DK2364769T3 - Apparater og fremgangsmåder til biomaterialeproduktion - Google Patents
Apparater og fremgangsmåder til biomaterialeproduktion Download PDFInfo
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- DK2364769T3 DK2364769T3 DK10075640.2T DK10075640T DK2364769T3 DK 2364769 T3 DK2364769 T3 DK 2364769T3 DK 10075640 T DK10075640 T DK 10075640T DK 2364769 T3 DK2364769 T3 DK 2364769T3
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Claims (19)
1. Fremgangsmåde til lysering af celler med en blandeindretning med høj forskydning og kort opholdstid, omfattende: - strømning af en lysisopløsning og en cellesuspension gennem blandeindretnin-gen med høj forskydning og kort opholdstid for at frembringe en blandet celle-opslemning og lysisopløsning, hvori opholdstiden er mindre end eller lig med ca. et sekund, - transportering af den blandede celleopslemning og lysisopløsning til en boble-blandeindretning gennem en holdespiral, hvori transporten sker over en defineret overføringstid, som er tilstrækkelig til at tillade i det væsentlige fuldstændig cellelysis uden permanent denaturering af cellekomponenter af interesse, til fremstilling af en cellelysatopløsning, - strømning af cellelysatopløsningen og en neutraliserings-/udfældningsopløsning gennem bobleblandeindretningen, hvori gasboblerne indføres i en takt, som er tilstrækkelig til at forårsage væsentlig blanding af cellelysatopløsningen med neu-traliserings-/udfældningsopløsningen, for at frembringe en udfældet lysatopløs-ning indeholdende udfældede cellekomponenter, - holde den udfældede lysatopløsning en tid, som er tilstrækkelig til at tillade den største del af de udfældede cellekomponenter at danne et flydelag, - valgfrit påtrykke den udfældede lysatopløsning et undertryk for at frembringe en blanding af udfældede cellekomponenter og klaret cellelysat, og - genvinding af det klarede cellelysat indeholdende cellekomponenter af interesse ved at dræne eller pumpe det klarede cellelysat, mens de udfældede cellekomponenter efterlades.
2. Fremgangsmåde ifølge krav 1, hvori blandeindretningen med høj forskydning og kort opholdstid er en inline rotor-/statorblander.
3. Fremgangsmåde ifølge krav 1, hvori bobleblandeindretningen er en indretning til blanding af et første fluid med mindst et yderligere fluid, omfattende: - et blandekammer, - et første indløb gennem hvilket det første fluid indføres i blandekammeret, - et eller flere indløb, gennem hvilket det yderligere fluid indføres i blandekammeret, - et middel til at indføre gasbobler i blandekammeret, hvori gasboblerne indføres i en takt, som er tilstrækkelig til at forårsage væsentlig blanding af det første fluid med det yderligere fluid, og - et udløb gennem hvilket det første fluid og det yderligere fluid udgår efter at være blandet i væsentlig grad, hvori det første fluid omfatter opslemmede celler, lyserede celler eller cellekomponenter, og det yderligere fluid omfatter lysisopløsning, neutraliseringsopløsning, udfældningsopløsning eller en kombination deraf.
4. Fremgangsmåde ifølge krav 1, yderligere omfattende det trin at rense det klarede cellelysat for at genvinde en cellekomponent af interesse, såsom et plasmid.
5. Fremgangsmåde ifølge krav 4, hvori rensning af det klarede cellelysat omfatter - at udsætte det klarede cellelysat for en ionbyttermembran, og - at udsætte det klarede cellelysat for en hydrofobisk interaktionsmembran.
6. Fremgangsmåde til isolering af et plasmid-DNA fra celler, omfattende: (a) blanding af en opslemning af celler med plasmid-DNA'et med en lysisopløsning i en blandeindretning med høj forskydning og kort opholdstid i en første tidsperiode til dannelse af et cellelysatfluid, (b) inkubering af cellelysatfluidet i en anden tidsperiode i en holdespiral til dannelse afen cellelysatopslemning, (c) levering af cellelysatopslemningen ind i et kammer, (d) levering af et udfældningsfluid, en neutraliseringsopløsning eller en kombination deraf ind i kammeret, (e) blanding af cellelysatopslemningen og udfældningsopløsningen, neutraliseringsopløsningen eller kombinationen deraf i kammeret med gasbobler, der danner en gasblandet opslemning, hvori den gasblandede opslemning omfatter et uklaret lysat og et udfældningsprodukt med plasmid-DNA'et i det uklarede lysat, hvorved cellerester er i udfældningsproduktet, og hvori udfældningsproduktet har mindre vægtfylde end det uklarede lysat, (f) flydning af udfældningsproduktet ovenpå det uklarede lysat, (g) fjernelse af det uklarede lysat fra udfældningsproduktet, som danner et klaret lysat, hvorved plasmid-DNA'et i det væsentlige skilles fra cellerester, (h) udfældning af plasmid-DNA'et fra det klarede lysat, der danner et udfældet plasmid-DNA, og (i) genopslemning af det udfældede plasmid-DNA i en vandig opløsning, hvori - den første tidsperiode er lig med eller mindre end ca. 1 sekund, og - den anden tidsperiode er i et interval fra ca. 2 til 8 minutter.
7. Fremgangsmåde ifølge krav 6, yderligere omfattende rensning af det klarede lysat, eller fremgangsmåden ifølge krav 4, hvori rensningen omfatter at underkaste det klarede lysat et eller flere rensetrin valgt fra gruppen bestående af ionbytning, hydrofobisk interaktion, størrelseseksklusion, omvendt faserensning, endo-toksinforarmning, affinitetsrensning, adsorption til siliciumoxider, glas eller polymermaterialer, expanded-bed kromatografi, mixed-mode kromatografi, forskydningskromatografi, hydroxyapatitrensning, selektiv udfældning, vandig tofase-rensning, DNA kondensering, thiofil rensning, ionparrensning, metalchelatrens-ning, filtrering gennem nitrocellulose eller ultrafiltrering.
8. Fremgangsmåde ifølge krav 1 eller 6, hvori lysisopløsningen yderligere omfatter et lyseringsmiddel valgt fra en gruppe bestående af et alkali, en syre, et afspændingsmiddel, et organisk opløsningsmiddel, et enzym, en kaotrop eller et denatureringsmiddel.
9. Fremgangsmåde ifølge krav 1 eller 6, hvori udfældningsopløsningen eller neutraliseringsopløsningen er en opløsning af kaliumacetat, ammoniumacetat eller en blanding deraf.
10. Fremgangsmåde ifølge krav 6, hvori gasboblerne er trykluft eller en inaktiv gas.
11. Fremgangsmåde ifølge krav 6, hvori kammeret omfatter: - en første indløbsport til levering af det første fluid ind i kammeret, - en anden indløbsport til levering af det andet fluid ind i kammeret, og - en tredje indløbsport til levering afen blandegas ind i kammeret, - en udluftning, og - en udløbsport til udføring af den gasblandede opslemning fra kammeret.
12. Apparat til lysering af celler ifølge fremgangsmåden ifølge krav 1, omfattende: (a) en første tank i flydende forbindelse med en blander, hvori den første tank er egnet til at holde en opslemning af celler med en cellekomponent af interesse, såsom et DNA-plasmid, (b) en anden tank i flydende forbindelse med blanderen, hvori den anden tank er egnet til at holde en lysisopløsning, (d) en holdespiral i flydende forbindelse med blanderen, og (e) et bobleblandekammer med en top og en bund, omfattende: (i) et første indløb i flydende forbindelse med holdespiralen, (ii) et andet indløb i flydende forbindelse med en tredje tank, hvori den tredje tank er egnet til at holde et udfældningsopløsning, en neutraliseringsopløsning eller en blanding deraf, (iii) et tredje indløb i flydende forbindelse med en gaskilde, (iv) en udluftning, og (v) et udløb i flydende forbindelse med en fjerde tank, hvori den fjerde tank er egnet til at skille udfældede cellekomponenter fra flydende cellelysat, hvori blanderen er blandeindretning med høj forskydning og lav opholdstid med en opholdstid på mindre end eller lig omkring et sekund, og apparatet er arrangeret sådan, at - celleopslemningen med plasmid-DNA'et fra den første tank tillades at flyde ind i blanderen, - lysisopløsningen fra den anden tank tillades at flyde ind i blanderen, - en lysatblanding tillades at flyde fra blanderen og ind i holdespiralen, - lysatblandingen fra holdespiralen tillades at flyde ind i bobleblandekammeret, - udfældningsopløsningen, neutraliseringsopløsningen eller blandingen deraf fra den tredje tank tillades at flyde ind i bobleblandekammeret, og - en opslemning indeholdende cellekomponenten af interesse tillades at flyde fra bobleblandekammeret og ind i den fjerde tank.
13. Apparat ifølge krav 12, yderligere omfattende en første pumpe til transport af celleopslemningen med cellekomponenten af interesse fra den første tank til blanderen, en anden pumpe til transport af lysisopløsningen fra den anden tank til blanderen, en tredje pumpe til transport af udfældningsopløsningen, neutraliseringsopløsningen eller blandingen deraf fra den tredje tank og ind i bobleblandekammeret, hvori den første pumpe og den anden pumpe valgfrit er kombineret i en dobbeltpumpe, som tillader celleopslemningen med cellekomponenten af interesse og lysisopløsningen samtidigt at pumpes til blanderen med en lineær strømningshastighed på ca. 0,1-1 fod/sekund, og valgfrit yderligere omfattende et Y-samlestykke med en første tvegrenet tilslutningsstuds, en anden tvegrenet tilslutningsstuds og en afgangsstuds, hvori den første tank er i flydende forbindelse med den første tvegrenede tilslutningsstuds af Y-samlestykket gennem den første pumpe, den anden tank er i flydende forbindelse med den anden tvegrenede tilslutningsstuds af Y-samlestykket gennem den anden pumpe, og blanderen er i flydende forbindelse med Y-samlestykkets afgangsstuds, hvori den første og anden pumpe tilvejebringer en lineær strømningshastighed på ca. 0,2 til 2 fod/sekund for et fluid i kontakt, som udgår fra Y-samlestykket.
14. Apparat ifølge krav 12, hvori apparatet er arrangeret til at anvende tyngdekraft, tryk, undertryk eller en blanding deraf, til - transport af celleopslemningen med cellekomponenten af interesse fra den første tank til blanderen, - transport af lysisopløsningen fra den anden tank til blanderen, og - transport af udfældningsopløsningen, neutraliseringsopløsningen eller blandingen deraf fra den tredje tank og ind i bobleblandekammeret.
15. Apparat ifølge krav 12 eller fremgangsmåde ifølge krav 11, hvori kammeret omfatter - en lukket, lodret søjle med udluftningen ved søjlens øverste ende, - det første indløb går ind i bobleblandekammeret nærmest bunden af en første side af den lukkede, lodrette søjle, - det andet indløb går ind i bobleblandekammeret nærmest bunden på en anden side og modsat det første indløb, - det tredje indløb går ind i bobleblandekammeret nærmest bunden og omkring midt mellem de første og andre indløb, og - udløbet udgår fra bobleblandekammeret nærmest den lukkede, lodrette søjles øverste ende, - hvori det tredje indløb valgfrit omfatter en sintret gasfordeler inde i den lukkede, lodrette søjle.
15. Apparat ifølge krav 12, hvori blanderen omfatter en indretning, der blander ved gennemstrømning, og som har en rotor-/statorblander eller emulgator og med lineære strømningshastigheder fra ca. 0,1 l/min til ca. 20 l/min.
17. Apparat ifølge krav 12, hvori holdespiralen ved en fast lineær strømningshastighed omfatter rør med længde og diameter, som er tilstrækkelig til at tillade den lysatblanding, der forlader blanderen, ca. 2 til ca. 8 minutters kontakttid med holdespiralen, før lysatblandingen kan gå ind i bobleblandekammeret.
18. Apparat ifølge krav 12, hvori den fjerde tank endvidere omfatter en omrører, som kan tilvejebringe ensartet blanding af fluid uden at forstyrre et flokkulent udfældningsprodukt.
19. Apparat ifølge krav 12, yderligere omfattende en vakuumpumpe i forbindelse med den fjerde tank.
20. Apparat ifølge krav 12, yderligere omfattende - en tredje pumpe i flydende forbindelse med den fjerde tank, - et første filter i flydende forbindelse med den tredje pumpe, - et andet filter i flydende forbindelse med det første filter, og - en femte tank til at holde et klaret lysat, hvori det første filter valgfrit har en partikelstørrelsesgrænse på ca. 5-10 pm, og det andet filter har en grænse ved ca. 0,2 pm.
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| DE102004052254A1 (de) * | 2004-05-03 | 2005-12-01 | Plasmid Factory Gmbh & Co. Kg | Verfahren zum Aufschluss von Zellen und zur Abtrennung von Zellbestandteilen |
| US7846720B2 (en) * | 2005-01-26 | 2010-12-07 | Vgx Pharmaceuticals, Inc. | Optimized high yield synthetic plasmids |
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| JP5439183B2 (ja) | 2006-10-17 | 2014-03-12 | ヴィージーエックス ファーマシューティカルズ，インコーポレイテッド | 哺乳動物の細胞の電気穿孔のための電気穿孔装置および該電気穿孔装置を使用する方法 |
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| US20080299621A1 (en) * | 2007-05-31 | 2008-12-04 | Ge Healthcare Uk Limited | Miniprep system for simple and rapid plasmid dna extraction |
| AU2008256411B2 (en) | 2007-06-01 | 2013-08-22 | F. Hoffmann-La Roche Ag | Immunoglobulin purification |
| US7989615B2 (en) * | 2007-07-13 | 2011-08-02 | The Penn State Research Foundation | Separation of different isoforms of plasmid DNA using ultrafiltration |
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- 2004-05-27 EP EP10075640.2A patent/EP2364769B1/en not_active Expired - Lifetime
- 2004-05-27 TW TW093115170A patent/TWI303275B/zh not_active IP Right Cessation
- 2004-05-27 EP EP04753608.1A patent/EP1628749B1/en not_active Expired - Lifetime
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- 2004-05-27 CA CA2567324A patent/CA2567324C/en not_active Expired - Lifetime
- 2004-05-27 WO PCT/US2004/016806 patent/WO2004108260A2/en active Application Filing
- 2004-05-27 US US10/857,439 patent/US7238522B2/en not_active Expired - Lifetime
- 2004-05-27 DK DK10075640.2T patent/DK2364769T3/da active
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| EP2364769A1 (en) | 2011-09-14 |
| TW200504207A (en) | 2005-02-01 |
| CA2567324C (en) | 2012-01-03 |
| EP2364769B1 (en) | 2016-01-13 |
| TWI303275B (en) | 2008-11-21 |
| WO2004108260A2 (en) | 2004-12-16 |
| EP1628749A2 (en) | 2006-03-01 |
| EP1628749B1 (en) | 2019-07-31 |
| WO2004108260A3 (en) | 2005-05-12 |
| US7238522B2 (en) | 2007-07-03 |
| DK1628749T3 (da) | 2019-09-16 |
| ES2748130T3 (es) | 2020-03-13 |
| US20050014245A1 (en) | 2005-01-20 |
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