Integration of NMR Spectroscopy in an Analytical Workflow to Evaluate the Effects of Oxidative Stress on Abituzumab: Beyond the Fingerprint of mAbs.

The assessment of the higher-order structure (HOS) by NMR is a powerful methodology to characterize the structural features of biologics. Forced oxidative stress studies are used to investigate the stability profile, to develop pharmaceutical formulations and analytical methods. Here, the effects of forced oxidative stress by H2O2 on the monoclonal antibody Abituzumab have been characterized by a multianalytical approach combining NMR spectroscopy, mass spectrometry, differential scanning calorimetry, surface plasmon resonance, computational tools, and bioassays. This integrated strategy has provided qualitative and semiquantitative characterization of the samples and information at residue level of the effects that oxidation has on the HOS of Abituzumab, correlating them to the loss of the biological activity.

Rapid cGMP manufacturing of COVID-19 monoclonal antibody using stable CHO cell pools.

Therapeutic proteins, including monoclonal antibodies, are typically manufactured using clonally derived, stable host cell lines, since consistent and predictable cell culture performance is highly desirable. However, selecting and preparing banks of stable clones takes considerable time, which inevitably extends overall development timelines for new therapeutics by delaying the start of subsequent activities, such as the scale-up of manufacturing processes. In the context of the coronavirus disease 2019 (COVID-19) pandemic, with its intense pressure for accelerated development strategies, we used a novel transposon-based Leap-In Transposase® system to rapidly generate high-titer stable pools and then used them directly for large scale-manufacturing of an anti-severe acute respiratory syndrome coronavirus 2 monoclonal antibody under cGMP. We performed the safety testing of our non-clonal cell bank, then used it to produce material at a 200L-scale for preclinical safety studies and formulation development work, and thereafter at 2000L scale for supply of material for a Phase 1 clinical trial. Testing demonstrated the comparability of critical product qualities between the two scales and, more importantly, that our final clinical trial product met all pre-set product quality specifications. The above expediated approach provided clinical trial material within 4.5 months, in comparison to 12-14 months for production of clinical trial material via the conventional approach.

Glycoengineered antibodies: towards the next-generation of immunotherapeutics.

Monoclonal antibodies (mAbs) are currently the largest and fastest growing class of biopharmaceuticals, and they address unmet medical needs, e.g., in oncology and in auto-immune diseases. Their clinical efficacy and safety is significantly affected by the structure and composition of their glycosylation profile which is commonly heterogeneous, heavily dependent on the manufacturing process, and thus susceptible to variations in the cell culture conditions. Glycosylation is therefore considered a critical quality attribute for mAbs. Commonly, in currently marketed therapeutic mAbs, the glycosylation profile is suboptimal in terms of biological properties such as antibody-dependent cell-mediated cytotoxicity or may give rise to safety concerns due to the presence of non-human glycans. This article will review recent innovative developments in chemo-enzymatic glycoengineering, which allow generating mAbs carrying single, well-defined, uniform Fc glycoforms, which confers the desired biological properties for the target application. This approach offers significant benefits such as enhanced Fc effector functions, improved safety profiles, higher batch-to-batch consistency, decreased risks related to immunogenicity and manufacturing process changes, and the possibility to manufacture mAbs, in an economical manner, in non-mammalian expression systems. Overall, this approach could facilitate and reduce mAb manufacturing costs which in turn would translate into tangible benefits for both patients and manufacturers. The first glycoengineered mAbs are about to enter clinical trials and it is expected that, once glycoengineering reagents are available at affordable costs, and in-line with regulatory requirements, that targeted remodeling of antibody Fc glycosylation will become an integral part in manufacturing the next-generation of immunotherapeutics.

How unique is interferon-ß within the type I interferon family?

All type I interferons share structural homology and bind to a common heterodimeric receptor consisting of the IFNAR1 and IFNAR2 subunits, which are expressed on most cell types. Although binding to the same receptor pair, they evoke a broad range of activities within the cell affecting the expression of numerous genes and resulting in profound cellular changes. Differential activation results from multiple levels of cellular and molecular events including binding affinity, receptor density, cell type-specific variations, and post-translational modification of signaling molecules downstream. Within the type I interferon family the Asn-Gly-Arg (NGR) sequence motif is unique to interferon-ß and, together with its deamidated variants Asp-Gly-Arg (DGR) and iso-Asp-Gly-Arg (iso-DGR), imparts additional binding specificities that go beyond that of the canonical IFNAR1/IFNAR2. These warrant further investigations and functional studies and may eventually shed new light on differential effects observed for this molecule in oncology and autoimmune diseases.

A deamidated interferon-ß variant binds to integrin αvß3.

Human type I interferons are a family of pleiotropic cytokines with antiviral, anti-proliferative and immunomodulatory activities. They signal through the same cell surface receptors IFNAR1 and IFNAR2 yet evoking markedly different physiological effects. One differentiating factor of interferon-beta (IFN-ß) from other type I interferons is the presence of theAsn-Gly-Arg (NGR) sequence motif, which, upon deamidation, converts to Asp-Gly-Arg (DGR) and iso-Asp-Gly-Arg (iso-DGR) motifs. In other proteins, the NGR and iso-DGR motifs are reported as CD13- and αvß3, αvß5, αvß6, αvß8 and α5ß1 integrin-binding motifs, respectively. The scope of this study was to perform exploratory surface plasmon resonance (SPR) experiments to assess the binding properties of a deamidated IFN-ß variant to integrins. For this purpose, integrin αvß3 was selected as a reference model within the iso-DGR- integrin binding members. The obtained results show that deamidated IFN-ß binds integrin αvß3 with nanomolar affinity and that the response was dependent on the deamidation extent. Based on these results, it can be expected that deamidated IFN-ß also binds to other integrin family members that are able to bind to the iso-DGR binding motif. The novel binding properties could help elucidate specific IFN-ß attributes that under physiological conditions may be modulated by the deamidation.

In vitro biological characterization of IFN-ß-1a major glycoforms.

Recombinant human interferon ß-1a (IFN-ß-1a) is extensively used as the first-line treatment of relapsing forms of multiple sclerosis. Its glycosylation is recognized as having a complex impact on a wide range of molecule characteristics and functions. The present study reports the enrichment of IFN-ß-1a glycoforms and their physicochemical and biological characterization by means of electrospray ionization-mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays (antiproliferative, antiviral, immunomodulatory and reporter gene assay). The glycoforms were fractionated by means of cation-exchange chromatography using recombinant IFN-ß-1a derived from Chinese Hamster Ovary cell culture as starting material. The obtained fractions contained bi- and higher-antennarity glycans as described in the European Pharmacopoeia monograph (Nr. 1639E, Interferon beta 1a concentrated solution). The in vitro bioassay responses revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms have biological activity and play a role in modulating the overall IFN-ß biological activity with higher-antennarity glycoforms being able to better sustain IFN-ß-1a bioactivity over time. These data indicate the role of IFN-ß-1a glycosylation in vivo and shed new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.

Effect of Fc core fucosylation and light chain isotype on IgG1 flexibility.

N-glycosylation plays a key role in modulating the bioactivity of monoclonal antibodies (mAbs), as well as the light chain (LC) isotype can influence their physicochemical properties. However, investigating the impact of such features on mAbs conformational behavior is a big challenge, due to the very high flexibility of these biomolecules. In this work we investigate, by accelerated molecular dynamics (aMD), the conformational behavior of two commercial immunoglobulins G1 (IgG1), representative of κ and λ LCs antibodies, in both their fucosylated and afucosylated forms. Our results show, through the identification of a stable conformation, how the combination of fucosylation and LC isotype modulates the hinge behavior, the Fc conformation and the position of the glycan chains, all factors potentially affecting the binding to the FcγRs. This work also represents a technological enhancement in the conformational exploration of mAbs, making aMD a suitable approach to clarify experimental results.

Asn25 Deamidation as an Allosteric Tool to Increase IFNß-1a Biological Activity.

Interferon beta (IFNß) is a well-known cytokine, belonging to the type I family, that exerts antiviral, immunomodulatory, and antiproliferative activity. It has been reported that the artificially deamidated form of recombinant IFNß-1a at Asn25 position shows an increased biological activity. As a deepening of the previous study, the molecular mechanism underlying this biological effect was investigated in this work by combining experimental and computational techniques. Specifically, the binding to IFNAR1 and IFNAR2 receptors and the canonical pathway of artificially deamidated IFNß-1a molecule were analyzed in comparison to the native form. As a result, a change in receptor affinity of deamidated IFNß-1a with respect to the native form was observed, and to better explore this molecular interaction, molecular dynamics simulations were carried out. Results confirmed, as previously hypothesized, that the N25D mutation can locally change the interaction network of the mutated residue but also that this effect can be propagated throughout the molecule. In fact, many residues not involved in the interaction with IFNAR1 in the native form participate to the recognition in the deamidated molecule, enhancing the binding to IFNAR1 receptor and consequently an increase of signaling cascade activation. In particular, a higher STAT1 phosphorylation and interferon-stimulated gene expression was observed under deamidated IFNß-1a cell treatment. In conclusion, this study increases the scientific knowledge of deamidated IFNß-1a, deciphering its molecular mechanism, and opens new perspectives to novel therapeutic strategies.

Current views on N-glycolylneuraminic acid in therapeutic recombinant proteins.

The incorporation of the non-human N-glycolylneuraminic acid (Neu5Gc) in therapeutic recombinant proteins raises clinical concerns due to its immunogenic potential and the high prevalence of pre-existing anti-Neu5Gc antibodies in humans. The scientific literature is ambiguous regarding the actual impact of Neu5Gc-containing biotherapeutics as no severe adverse clinical manifestations were unequivocally attributed to Neu5Gc for currently marketed biotherapeutics. This review discusses structural and functional considerations of Neu5Gc-containing glycans regarding the potential impact on drug clearance, their recognition by pre-existing antibodies, and recent hypotheses regarding the tolerance to low Neu5Gc levels. Furthermore, it provides recommendations regarding the standardization of analysis and reporting, analytical aspects relevant for assessing risks associated with Neu5Gc-containing biotherapeutics, and approaches to minimize Neu5Gc incorporation in recombinant protein manufacturing.

The Formidable Challenge of Controlling High Mannose-Type N-Glycans in Therapeutic mAbs.

The clinical efficacy and safety of therapeutic monoclonal antibodies (mAbs) are significantly affected by their Fc-glycosylation profile. High mannose-type N-glycans (HM) affect efficacy (in terms of antibody-dependent cell cytotoxicity), pharmacokinetics, and stability. While in endogenous IgGs the HM levels are very low, they are significantly higher in marketed therapeutic mAbs. In order to meet the demands for late-phase clinical trial and market supply, process intensification is required. Since glycosylation profiles are sensitive to process variations and changes, controlling HM levels in robust manufacturing processes presents a formidable challenge and requires a thorough understanding of the cellular processes as well as the biotechnical aspects that govern the production of HM glycans.

Site-specific N- and O-glycosylation analysis of atacicept.

The Fc-fusion protein atacicept is currently under clinical investigation for its biotherapeutic application in autoimmune diseases owing to its ability to bind the two cytokines B-Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL). Like typical recombinant IgG-based therapeutics, atacicept is a glycoprotein whose glycosylation-related heterogeneity arises from the glycosylation-site localization, site-specific occupation and structural diversity of the attached glycans. Here, we present a first comprehensive site-specific N- and O-glycosylation characterization of atacicept using mass spectrometry-based workflows. First, N- and O-glycosylation sites and their corresponding glycoforms were identified. Second, a relative quantitation of the N-glycosylation site microheterogeneity was achieved by glycopeptide analysis, which was further supported by analysis of the released N-glycans. We confirmed the presence of one N-glycosylation site, carrying 47 glycoforms covering 34 different compositions, next to two hinge region O-glycosylation sites with core 1-type glycans. The relative O-glycan distribution was analyzed based on the de-N-glycosylated intact protein species. Overall, N- and O-glycosylation were consistent between two individual production batches.

In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f).

Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated.

Data for the crystal structure of APRIL-BAFF-BAFF heterotrimer.

The TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) modulate B cell function by forming homotrimers and heterotrimers. To determine the structure of a heterotrimer of BAFF and APRIL, these ligands were expressed as a single chain protein in HEK 293 cells, purified by affinity and size exclusion chromatographies, and crystallized. Crystals belonging to the orthorhombic crystal system with a space group of C2221 diffracted to 2.43 Å. Initial structural solution was obtained by the molecular replacement method, and the structure was further refined to an R factor of 0.179 and free R factor of 0.234. The atomic coordinates and structure factors have been deposited into the Protein Data Bank (accession code 4ZCH).

Biological Functions of Interferon ß-1a Are Enhanced By Deamidation.

Human type I Interferons (IFN-ß, IFN-ɛ, IFN-κ, IFN-ω, and 12 subtypes of IFN-α) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory activities. They signal through the same cell surface receptors, IFNAR1 and IFNAR2, yet evoking markedly differential potency. One differentiating factor of IFN-ß from other type I interferons is the presence of a consensus sequence (NG) for deamidation. Comparing almost completely deamidated IFN-ß-1a with untreated IFN-ß-1a, this present study reports the increased activities in 3 in-vitro bioassays testing the antiviral, antiproliferative, and immunomodulatory properties, respectively, of the molecule. Deamidated IFN-ß-1a has the potential to improve current therapies in multiple sclerosis, and its ability to potentiate the MHC-Class I expression suggests a clinical benefit in diseases where the downmodulation of the MHC-class I expression plays a role (eg, in immuno-oncology combination therapies or antiviral agents). The present study on IFN-ß deamidation adds a new prospective on deamidation as part of a posttranslational modification code that allows the modulation of the biological properties of proteins. Moreover, it underlines the unique IFN-ß-1a properties that differentiate this molecule from other members of the type I interferon family.