Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins

Abstract

Recombinant glycosylated biotherapeutic agents are usually produced in non-human mammalian cell lines, which can synthesize and/or metabolically incorporate the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). This contamination was previously known but ignored, as normal humans were thought to not react against Neu5Gc. However, recent findings indicate that humans have variable spectra of sometimes high levels of circulating anti-Neu5Gc antibodies. We studied two monoclonal antibodies in clinical use (Cetuximab and Panitumumab), and show covalently-bound Neu5Gc on Cetuximab. Anti-Neu5Gc antibodies from normal humans interact with Cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate anti-Neu5Gc antibodies upon injection with Cetuximab. Circulating anti-Neu5Gc antibodies enhance Cetuximab clearance. These findings have potential relevance to half-life, efficacy and immune reactions in patients given such drugs. Finally, we show a method to reduce the Neu5Gc content of cultured cell lines and their secreted glycoproteins.

Local Inhibition of Complement Improves Mesenchymal Stem Cell Viability and Function After Administration

“…Consistent with previous reports, 18 the addition of Neu5AC to the culture medium significantly reduced Neu5GC levels on the MSC surface (Figure 1b, left panel). We then incubated these cells with pooled normal human serum and, using flow cytometry, examined whether human immunoglobulins (IgGs and IgMs) were present on the MSCs.…”

“…Detection of Neu5GC was performed as described previously. 18 In brief, after blocking with a 0.5% solution of fish skin gelatin (Sigma-Aldrich, St. Louis, MO), MSCs were incubated for 30 minutes on ice with 5 µg/ml of chicken anti-Neu5GC IgY or control IgY, then were washed, stained with 5 µg/ml of FITC-labeled anti-IgY antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), and analyzed on a flow cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA).…”

“…The activity of the alternative complement pathway was assessed using a zymosan C3b deposition assay and that of the classical complement pathway using a C3b deposition assay employing E shA , following previously established protocols. 18 Heparin painting. Heparin was first activated by incubation for 15 minutes at room temperature with 2 mmol/l EDC and 5 mmol/l NHS in a 2-(N-morpholino)-ethanesulfonic acid/NaCl buffer (all from Thermo Fisher Scientific), then 2-mercaptoethanol (final concentration 20 mmol/l) was added to quench the reaction and excess EDC, EDC byproducts, NHS, and 2-mercaptoethanol removed on a PBS-equilibrated desalting column (Thermo Fisher Scientific).…”

Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium

“…These contributing factors are reviewed extensively elsewhere 13, 14, 15, 16, 17, 18. Key concepts and recommended definitions for standardizing the description and interpretation of immunogenicity are described further below in relation to practices for monitoring and mitigating immunogenicity during clinical use of BPs.…”

Glycosylation profiles determine extravasation and disease-targeting properties of armed antibodies

“…Another receptor involved in glycoprotein homeostasis, primarily expressed by macrophages and dendritic cells, is the mannose receptor, recognizing terminal mannose or N-acetylglucosamine (11). Certain glycan epitopes are also known to be immunogenic and can lead to antidrug antibody responses in humans (12).…”