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Uporaba lektinov za prepoznavanje glikanskih struktur terapevtskih proteinov
Cvijić, Tamara (Author), Marinšek Logar, Romana (Mentor) More about this mentor... This link opens in a new window, Marušič, Jaka (Co-mentor)

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Abstract
Biološka zdravila so proizvedena s tehnikami molekulske in celične biologije in so proteinske narave. Med rekombinantnimi proteini se v zadnjem času vse bolj uveljavljajo monoklonska protitelesa (mAb) pridobljena s tehnologijo rekombinantne DNA. Protitelesa (Ab) so glikoproteini, ki so bistvenega pomena pri protitelesnem imunskem odzivu. Med njihovo sintezo prihaja do post-translacijskih sprememb in ena izmed glavnih je glikozilacija. Pri slednji celice ovarijev kitajskega hrčka (CHO), ki so trenutno prevladujoč ekspresijski sistem, na protein pripenjajo različne glikanske strukture. Le-te lahko znatno vplivajo tako na fizikalno-kemijske lastnosti kot tudi na efektorske funkcije Ab: od protiteles posredovana celična citotoksičnost (aktivnost ADCC), nizka imunogenost, od komplementa odvisna citotoksičnost (aktivnost CDC) ter primeren razpolovni čas Ab v telesu pacienta. Pri razvoju in proizvodnji terapevtskih proteinov je zato merjenje in uravnavanje glikanskih struktur ključnega pomena. Namen raziskovalne naloge je bil razviti metodo za kvantifikacijo glikanskih struktur s proteini, ki specifično prepoznavajo in se reverzibilno vežejo na glikanske strukture proteinov. Poznamo jih pod imenom lektini. Pri raziskovalnem delu smo uporabljali tehniko BLI (ang. Bio-Layer Interferometry), s katero je mogoče spremljati vezavo v realnem času. Razvili smo uporabno orodje za kvantifikacijo glikanov v prečiščenih denaturiranih vzorcih, ki se je izkazalo kot odlična alternativa relativno dolgotrajnim in dragim konvencionalnim metodam.

Language:Slovenian
Keywords:celična linija CHO, rekombinantna terapevtska protitelesa, terapevtski proteini, lektini, glikozilacija, BLI
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2018
Publisher:[T. Cvijić]
UDC:577.27.083:547.96:615.32
COBISS.SI-ID:4922488 This link opens in a new window
Views:942
Downloads:371
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Secondary language

Language:English
Title:Lectin based glycan identification in therapeutic proteins
Abstract:
Biopharmaceuticals are therapeutic proteins produced using biotechnology. Monoclonal antibodies (mAb) are glycoproteins, which are also an important part of our immune system. They are the most common type of bipharmaceuticals currently in the market and are produced using recombinant DNA technology by Chinese Hamster Ovary (CHO) cells. During their synthesis, various post-translation modifications take place. One of the most important modifications is glycosylation, where glycal structures are added to mAb. These structures have a major impact on physical, chemical and effector functions of antibodies (Ab) such as: antibodies cell dependent citotoxicity (ADCC), low immunogenicity, complement dependent citotoxicity (CDC) and appropriate Ab half-life. Therefore, characterisation and quantification of glycans is of critical importance during developement and manufacture of therapeutic proteins. The main scope of our work was the development of a method for characterisation of glycan structures using specific proteins, called lectins. The primary analitical technique used was BLI (Bio-Layer Interferometry), which enables real-time measurements of molecular binding. We have developed a practical method for quantification of glycans in purified and denaturated samples as a major improvement over convetional expensive and time-consuming procedures.

Keywords:CHO cell lines, recombinant monoclonal antibodies, therapeutic proteins, lectins, glycosylation, BLI

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