Increased in vitro neutralizing activity of SARS-CoV-2 IgA1 dimers compared to monomers and IgG

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineered Nicotiana benthamiana plants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA’s structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins.

Longitudinal Pathogenic Properties and N-Glycosylation Profile of Antibodies from Patients with Pemphigus after Corticosteroid Treatment

Pemphigus vulgaris is an autoimmune disease that occurs due to pathogenic autoantibodies that recognize the following epidermal adhesion proteins: desmogleins. Systemic corticosteroids usually decrease the titers of anti-desmoglein autoantibodies and improve patients’ conditions. Since modifications of IgG N-glycosylation have been described in some autoimmune diseases, we hypothesized that changes in the pathogenic activity of pemphigus IgG could be related to changes in their N-glycosylation profile. The purpose of this study was to assess, longitudinally, the pathogenicity of pemphigus serum IgG and their N-glycosylation profile during phases of disease activity and clinical remission. The pathogenic activity of serum IgG was measured in vitro on immortalized keratinocytes, by immunofluorescence and dissociation assays, and IgG N-glycans were analyzed by mass spectrometry. We showed (i) a correlation between pemphigus clinical activity and the pathogenicity of serum IgG at baseline and at month 6, while the persistence of the in vitro pathogenic activity of IgG during its evolution, even in patients in clinical remission, seemed to be predictive of relapse; (ii) that modifications of the N-glycan structure were altered the in vitro pathogenicity of patients’ autoantibodies; (iii) that the pathogenic properties of pemphigus IgG did not appear to be related to the disparity in IgG N-glycans during the course of pemphigus.

P–661 Comparative assessment of the structural features of human follicle-stimulating hormone in products from multiple markets

Study question The aim of the study is to explore the structural differences occurring in recombinant human follicle-stimulating hormone alfa (r-hFSH- α), originator and its biosimilars, from various countries. Summary answer When compared with r-hFSH-α originator (Gonal-f), its biosimilars presented structural differences, namely Primapur showed a significant different glycosylation profile. What is known already FSH is part of cystine knot growth factor superfamily and plays a central role in reproduction, as FSH stimulates follicular development and estrogen synthesis. R-hFSH- α is commonly used in assisted reproductive technologies to achieve multifollicular development. At the present r-hFSH- α biosimilars are available in Europe and other regions. R-hFSH-α is a complex glycoprotein, that possesses several structural features critical for its efficacy and safety1–2. Glycosylation profile is one of the most impactful attributes of the molecule defining a moiety of FSH isoforms with impact on its biological net effect3. Efficacy and safety of r-hFSH- α are strictly correlated with glycoforms’ composition3–8. Study design, size, duration At least two different batches of each r-hFSH- α originator and its biosimilars have been included in the study. Participants/materials, setting, methods The structural features of products from six different marketed r-hFSH α (Gonal-f, Primapur, Folisurge Intas, Corneumon, Jin Sai Heng, Follitrope LG) have been investigated with a variety of analytical techniques in order to evaluate the presence of molecular differences, which could have a severe impact on the efficacy and safety of the product. The attributes which have been investigated in-depth include primary, secondary and tertiary structure as well as post-translational modifications (PTMs), including glycosylation and contaminants. Main results and the role of chance All r-hFSH- α biosimilars analyzed presented differences compared to the originator. We firstly investigated Primapur and found significant differences regarding multiple structural attributes, particularly in the glycosylation profile. Gonal-f exhibited lower glycan branching, expressed by an A-index* of 2.5, while Primapur showed an A-index of 2.4. Furthermore, Primapur showed a lower level of sialylation in comparison with Gonal-f, as measured by their respective S-index* of 1.8 and 2.1. FSH glycosylation exhibits both macroheterogeneity and microheterogeneity, impacting both FSH protein’s half-life and affinity with the follicle-stimulating hormone receptor (FSHR). Antennarity, representing FSH microheterogeneity, influence r-hFSH-α activity since it has been shown that bulky and extended glycans may take longer to fit into the FSHR cavity compared to less sterically hindering glycans, resulting in a delayed response 7,8.Additionaly, sialylation has been shown in-vivo to correlate with plasma half-life and effect on granulosa cells proliferation 1,2,3. The slower clearance of highly sialylated r-hFSH has been shown to lead to a higher in-vivo activity, despite the lower in-vitro bioactivity 1,2,3. *A-index and S-index express respectively a measure of the number of antennae and sialic acid per glycan. Final values are generated from many relative abundances normalized to 100, highlighting the significance of small numerical differences. Limitations, reasons for caution More batches should be tested for each product. The authors are presenting full characterization of only one of the biosimilars since the rest of the products are under characterization. Wider implications of the findings: r-hFSH-α originator and its biosimilar showed differences in terms of glycosylation profile that is well known as the major protein characteristic impacting FSH activity as extensively demonstrated in in-vivo and in-vitro models. This structural difference could have impact also on product efficacy and safety. Trial registration number ‘not applicable’

Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method

Glycosylation is critical for monoclonal antibody production because of its impact on pharmacokinetics and pharmacodynamics. Modulation of glycan profile is frequently needed in biosimilar development. However, glycosylation profile is not a single value like that of cell culture titer, hence making it challenging for the Design of Experiment (DoE) methodology to be directly applied. In this study, a Her2-binding antibody was developed as a biosimilar to Herceptin. Cluster analysis was introduced to demonstrate the similarity of glycan profiles between the samples and the reference with specific value—distance. The glycosylation was subsequently optimized with the DoE method. Basal medium and feed medium were found to be the significant factors to the glycosylation pattern. Moreover, a combination of medium and feed strategy was developed to attain the most similar glycoprotein molecule to that of the originator biologic drug. This study may provide an additional option to evaluate multivariable factors and assess biosimilarity and/or comparability in monoclonal antibody production.

A46 LOSS OF BMP-SIGNALING IN MESENCHYMAL TELOCYTES TRIGGERS MODIFICATION OF THE GLYCOSYLATION PROFILE OF COLONIC MUCINS.

NOT PUBLISHED AT AUTHOR’S REQUEST Funding Agencies: CIHR

Variation of IgG N-linked glycosylation profile in diabetic retinopathy

Background: The relationship of IgG glycosylation with diabetes and diabetic nephropathy has been reported, while its role in diabetic retinopathy (DR) remained unclear. We aimed to investigate and validate the association of IgG glycosylation with DR. Methods: We analyzed the IgG N-linked glycosylation profile and identified the specific panel in the discovery population using binary logistics model. Findings were validated in the replication population. The discriminative capacity of IgG glycosylation panel was explored by ROC analysis using cross validation and Brier score. Multiple sensitive analyses were performed on the whole population. Results: 2 IgG glycans (GP15, GP20) and 2 derived traits (IGP32, IGP54) were identified and validated significantly associated with DR (P<0.05), and the adjusted OR were 0.676, 0.671, 1.770, 0.681 in combined population, respectively. The glycosylation panel achieved an average AUC of 0.67 and 0.60 in the discovery and replication population. The association was independent of blood pressure, glucose and lipids, thus improving the ROC and Brier score when the panel added. In addition, the results remained consistent when the controls were re-defined and 1:3 re-matched. Conclusions: IgG glycosylation profile reflecting a pro-inflammatory status were associated with DR. The variation of IgG glycome deserves more attention in the aggravation of diabetes and the underlying mechanism warrants further research.