Glycosylation profiles of therapeutic antibody pharmaceuticals

Wacker, Christoph; Berger, Christoph; Girard, Philippe; Meier, Roger

doi:10.1016/j.ejpb.2011.06.010

Cited by 57 publications

(49 citation statements)

References 25 publications

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“…1 The galactosylation pattern of a panel of six marketed rIgGs (Tables 1 and 2) clearly shows product-specific variability known to exist in such rIgGs. 22,23 At least to some extent, the variability appears to result from use of different cell lines for their expression. 22,23 Terminal galactosylation may also vary by lot, suggesting that even minor variations in cell culture conditions may have an effect, although these are generally smaller than the variations observed in products manufactured in different cell cells.…”

Section: Resultsmentioning

confidence: 96%

“…22,23 At least to some extent, the variability appears to result from use of different cell lines for their expression. 22,23 Terminal galactosylation may also vary by lot, suggesting that even minor variations in cell culture conditions may have an effect, although these are generally smaller than the variations observed in products manufactured in different cell cells. They are marketed for the same indications by two different manufacturers.…”

Section: Resultsmentioning

confidence: 96%

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Galactosylation variations in marketed therapeutic antibodies

Raju

Jordan

2012

mAbs

106

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There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 96%

Galactosylation variations in marketed therapeutic antibodies

Raju

Jordan

2012

mAbs

106

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“…The largest fragment peak in the spectrum corresponds to a charge separation process leading to 'antenna loss' -as the compound contains a truncated antenna, this is a monosaccharide unit, [ 6c). This indicates, that Cetuximab is predominantly core fucosylated [22,23]; antenna fucosylation is present in very small (very approximately 10%) amount.…”

Section: The Breakdown Diagram Of [Bis2·neeynk]mentioning

confidence: 99%

Fragmentation characteristics of glycopeptides

Vékey

Ozohanics

Tóth

et al. 2013

International Journal of Mass Spectrometry

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Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, "B" type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna-and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant.

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“…For the production of therapeutic glycoproteins mammalian cell lines are typically used to minimize any potential immunogenicity caused by the presence of nonhuman glycoforms [ 18 ]. However, the use of mammalian cell lines themselves raises several concerns.…”

Section: Major Differences Between Human and Yeast Glycosylationmentioning

confidence: 99%

Progress in Yeast Glycosylation Engineering

Hamilton

Zha

2015

Glyco-Engineering

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While yeast are lower eukaryotic organisms, they share many common features and biological processes with higher eukaryotes. As such, yeasts have been used as model organisms to facilitate our understanding of such features and processes. To this end, a large number of powerful genetic tools have been developed to investigate and manipulate these organisms. Going hand-in-hand with these genetic tools is the ability to efficiently scale up the fermentation of these organisms, thus making them attractive hosts for the production of recombinant proteins. A key feature of producing recombinant proteins in yeast is that these proteins can be readily secreted into the culture supernatant, simplifying any downstream processing. A consequence of this secretion is that the proteins typically pass through the secretory pathway, during which they may be exposed to various posttranslational modifications. The addition of glycans is one such modification. Unfortunately, while certain aspects of glycosylation are shared between lower and higher eukaryotes, significant differences exist. Over the last two decades much research has focused on engineering the glycosylation pathways of yeast to more closely resemble those of higher eukaryotes, particularly those of humans for the production of therapeutic proteins. In the current review we shall highlight some of the key achievements in yeast glyco-engineering which have led to humanization of both the N- and O-linked glycosylation pathways.

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Glycosylation profiles of therapeutic antibody pharmaceuticals

Cited by 57 publications

References 25 publications

Galactosylation variations in marketed therapeutic antibodies

Galactosylation variations in marketed therapeutic antibodies

Fragmentation characteristics of glycopeptides

Progress in Yeast Glycosylation Engineering

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