Successive remodeling of IgG glycans using a solid-phase enzymatic platform

ARTICLE https://doi.org/10.1038/s42003-022-03257-4 OPEN Successive remodeling of IgG glycans using a solid-phase enzymatic platform 1234567890():,; Yen-Pang Hsu 1,4, Deeptak Verma Benjamin F. Mann 1 ✉ 2, Shuwen Sun1, Caroline McGregor3, Ian Mangion1 & The success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering strategies to control the glycan structures. With the accumulation of knowledge about the glycan biosynthesis enzymes, enzymatic glycan remodeling provides a potential strategy to construct highly ordered glycans with improved efficiency and biocompatibility. In this study, we quantitatively evaluate more than 30 enzymes for glycoengineering immobilized immunoglobulin G, an impactful glycoprotein class in the pharmaceutical field. We demonstrate successive glycan remodeling in a solid-phase platform, which enabled IgG glycan harmonization into a series of complex-type N-glycoforms with high yield and efficiency while retaining native IgG binding affinity. 1 Analytical Research and Development, Merck & Co., Inc, Rahway, NJ 07065, USA. 2 Computational and Structural Chemistry, Discovery Chemistry, Merck & Co., Inc, Rahway, NJ 07065, USA. 3 Process Research & Development, Merck & Co., Inc, Rahway, NJ 07065, USA. 4Present address: Exploratory Science Center, Merck & Co., Inc, Cambridge, MA 02141, USA. ✉email: COMMUNICATIONS BIOLOGY | (2022)5:328 | https://doi.org/10.1038/s42003-022-03257-4 | www.nature.com/commsbio 1 ARTICLE P COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03257-4 rotein glycosylation directly affects the physical and biochemical properties of proteins in eukaryotic systems1. According to glycoproteomic analyses, over 1% of the human genome encodes glycosylation-related enzymes and more than 50% of human proteins are glycosylated2. Glycoproteins carry structurally diverse oligosaccharides, called glycans, that are involved at the interface of protein-biomolecular interactions and thus determine protein stability, selectivity, and activity. The significance of protein glycosylation to biological systems has been exemplified by several diseases associated with various cancers and the immune system3,4. For example, patients with rheumatoid arthritis were found to have an increased galactosylation level in their serum immunoglobulin G (IgG), though the mechanism remains elusive5. Unsurprisingly, it follows that insights into the structure and function of glycans have yielded a profound impact on the development of therapeutic glycoproteins6. Manipulating glycan structures present an effective strategy to improve their efficacy and safety by modulating immunological responses, circulatory half-life, and effector functions7,8. Thus, glycoengineering represents a versatile tool and a great opportunity to create better medicines. To achieve this goal, technologies that enable the control of protein glycosylation profiles are essential. However, tools to access the diverse array of glycan structures displayed in nature remain scarce, and methods that provide a high yield of the desired glycoforms have proven to be a still greater challenge to develop despite decades of study9,10. Through synthetic and chemoenzymatic approaches, various glycoforms have been accessed11–13. These structurally defined glycans can be installed onto glycoproteins through endoglycosidase and glycosynthase activities14,15. While this approach has advanced our ability to control protein glycosylation, the preparation of synthetic glycans becomes increasingly difficult as the number of saccharide units increases. As a result, the installation of synthetic glycans is not practical for many applications. On the other hand, genetic engineering has been applied for controlled glycan biosynthesis by either knocking out or introducing certain glycoengineering enzymes in the host cells16. This strategy enables in vivo glycan remodeling and has been demonstrated in nonhuman cell lines17. However, the optimization of this strategy has been impeded by the complexity of engineering glycosylation pathways. Also, microheterogeneity is often generated during glycan formation, which, although it is comparable to the natural phenomenon, does not provide exquisite control over the molecular structure18. In recent decades, our understanding of the in vitro activity of glycoengineering enzymes is growing rapidly19–22. Some of the enzymes can even function on intact glycoproteins, which opens a new window for glycan remodeling23,24. A remarkable example comes from the use of endoglycosidase S (Endo S) and its mutants to replace native IgG glycans with synthetic ones25,26. To further leverage the use of more glycoengineering enzymes, three primary challenges need to be addressed. First, characterization of enzyme activities on intact glycoproteins is required21,27. A comprehensive understanding of their activity, selectivity, and stability would allow researchers to design and execute glycan remodeling enzymatically. Second, preserving the integrity and functions of the substrates after the enzymatic reactions is critical, especially for therapeutic glycoproteins. Protocols with high biocompatibility are thus required. Third, to construct complex glycan structures, successive reactions using different enzymes are needed. These enzymes might require very different working conditions, such as pH and temperature. Therefore, one would need to repeat the buffer swapping and product purification processes between the enzymatic reactions, which is highly laborintensive and time-consuming. Together, to address these needs, platforms that enable efficient, successive enzymatic glycan 2 remodeling with high biocompatibility to the substrates are in great demand. Inspired by solid-phase peptide synthesis (SPPS), herein, we introduce solid-phase glycan remodeling (SPGR) where enzymatic reactions are carried out on the substrates immobilized on resins28. This approach enables efficient reaction swapping, substrate purification, and the recovery of both products and engineering enzymes. We use human IgG as the substrate in this study because it is a major class of glycoproteins that have been applied in therapeutic development6,29. We quantitatively examined more than 30 glycan engineering enzymes for their activities on intact IgG immobilized on resins and then applied them in SPGR. This method has allowed us to harmonize IgG glycans into ten different glycoforms, including noncanonical structures, in 48 h with an average conversion ratio of over 95%. Physical and biochemical (...truncated)