C-terminal lysine clipping of IgG1: impact on binding to human FcγRIIIa and neonatal Fc receptors.

Monoclonal antibodies (mAbs) display numerous structural attributes, some of them may impact their safety and/or efficacy profiles. C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants. If Fc-glycosylation has been by far the most documented critical quality attribute (CQA), the potential impacts of mAb C-terminal lysine content is far less reported, particularly on the ability of these basic variants to bind human Fc receptors. To address this question, three charge variant species having zero (K0), one (K1) and two (K2) C-terminal lysine(s) were isolated with high purity from an in-house human IgG1 by preparative strong-cation exchange (SCX) chromatography. A comprehensive biophysical characterization of these three fractions was undertaken, demonstrating their high similarity in terms of structural homogeneity, with a particular attention paid on their respective N-glycosylation profiles. The binding affinity of the fractions to human FcγRIIIa-Val176 was assessed both by affinity chromatography and surface plasmon resonance (SPR), and to human neonatal Fc receptor (FcRn) by affinity chromatography. Results demonstrate that the three charge variants did not show any significant binding difference for the two tested human Fc receptors, translating certainly to comparable biological properties. As a consequence, C-terminal lysine clipping of the present therapeutic IgG1 should not impact both FcRn-dependent pharmacokinetic profiles and FcγRIIIa-driven cytotoxic activities. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA.

Aggregates Dramatically Alter Fibrin Ultrastructure.

Among the many factors influencing fibrin formation and structure (concentration, temperature, composition, pH, etc.), it has been suggested that the polydispersity of fibrinogen may play an important role. We propose here a detailed investigation of the influence of this parameter on fibrin multiscale structure. Two commercial fibrinogen preparations were used, a monodisperse and a polydisperse one. First, the respective compositions of both fibrinogen preparations were thoroughly determined by measuring the fibrin-stabilizing factor; fibronectin; α, ß, and γ intact chain contents; the γ/γ' chains ratio; the N-glycosylation; and the post-translational modifications. Slight variations between the composition of the two fibrinogen preparations were found that are much smaller than the compositional variations necessary to alter significantly fibrin multiscale structure as observed in the literature. Conversely, multiangle laser light scattering-coupled size exclusion chromatography and dynamic light scattering measurements showed that the polydisperse preparation contains significant amounts of aggregates, whereas the other preparation is essentially monodisperse. The multiscale structure of the fibrins produced from those two fibrinogen preparations was determined by using x-ray scattering, spectrophotometry, and confocal microscopy. Results show that fibers made from the aggregate-free fibrinogen present a crystalline longitudinal and lateral structure and form a mikado-like network. The network produced from the aggregates containing fibrinogen looks to be partly built around bright spots that are attributed to the aggregate. The multiscale structure of mixtures between the two preparations shows a smooth evolution, demonstrating that the quantity of aggregates is a major determining factor for fibrin multiscale structure. Indeed, the effect of a few percent in the mass of aggregates is larger than any other effect because of compositional differences under the same reaction conditions. Finally, we propose a mechanistic interpretation of our results, which points at a direct role of the aggregates during polymerization, which disrupts the ideal ordering of monomers inside fibrin protofibrils and fibers.

Human serum albumin presents isoform variants with altered neonatal Fc receptor interactions.

Human serum albumin (HSA) is the most abundant protein in plasma and presents the particularity, with IgG, to have an extraordinary long serum half-life conferred by its interaction with the neonatal Fc receptor (FcRn). If the impact of IgG post-translational modifications (PTMs) on FcRn binding is well documented, it is far less reported for HSA despite numerous PTMs occurring on the protein in plasma. HSA is susceptible to numerous degradation reactions in plasma, because of aging, oxidative stress or liver and pancreas related pathologies. In the present study, we combined FcRn affinity chromatography and mass spectrometry to investigate the impact of HSA PTMs upon FcRn binding. This methodology presents the advantage to distinguish the effect of a single modification from a plasma HSA preparation made of a mixture of different isoforms. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation, which are modifications related to several pathological conditions, were demonstrated to act negatively on HSA-FcRn interaction. The HSA-FcRn binding alteration generated by these modifications is consistent with their vicinity with the interaction interface of the two proteins. Results were discussed regarding altered half-life of HSA observed in several disease states and pave the way toward new understandings of the hypoalbuminemia pathogenesis. SIGNIFICANCE STATEMENT: In this study, we investigated the impact of several post-translational modifications of HSA toward its ability to bind to the neonatal Fc receptor using in vitro affinity chromatography, mass spectrometry, and surface plasmon resonance. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation were demonstrated to decrease HSA-FcRn binding. These modifications occurring in circulating HSA were discussed in relation to several pathologies as well as for the use of HSA as a therapeutic protein.

Quantification of proteins by data independent acquisition: Performance assessment of the Hi3 methodology.

Proteomics greatly benefited from the development of mass spectrometry. Over the last years, data-independent acquisitions increased in popularity in an effort to provide routine label free quantitative information. In this report, the performance of the Hi3 label free method was assessed based on the analysis of a plasma-derived protein mixture. The following parameters of the method (CVs) were determined: repeatability 13.8%, intermediate precision 27.6%, bias 32.3% and linearity observed over 3 orders of magnitude. Finally an accuracy of 42.5% corresponding to a confidence interval within 2 fold the expected protein abundance should be a good approximation of the method performance.

Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls.

Despite significant analytical improvements during this last decade, characterizing the whole integrity of monoclonal antibodies during their bioproduction remains a challenge. In this study, we report a new analytical approach to evaluate the overall heterogeneity/integrity of mAbs by LC-MS after combined proteolysis at their lower- and upper-hinge sites using the immunoglobulin-degrading enzymes IdeS and IgdE respectively. The whole sample preparation did not use any harsh conditions such as low pH, high temperature or reductive conditions and enables the splitting of mAbs structure into three fragments, namely the hinge dimer, Fab and Fc/2. Using the NIST mAb reference material, this method was demonstrated to be particularly suited for the analysis of mAbs disulfide bridges. The three fragments as well as their corresponding free sulfhydryl forms were well separated by chromatography and identified online by mass spectrometry. The method was then successfully applied to several mAbs of variable hydrophobicities.

DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.

Mass spectrometry based analysis of human plasma-derived factor X revealed novel post-translational modifications.

Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by-passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post-translational modifications of human factor X such as γ-carboxylation/ß-hydroxylation of the N-terminal light chain and N-/O-glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post-translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O-glucosylated on its light chain at Ser106 position, Ser9 of its activation peptide is phosphorylated at about 30% and its C-terminal heavy chain is fully O-glycosylated at Thr249 by a mucin-type O-glycan (HexNAc-Hex-NeuAc). The knowledge of these post-translational modifications is mandatory for the development of recombinant molecules.

Site-specific N-glycosylation analysis of human factor XI: Identification of a noncanonical NXC glycosite.

Human factor XI (hFXI) is a 160-kDa disulphide-linked homodimer zymogen involved in the coagulation cascade. Its deficiency results in bleeding diathesis referred to as hemophilia C. hFXI bears five N-glycosylation consensus sites per monomer, N72 , N108 , N335 on the heavy chain and N432 , N473 on the light chain. This study reports the first in-depth glycosylation analysis of hFXI based on advanced MS approaches. Hydrophilic interaction LC and MS characterization and quantification of the N-glycans showed that the two major forms are complex biantennary mono-α2,6-sialylated (A2 S1 , 20%) and bis-α2,6-sialylated structures (A2 S2 , 66%). Minor triantennary structures (A3 S3 F, ∼1.5%; A3 S3 , ∼2%) were also identified. MS analyses of intact hFXI revealed full occupation of two of the three heavy-chain glycosites and almost full-site occupancy of the light chain. Analysis of hFXI glycopeptides by LC-MS/MS enabled site-specific glycan profiling and occupancy. It was evidenced that N335 was not glycosylated and that N72 and N108 were fully occupied, whereas N432 and N473 were occupied at about 92 and 95%, respectively. We also identified a new glycosite of the noncanonical format NXC at N145 , occupied at around 5%. These data provide valuable structural information useful to understand the potential roles of N-glycosylation on hFXI function and could serve as a structural reference.

LC-MS analysis of polyclonal IgGs using IdeS enzymatic proteolysis for oxidation monitoring.

Susceptibility of IgGs to oxidation is a significant issue for intravenous immunoglobulin preparations (IVIG) in liquid solution and raises both safety and efficacy concerns. Here we present an optimized chromatography method coupled to mass spectrometry (MS) to determine the oxidation of Fc/2 fragments derived from polyclonal IgGs after IdeS treatment. Separation of the four IgG subclasses was achieved using a diphenyl column and UV/MS detections were used for quantification and characterization. Several oxidized Fc/2 fragments generated by stress conditions were resolved and oxidized methionines were identified. This procedure can be used to monitor the oxidative status of IVIG preparations during formulation or stability studies.

N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits.

Human coagulation factor VIIa is a glycoprotein that promotes haemostasis through activation of the coagulation cascade extrinsic pathway. Most haemophilia A/B patients with inhibitors are treated by injection of plasma-derived or recombinant FVIIa. The use of recombinant products raises questions about the ability of the host cell to produce efficiently post-translationally modified proteins. Glycosylation is especially critical considering that it can modulate protein safety and efficacy. The present paper reports the N-/O-glycosylation pattern of a new recombinant human factor VIIa expressed in the mammary glands of transgenic rabbits. Glycosylation was investigated by chromatography and advanced mass spectrometry techniques for glycan identification and quantitation. Mass spectrometry (MS)/MS analyses were performed to confirm the glycan structures as well as the position and branching of specific monosaccharides or substituents. The two N-glycosylation sites were found to be fully occupied mostly by mono- and bi-sialylated biantennary complex-type structures, the major form being A(2)G(2)S(1). Some oligomannose/hybrid structures were retrieved in lower abundance, the major ones being GlcNAcα1,O-phosphorylated at the C6-position of a Man residue (Man-6-(GlcNAcα1,O-)phosphate motif) as commonly observed on lysosomal proteins. No immunogenic glycotopes such as Galili (Galα1,3Gal) and HD antigens (N-glycolylneuraminic acid (NeuGc)) were detected. Concerning O-glycosylation, the product exhibited O-fucose and O-glucose-(xylose)(0, 1, 2) motifs as expected. The N-glycosylation consistency was also investigated by varying production parameters such as the period of lactation, the number of consecutive lactations and rabbit generations. Results show that the transgenesis technology is suitable for the long-term production of rhFVIIa with a reproducible glycosylation pattern.

Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry.

We describe a fast and informative method to investigate the posttranslational modifications of monoclonal antibodies (MAbs). The MAb is first digested by a specific enzyme that cleaves heavy chains under the hinge domain. After reduction of disulfide bridges, three polypeptide chains of approximately 25 kDa are released and analyzed by liquid chromatography-mass spectrometry (LC-MS). By bisecting the heavy chains prior to MS analysis, this method provides a better MS resolution and facilitates the study of the N-linked glycans as well as of other modifications (loss of C-terminal lysine, pyroglutamination, and oxidation). The sample preparation and analysis can be performed within few hours.

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII.

Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145). Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.

Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16.

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcgamma receptor IIIA (FcgammaRIIIA)/CD16 binding and FcgammaRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcgammaRIIIA-mediated interleukin-2 production by FcgammaRIIIA(+) Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.

Site-specific N-glycan characterization of human complement factor H.

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.

Selection of a human anti-RhD monoclonal antibody for therapeutic use: impact of IgG glycosylation on activating and inhibitory Fc gamma R functions.

The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety concerns. Since it has been suggested that FcgammaR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcgammaR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcgammaRIII and inhibitory FcgammaRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD+ RBCs and a potent FcgammaRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.