Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

Motivation Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii . SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae . Availability The source code is freely available on GitHub ( https://github.com/SugarPy/SugarPy ), and its implementation in Python ensures support for all operating systems. Contact mhippler@uni-muenster.de and pohlschr@uni-muenster.de Supplementary information Supplementary data are available online.

Ulvans from green algae are promising compounds for plant protection because they are environmentally friendly and induce plant defense responses. We analyzed the structure-function relationship of ulvan polymers and oligomers for their elicitor activity in suspension-cultured cells of three dicot species. The polysaccharide from Ulva fasciata was characterized regarding its monosaccharide composition, degree of sulfation, and molecular mass. The polymer was partially depolymerized using acid hydrolysis, and the oligomers were separated using size exclusion chromatography. The oligomeric fractions were analyzed revealing mostly sulfated and de-sulfated ulvan dimers. Both the polymer and the oligomer fractions induced an NADPH oxidase-dependent oxidative burst in plant cells. The elicitor activity of the ulvan dimers did not require sulfation. By identifying the smallest elicitor-active unit, HexA-Rha, we took an important next step to understand how the structure influences ulvan elicitor responses. The desulfated ulvan dimer is discussed as a promising agro-biologic for sustainable agriculture.

Nachrichten aus der ChemieVolume 67, Issue 7-8 p. 85-85 Service Rezension: Massenspektrometrie -- guter Einstieg Michael Mormann, Michael MormannSearch for more papers by this author Michael Mormann, Michael MormannSearch for more papers by this author First published: 01 July 2019 https://doi.org/10.1002/nadc.20194089138Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume67, Issue7-8Juli ‐ August 2019Pages 85-85 RelatedInformation

Hot-water soluble polysaccharides H-1-3 and H-2-1 were isolated from the thalli of the lichen Xanthoria parietina (L.) Th. Fr. and purified by ion exchange and gel permeation chromatography. Structure elucidation was mainly based on 2D-NMR and nano-ESI-Q-TOF MS/MS experiments. H-1-3 (13.7 kDa) was shown to be linear α-glucan with α-d-Glcp-(1 → [→[4)-α-d-Glcp-(1]2 → [6)-α-d-Glcp-(1]3 → 4)]n core backbone. The (1,4)- and (1,6)-α-d-Glcp linkages were in a 2:3 M ratio. H-2-1 (525 kDa) was characterized as a complex branched β-galacto-α-mannan with →[6)-α-d-Manp-(1 → [2,6)-α-d-Manp-(1]2 → [2)-α-d-Manp-(1]2→]n core units and main side chains of (1,3)-β-d-Galf linked at O-6 to →2)-α-d-Manp-(1→, together with minor terminal units of 1,4/1,6-α-D -Glcp units attached to the core chain at O-6 position and α-L-Rhap linked to Galf side chain at O-2 position (Manp: Galf: Glcp: Rhap linkage ratio = 9:3:2:1). H-2-1 exerted strong immunoactivity in vitro and activated murine RAW macrophages 264.7 towards significantly increased phagocytosis, TNF-α and IL-1β secretion. These effects are due to an interaction of the galactomannan with the transmembrane pattern-recognition protein Dectin-2 of the macrophages.

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra ( Abelmoschus esculentus ) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains ~39% β-sheets but only ~5% α-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at ~70°C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor . An intramolecular disulfide linkage between Cys 149 and Cys 156 was also detected. The protein showed ~24 and ~25% sequence identity with α-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys 149 –Cys 156 disulfide bond as detected by MS and a second disulfide bond connecting Cys 44 –Cys 91 , conserved in all Kunitz-type TIs, is also identified in the model.

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards β-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble β-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.

Lichenan (molecular weight 275 kDa, beta-D-1,3/1,4-glucopyranose ratio 1:3) from Cetraria islandica at a concentration of 100 mu g/mL induced terminal cellular differentiation of primary human keratinocytes as demonstrated by immunofluorescence staining using cytokeratin 10 and involucrin as marker proteins. Lichenan-derived oligosaccharides (DP3 to 8), obtained by acid-catalyzed partial hydrolysis of the polymer, did not influence cellular differentiation. Cytokeratin, filaggrin, involucrin, loricrin and transglutaminase gene expression as typical differentiation markers was increased by lichenan in a time-dependent manner. Lichenan upregulated gene cluster which were mostly related to cellular differentiation with focus on MAPK signaling as was shown by Whole Human Genome Microarray. These gene expression data from the array experiments were subsequently confirmed by qPCR for selected genes. For identification of the molecular binding structures of lichenan 1- and 2-D PAGE of keratinocyte protein membrane preparations was performed, followed by blotting with FITC-labeled lichenan and subsequent mass spectrometric identification of the pinpointed proteins. Epidermal growth factor receptor (EGFR) and integrin beta 4, both proteins being strongly involved in induction of keratinocyte differentiation were identified. In addition, protein disulfide isomerase A3 (PDIA3) showed strong binding to FITC-lichenan, indicating this enzyme to be an intracellular target of the glucan for induction of the cellular differentiation of keratinocytes. As lichenan did not influence the EGFR phosphorylation and the phosphorylation of CREB transcription factor but strongly interacted with cytosolic proteins it is hypothized that the glucan may interact with EGFR and is subsequently internalized into the cell via endosomal uptake, interacting with PDIA3, which again alters TGF beta 1 signaling towards keratinocyte differentiation.

PP2-like chitin binding phloem exudate lectins, abundant in the sieve tube of cucurbits, have been implicated to play key roles in wound sealing and antipathogenic responses of the plant. Here we report the affinity purification, macromolecular characterization and carbohydrate binding properties of a new chitooligosaccharide-specific lectin from the phloem exudate of ivy gourds (Coccinia indica). The protein, CIA24, has a subunit mass of 24 kDa. Partial sequence analysis indicated that CIA24 exhibits high homology with CIA17 and other Cucurbitaceae PP2 proteins whereas CD spectroscopic studies suggested that β-sheets constitute the predominant secondary structure. Temperature dependent CD spectroscopic and differential scanning calorimetric studies revealed that CIA24 is a highly thermostable protein, which undergoes complete unfolding at ∼105 °C. Isothermal titration calorimetric studies suggested that binding of chitooligosaccharides to CIA24 is a highly exothermic process. The lectin combining site can accommodate upto a tetrasaccharide with the binding stoichiometry (n) close to unity with respect to each protein subunit, whereas for chitohexaose a sharp decrease in the binding stoichiometry (n) to ∼1:0.5 was observed. This suggests that the protein probably undergoes dimerisation in presence of chitohexaose, wherein two protein molecules bind to the oligosaccharides from the reducing and non-reducing end, respectively.

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.

Phloem protein-2 (PP2) is an abundant soluble protein in the sieve elements in plants. Its lectin property was reported in various species. The primary structure of a 17 kDa PP2 from Coccinia indica (Coccinia indica agglutinin, CIA17), determined by mass spectrometry, shows extensive homology with PP2 super family phloem lectins. Analysis of mass spectrometric data indicated the presence of 16 potential allelic variants of CIA17 with insignificant divergence in the primary structure. The primary structure contains an intramolecular disulfide bridge between Cys-34 and Cys-51, which is conserved across various cucurbit species and hence likely to be important for carbohydrate binding. CD spectroscopic studies revealed that CIA17 is rich in antiparallel β-sheets, similar to PP2 proteins from Cucurbita maxima and Arabidopsis thaliana. CD spectra recorded at various temperatures showed very little change in the spectral intensity and shape up to 90 °C, suggesting that CIA17 is a highly thermostable protein. Atomic force microscopic studies revealed that CIA17 forms filamentous structures at higher concentrations. In light of these results, we propose that CIA17 and other PP2 proteins play a role in the plant defense against pathogens by directly binding with the chitin cell wall, and also promote wound healing by forming self-assembled filaments.

Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing submicrogram amounts of paCOS using quantitative mass spectrometry, allowing one to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an 18O-tag; (iii) quantifying paCOS using [13C2, 2H3]-labeled isotopologs as internal standards; (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

An α-D-galactose specific lectin belonging to the family of jacalin-related lectins (JRL) has been purified by affinity chromatography on cross-linked guar-gum. Mass spectrometric data revealed that the protein harbors two chains like all the members of galactose-specific jacalin-related lectins (gJRL). De novo sequencing of proteolytic peptides demonstrated that the heavier chain consists of 133 amino acids and the lighter chain comprises of 21 or 24 amino acids. The heavier chain contains one N-glycosylation site (Asn47) occupied with either pauci-mannose type [GlcNAc2(Fuc)Man3(Xyl)] or complex type [GlcNAc2(Fuc)Man3(Xyl)GlcNAc(Fuc)Gal] N-glycans. Circular dichroism spectroscopy indicated that the secondary structure of the lectin is predominantly made up of β-sheets, and differential scanning calorimetry revealed a thermal denaturation temperature of 77.6 °C. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assays on MCF-7 and MDCK cells showed that the lectin is highly cytotoxic towards both cell lines when dosed at micromolar concentrations, suggesting that it may play a role in the defense mechanism of the plant.

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.

The cytokine secretion of primary cells of human macrophages during the interaction of TiO2 nanoparticles (with an average primary size of 100-120 nm) is investigated down to concentration levels suggested to be relevant for in vivo conditions. We find that high TiO2 concentrations induce the cytokines Interleukin IL-1 beta, IL-6, and IL-10 secretion, while at low concentrations only IL-6 secretion is observed. To obtain further evidence on in vivo conditions we investigated the development and structure of the protein corona of the nanoparticles. We demonstrated that the surface of TiO2 particles attract preferably secondary modified proteins which then induce cytokine secretion of macrophages. Our results indicate that concentration of corona covered TiO2 particles below 1 mu g/ml induce IL-6 secretion which is reported to be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins.From the Clinical Editor: This study investigates the effects of protein corona covered titanium dioxide nanoparticles on human macrophages, concluding that concentration of such particles below 1 mu g/ml induces IL-6 secretion, which may be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. This finding has important implications on future applications of such nanoparticles. (C) 2015 Elsevier Inc. All rights reserved.

Xyloglucan XG (molecular weight 462 kDa, 1,4-/1,4,6-(pGlc) linked backbone, side chains of 1-pXyl, 1,2-pXyl, 1-p-Gal) was isolated from the seeds of Tropaeolum majus. XG (100 μg/mL) induced terminal cellular differentiation of human keratinocytes, as demonstrated by immunofluorescence staining and Western blot using cytokeratin 10 and involucrin as marker proteins. Differentiation was also induced by XG-derived oligosaccharides (degree of polymerization 7-9). Quantitative real-time polymerase chain reaction (qPCR) revealed the induction of gene expression of typical differentiation markers (cytokeratin, filaggrin, involucrin, loricrin, transglutaminase) in a time-dependent manner. Whole human genome microarray indicated that most of upregulated genes were related to differentiation processes. Microarray findings on selected genes were subsequently confirmed by qPCR. For identification of the molecular target of xyloglucan PAGE of keratinocyte membrane preparations was performed, followed by blotting with fluorescein isothiocyanate-labeled XG. XG interacting proteins were characterized by MS. Peptide fragments of epidermal growth factor receptor (EGFR) and integrin β4 were identified. Subsequent phospho-kinase array indicated that phosphorylation of EGFR and transcription factor cAMP response element-binding protein (CREB) was decreased in the XG-treated cells. Thus, the XG-induced differentiation of keratinocytes is proposed to be mediated by the inhibition of the phosphorylation of EGFR, leading to a dimished CREB activation, which is essential for the activation of cellular differentiation.