Ekspresija i karakterizacija rekombinantnoga folikul-stimulirajućega hormona (FSH) u CHO staničnoj liniji u mediju bez seruma

doctoral thesis

• Title (english): Expression and characterization of recombinant follicle-stimulating hormone (FSH) in CHO cell line in serum-free medium
• Author: Željka Antolović; MBZ: 193234
• Mentor: Vladimir Mrša (mentor)
• Committee member: Višnja Gaurina-Srček (predsjednik povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; Zagreb
• Defense date and country: 2012-09-19, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

FSH, član porodice gonadotropnih peptidnih hormona, nastaje u adenohipofizi i ima ključnu ulogu u reprodukciji sisavaca. Humani FSH se danas proizvodi rekombinantnom DNA tehnologijom u kulturi animalnih stanica. Zbog niza problema i rizika vezanih za korištenje seruma u mediju, za biotehnološku proizvodnju terapijskih proteina poželjno je koristiti medij bez seruma odnosno kemijski definirani medij. U ovom radu stanice CHO DG44, koje rastu u suspenziji i kemijski definiranom mediju, transfektirane su zasebnim ekspresijskim vektorima od kojih svaki sadržava gen za jednu od podjedinica humanog FSH. Tijekom uzgoja u selektivnom mediju s metotreksatom i nakon nekoliko ciklusa umnožavanja FSHα i FSHβ gena, dobivene su stanice koje proizvode heterodimer rFSH. Metodom graničnog razrijeđivanja stanica izdvojeni su pojedinačni klonovi. Napravljen je probir klonova s obzirom na ekspresiju rFSH. Odabrani klonovi uzgajani su u suspenziji u kemijski definiranom mediju, a tijekom procesa uzgoja u različitim kultivacijskim sustavima laboratorijskog mjerila praćen je udio živih stanica i specifična brzina rasta, kinetika potrošnje nutritijenata i nastajanja metabolita te proizvodnja eksprimiranog rFSH. Optimiranjem uzgoja dodatkom kemijski definiranih hranjivih smjesa u medij četiri puta je povećan prinos rFSH u odnosu na uzgoj u početnim uvjetima. Dokazano je da se rFSH izlučuje u medij kao cjeloviti heterodimer čiji glikozilacijski profil odgovara referentnom rFSH.

Abstract (english)

FSH, a member of gonadotropin family of peptide hormones, is secreted by the anterior pituitary gland and has a key role in reproduction. FSH can be produced in animal cell expression systems by recombinant DNA technology. In biopharmaceutical production of therapeutic proteins it is preferred to use growth medium without serum. In this work, CHO DG44 cells were cultivated in a chemically-defined medium in suspension and transfected by two separate expression vectors containing FSHα and FSHβ genes to achieve stable expression of recombinant human FSH. After gene amplification process and cloning, the clones producing rFSH were selected. The production of rFSH was increased 4 times by optimization of cell growth in suspension and various cultivation systems. Analysis of metabolites present in media led to define the best ratio among the used chemically-defined feedings. Additionally, their influence on cell growth in suspension and production of rFSH was determined. The rFSH was purified by immuno-affinity chromatography. It was confirmed that the rFSH heterodimer, expressed in CHO DG44 cells and secreted into medium, had the glycosylation pattern of isoformes corresponding to that of the rFSH reference product.

• Keywords: FSH; CHO stanična kultura; stabilna ekspresija; medij bez seruma
• Keywords (english): FSH; CHO cell line; stable expression; serum-free media
• Language: croatian
• URN:NBN: urn:nbn:hr:159:761874
• Promotion: 2012-10-21
• Study programme: Title: Postgraduate PhD Study in Biotechnology and Bioprocess Engineering; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti (doktor/doktorica znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2018-05-16 11:50:14