A robust nanoscale RP HPLC-MS approach for sensitive Fc proteoform profiling of IgG allotypes

The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes.To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness.This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs.Pathophysiology and treatment of rheumatic disease

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh resolution MALDI FT-ICR mass spectrometry

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in formulation or in vivo after application of the drug. Glycation affects the structure, function and stability of monoclonal antibodies, and consequently, detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd’ regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS

Chemical Synthesis and Immunological Evaluation of Fragments of the Multiantennary Group-Specific Polysaccharide of Group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their different capsular polysaccharides. The Group B carbohydrate (GBC) is shared by all serotypes and therefore attractive be used in a glycoconjugate vaccine. The GBC is a highly complex multiantennary structure, composed of rhamnose rich oligosaccharides interspaced with glucitol phosphates. We here report the development of a convergent approach to assemble a pentamer, octamer, and tridecamer fragment of the termini of the antennae. Phosphoramidite chemistry was used to fuse the pentamer and octamer fragments to deliver the 13-mer GBC oligosaccharide. Nuclear magnetic resonance spectroscopy of the generated fragments confirmed the structures of the naturally occurring polysaccharide. The fragments were used to generate model glycoconjugate vaccine by coupling with CRM197. Immunization of mice delivered sera that was shown to be capable of recognizing different GBS strains. The antibodies raised using the 13-mer conjugate were shown to recognize the bacteria best and the serum raised against this GBC fragment-mediated opsonophagocytic killing best, but in a capsule dependent manner. Overall, the GBC 13-mer was identified to be a highly promising antigen for incorporation into future (multicomponent) anti-GBS vaccines.Bio-organic Synthesi

Электроснабжение установки перекачки нефти п. Пионерный ОАО «Томскнефть»

РЕФЕРАТ Выпускная квалификационная работа 149 с., 23 рис., 32 табл., 29 источников, 6 прил. Ключевые слова: нефтепровод, насос, электрооборудование, схема электроснабжения, линия, сеть, электроприемник, нагрузка, оборудование, защита, ток, напряжение, мощность. Объектом исследования является электрическая часть УПН п. Пионерный ОАО «Томскенефть». Цель работы – проектирование схемы электроснабжения предприятия, выбор оборудования. В процессе исследования проводился сбор исходных данных в ходе производственной практики на объекте исследования. В результате была спроектирована схема электроснабжения от подстанции энергосистемы, до конечного электроприемника. Были выбраны кабели и провода, коммутационное оборудование, были сделаны необходимые проверки. Также результатом работы сталESSAY Final qualifying work 149 p., 23 fig., 32 tab., 29 sources, 6 adj. Keywords: oil, pump, electrical equipment, power supply circuit, line, network, power-consuming equipment, load equipment, protection, current, voltage, power. The object of research is the electrical part of UPN claim. Pionerny of "Tomskeneft". The purpose of work - designing enterprise power scheme, the choice of equipment. The study was conducted to collect baseline data in the course of practical training on the subject of the study. As a result, power supply circuit has been designed from the substation grid, appliance, to the end. Were selected cables and wires, switching equipment, the necessary checks have been made. It is also the result of the work became an economic calculation of capital costs for the con

Serum-PGE2 als Indikator früher Tumorstadien bei Patienten mit Plattenepithelkarzinomen

Einleitung: Einige Studien konnten bisher zeigen, dass die Konzentration von PGE2 in humanem Serum von Tumorpatienten gegenüber der Konzentration bei gesunden Probanden erhöht ist. Ein klinischer Bezug zu Ausmaß und Verlauf des jeweiligen Erkrankungsfalles konnte jedoch bislang nicht hergestellt werden. Methoden: Wir analysierten die PGE2-Serumkonzentrationen von 100 Patienten mit Plattenepithel-karzinomen des Kopf-Hals-Bereiches unterschiedlicher T-Stadien vor und 1 Jahr nach Therapie und verglichen die Werte mit den Serumkonzentrationen gesunder Probanden. Die Ergebnisse wurden in Relation zum Sekretionsprofil von 8 verschiedenen plattenepithelialen Karzinomzellinien gestellt.Ergebnisse: Es zeigte sich eine signifikante Korrelation zwischen PGE2 Serumkonzentration und T-Stadium bei Diagnose. Die Serumkonzentrationen waren 12 Monate nach Therapie signifikant erniedrigt. Im Zellkultursystem zeigte sich eine Abnahme der PGE2-Sekretion mit steigender Zellzahl. Schlussfolgerungen: Die PGE2-Serumkonzentration ist ein Indikator für frühe Stadien plattenepithelialer Kopf-Hals-Karzinome und kann daher ein nützlicher Parameter in der Tumornachsorge sein

Ein neues Hörsystem für die elektroakustische Stimulation - Sprachergebnisse nach mehrmonatiger Nutzung

Patienten, die nach hörerhaltender Cochlea Implantation die elektro-akustische Stimulation (EAS) nutzen, hatten bislang die Möglichkeit das CI alleine zu nutzen oder das Tieftonrestgehör akustisch durch zusätzliches Tragen eines Im-Ohr-Hörgerätes (IdO) zu verstärken.Das neue DUET® Hörsystem wurde speziell für EAS Patienten entwickelt und bietet nun die Kombination eines CI-Sprachprozessors mit den Vorteilen eines Hinter-dem-Ohr-Hörgerätes (HdO) in nur einem Gerät an.9 Patienten wechselten vom Tempo+® -Spachprozessor zum DUET® , wobei 6 von ihnen das IdO mittlerweile aus Bequemlichkeit oder medizinischen Gründen ablehnten.Vor und 2 Monate nach Umrüstung wurde das Sprachverstehen mittels Freiburger Einsilber-Testung und Hochmaier-Schulz-Moser (HSM)-Satztestungen in Ruhe und im Störschall (0dB, 5dB und 10dB Schall-Rausch-Abstand) überprüft.Zudem wurden subjektive Veränderungen mit dem "Abbreviated Profile of Hearing Aid Benefit" (APHAB) erfasst.Alle Patienten akzeptieren das neue Hörsystem 2 Monate nach dem Wechsel gut, obwohl die Auswertung des APHAB-Fragebogens keinen signifikanten subjektiven Unterschied nachweisen konnte.Die objektiven Testungen belegten ein signifikant besseres Sprachverstehen mit dem neuen Hörsystem im Vergleich zu Messungen mit dem alten Sprachprozessor.Die 3 Patienten, die bislang konsequent IdO und CI kombiniert haben, zeigen tendenziell ein besseres Verstehen mit dem neuen Gerät.Das System wird gut akzeptiert und ermöglicht es nun mehr Patienten als zuvor von der EAS zu profitieren.Unterstützt durch: MedEl Deutschland GmB