WO2017069781A1 - Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension - Google Patents

Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension Download PDF

Info

Publication number
WO2017069781A1
WO2017069781A1 PCT/US2015/057217 US2015057217W WO2017069781A1 WO 2017069781 A1 WO2017069781 A1 WO 2017069781A1 US 2015057217 W US2015057217 W US 2015057217W WO 2017069781 A1 WO2017069781 A1 WO 2017069781A1
Authority
WO
WIPO (PCT)
Prior art keywords
filter
lysis
workflow
cells
cell
Prior art date
Application number
PCT/US2015/057217
Other languages
English (en)
Inventor
Philip Martin
Original Assignee
Life Technologies Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Life Technologies Corporation filed Critical Life Technologies Corporation
Priority to PCT/US2015/057217 priority Critical patent/WO2017069781A1/fr
Publication of WO2017069781A1 publication Critical patent/WO2017069781A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/147—Microfiltration

Definitions

  • This application relates to methods for extracting protein from cells suspended in a liquid cell culture medium, and, more particularly, to automated systems and methods for cell capture, lysis, and protein clarification from liquid cell cultures.
  • a cell lysis workflow is disclosed.
  • a cell suspension is passed through a filter in a first flow direction from a first side of the filter toward a second side thereof.
  • the filter captures a plurality of cells on the first side.
  • a lysis solution is passed through the filter in a second flow direction opposite the first direction, thereby dislodging the plurality of cells captured on the filter, and resuspending the plurality of cells in the lysis solution on the first side of the filter.
  • the lysis solution lyses the plurality of cells to produce a cellular lysate.
  • the cellular lysate is passed through the filter in the first flow direction.
  • a cell lysis system in another aspect, includes a filter sized to allow fluid to pass therethrough and to filter out cells, a reservoir in fluid communication with the filter and positioned on a first side thereof, a cell suspension intake in fluid communication with the filter and positioned on the first side thereof, a lysis solution intake in fluid communication with the filter and positioned on a second side thereof, and a reversible pump in fluid communication with the filter.
  • the reversible pump has a first operating state to facilitate fluid flow in a first flow direction through the filter from the first side toward the second side thereof, and a second operating state to facilitate fluid flow in a second flow direction through the filter from the second side toward the first side thereof.
  • the cell suspension intake is configured to receive a cell suspension therethrough such that the cell suspension is filtered through the filter and a plurality of cells of the cell suspension are captured on the first side of the filter when the reversible pump is in the first operating state.
  • the lysis solution intake is configured to receive a lysis solution therethrough such that the lysis solution passes through the filter in the second flow direction when the reversible pump is in the second operating state to lyse and suspend the plurality of cells captured on the first side of the filter to produce a cellular lysate.
  • the reservoir is configured to receive the cellular lysate when the reversible pump is in the second operating state.
  • FIG. 1 schematically depicts one embodiment of a cell lysis workflow.
  • FIG. 2 schematically depicts one embodiment of a cell lysis and protein purification workflow.
  • FIG. 3 depicts an embodiment of a filter suitable for use with the workflows of Figs. 1 and Fig. 2.
  • Figs. 4 depicts an embodiment of a reservoir-filter compatible with the workflows of Fig. 1 and Fig. 2.
  • Figs. 5 depicts an alternate embodiment of a reservoir-filter compatible with the workflows of Fig. 1 and Fig. 2.
  • Figs. 6 depicts a further alternate embodiment of a reservoir-filter compatible with the workflows of Fig. 1 and Fig. 2.
  • Fig. 7 depicts embodiments of portions of an instrument suitable for automatically performing the workflow of Fig. 2.
  • Fig. 1 an embodiment of a cell lysis system or workflow, generally designated 100, is shown.
  • the alternative pathways represented in the schematic diagram by various junctions 101 may include manual or automatic valves or the like to control and direct the flow of fluid, or alternately, distinct fluid lines may be included to facilitate some or all of the different pathways.
  • a cell suspension 102 is provided and may include any of a variety of eukaryotic and/or prokaryotic cells.
  • the cells are mammalian cells, or more particularly, rodent (e.g., mouse, rat, or hamster) such as CHO cells, or primate/human cells such as 293 or COS cells.
  • rodent e.g., mouse, rat, or hamster
  • the cells are prokaryotic, for example bacterial cells such as E. coli.
  • the cells may contain an expressed protein desired to be purified and collected.
  • Cells from the suspension 102 are passed through a filter 1 04 and captured by filtration on a first side 106 of the filter 104.
  • the filtrate 126 (Fig. 3) passes through the filter 104 and past a second side 108 thereof, and may be directed toward a waste receptacle 1 1 0.
  • Completion of this and each subsequent step may optionally be detected by an air/bubble sensor (not shown), or by any other methods for detecting the cessation of fluid flow or sufficiently low flow rates known to the skilled practitioner, and in the case of an automated workflow, a controller is then directed to perform the subsequent step.
  • a reversible pump 1 12 which in one embodiment may be a peristaltic pump, is positioned to create negative pressure on the filter 104/suspension 102 and cause the cells and media to be drawn in and through the filter 104 in a first flow direction, generally designated by arrow 1 14, when the pump 1 12 is in a first operating state.
  • the reversible pump 1 12 also has a second operating state to generate positive pressure on the filter 104, which facilitates liquid flow in an opposite, second flow direction, generally designated by arrow 1 1 6.
  • a suitable filter system 104 is selected primarily based on its flow rate and filter capacity. Additionally, a suitable filter system 104 should have an adequate pore size to capture the cells to be lysed. In one embodiment in which E. coli are captured on a membrane system and subsequently subjected to lysis, a 0.2 ⁇ hollow fiber filter membrane may be used. Referring additionally to Fig. 3, in one embodiment, the filter 104 includes a plurality of cellulose acetate-based porous hollow fibers 1 20, bent into a series of loops 1 18. This collection of loops 1 18 gives the filter 104 a large surface area.
  • the hollow fiber filter 104 may be a MEDIAKAP ® filter commercially available from SPECTRUM ® Laboratories, Inc., such as the MEDIAKAP-25 filter.
  • filtration of the suspension 102 is achieved by adding the cell suspension 102 to the outside of the hollow fiber filter 104 and subjecting the suspension to a vacuum applied across the filter 104 in the first flow direction 1 14. This action draws liquid and sufficiently-small particles of the cell suspension 102 through the wall 128 of the hollow fibers 120 of the filter 104 and down internal lumens 130 within the hollow fibers 120. The cells 132 of the cell suspension 102 are captured in filter fibers 134 that form the external surface of the wall 128. The filtrate 126 then passes out of the lumen 1 30 and beyond the polymer plug 124.
  • the outside 1 1 9 of the wall 128 defines the first side 106 of the filter 104 (Fig. 1 ), and the inside 1 21 of the wall 128 (bounding the internal lumen 130) (Fig. 3) defines the second side 1 08 of the filter 104 (Fig. 1 ).
  • any number of other filter configurations may be employed to capture the cells 132 on the first side 1 06 of the filter 104 and permit passage of the filtrate 126 through to the second side 108 the filter 104.
  • a lysis buffer or solution 1 36 (Fig. 1 ) is added to the cells 1 32 captured on the filter 1 04 to lyse the cells 132.
  • a requisite amount of a lysis buffer 136 suitable for lysing the captured cells 132 on the first side 106 of the filter 1 04 is dispensed manually and/or by an automated pump system 138.
  • the pump system 138 is a syringe pump with two way valving to direct flow and provide precise control of the dispensed volume of lysis buffer 136.
  • One or more optional holding chambers 140 may be positioned between the pump system 138 and the reversible pump 1 12 to temporarily retain dispensed lysis buffer 136 (and/or other buffers, wash solutions, and the like for use in the workflow 100) before the lysis buffer 136 is added to the cells 132, such that the pump system 138 controls flow of the lysis buffer 136 to the holding chamber 140, but the reversible pump controls flow of the lysis buffer 136 to the filter 104.
  • the lysis buffer 136 may be supplied to the first side 106 of the filter 1 04 in any of a variety of ways to lyse the cells 132 captured on the filter 104, which releases their contents, to produce a lysate including the target protein or other target material.
  • the lysis buffer 136 may be retained on the first side 106 of the filter 104 for an optional incubation period, which, in one embodiment, may be about 1 -30 minutes.
  • the reversible pump 1 12 is operated to pump the lysate 136 in the first flow direction 1 14 to filter the lysate through the filter 1 04 to produce a clarified lysate 154 for purification or further downstream workflows.
  • the lysis buffer 1 36 is added to the cells 132 in multiple passes, using either the entire volume of the lysis buffer 136, and/or portions thereof that are ultimately pooled together as the clarified lysate 154.
  • a reservoir 144 (Figs. 1 and 4-7) positioned on the first side 106 of the filter 104 provides sufficient volumetric capacity on the first side 106 of the filter 104 to contain the added lysis buffer 136, or at least portion thereof in an embodiment involving multiple lysis buffer addition steps.
  • the reservoir 144 may be sized to retain up to about 1 0-500 ml_, or about 10, 20, 50, 75, 1 00, 250, 300, 350, 400, 450, or up to about 500 ml_ of fluid therein or any volume falling therebetween.
  • the reservoir 144 has a generally cylindrical body 146 with a conical portion 148 that tapers to direct fluid toward the filter 104.
  • the reservoir 144 and the filter 1 04 are arranged together within a unitary housing 150, which may be a user-replaceable lysis consumable 153 for an instrument 151 , such as a manually-carryable bench top instrument, for performing the workflow 100.
  • the instrument 151 may include any of a variety of ports 142 which can serve as intakes and/or outlets for fluids to and from the reservoir 144 and/or the filter 104.
  • the ports 142 may also interface with an air supply 145 and/or a pressure sensor 147 (Fig. 7) to monitor the workflow.
  • Check valves 155 may be included to regulate fluid through the ports 142.
  • the lysis buffer 136 may be supplied from the holding chamber 140 to the reservoir 144 and the cells 132 on the first side 106 of the filter 104 using any of a variety of approaches. As shown in Fig. 4, in one case, the lysis buffer 136 is supplied to the reservoir 144 by "back-flushing" the lysis buffer 136 through the filter 104. The lysis buffer 136 is supplied from the holding chamber 140 by operating the reversible pump 1 12 in the second flow direction 1 16 to introduce the lysis buffer 136 through a port 152 that is in direct fluid communication with the second side 108 of the filter 104.
  • the pump 1 12 drives the lysis buffer 136 into the lumens 130 of the hollow fibers 120 of the filter 104, and through the walls 128 thereof to reach the first side 106 of the filter 104 and the reservoir 144. Accordingly, the lysis buffer 136 passes through the filter 104 opposite the filtering direction through the filter 104 that was used to capture the cells 1 32. Back-flushing the lysis buffer 136 through the filter 104 in this manner simultaneously lyses the cells 1 32 and dislodges the cells 132 from the filter fibers 134 of the wall 128 of the filter 104 (Fig. 3), thereby resuspending the cells 132 into the lysis buffer 136 for incubation in the reservoir 144 on the first side 106 of the filter 1 04.
  • the reversible pump 1 12 is operated in the first flow direction 1 14 to filter the lysate back through the filter 104 and out through the port 152, as earlier described, to obtain the clarified lysate 154 for purification or further downstream workflows.
  • the back-flushing procedure is repeated one or more additional times to improve yield.
  • Fig. 5 depicts an alternate "top-loading" approach for supplying the lysis buffer 136 to the cells 132 on the filter 104.
  • the lysis buffer 136 is supplied directly to the first side 106 of the filter 1 04 from above, for example through a port 142 that is an intake 1 50 into the reservoir 144.
  • the lysis buffer 136 may be supplied by operating the reversible pump 1 1 2 in the second flow direction 1 16 (Fig. 1 ). After incubation, the reversible pump 1 12 is operated in the first flow direction to drive and clarify the lysate through the filter 104 and out through the port 152 as earlier described.
  • the cells 132 are not resuspended and agitated in the lysis buffer 136 to the same degree as in the back-flushing embodiment (Fig. 4), so multiple passes of the lysis buffer 136 through the filter 104 may be necessary to achieve a suitable yield.
  • Fig. 6 depicts a "side-loading" approach for supplying the lysis buffer 136 to the cells 132 on the filter 1 04.
  • the lysis buffer 136 is supplied to the first side 106 of the filter 104, near the base of the filter 1 04 (e.g., near the end plug 1 22 in the embodiment of Fig. 3).
  • the lysis buffer 136 is added through an intake 156.
  • Adding the lysis buffer 136 at this location generates a vortex effect within the filter 104 and increases agitation to dislodge the cells 132 from the wall 28 of the fibers 1 18 of the filter 1 04, without the need to back-flush fluid through the filter 104 from the second side 108 to the first side 106 thereof.
  • the workflow 100 may incorporate features of more than one of the lysis buffer 136 supply methods of Figs. 4-6.
  • the lysis step may begin with one or more back-flushing steps and incubation steps to simultaneously lyse and dislodge the cells 132 from the filter 104.
  • additional lysis buffer 136 (or, alternately, some or all of the lysate from the back-flushing step) may be added back through the reservoir 144 using the "top loading" methodology to rinse the filter 104 and avoid or minimize lysate loss from residual material left behind on the filter 104 after the back-flushing step(s).
  • any acceptable lysis buffer known in the art may be used for the workflow 100 described herein.
  • the selection of a suitable lysis buffer to properly lyse various types of cells is well within the skill level of the practitioner, and lysis buffers suitable for lysing bacterial cells, plant cells, fungal cells, yeast cell, animal cells, insect cells, dispersed cells and the like are known in the art.
  • the lysis buffer 136 is substantially free of proteases and contains at least one nuclease.
  • the nuclease may be a DNase, or a combination of a DNase with an RNase, which can prevent problems with filter performance stemming from the release of double-stranded DNA (dsDNA) and the subsequent increase in cell lysate viscosity.
  • Suitable enzymes include BENZONASE® (EMD), TURBEDNASE® (Life Technologies), and CYANASE® (RiboSolutions).
  • EMD BENZONASE®
  • TURBEDNASE® Life Technologies
  • CYANASE® RaboSolutions
  • One benefit of using CYANASE® is that the enzyme may be deactivated if desired upon completion of the lysis step.
  • the lysis buffer 136 may further include any of a variety of additional additives and/or detergents as appropriate for the particular type of cell included in the cell suspension 102 and the target protein.
  • the lysis buffer includes pH 8.0 Tris buffer, NaCI, 3-[(3- cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS), ⁇ -D- thioglucopyranoside (OTGP), imidazole, and BENZONASE®.
  • the workflow 1 00 may optionally include one or more wash steps after the filtration of the cell suspension 102 through the filter 104 and before the addition of the lysis buffer 136.
  • a suitable aqueous wash buffer 158 is passed through the filter 1 04 containing the capture cells 1 32 to remove protein or other components contaminating the captured cells 132.
  • the wash buffer 158 may be supplied using any of the methodologies described above with respect to the lysis buffer 136.
  • the wash buffer 158 may first be dispensed using the pump system 138 to the holding chamber 140, and then supplied to the filter 104 and reservoir 144 by the pump 1 12 in the second flow direction 1 16.
  • the wash buffer 158 may rinse the cells 132 using the "back-flushing," "top-loading,” and/or “side-loading” methodologies, or combinations thereof.
  • the workflow 100' may further include purifying a protein or other target material from the clarified lysate 154.
  • the protein may be purified using any method known in the art, such as chromatography through the use of a purification column 160, including but not limited to affinity chromatography, gel filtration chromatography, and ion exchange chromatography, or a combination thereof.
  • the protein is purified using affinity chromatography methodology, where the purification column 1 60 is loaded with a charged resin, such as MC20 POROS ® (Applied Biosystems, LLC) resin activated with nickel sulfate.
  • the reversible pump 1 1 2 and/or the pump system 1 38 can be activated to direct the clarified lysate 154 from the filter 104 and to load the clarified lysate 154 onto the purification column 160.
  • the target protein product is retained on the column 160, while the remainder of the lysate fluid passes through the column 160.
  • the clarified lysate 154 may be cycled through the purification column 160 multiple times to improve the yield. Once purification is complete, the remaining liquid may be pumped to the waste receptacle 1 10 and discarded. Wash buffer 158 may optionally be cycled through the purification column 160 one or more times.
  • the target protein may then be eluted from the purification column 160 with an elution buffer 162 of any suitable type known in the art, which in one embodiment may include pH 8.0 Tris buffer, NaCI, and imidazole. In one embodiment, the target protein is eluted in about 5 ml_ of elution buffer, or less.
  • the protein-containing eluent may then be directed via the reversible pump 1 12 and/or pump system 138, and/or manually, for collection in an elution tube 164 for further downstream workflows.
  • an automated embodiment incorporating the workflow 100' may be contained with a single instrument 151 , which in one case may be a manually-carryable bench top instrument.
  • the purification column 160 may be included in a user-replaceable purification consumable 1 66 for insertion into the instrument 151 .
  • the purification consumable 166 has a housing 168 that includes the purification column 160 and a reservoir 170 to receive and retain a volume of clarified lysate 154 or other fluid to loaded to or otherwise passed through the column 160.
  • the reservoir 170 may be generally cylindrical in shape, and include a tapered conical portion 171 to facilitate liquid flow from the reservoir 170 into the purification column 1 60.
  • the portion of the instrument 151 which receives the purification consumable 1 66 may include features similar to those described above with respect to the portion of the instrument 1 51 that receives the lysis consumable 153.
  • this portion of the instrument 151 may include any of a variety of ports 172 to serve as intakes for fluids to enter the reservoir 170 or to interface with an air supply and/or a pressure sensor to control and monitor the workflow 1 00'.
  • An outlet 174 is positioned to facilitate flow of liquids out of the column 160.
  • any of the individual steps of the workflows 1 00, 100' described above may be performed manually by a user or users who may monitor the progression and/or the completion thereof.
  • any one or more of the individual steps of the workflows 100, 100' may be automated and may operate independently of any user monitoring or input, aside from, in one case, stocking the liquid components 1 02,136, 1 58, 162.
  • the process may be controlled by a touch screen computer running LABVIEW® (National Instruments corporation) software, or other suitable software capable of directing and controlling the various steps of the workflow 100, 100'.
  • an automated workflow 100, 1 00' the workflow is performed substantially entirely within the instrument 151 , for example a manually- carryable bench top instrument that is configured to receive the lysis consumable 153 and/or the purification consumable 166.
  • the sources of the various fluids and buffers 102, 136, 158, 162, and/or various collection vessels such as the holding chamber 140, elution tube 164, and/or a container for the clarified lysate 154, may be positioned external to the instrument 151 but fluidly connected thereto through suitable ports and valve systems.
  • the instrument 151 may be operatively connected to a controller loaded with software for automating the workflow 100, 100' and a user interface to provide control and monitoring capabilities over the progress and specific functional parameters of the workflow 100, 100'.
  • the entire lysis process including filtration of up to at least 1 liter of the cell suspension 1 02, any optional wash steps using the wash buffer 158, all steps related to loading the lysis buffer 136 into the reservoir 144 for incubation and/or resuspension of captured cells 1 32 and lysing the cells 1 32, and collection of the clarified lysate 1 54 are completed in 2 hours or less.
  • all steps involved in collecting a purified target protein from the clarified lysate 1 54 including purification of the target protein through the purification column 160, with all wash and elution steps, are completed in about 2 hours or less.
  • filtration, lysis, clarification, and purification of a target protein from up to at least 1 liter of cell suspension 102 may be completed in a single automated process in about 4 hours or less.
  • the workflow 100, 1 00' is scalable to accommodate filtration and/or purification requirements for volumes of cell suspension 102 greater than 1 liter by increasing the size and/or number of the various system components.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un flux de travail de lyse cellulaire impliquant une suspension cellulaire qui passe à travers un filtre dans une première direction d'écoulement depuis un premier côté du filtre vers un deuxième côté correspondant. Le filtre recueille une pluralité de cellules sur le premier côté. Une solution de lyse passe à travers le filtre dans une deuxième direction d'écoulement opposée à la première direction, ce qui permet de déloger la pluralité de cellules recueillies sur le filtre et de remettre en suspension la pluralité de cellules dans la solution de lyse sur le premier côté du filtre. La solution de lyse lyse la pluralité de cellules pour produire un lysat cellulaire. Le lysat cellulaire passe à travers le filtre dans la première direction d'écoulement.
PCT/US2015/057217 2015-10-23 2015-10-23 Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension WO2017069781A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2015/057217 WO2017069781A1 (fr) 2015-10-23 2015-10-23 Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2015/057217 WO2017069781A1 (fr) 2015-10-23 2015-10-23 Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension

Publications (1)

Publication Number Publication Date
WO2017069781A1 true WO2017069781A1 (fr) 2017-04-27

Family

ID=54478256

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/057217 WO2017069781A1 (fr) 2015-10-23 2015-10-23 Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension

Country Status (1)

Country Link
WO (1) WO2017069781A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010886A2 (fr) * 1999-08-06 2001-02-15 Qiagen Gmbh Purification automatisee de proteines par filtration sous vide au moyen d'une plaque de microtitration a puits multiples
WO2001077316A2 (fr) * 2000-04-11 2001-10-18 Bureco Ag Procede de recuperation d'acides nucleiques
WO2002006456A1 (fr) * 2000-07-13 2002-01-24 Invitrogen Corporation Methodes et compositions d'extraction et d'isolation rapides de proteines et de peptides au moyen d'une matrice de lyse
US20090325269A1 (en) * 2008-06-30 2009-12-31 General Electric Company Bacteria/RNA extraction device
US20110129931A1 (en) * 2009-10-20 2011-06-02 Agency For Science, Technology And Research Microfluidic system for detecting a biological entity in a sample
EP2602315A1 (fr) * 2011-12-05 2013-06-12 Pall Corporation Purification de leucocytes
WO2014151177A1 (fr) * 2013-03-15 2014-09-25 3M Innovative Properties Company Concentrateur d'échantillon et méthode d'utilisation
US20140356890A1 (en) * 2013-06-03 2014-12-04 Samsung Electronics Co., Ltd. Method of re-collecting target material

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010886A2 (fr) * 1999-08-06 2001-02-15 Qiagen Gmbh Purification automatisee de proteines par filtration sous vide au moyen d'une plaque de microtitration a puits multiples
WO2001077316A2 (fr) * 2000-04-11 2001-10-18 Bureco Ag Procede de recuperation d'acides nucleiques
WO2002006456A1 (fr) * 2000-07-13 2002-01-24 Invitrogen Corporation Methodes et compositions d'extraction et d'isolation rapides de proteines et de peptides au moyen d'une matrice de lyse
US20090325269A1 (en) * 2008-06-30 2009-12-31 General Electric Company Bacteria/RNA extraction device
US20110129931A1 (en) * 2009-10-20 2011-06-02 Agency For Science, Technology And Research Microfluidic system for detecting a biological entity in a sample
EP2602315A1 (fr) * 2011-12-05 2013-06-12 Pall Corporation Purification de leucocytes
WO2014151177A1 (fr) * 2013-03-15 2014-09-25 3M Innovative Properties Company Concentrateur d'échantillon et méthode d'utilisation
US20140356890A1 (en) * 2013-06-03 2014-12-04 Samsung Electronics Co., Ltd. Method of re-collecting target material

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Manual of Aquatic Viral Ecology", 13 May 2010, AMERICAN SOCIETY OF LIMNOLOGY AND OCEANOGRAPHY, ISBN: 978-0-9845591-0-7, article GRIEG F. STEWARD ET AL: "Extraction and purification of nucleic acids from viruses", pages: 154 - 165, XP055233334, DOI: 10.4319/mave.2010.978-0-9845591-0-7.154 *
J. A. MUELLER ET AL: "Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses, Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 80, no. 13, 18 April 2014 (2014-04-18), US, pages 3930 - 3942, XP055233031, ISSN: 0099-2240, DOI: 10.1128/AEM.00245-14 *
KESBERG ANNA ISABELLA ET AL: "Improved protocol for recovery of bacterial DNA from water filters: Sonication and backflushing of commercial syringe filters", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM, NL, vol. 93, no. 1, 16 February 2013 (2013-02-16), pages 55 - 57, XP028997979, ISSN: 0167-7012, DOI: 10.1016/J.MIMET.2013.02.001 *
O'MAHONY K ET AL: "Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 119, no. 2, 23 September 2005 (2005-09-23), pages 118 - 132, XP027663503, ISSN: 0168-1656, [retrieved on 20050923] *

Similar Documents

Publication Publication Date Title
AU2012347741B2 (en) Method and device for sample processing
US20080153078A1 (en) System for isolating biomolecules from a sample
EP2870233B1 (fr) Méthode de filtration d'un échantillon biologique
KR20140147805A (ko) 지방조직으로부터 비지방 세포의 단리를 위한 방법 및 장치
US10364414B2 (en) Filter based system for capture of lysis of suspended cells
EP3199617B1 (fr) Systèmes et procédés de séparation de cellules à partir des microsupports à l'aide d'une membrane rotative
WO2017069781A1 (fr) Système et procédé à base de filtre pour la capture et la lyse efficaces de cellules en suspension
AU2020329234A1 (en) Control systems and methods for automated clarification of cell culture with high solids content
US9522350B2 (en) Method and device for the concentration of multiple microorganisms and toxins from large liquid toxins
US11946033B2 (en) Micro alternating tangential flow perfusion filter, micro bioreactor, and methods of use thereof
CN206635330U (zh) 一种病毒颗粒抗原纯化浓缩设备
KR20230131239A (ko) 핵산 추출 장치, 키트 및 방법
CN108441471B (zh) 一种血液中稀有细胞的分离方法
US20150104825A1 (en) Capture, disruption, and extraction apparatus and method
EP3893956B1 (fr) Appareil de lavage de cellules
US20240027424A1 (en) Sample preparation device and sample preparation system
CN108067147A (zh) 一种液体混合机及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15791441

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15791441

Country of ref document: EP

Kind code of ref document: A1