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Author:Siivinen, Johannes
Title:DNA-plasmidin puhdistusprosessin alkuvaiheen kehittäminen
Development of the initial phase of a DNA plasmid purification process
Publication type:Master's thesis
Publication year:2009
Pages:ix + 96 (+42)      Language:   fin
Department/School:Biotekniikan ja kemian tekniikan laitos
Main subject:Bioprosessitekniikka   (Kem-70)
Supervisor:Leisola, Matti
Instructor:Koskinen, Jani
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Location:P1 Ark TKK  4057   | Archive
Keywords:plasmid purification
chromatography
filtration
plasmidien puhdistus
kromatografia
suodatus
Abstract (eng): The objective of this work was to compare chromatographic procedures in plasmid purification as well as to develop the lysis stage of the purification process.

Plasmid containing bacterial pellet was lysed by an alkali procedure.
Precipitate that was generated during the lysis stage and which contained mainly impurities was removed by centrifugation.
Chromatographic procedures were utilized for purification of plasmid DNA.
Six different chromatography columns (ii Prep 16/10 ANX FF (high sub), Poros 50 P1, Poros 50 D, Sartobind D 75, CHT Ceramic Hydroxyapatite Type II, Capto DEAE) were compared with a well established plasmid purification procedure (Qiagen EndoFree Plasmid Mega Kit).
The results were evaluated on the basis of product purity, process yield and time consumption.
Product purity was investigated for endotoxins, RNA, genomic DNA, proteins and plasmid homogeneity.
The centrifugation in lysis stage was replaced with filtration and obtained results were compared with the original process.

Of the evaluated chromatographic procedures Sartobind D 75 -membrane was found to be best.
Product purity was superior in comparison with the other procedures.
Process yield was 40 % which was comparable to the established plasmid purification procedure.
In addition Sartobind D 75 -membranes were fast and convenient to use.
Results from Capto DEAE -column was very promising.
Process yield was 63 % which was 20 % higher than the established plasmid purification process.
The product purity should be further investigated.
Process yield was below 30 % for HiPrepl6/10 ANX FF (high sub) and Poros 50 P1 columns.
The product was also contaminated with significant amounts of RNA.
The product purity of CHT Ceramic Hydroxyapatite Type II -column was insufficient according to all criteria.
The procedure was also more labour-consuming than the others.
Poros 50 D -column did not purify the plasmid in the process conditions.
The filters which were used to replace centrifugation were found to be able to remove precipitate from the lysate.
However the process yield was somewhat lower.
Between the different filters differences were discovered in usability and process yield.
To confirm the results for the filtration studies further research is needed.
Abstract (fin): Työn tarkoituksena oli sekä vertailla kromatografisia menetelmiä plasmidin puhdistusprosessissa että kehittää prosessin lyysivaihetta.

Plasmidia sisältävä bakteeripelletti hajotettiin alkalilyysimenetelmällä.
Lyysin aikana muodostuva sakka, joka sisälsi pääasiassa epäpuhtauksia, erotettiin sentrifugoimalla: Plasmidi puhdistettiin kromatografisin menetelmin.
Kuutta eri kromatografiakolonnia (Hi Prep 16/10 ANX FF (high sub), Poros 50 P1, Poros 50 D, Sartobind D 75, CHT Ceramic Hydroxyapatite Type II, Capto DEAE) vertailtiin vakiintuneeseen plasmidin puhdistusmenetelmään (Qiagen EndoFree Plasmid Mega Kit).
Tuloksia arvioitiin tuotteen puhtauden, prosessin saannon ja ajan kulutuksen perusteella.
Tuotteen puhtaus tutkittiin endotoksiinien, RNA:n, genomisen DNA:n, proteiinien ja plasmidin homogeenisuuden osalta.
Lyysivaiheen sentrifugointi korvattiin suodatuksella ja saatuja tuloksia verrattiin alkuperäiseen prosessiin.

Kromatografisista menetelmistä parhaaksi arvioitiin Sartobind D 75 -membraani.
Tuotteen puhtaus oli ylivoimainen muihin menetelmiin verrattuna.
Puhdistusprosessin saanto oli 40 %, mikä vastasi vakiintunutta plasmidin puhdistusmenetelmää.
Lisäksi Sartobind D 75 -membraanien käyttö oli nopeaa ja vaivatonta.
Capto DEAE -kolonnin tulokset olivat erittäin lupaavia.
Puhdistusprosessin saanto 63 % oli peräti 20 % korkeampi kuin vakiintuneen plasmidin puhdistusprosessin.
Tutkimuksia on syytä jatkaa tuotteen puhtauden selvittämiseksi.
HiPrepl6/l0 ANX FF (high sub) - ja Poros 50 P1 -kolonnien saanto jäi alle 30 %.
Lisäksi tuotteen puhtaus kärsi merkittävästä RNA kontaminaatiosta.
CHT Ceramic Hydroxyapatite Type II -kolonnilla valmistetun tuotteen puhtaus oli riittämätön kaikkien kriteerien suhteen.
Menetelmä oli myös muihin verrattuna työläs.
Poros 50 D -kolonni ei kyennyt puhdistamaan plasmidia prosessiolosuhteissa.
Sentrifugoinnin korvanneilla suodattimilla pystyttiin erottamaan sakkaa lysaatista.
Puhdistusprosessin saanto kuitenkin laski hieman.
Eri suodattimien välillä havaittiin eroja sekä käytettävyydessä että tuotteen saannossa.
Tutkimuksia on jatkettava myös suodatuksen osalta tulosten vahvistamiseksi.
ED:2009-12-11
INSSI record number: 38658
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