Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N‐glycosylation sites, and alteration of N‐glycan micro‐ and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N‐glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin–Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N‐glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N‐glycan microheterogeneity showed an increasing variability and a higher complexity for N‐glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N‐glycosylated at site N73. Almost all N‐glycan structures were fucosylated. Furthermore, HA and NA N‐glycan structures were exclusively hybrid‐ and complex‐type structures, to some extent terminated with alpha‐linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose‐type, alpha‐linked galactose, and multiantennary complex‐type N‐glycans.

Background: Sulfate modification ofN-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of thisN-glycan modification and its functions remain largely unexplored. Characterization ofN-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection ofN-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO₄onN-glycans.Results: Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO₄when it is in a terminal position on anN-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfatedN-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO₄(but not asulfated GlcNAc) from a terminal position onN-glycans. Finally, the use of these enzymes to detect the presence of sulfatedN-glycans by xCGE-LIF is demonstrated.Conclusion: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO₄inN-glycan structural analyses.

The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.

Background: Malignant Ascites (MA) is a therapeutic dilemma significantly impairing patients’ quality of life (QoL). The Sequana Medical alfapump System (AP), a subcutaneous, externally rechargeable, implantable device, continually draining ascites via the urinary bladder, has been well established in liver cirrhosis, but not yet in MA. The AP-system was evaluated in cancer patients in reducing the need for large volume paracentesis (LVP). Methods: A retrospective multicentre evaluation of all eligible patients who received an AP for MA-palliation was performed. AP was evaluated for its ability to reduce LVP and cross-correlated with adverse events (AE), survival and retrospective physician-reported QoL. Results: Seventeen patients with median age of 63 years (range: 18–81), 70.6% female, across 7 primary tumour types were analysed. Median duration of AP-implantation was 60 min (range: 30–270) and median post-implantation hospital stay: 4 days (range: 2–24). Twelve protocol-defined AE occurred in 5 patients (29.4%): 4 kidney failures, 4 pump/catheter-related blockages, 3 infections/peritonitis and 1 wound dehiscence. Median ascitic volume (AV) pumped daily was 303.6 ml/day (range:5.6–989.3) and median total AV drained was 28 L (range: 1–638.6). Median patient post-AP-survival was 111 days (range:10–715) and median pump survival was 89 days (range: 0–715). Median number of paracenteses was 4 (range: 1–15) per patient pre-implant versus 1 (range: 0–1) post-implant (p = 0.005). 71% of patients were reported to have an improvement of at least one physician reported QoL-parameters. Conclusions: AP appears to be effective in palliating patients with MA by an acceptable morbidity profile. Its broader implementation in oncology services should be further explored. Trial registration: NCT03200106; June 27, 2017.