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The effects of multivalent phosphate and sulphonate ions on protein aggregation for use in biopharmaceuticals
Get PDFV zadnjih desetletjih se je biofarmacevtska industrija osredotočila na razvoj bioloških zdravil. Večinoma gre za kompleksne beljakovinske molekule, ki so zahtevne in drage za izdelavo ter imajo omejen rok uporabe, ki je posledica nestabilnosti samih beljakovin. Na nestabilnost proteinov in agregacijo lahko vplivamo z dodatkom malih molekul in pomožnih snovi kot so površinsko aktivne snovi, pufri, sladkorji, soli itd., ki se na protein neposredno vežejo in ga tako stabilizirajo. Dosedanje raziskave so že preučile vpliv adenozin tripolifosfata in tripoli fosfata na agregacijo biofarmacevtskih proteinov, zato smo v diplomskem delu preučili še vpliv petih drugih že priznanih fosfatnih ionov in za primerjavo izbrali še deset ATP- ju podobnih sulfonatnih spojin, saj so polifosfati nagnjeni k hidrolizi in posledično niso primerni za uporabo kot pomožne snovi. Uporabili smo modelni protein lizocim kokošjega beljaka ter preverjali vpliv pomožnih snovi pri različnih koncentracijah na kinetiko agregacije proteina, ki smo jo spremljali z UV- Vis spektrometrijo. S pomočjo UV-Vis spektrometrije smo opravili tudi presejalni test pomožnih snovi na agregacijo proteina in izračunali delež monomernega proteina. Ugotovili smo, da fosfatne pomožne snovi in večina sulfonatnih učinkovito zaščitijo in stabilizirajo protein in ga tako zaščitijo pred agregacijo kot je ta podvržen temperaturnemu stresu ter ga zaščitijo pred agregacijo v daljšem časovnem obdobju, prav tako tudi zmanjšajo delež monomernega proteina v vzorcu. Preverili smo tudi vpliv pomožnih snovi na agregacijo delno razvitih beljakovin, kar smo dosegli z dodatkom ditiotreitola ali DTT, ki cepi disulfidne mostičke v beljakovini. V povprečju se je izkazalo, da dajejo boljše rezultate fosfatne pomožne snovi in ne sulfonatne. Najboljše rezultate sta med pomožnimi snovmi dala ATP in TPP, preostale pomožne snovi pa dale primerljive rezultate.In recent decades, the biopharmaceutical industry has focused on developing biological drugs. These are complex protein , nucleic acids or carbohydrate molecules. They are difficult and expensive to manufacture and have a limited shelf life, which is a result of the instability of the proteins themselves. Protein instability and aggregation can be influenced by adding small molecules and auxiliary substances, also called excipients,such as surfactants, buffers, sugars, salts, etc., which can directly interact with the protein and thus stabilize it. Previous research has already examined the influence of adenosine tripolyphosphate and tripolyphosphate on the aggregation of biopharmaceutical proteins. In the thesis, we also studied the influence of five other phosphate ions and selected ten ATP-like sulfonate compounds for comparison, since polyphosphates are prone to hydrolysis and consequently, they are not suitable for use as excipients. We used the model protein lysozyme of chicken egg white and checked the influence of excipients at different concentrations on the kinetics of protein aggregation, which was monitored by UV-Vis spectrometry. Utilizing UV-Vis spectrometry, we also performed a screening test of excipients against protein aggregation and calculated the fraction of monomeric protein. We have found that phosphate excipients and most sulfonate excipients effectively protect and stabilize the protein, shielding it from aggregation when subjected to temperature stress, and also prevent aggregation over a longer period of time. We also checked the influence of excipients on the aggregation of partially unfolded proteins, which was achieved by adding dithiothreitol or DTT, which cleaves disulfide bridges in the protein. On average, phosphate excipients have been shown to yield better results compared to sulfonate excipients. Among the excipients tested, ATP and TPP have shown the best results, while the other excipients yielded comparable outcomes
Comparison of different types of glass for in situ characterization of biological drugs
Get PDFBiološka zdravila na osnovi proteinov se soočajo s problematiko stabilnosti pri različnih pogojih shranjevanja. Proteini so podvrženi agregaciji in razvijanju sekundarne strukture, ki povzročijo izgubo njihove primarne funkcije in lahko vodi do ogrožanja zdravja pacientov. Ti procesi so pospešeni pod stresnimi pogoji, kot sta povišana temperatura in , izpostavljenosti svetlobi. Trenutne metode za ocenjevanje strukturne integritete proteinov namreč zahtevajo predpripravo vzorcev in redčenje, pri čemer je povečana možnost človeške napake. Mednje spadajo spektroskopske metode kot so krožni dihroizem (CD), ultravijolična-vidna spektroskopija (UV-Vis) in Fourierjeva transformacijska infrardeča spektroskopija (FTIR). Zaradi inertnosti, so biološka zdravila najpogosteje shranjena v steklenih vialah, ki omogočajo izvajanje spektroskopskih metod. Naš cilj je bil preveriti, katere vrste stekla omogočajo razvoj metode na podlagi CD spektroskopije, s katero bo omogočena in situ analiza proteinov. Primerjali smo vpliv različnih vrst stekla na razvijanje sekundarne strukture proteina.V diplomskem delu smo preučevali tudi vpliv robnih pogojev na strukturo lizocima. To smo opazovali s snemanjem v kvarčnih kivetah, HPLC in farmacevtskih vialah.
Ugotovili smo, da pri spremenjenih pogojih, ne pride do razvijanja sekundarne strukture lizocima, ko pa se struktura spremeni, takoj nastopi agregacija. Primerjava stekla pripelje do zaključka, da je za snemanje v CD spektrometru pričakovano najbolj ugodna kvarčna kiveta. HPLC viala zaradi samo absorpcije in napetosti nad mejno vrednostjo nasičenja detektorja, ne omogoča izvajanje meritev.
Tako kvarčna kiveta, ki je specializirana za merjenje v CD spektru, kot tudi farmacevtska viala zadovoljita pogoje za snemanje v CD spektrometru.Protein-based biologics face stability issues under various storage conditions. Proteins are prone to aggregation and the development of secondary structure, which impair their primary function and pose a risk to patient health. These processes are accelerated by elevated temperatures, exposure to light, etc. Current methods for assessing protein structural integrity require sample preparation and dilution, which increases the potential for human error. These include spectroscopic methods such as circular dichroism (CD), ultraviolet-visible spectroscopy (UV-Vis), and Fourier transform infrared spectroscopy (FTIR). Due to their inertness, biologics are most often stored in glass vials, which enable the use of spectroscopic methods. Our goal was to determine which types of glass enable the development of a CD spectroscopy-based method for in situ protein analysis. We compared the effects of different glass types on CD spectroscopy.We compared quality of spectra obtained in quartz cuvettes, pharmaceutical grade vials and HPLC vials. In this thesis we also studied the influence of stress conditions on the structure of lysozyme.
We found that under altered conditions, lysozyme does not develop a secondary structure, and when a change in structure does occur, it leads to aggregation. Comparison of the spectra quality, indicated thatthe best results are obtained in quartz cuvettes, as one would expect. Nevertheless, the tested pharmaceutical grade vials performed well, allowing for in-situ analysis of tertiary and secondary structure. The HPLC vial, however, does not allow measurements of CD spectra due to absorption of UV spectra exceeding the saturation limit of the detector
Experimental characterisation of protein-protein and protein-excipient interactions and their impact on protein conformation and aggregation
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Nonspecific Binding of Adenosine Tripolyphosphate and Tripolyphosphate Modulates the Phase Behavior of Lysozyme
No full textAdenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme-ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein-protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.</p
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