcγRIIIa, while GlcNAc-Fc had weak affinity, and the non-glycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These four IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles, and then used as model system to mathematically assess overall biosimilarity, as described in a series of companion papers.

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Section: Discussionmentioning

confidence: 91%

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

Okbazghi

White

et al. 2016

Journal of Pharmaceutical Sciences

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“…Different isoforms of dimeric hCG were detected using the SDS-PAGE technique [27,28,30]. As reported, for example, by Gam and Latiff [28], many hCG bands were observed with main apparent MWs around 43.5 kDa and 38.5 kDa.…”

Section: Gel-electrophoretic Analysis Of Hcgmentioning

confidence: 78%

“…The analysis of intact hCG or its subunits by electrokinetic techniques was mainly carried out at the non-miniaturized scale, i.e. SDS-PAGE or isoelectric focusing [27][28][29][30]. These approaches require the use of toxic products, show a long analysis times, use expensive materials, high sample volumes while the separation resolution is low [31].…”

Section: Introductionmentioning

confidence: 99%

“…This is why the glycan moieties confer an additional electrophoretic contribution to that of the protein, which may decrease or increase its total apparent electrophoretic mobility. This explains why some authors observed what they called abnormal migration shifts in some cases when they implemented successive digestions with different enzymes to remove gradually the glycans from hCG[27]. To overcome this limitation, Magnelli et al used a glycoprotein standard ladder (Candy-Cane from Invitrogen)[27].…”

mentioning

confidence: 99%

“…This explains why some authors observed what they called abnormal migration shifts in some cases when they implemented successive digestions with different enzymes to remove gradually the glycans from hCG[27]. To overcome this limitation, Magnelli et al used a glycoprotein standard ladder (Candy-Cane from Invitrogen)[27]. However, it cannot lead to a correct MW determination as the glycan content of the standard glycosylated proteins may be dramatically different in nature (size)…”

mentioning

confidence: 99%

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First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level

Camperi

Cock

Pichon

et al. 2019

Talanta

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In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a heterodimeric glycoprotein, specific to the human pregnancy, consisting of an α and a β subunit, socalled hCGα and hCGβ, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize. First, Capillary Gel Electrophoresis (CGE) was used to separate the isoforms of two hCG-based drugs: Ovitrelle® (recombinant r-hCG) and Pregnyl (hCG isolated from the urine of pregnant women, u-hCG). As expected, CGE led to a better resolution than SDS-PAGE and confirmed the large heterogeneity of hCG. Different CGE profiles were obtained for the two hCG-based drugs, varying in number of peaks, migration times, and peak intensities, thus demonstrating that the drugs contain isoforms, different in nature and proportion. This result was confirmed by Capillary IsoElectrophoretic Focusing (CIEF). The pI ranges of the hCG isoforms were found between 3.4 and 4.7, and 4.5 and 5.2 for r-hCG and u-hCG, respectively. This information was further used to develop the separation of the hCG isoforms by Capillary Zone Electrophoresis (CZE). The pH, the nature, and the concentration of the background electrolyte as well as the nature and the content of its organic modifier were optimized. The use of a coated capillary to avoid protein adsorption was also evaluated. The final CZE-UV method allowed distinguishing at least 6 peaks, corresponding to different hCG isoforms. To significantly improve the level of information obtained, the CZE instrument was then coupled by means of an electrospray ionization source to a triple quadrupole (TQ) mass spectrometer. Two detection strategies were used, one focusing on the lower m/z values (100-1000) in order to identify some sugar residues as diagnostic ions to confirm the presence of glycan chains, and the second focusing on the higher m/z values (1000-2000), corresponding to the multiple charged intact protein isoforms. For both approaches, the fragmentor and capillary voltage values were optimized. The composition and the flow-rate of the sheath liquid were then optimized for the strategy focusing on the higher m/z values in order to both increase the charge state of the ionized intact isoforms and the signal-to-noise ratio. The final method was used to compare the two hCG-based drugs, demonstrating the potential of the developed CZE-MS method for isoforms fingerprinting.

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Separation and Purification of Glycans Out of Glycoproteins

Gornik

Keser

Lauc

2016

Springer Protocols Handbooks

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Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Abstract: Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction [1][2][3][4]

Cited by 23 publications

References 26 publications

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level

Separation and Purification of Glycans Out of Glycoproteins

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