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In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs.

Song T, Ozcan S, Becker A, Lebrilla CB - Anal. Chem. (2014)

Bottom Line: The complete structures were obtained through exoglycosidase sequencing.The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances.The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California , One Shields Avenue, Davis, California 95616, United States.

ABSTRACT
The glycosylation in recombinant monoclonal antibody (rMab) drugs is a major concern in the biopharmaceutical industry as it impacts the drugs' many attributes. Characterization is important but complicated by the intricate structures, microheterogeneity, and the limitations of current tools for structural analysis. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) N-glycan library based on eight commercial rMab drugs. A library of over 70 structures was developed for the rapid characterization of rMab. N-Glycans were separated on a porous graphitized carbon (PGC) column incorporated on a chip and then analyzed by an electrospray ionization hybrid quadrupole time-of-flight (ESI-Q-TOF) MS. The retention time and accurate mass for each N-glycan were recorded in the library. The complete structures were obtained through exoglycosidase sequencing. The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances. The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

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Illustration of the identification procedure for antibodyN-glycansusing a previously annotated reference library. (a) Extracted compoundchromatogram (ECC) of composition N54110 from an N-glycan referencelibrary and from the rMab. Four isomers were found in the referencelibrary for the monofucosylated monosialylated structure. On the basisof the similarities in accurate mass and retention times, the structurewas determined as shown (inset). (b) Two isomers with similar retentiontimes corresponding to monofucosylated biantennary structures. TherMab glycans matched those corresponding to the library and the structureswere determined (inset). (c) Two isomers corresponding to terminalmonogalactosylated structures. The structures were determined andare shown (inset). (d) A bisecting GlcNAc with monosialylated andmonofucosylated structure as determined by comparison to the referencelibrary. Compounds were reduced to the alditol prior to the LC–MSanalysis. Symbolic N-glycan structures correspond to (blue ■)N-acetylglucosamine, (green ●) mannose, (yellow ●) galactose,(○) hexose, (red ▲) fucose, (purple ◆) N-acetylneuraminic acid, (◊) N-glycolylneuraminic acid. Linkages areprovided at the glycosidic bond.
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fig1: Illustration of the identification procedure for antibodyN-glycansusing a previously annotated reference library. (a) Extracted compoundchromatogram (ECC) of composition N54110 from an N-glycan referencelibrary and from the rMab. Four isomers were found in the referencelibrary for the monofucosylated monosialylated structure. On the basisof the similarities in accurate mass and retention times, the structurewas determined as shown (inset). (b) Two isomers with similar retentiontimes corresponding to monofucosylated biantennary structures. TherMab glycans matched those corresponding to the library and the structureswere determined (inset). (c) Two isomers corresponding to terminalmonogalactosylated structures. The structures were determined andare shown (inset). (d) A bisecting GlcNAc with monosialylated andmonofucosylated structure as determined by comparison to the referencelibrary. Compounds were reduced to the alditol prior to the LC–MSanalysis. Symbolic N-glycan structures correspond to (blue ■)N-acetylglucosamine, (green ●) mannose, (yellow ●) galactose,(○) hexose, (red ▲) fucose, (purple ◆) N-acetylneuraminic acid, (◊) N-glycolylneuraminic acid. Linkages areprovided at the glycosidic bond.

Mentions: Figure 1 illustrates how the structureswere assigned for rMab via LC retention time and accurate mass. Referencecompounds in the library are obtained from serum glycoproteins. Shownin Figure 1 are extracted compound chromatograms(ECC) of several compounds from the reference library and from theantibody drug. In Figure 1a, the upper chromatogramshows the four isomers of N54110 in the reference compound. The nomenclatureuses the letter “N” for N-glycan with each digit representingthe composition Hex:HexNac:Fuc:NeuAc:NeuGc. The mass spectrometerdoes not distinguish between mannose and galactose (Hex) or betweenN-acetylgalactose and N-acetylglucose amine (HexNAc). However, fucose(Fuc) as well as N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminicacid (NeuGc) are readily determined by mass. NeuGc is not typicallyfound in humans but is found in other mammals.33 The abundances of NeuGc residues in rMab are low but measurableand are due to the production in CHO/SP 20/NS0 cells.21 The lower case letters following the numbers representthe order in which the compound was elucidated and roughly followsits relative abundance in serum or its relative abundance in rMabfor those not present in serum. The lower chromatogram in Figure 1a shows the ECC of one isomer of N54110 found inTrastuzumab that is aligned with a structure originally found in serumglycoproteins and whose structure is known. Therefore, the compoundin rMab is identified as the same structure N54110a in the N-glycanlibrary. The symbolic structure is shown in the inset. Figure 1b demonstrates the identification of N44100a andN44100b in rMab. The complete structures for N44100a and N44100b aregiven with the isomer “a” having a Gal on the 1-6 armand isomer “b” with the terminal Gal on the 1-3 arm(structure inset). N44000a and N44000b in Figure 1c and N55110a in Figure 1d were identifiedin the same way.


In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs.

Song T, Ozcan S, Becker A, Lebrilla CB - Anal. Chem. (2014)

Illustration of the identification procedure for antibodyN-glycansusing a previously annotated reference library. (a) Extracted compoundchromatogram (ECC) of composition N54110 from an N-glycan referencelibrary and from the rMab. Four isomers were found in the referencelibrary for the monofucosylated monosialylated structure. On the basisof the similarities in accurate mass and retention times, the structurewas determined as shown (inset). (b) Two isomers with similar retentiontimes corresponding to monofucosylated biantennary structures. TherMab glycans matched those corresponding to the library and the structureswere determined (inset). (c) Two isomers corresponding to terminalmonogalactosylated structures. The structures were determined andare shown (inset). (d) A bisecting GlcNAc with monosialylated andmonofucosylated structure as determined by comparison to the referencelibrary. Compounds were reduced to the alditol prior to the LC–MSanalysis. Symbolic N-glycan structures correspond to (blue ■)N-acetylglucosamine, (green ●) mannose, (yellow ●) galactose,(○) hexose, (red ▲) fucose, (purple ◆) N-acetylneuraminic acid, (◊) N-glycolylneuraminic acid. Linkages areprovided at the glycosidic bond.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4066919&req=5

fig1: Illustration of the identification procedure for antibodyN-glycansusing a previously annotated reference library. (a) Extracted compoundchromatogram (ECC) of composition N54110 from an N-glycan referencelibrary and from the rMab. Four isomers were found in the referencelibrary for the monofucosylated monosialylated structure. On the basisof the similarities in accurate mass and retention times, the structurewas determined as shown (inset). (b) Two isomers with similar retentiontimes corresponding to monofucosylated biantennary structures. TherMab glycans matched those corresponding to the library and the structureswere determined (inset). (c) Two isomers corresponding to terminalmonogalactosylated structures. The structures were determined andare shown (inset). (d) A bisecting GlcNAc with monosialylated andmonofucosylated structure as determined by comparison to the referencelibrary. Compounds were reduced to the alditol prior to the LC–MSanalysis. Symbolic N-glycan structures correspond to (blue ■)N-acetylglucosamine, (green ●) mannose, (yellow ●) galactose,(○) hexose, (red ▲) fucose, (purple ◆) N-acetylneuraminic acid, (◊) N-glycolylneuraminic acid. Linkages areprovided at the glycosidic bond.
Mentions: Figure 1 illustrates how the structureswere assigned for rMab via LC retention time and accurate mass. Referencecompounds in the library are obtained from serum glycoproteins. Shownin Figure 1 are extracted compound chromatograms(ECC) of several compounds from the reference library and from theantibody drug. In Figure 1a, the upper chromatogramshows the four isomers of N54110 in the reference compound. The nomenclatureuses the letter “N” for N-glycan with each digit representingthe composition Hex:HexNac:Fuc:NeuAc:NeuGc. The mass spectrometerdoes not distinguish between mannose and galactose (Hex) or betweenN-acetylgalactose and N-acetylglucose amine (HexNAc). However, fucose(Fuc) as well as N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminicacid (NeuGc) are readily determined by mass. NeuGc is not typicallyfound in humans but is found in other mammals.33 The abundances of NeuGc residues in rMab are low but measurableand are due to the production in CHO/SP 20/NS0 cells.21 The lower case letters following the numbers representthe order in which the compound was elucidated and roughly followsits relative abundance in serum or its relative abundance in rMabfor those not present in serum. The lower chromatogram in Figure 1a shows the ECC of one isomer of N54110 found inTrastuzumab that is aligned with a structure originally found in serumglycoproteins and whose structure is known. Therefore, the compoundin rMab is identified as the same structure N54110a in the N-glycanlibrary. The symbolic structure is shown in the inset. Figure 1b demonstrates the identification of N44100a andN44100b in rMab. The complete structures for N44100a and N44100b aregiven with the isomer “a” having a Gal on the 1-6 armand isomer “b” with the terminal Gal on the 1-3 arm(structure inset). N44000a and N44000b in Figure 1c and N55110a in Figure 1d were identifiedin the same way.

Bottom Line: The complete structures were obtained through exoglycosidase sequencing.The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances.The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California , One Shields Avenue, Davis, California 95616, United States.

ABSTRACT
The glycosylation in recombinant monoclonal antibody (rMab) drugs is a major concern in the biopharmaceutical industry as it impacts the drugs' many attributes. Characterization is important but complicated by the intricate structures, microheterogeneity, and the limitations of current tools for structural analysis. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) N-glycan library based on eight commercial rMab drugs. A library of over 70 structures was developed for the rapid characterization of rMab. N-Glycans were separated on a porous graphitized carbon (PGC) column incorporated on a chip and then analyzed by an electrospray ionization hybrid quadrupole time-of-flight (ESI-Q-TOF) MS. The retention time and accurate mass for each N-glycan were recorded in the library. The complete structures were obtained through exoglycosidase sequencing. The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances. The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

Show MeSH