The quest for down scale representativeness: how to exploit CFD to design a shear study for vaccines.

In this work, we exploit computational fluid dynamics (CFD) to evaluate stirred tank reactor (STR) process engineer parameters (PEP) and design a scale-down system (SDS) to be representative of the formulation and filling process steps for an Aluminum adjuvanted vaccine drug product (DP). To study the shear history in the SDS we used the concept of number of passages, combined with an appropriate stirring speed down scale strategy comprising of either (i) tip speed equivalence, widely used as a scale-up criterion for a shear-sensitive product, or (ii) rotating shear, a shear metric introduced by Metz and Otto in 1957 but never used as scaling criterion. The outcome of the CFD simulations shows that the tip equivalence generates a worst-case SDS in terms of shear, whereas the rotating shear scaling approach could be used to design a more representative SDS. We monitored the trend over time for "In Vitro Relative Potency" as DP Critical Quality Attribute for both scaling approaches, which highlighted the crucial role of choosing the appropriate scaling-down approach to be representative of the manufacturing scale during process characterization studies.

Biological Assay to Determine Gonadotropin Potency: From In Vivo to In Vitro Sustainable Method.

Various preparations of follicle-stimulating hormone (FSH) are commercially available; however, they differ in glycoforms composition and purity owing to their respective sources. Additional chemical/physical changes can also be introduced during manufacturing and can impact their biological activity (biopotency), which is routinely assessed using an in vivo bioassay (Steelman-Pohley). This study aimed to determine whether an in vitro bioassay could assess biopotency by distinguishing between r-hFSH chemical/physical variants with similar ability to the in vivo bioassay. The specific activity (units of biological activity per mg of product) of variants of r-hFSH generated through enrichment (acidic/basic), stress (oxidative/acidic pH) and enzymatic treatment (desialylation and desialylation/degalactosylation) was compared using the in vivo and in vitro bioassays. The in vitro bioassay reliably detected potential chemical/physical modifications in r-hFSH variants that may impact biopotency. Overall, the methods demonstrated a comparable ability to detect changes in specific activities due to chemical/physical differences in r-hFSH variants. These data indicate that the in vitro bioassay is suitable to replace the in vivo bioassay.

Comprehensive investigation of a structural variant in a bi-specific, N-and C-terminal Fc-fusion molecule, and its monitoring with LC-MS based method.

There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N- and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N- and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.

Comparative Assessment of the Structural Features of Originator Recombinant Human Follitropin Alfa Versus Recombinant Human Follitropin Alfa Biosimilar Preparations Approved in Non-European Regions.

Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed.

Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm.

Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.

LC-MS/MS-Based Quantification Method of Polyphenols for Valorization of Ancient Apple Cultivars from Cilento.

Safeguarding the biodiversity of plant species is of fundamental importance for their defense against pests and diseases even through the maintenance and dissemination of ancient agricultural traditions rooted within the small rural environments. The investigation area of the current research covered some municipalities belonging to the "Parco Nazionale del Cilento e Vallo di Diano" including the sub-mountainous part of "Comunità Montana del Vallo di Diano (Salerno, Campania)". Fifteen ancient apple varieties were collected from local communities to be analyzed and compared to some commercially available apples. To this aim, a Folin-Ciocâlteu assay was preliminarily used to measure the total polyphenol content in both ancient and commercial apple cultivars. Then, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis in the multiple reaction monitoring (MRM) ion mode was then implemented to detect and quantify specific polyphenols and to obtain a molecular comparison of a wide panel of polyphenols. The main finding of the present work pointed out that ancient apple cultivars are richer than commercial ones in anthocyanins, dihydrochalcones, and chlorogenic acid, whose beneficial effects on health are widely known. Thus, the safeguarding of these ancient varieties is greatly encouraged for the richness of polyphenols crucial both for the defense of plants from insects and for remarkable nutraceutical properties, in addition to the need for germplasm conservation as a source of genetic variability.

Characterization of disulfide bridges pattern of recombinant human interleukin 12 fusion protein p40 subunit and identification and quantification of cysteinylated free cysteine 252.

RATIONALE: We show evidence of cysteinylation on Cys252 of recombinant human p40 subunit of interleukin 12 (IL-12). This was reported in 1996. However, no paper detailing this concept has been published yet. Our paper reports the quantification of Cys252 cysteinylation as well as the full disulfide bridges assignment by nonreducing peptide mapping using mass spectrometry (MS) detection. METHODS: Nonreducing peptide mapping was applied for disulfide bridges assignment. This study presents an ad hoc method in which applying a neutral pH in the presence of an alkylating agent allowed to mitigate the formation of artifacts such as reshuffled disulfide bridges and permitted the detection of free cysteine. Ultra-high-performance liquid chromatography-MS analysis was performed on a Waters quadrupole time-of-flight Xevo G2-XS mass spectrometer acquiring data in MSE mode. MS data were processed using Expressionist MS Refiner 13.5 (Genedata). RESULTS: Scouting experiments were performed using two batches of drug substance. An in-depth study of the LC tandem mass spectrometry profiles revealed the presence of additional species related to "free" Cys252; this cysteine residue was also detected in its S-cysteinylated and S-homocysteinylated forms. This result is consistent with that reported in literature so far. The relative abundance of overall "cysteinylated" species resulted in the range between 46% and 36%, which has also been confirmed using orthogonal techniques such as Ellman's assay. CONCLUSIONS: Our data clearly demonstrate that the free cysteine (Cys252) on the p40 subunit of recombinant IL-12 is also present in its cysteinylated and homocysteinylated forms at a considerable rate. Our observations, although based on results obtained on an IL-12-derived fusion protein, are consistent with the current literature.

Impact of four inorganic impurities - iron, copper, nickel and zinc - on the quality attributes of a Fc-fusion protein upon incubation at different temperatures.

A key aspect that must be supervised during the development of a biotherapeutic is the presence of elemental impurities in the final drug product: they must be quantified as to ensure that their concentrations does not affect patients' safety. Regulatory guidelines such as ICH Q3D provides Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes (QA) of a biologic may also take place. In order to understand how certain impurities could affect not only the safety of patients, but also the physicochemical properties of biotherapeutics, here we present a study in which we examined how four commonly observed elemental impurities could impact the QAs of a Fc-fusion protein, under normal storage conditions and after six weeks of incubation at +25 °C and +40 °C. The molecule was indeed treated with increasing concentrations of Ni2+, Cu2+, Zn2+ and Fe3+ and the potential changes in conformation, oxidation, aggregation, and fragmentation were monitored. Our data suggest that keeping the levels of these impurities under the safety threshold limits does not guarantee the product quality. While nickel and zinc slightly altered the physicochemical properties of our Fc-fusion protein, iron and copper appeared to be more harmful for the QAs stability. Indeed, these latter elements might cause significant alterations of the product quality such as to potentially alter its efficacy.

Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars.

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10-3-1 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and ß-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and ß-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9-24 ± 1.7 ng/ml; ß-arrestin 2 EC50 range = 140 ± 14.1-313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 µg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

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.

Generation of site-distinct N-glycan variants for in vitro bioactivity testing.

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

Demonstration of physicochemical and functional similarity between the proposed biosimilar adalimumab MSB11022 and Humira®.

Biosimilars are biological products that are highly similar to existing products approved by health authorities. Demonstration of similarity starts with the comprehensive analysis of the reference product and its proposed biosimilar at the physicochemical and functional levels. Here, we report the results of a comparative analysis of a proposed biosimilar adalimumab MSB11022 and its reference product, Humira®. Three batches of MSB11022 and up to 23 batches of Humira® were analyzed by a set of state-of-the-art orthogonal methods. Primary and higher order structure analysis included N/C-terminal modifications, molecular weight of heavy and light chains, C-terminal lysine truncation, disulfide bridges, secondary and tertiary structures, and thermal stability. Purity ranged from 98.4%-98.8% for MSB11022 batches (N = 3) and from 98.4%-99.6% for Humira® batches (N = 19). Isoform analysis showed 5 isoform clusters within the pI range of 7.94-9.14 and 100% glycan site occupancy for both MSB11022 and Humira®. Functional analysis included Fab-dependent inhibition of tumor necrosis factor (TNF)-induced cytotoxicity in L929-A9 cell line and affinity to soluble and transmembrane forms of TNF, as well as Fc-dependent binding to Fcγ and neonatal Fc receptors and C1q complement proteins. All tested physicochemical and functional parameters demonstrated high similarity of MSB11022 and Humira®, with lower variability between MSB11022 and Humira® batches compared with variability within individual batches of Humira®. Based on these results, MSB11022 is anticipated to have safety and efficacy comparable to those of Humira®.

Effective mutations in a high redox potential laccase from Pleurotus ostreatus.

Since the first report on a laccase, there has been a notable development in the interest towards this class of enzymes, highlighted from the number of scientific papers and patents about them. At the same time, interest in exploiting laccases-mainly high redox potential-for various functions has been growing exponentially over the last 10 years. Despite decades of work, the molecular determinants of the redox potential are far to be fully understood. For this reason, interest in tuning laccase redox potential to provide more efficient catalysts has been growing since the last years. The work herein described takes advantage of the filamentous fungus Aspergillus niger as host for the heterologous production of the high redox potential laccase POXA1b from Pleurotus ostreatus and of one of its in vitro selected variants (1H6C). The system herein developed allowed to obtain a production level of 35,000 U/L (583.3 µkat/L) for POXA1b and 60,000 U/L (1,000 µkat/L) for 1H6C, corresponding to 13 and 20 mg/L for POXA1b and 1H6C, respectively. The characterised proteins exhibit very similar characteristics, with some exceptions regarding catalytic behaviour, stability and spectro-electrochemical properties. Remarkably, the 1H6C variant shows a higher redox potential with respect to POXA1b. Furthermore, the spectro-electrochemical results obtained for 1H6C make it tempting to claim that we spectro-electrochemically determined the redox potential of the 1H6C T2 site, which has not been studied in any detail by spectro-electrochemistry yet.

Subtype-selective activation of K(v)7 channels by AaTXKß2₋64, a novel toxin variant from the Androctonus australis scorpion venom.

K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKß2₋64, a novel variant of AaTXKß1₋64, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKß2₋64, but not AaTXKß1₋64, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKß2₋64 also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKß2₋64-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels.

Privileged incorporation of selenium as selenocysteine in Lactobacillus reuteri proteins demonstrated by selenium-specific imaging and proteomics.

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS ((78)Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Novel method to investigate protein carbonylation by iTRAQ strategy.

This paper reports a novel methodology for relative quantitative analysis of carbonylation sites in proteins by exploiting a new isobaric tag for relative and absolute quantitation (iTRAQ) derivative, iTRAQ hydrazide (iTRAQH), and the analytical power of linear ion trap instruments (QqLIT). Because of its operational simplicity, avoiding time-consuming enrichment procedures, this new strategy seems to be well suited for quantitative large-scale proteomic profiling of carbonylation.

A family GH51 α-L-arabinofuranosidase from Pleurotus ostreatus: identification, recombinant expression and characterization.

An α-L-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-L-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L(-1)) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-L-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-L-arabinofuranoside it follows Michaelis-Menten kinetics with a K (M) of 0.64 mM and a k (cat) of 3,010 min(-1). The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-L-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.

Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation.

Carbonylation is a non-enzymatic irreversible post-translational modification. The adduction of carbonyl groups to proteins is due to the presence of excess of ROS in cells. Carbonylation of specific amino acid side chains is one of the most abundant consequences of oxidative stress; therefore, the determination of carbonyl groups content in proteins is regarded as a reliable way to estimate the cellular damage caused by oxidative stress. This paper reports a novel RIGhT (Reporter Ion Generating Tag) (A. Amoresano, G. Monti, C. Cirulli, G. Marino. Rapid Commun. Mass Spectrom. 2006, 20, 1400) approach for selective labeling of carbonyl groups in proteins using dansylhydrazide, coupled with selective analysis by bidimensional mass spectrometry. We first applied this approach to ribonuclease A and lysozyme as model proteins. According to the so-called 'gel-free procedures', the analysis is carried out at the level of peptides following tryptic digest of the whole protein mixture. Modified RNaseA was analyzed in combined MS(2) and MS(3) scan mode, to specifically select the dansylated species taking advantage of the dansyl-specific fragmentation pathways. This combination allowed us to obtain a significant increase in signal/noise ratio and a significant increase in sensitivity of analysis, due to the reduction of duty cycle of the mass spectrometer. The unique signal obtained was correlated to peptide 1-10 of RNaseA carbonylated and labeled by dansylhydrazide. This strategy represents the first method leading to the direct identification of the carbonylation sites in proteins, thus indicating the feasibility of this strategy to investigate protein carbonylation in a proteomic approach.

Plasma nitroproteome of kidney disease patients.

3'-Nitrotyrosine (3NT) is a post-translational modification (PTM) of body fluids and tissues that is sustained by chronic inflammation and oxidative stress, two main clinical traits of chronic kidney disease (CKD). Despite this background, protein targets and their differential susceptibility to in vivo nitration remain almost completely unexplored in CKD. This study reports a first investigation of plasma nitroproteome in these patients, carried out by both immunorecognition and LC-MS/MS techniques. Plasma proteins of chronic and end-stage KD patients showed a higher burden of nitration than in healthy controls, but main nitration targets appeared to be the same in these populations. Immunoblotting data showed that uremic albumin is largely represented in the uremic nitroproteome together with fibrinogen chains (A, B and C), transferrin, α1-antitrypsin, complement factor D, haptoglobin, and IgG light and heavy chains. However, immunopurification and affinity chromatography experiments demonstrated that the relative content of 3NT on the albumin molecule was very low when compared with that of the remaining plasma proteins. The uremic nitroproteome was investigated using also plasma proteins obtained by in vivo ultrafiltration from patients treated with protein leaking or standard high-flux hemodialyzers. The study of these samples revealed the possibility to selectively remove protein nitration products during hemodialysis. Identification of intramolecular sites of nitration was preliminarily obtained in IgG chains isolated by 2D PAGE and assessed by bidimensional tandem mass spectrometry after chemoselective tagging. Further studies are needed to confirm at the molecular level the presence of nitrated Tyr residues in other proteins tentatively identified as nitration targets in this study and to explore the biological meaning of such a selective modification of plasma proteins by reactive nitrogen species in uremia and dialysis patients.