Arabinosylation of recombinant human immunoglobulin-based protein therapeutics

Abstract

Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

Impact of Acetylated and Non-Acetylated Fucose Analogues on IgG Glycosylation

“…According to literature data, differences in the frequency of incorporation may be due to the relative position of the modification, the nature of the modification, or more likely the interplay of both. Modifications of fucose at carbon positions C-2, C-5 and C-6 have been frequently reported in the literature [14,15,21,28], while modifications at other carbon positions are likely undesirable, as these may be required for enzyme binding [28]. By comparing the incorporation data of 6F-fucose obtained by Okeley et al, to our data on 2F-fucose, we can gain an insight into the binding modality of some enzymes in the fucosylation pathway.…”

Interaction of cell culture process parameters for modulating mAb afucosylation

“…genetic engineering of recombinant cell lines (Louie et al, 2016;Yamane-Ohnuki et al, 2004); (b) addition of enzyme inhibitors (Allen et al, 2016;Okeley et al, 2015); (c) modification of the levels of cofactors and substrates involved in glycosylation, including supplementation with alternative sugars (Hossler et al, 2014;Hossler et al, 2017); and (d) fine-tuning of cell culture process parameters (Konno et al, 2012). Of the many cell culture process parameters known to impact glycosylation previously (Hossler, Khattak, & Li, 2009), osmolality was shown to affect afucosylation (Konno et al, 2012).…”

Glycoengineering of Antibodies for Modulating Functions

“…This study suggests that D-arabinose, which is different from L-fucose in lacking the 5-methyl group in the structure, can be efficiently incorporated into the biosynthetic pathway to compete with core fucosylation during antibody expression. Interestingly, these fully arabinosylated antibodies showed a similar increase in ADCC activity as that of nonfucosylated antibodies (59). Results suggest that the methyl group present in L-fucose may play an essential structural role for the adverse effects of core fucosylation on FcγIIIa receptor binding and ADCC activity.…”

“…Results suggest that the methyl group present in L-fucose may play an essential structural role for the adverse effects of core fucosylation on FcγIIIa receptor binding and ADCC activity. Removal of a methyl group from L-fucose, i.e., its conversion to D-arabinose, may be sufficient to increase the binding affinity of antibodies to FcγIIIa receptor and thus to enhance ADCC activity (59).…”