Characterization of FcγRIa (CD64) as a ligand molecule for site-specific IgG1 capture: A side-by-side comparison with protein a

Fc γreceptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification

Optimized Methods for Analytical and Functional Comparison of Biosimilar mAb Drugs: A Case Study for Avastin, Mvasi, and Zirabev

Bevacizumab is a humanized therapeutic monoclonal antibody used to reduce angiogenesis, a hallmark of cancer, by binding to VEGF-A. Many pharmaceutical companies have developed biosimilars of Bevacizumab in the last decade. The official reports provided by the FDA and EMA summarize the analytical performance of biosimilars as compared to the originators without giving detailed analytical procedures. In the current study, several key methods were optimized and reported for analytical and functional comparison of bevacizumab originators (Avastin, Altuzan) and approved commercial biosimilars (Zirabev and Mvasi). This case study presents a comparative analysis of a set of biosimilars under optimized analytical conditions for the first time in the literature. The chemical structure of all products was analyzed at intact protein and peptide levels by high-resolution mass spectrometry; the major glycoforms and posttranslational modifications, including oxidation, deamidation, N-terminal PyroGlu addition, and C-terminal Lys clipping, were compared. The SPR technique was used to reveal antigen and some receptor binding kinetics of all products, and the ELISA technique was used for C1q binding affinity analysis. Finally, the inhibition performance of the samples was evaluated by an MTS-based proliferation assay in vitro. Major glycoforms were similar, with minor differences among the samples. Posttranslational modifications, except C-terminal Lys, were determined similarly, while unclipped Lys percentage was higher in Zirabev. The binding kinetics for VEGF, FcRn, FcγRIa, and C1q were similar or in the value range of originators. The anti-proliferative effect of Zirabev was slightly higher than the originators and Mvasi. The analysis of biosimilars under the same conditions could provide a new aspect to the literature in terms of the applied analytical techniques. Further studies in this field would be helpful to better understand the inter-comparability of the biosimilars

Investigation of the role of brain extracellular matrix in neurodegeneration BY using alzheimer's disease mouse model

Alzheimer hastalığında (AD), sinaptik yanıt mekanizmalarının ve sinaptik bağlantı stabilitesinin hasar gördüğü bilinmektedir. Son yıllarda yapılan çalışmalar, nöron ve glialar tarafından üretilen bazı ekstrasellüler matriks bileşenlerinin, bu iki mekanizma üzerinde etkili olduğunu göstermiştir. Çalışmanın amacı, Alzheimer's hastalığının başlangıç ve gelişim evrelerinde, hastalık modeli olan 5XFAD farelerden elde edilen hipokampal ekstrasellüler matriks örneklerinde proteom profilini analiz ederek, nörodejenereasyon oluşumu ile ilgili bu evrelere ait protein seviyesindeki değişimleri ve posttranslasyonel modifikasyon değişimlerini tanımlamaktır. Bunlara ek olarak, sağlıklı ve Alzheimer's hastası bireylerden alınan beyin-omurilik sıvıları ve post mortem beyin dokusu örneklerinin proteom analizi yapılarak, hastalığın gelişiminde rol alan temel yolakları belirlemektir. Fareler 3 (3m), 6 (6m) ve 12 (12m) aylık iken, Morris Su Tankı deneyleri ile hafıza testlerini takiben, intraserebral mikrodiyaliz "push-pull" yöntemi ile hipokampal ekstrasellüler matriks örnekleri toplandı. Örneklerin protein ekspresyon profilleri nanoLC-MS/MS yöntemi kullanılarak analiz edildi. Kütle spektrometrisi ile elde edilen ham data, Progenesis QI yazılımı ile işlenerek protein tanımlamaları gerçekleştirildi. 3,6 ve 12 aylık ECM örneklerinde sırasıyla 251, 213 ve 238 protein tanımlandı. Bunlardan sırasıyla 68, 41 ve 33 protein istatistiksel olarak anlamlı (p<0.05) en az 1.4 kat ekspresyon değişikliği gösterdi. Beyin-omurilik sıvılarının analizinde 204ü anlamlı değişen 1041 protein, postmortem doku analizlerinde 311i anlamlı değişen 1737 protein tanımlandı. Anlamlı değişiklik gösteren proteinler, Ingenuity Pathway Analysis (IPA) yazılımı ile analiz edilerek ilgili yolaklar tespit edildi.In Alzheimer's disease (AD), the disruption of synaptic response and stability is known. In recent years, the studies showed that extracellular matrix produced by neurons and glial cells has several effects on those disruptions. The aim of this study is identifying the alterations in expression of extracellular matrix proteins and their posttranslational modifications in the early and progression stage of the disease by obtaining extracellular matrix from 5XFAD Alzheimer's disease mouse model via intracerebral push-pull perfusion. In addition, it has been aimed to identify the pathways related with the disease by studying on cerebro-spinal fluids (CSF) and postmortem brain tissues of healthy and AD subjects. Following the Morris Water Maze memory tests, the hippocampal ECM samples from each mice were collected by in-vivo intracerebral push-pull method at month 3 (3m), 6 (6m) and 12 (12m). The protein expression analysis were performed by nanoLC-MS/MS and ProgenesisQI were used for protein identification. In the extracellular matrix samples belong to 3, 6 and 12-month-old mouse, 251, 213 and 238 proteins were identified, respectively and 68, 41 and 33 proteins were statistically meaningful (p<0.05) and minimum 1.4-fold change. Additionally, 1041 proteins were identified in CSF samples and 204 of them were meaningful. 1737 proteins were identified in post mortem brain tissue samples and 311 of them were meaningful. These proteins were analyzed by Ingenuity Pathway Analysis (IPA) software and several pathways were determined

Mahmut Aydın, Hz. İsa’ya Ne Oldu?, (Otto, Ankara, 2017, 191 sayfa. )

Mahmut Aydın, tanıtmaya çalıştığımız eserinde genel olarak Hz. İsa'nın başına gelen olaylar silsilesini, özelde ise çarmıh hadisesi ve sonrasını ele almaktadır. Tarihi kaynaklarda, Hristiyan dini kaynaklarda ve Kur'an-ı Kerim'de verilen bilgileri okuyucuya sunarak, kulaktan dolma bilgilerle oluşan İsa portresini Kur'an ışığında değerlendirerek yeniden çizmeyi hedeflemektedir

Hz. İsa'ya Ne Oldu?

Mahmut Aydın, tanıtmaya çalıştığımız eserinde genel olarak Hz. İsa'nın başına gelen olaylar silsilesini, özelde ise çarmıh hadisesi ve sonrasını ele almaktadır. Tarihi kaynaklarda, Hristiyan dini kaynaklarda ve Kur'an-ı Kerim'de verilen bilgileri okuyucuya sunarak, kulaktan dolma bilgilerle oluşan İsa portresini Kur'an ışığında değerlendirerek yeniden çizmeyi hedeflemektedir

12 Eylül Darbesi öncesinde ve sonrasında yaşanan siyasi gelişmeler ve bunların gündelik yaşama etkileri

Ankara : İhsan Doğramacı Bilkent Üniversitesi İktisadi, İdari ve Sosyal Bilimler Fakültesi, Tarih Bölümü, 2014.This work is a student project of the The Department of History, Faculty of Economics, Administrative and Social Sciences, İhsan Doğramacı Bilkent University.by Ünsal, Mehmet Süha

Analytical investigation of forced oxidized anti-VEGF IgG molecules: a focus on the alterations in antigen and receptor binding activities

Alterations in the biological activity of the molecules under stress conditions have not been documented as widely in the literature yet. This study was designed to reveal the functional impacts of various oxidation conditions on a model mAb, a commercial anti-VEGF IgG molecule. The responses to antigen binding, cell proliferation, FcRn receptors, and C1q binding, which rarely appear in the current literature, were investigated. The authors report peptide mapping data, post-translational modification (PTM) analysis, cell proliferation performance, and antigen (VEGF), C1q, and FcRn binding activities of the mAb under various stress conditions. The oxidation-prone site of the mAb was determined as Met252 in the DTLMISR peptide. The VEGF binding activity and anti-cell proliferation activity of the mAbs did not alter, while C1q and FcRn binding capacity significantly decreased under oxidative stress conditions. The full report is vital for many scientific and industrial processes about mAbs. The authors recommend performing functional analyses in addition to the structural studies while investigating the impacts of stress factors on therapeutic mAbs

Neonatal neurodegeneration in alzheimer's disease transgenic mouse model

Alzheimer's disease (AD) is a progressive disorder characterized by a variety of molecular pathologies causing cortical dementia with a prominent memory deficit. Formation of the pathology, which begins decades before the diagnosis of the disease, is highly correlated with the clinical symptoms. Several proteomics studies were performed using animal models to monitor the alterations of the brain tissue proteome at different stages of AD. However, proteome changes in the brain regions of newborn transgenic mouse model have not been investigated yet. To this end, we analyzed protein expression alterations in cortex, hippocampus and cerebellum of transgenic mice carrying five familial AD mutations (5XFAD) at neonatal day-1. Our results indicate a remarkable difference in protein expression profile of newborn 5XFAD brain with region specific variations. Additionally, the proteins, which show similar expression alteration pattern in postmortem human AD brains, were determined. Bioinformatics analysis showed that the molecular alterations were mostly related to the cell morphology, cellular assembly and organization, and neuroinflammation. Moreover, morphological analysis revealed that there is an increase in neurite number of 5XFAD mouse neurons in vitro. We suggest that, molecular alterations in the AD brain exist even at birth, and perhaps the disease is silenced until older ages when the brain becomes vulnerable

Characterization of biological molecule–loaded nanostructures using circular dichroism and fourier transform infrared spectroscopy

Drug-loaded nanoparticles have many advantages in drug administration, which is an essential step for the impact of the drugs and their mechanism of action. Circular dichroism (CD) is a spectroscopy technique that measures the absorbance difference between right-circularly polarized light and left-circularly polarized light. Of several analytical techniques available, Fourier transform infrared spectroscopy is a powerful and widely employed technique explicitly for identifying chemical species. The peaks in the IR spectrum of a sample represent the molecular vibrations of the molecules present in the sample, signifying the various chemical bonds and functional groups. Various types of nanoparticles are being utilized for drug delivery applications because of their advantages such as controlled drug release, protection of the therapeutic payload, improved bioavailability, and targeted delivery. The discovery of novel nanoparticles and their application to the diagnosis and treatment of diseases have received considerable attention during the past decades