Method and apparatus for the purification of extra-chromosomal nucleic acids sequences

Abstract

The present invention relates to a new apparatus and a new method for the purification and the recovery of extra-chromosomal nucleic acids sequence(s).

Claims

1. A method for obtaining one or more extra-chromosomal nucleic acids from cells and comprising the steps of: a) optionally cultivating cells comprising the extra-chromosomal nucleic acids of interest, b) disintegrating the cells by mixing the cells in suspension with a lysis medium to form lysed cells, c) neutralizing the lysed cells by adding a neutralization solution to said lysed cells to produce a first cells lysate mixture comprising a soluble fraction and a precipitate, d) optionally further precipitating contaminants away from the extra-chromosomal nucleic acids of interest to obtain a second cells lysate mixture, by mixing the first cells lysate mixture with a solution containing one or more salt(s), wherein the salt(s) comprise one or more of CaCl.sub.2), MgCl.sub.2, ZnCl.sub.2, SrCl.sub.2, BaCl.sub.2, LiCl, ammonium acetate, ammonium sulfate, sodium sulfate and magnesium sulfate, e) collecting the cells lysate mixture obtained from step c) or from step d), f) separating the soluble fraction comprising the extra-chromosomal nucleic acids of interest of the cells lysate mixture from the precipitate in a separation tank, and g) recovering the separated soluble fraction comprising the extra-chromosomal nucleic acids of interest, wherein the separation step f) consists of dissolving a gas in a liquid aqueous medium under a pressure higher than atmospheric pressure followed by injecting the liquid aqueous medium comprising the dissolved gas into the cells lysate mixture contained in the separation tank.

2. The method of claim 1, wherein the gas is dissolved in the liquid aqueous medium by gas bubbling under pressure of at least 2 barg.

3. The method of claim 1, wherein the liquid aqueous medium comprising the dissolved gas is injected into the cells lysate mixture in a volume amount of between 0.2% and 25% v/v, relative to the volume of the cells lysate mixture.

4. The method of claim 1, wherein the liquid aqueous medium comprising the dissolved gas is injected into the cells lysate mixture under a pressure of at least 2 barg and wherein the cells lysate mixture is at atmospheric pressure in the separation tank.

5. The method of claim 1, wherein the liquid aqueous medium comprising the dissolved gas injected into the cells lysate mixture is saturated with the gas.

6. The method of claim 1, wherein the liquid aqueous medium comprising the dissolved gas is injected downwards towards the bottom of the separation tank.

7. The method of claim 1, wherein the injection of the liquid aqueous medium comprising a dissolved gas is a single injection or is successive injection.

8. The method of claim 1, wherein the neutralization of lysed cells from step c) with a neutralization solution is done by a static mixer having a mixing linear speed equal to or higher than 100 cm/min.

9. The method of claim 1, wherein the lysis medium is a mixture of NaOH and a detergent.

10. The method of claim 1, wherein the liquid aqueous medium comprising the dissolved gas is injected into the cells lysate mixture in a volume amount of between 0.5% and 15% v/v, relative to the volume of the cells lysate mixture.

11. The method of claim 9, wherein the detergent comprises Sodium Dodecyl Sulfate (SDS).

12. An apparatus for carrying out the method of claim 1 to obtain one or more extrachromosomal nucleic acids of interest from cells, the apparatus comprising: a preparation unit comprising one or more tanks configured to contain constituents of a lysis medium and comprising an outlet, a cells lysis unit comprising a cells suspension tank configured to contain cells with the extra-chromosomal nucleic acids of interest, and further comprising an inlet and an outlet, wherein the inlet is fluidly connected to the cells suspension tank and with the outlet of the preparation unit, a neutralization unit configured to generate a first cells lysate mixture, the neutralization unit comprising a neutralization solution tank, an inlet and an outlet, wherein the inlet is in fluid connection to the neutralization solution tank and with the outlet of the cells lysis unit, optionally a precipitation unit configured to obtain a second cells lysate mixture, the precipitation unit comprising a divalent ion salt(s) solution tank, an inlet and an outlet, wherein the precipitation unit inlet is in fluid communication with the outlet of the neutralization unit to receive the first cells lysate mixture, a separation unit configurated to collect the cells lysate mixture, the separation unit comprising a separation tank having an inlet fluidly connected to the outlet of the neutralization unit or the outlet of the precipitation unit, and an injection unit configured to inject a liquid aqueous medium comprising a dissolved gas into the separation tank, the injection unit comprising a liquid aqueous medium tank and a bubbling device configured to bubble a gas to be dissolved into the liquid aqueous medium tank, at a pressure higher than atmospheric pressure.

13. The apparatus of claim 12, wherein the injection unit comprises one or more injection pipe(s) having an exit disposed towards a bottom surface of the separation tank.

14. The apparatus of claim 13, wherein the one or more injection pipe(s) is (are) disposed to inject the liquid aqueous medium into the separation tank at a height located between ? and ? of the total height of the separation tank from the tank bottom.

15. The apparatus according to claim 12, wherein the bubbling device is configured to inject a gas to be dissolved into the liquid aqueous medium tank, at a pressure higher than or equal to 2 barg.

16. The apparatus according to claim 15, wherein the bubbling device is configured to keep the liquid aqueous medium tank under a pressure of at least 2 barg.

17. The apparatus according to claim 12, wherein the injection unit is configured to inject the liquid aqueous medium having the dissolved gas intermittently into the separation tank.

18. The apparatus of claim 12, wherein the preparation unit, the cells lysis unit, the neutralization unit and/or the precipitation unit comprise(s) one or more static mixer(s).

19. The apparatus of claim 13, wherein the one or more injection pipe comprises an exit nozzle and wherein the ratio between the injection pipe internal diameter and exit nozzle diameter is at least 2.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents schematically the apparatus of the invention.

(2) FIGS. 2 and 3 represent different configurations of the separation unit of the apparatus according to the invention.

(3) FIG. 4 represents the injection unit connected to the separation unit of the apparatus according to the invention.

(4) FIG. 5 represents a filtration unit connected to the separation unit of the apparatus according to the invention.

(5) FIG. 6 represents separation results obtained with methods and the apparatus of the state of the art.

(6) FIG. 7 represents separation results obtained with the method and apparatus according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

(7) The FIG. 1 illustrates the apparatus allowing to carry-out steps of the method of the present invention, being a pipe or tube of several meters long, made of several tubes or tubing elements having circular section and fluidly connected (or linked) together and connected to static mixers and tanks or reservoirs.

(8) Preferably, the apparatus and the method according to the invention are dedicated to the recovery and to the purification from cells, of extra-chromosomal nucleic acids sequence(s), such as DNA plasmid(s) that will be purified from cells impurities (or contaminants), especially from E. coli contaminants, such as host cells proteins, RNA sequences, genomic DNA sequences, endotoxins, . . . .

(9) The apparatus used in the method according to the invention comprises or consists of several units that are fluidly connected and include a preparation unit 1, a cells lysis unit 20, a neutralization unit 30, optionally a precipitation unit 40, a separation (decantation or clarification) unit 50, an injection unit 70 and a filtration unit.

(10) Furthermore, the cells lysis unit 20 could be fluidly connected to a production unit (including a reaction tank not represented in the figures), but used for the growth and multiplication of cells comprising the extra-chromosomal nucleic acids sequence(s) of interest to be recovered.

(11) In the passageway of the apparatus according to the invention the flow rates follow-up is obtained by chronometers, weight monitoring and/or by flow meters added to the different tubing(s) and connections between tubing(s) are of the Y type

(12) The first part of the apparatus according to the invention, is a preparation unit 1 which can present two different formats. According to a first embodiment, not presented in the figures, the apparatus according to the invention contains a lysis medium tank or reservoir with the (alkaline) lysis medium connected by a first tubing and a pump and possibly a flowmeter, to the lysis unit 20 to mix the cells with the added (alkaline) lysis medium.

(13) According to another embodiment of the present invention and as presented in the figures, the preparation unit 1 is advantageously made of at least two separate tanks (2 and 3), both maintained at room temperature, the first tank 2 containing NaOH (RM2-A) and the second tank 3 containing one or more detergents (RM2-B), preferably Sodium Dodecyl Sulfate (SDS).

(14) Both tanks 2 and 3 are in fluid connection respectively to a first set of tubing elements 4 and 5, preferably via a first pump 6 and a second pump 7, wherein these pumps (6 and 7) are controlling the adequate added amounts of each reactive product (NaOH, detergent and possibly one or more other molecule(s)) are mixed and fed to a first static mixer 9 to form the lysis solution.

(15) The reactive compounds present in the tubings of the first tubings set (4 and 5) meet in a Y type connection and are pushed, via an inlet or opening 8 into the first static mixer 9 used for the efficient homogeneous mixing of all the introduced compounds, to form the lysis buffer (RM2 buffer medium). This mixing insures the stability and constant composition quality of the (alkaline) lysis medium. A homogeneous mixing means a mixing that is homogeneous according to the naked human eye.

(16) The outlet or exit 10 of this first static mixer 9 of the preparation unit 1 is in fluid connection, via a second tubing 11 to the inlet or opening 21 of a second static mixer 22 which is part of the cells lysis unit 20. Preferably, the second static mixer 22 of the cells lysis unit 20 comprises between 12 and 24 mixing elements, preferably 18 mixing elements.

(17) The cells lysis unit 20 is the passageway described above and preferably includes the second static mixer 22, which is in fluid connection by a Y-Type connection at its inlet 21, to both the second tubing 11 of the preparation unit 1 and, via a third tubing 23 and a pump 24, to a cells suspension tank 25 containing at a suitable temperature, preferably at a temperature comprised between about 2? C. and about 8? C., the suspended cells with the extra-chromosomal nucleic acids sequence(s) to recover.

(18) The lysis or the cells disintegration is performed by addition of the cells lysis buffer, being preferably a mixture of NaOH+SDS, from the first static mixer 9 and the suspended cells from the cell suspension tank 25 to the second static mixer 22. Although the generated solution of disintegrated cells is viscous, an efficient homogenization was obtained without degrading the plasmid.

(19) The inventors have optimized the tubing diameter and lengths and the pump output in order to assess the optimal mean contact time of the cells and the lysis buffer. The apparatus that they developed allows surprisingly short mean contact times, such as less than 5 minutes preferably between (about) 1 minute and (about) 5 minutes, more preferably (about) 2 minutes, which advantageously prevents the degradation of the extra-chromosomal nucleic acids sequence(s) of interest, preferably the DNA plasmid.

(20) The outlet or exit 26 of this second static mixer 22 is in fluid connection to the features of the neutralization unit 30, via a fourth tubing 27 having an adequate length and diameter allowing to obtain the optimal contact time for the recovering of the cells lysate.

(21) The neutralization unit 30 comprises a neutralization tank 31 containing a neutralization medium (RM3), preferably made of a solution of acetic acid and acetate, preferably maintained at a pH comprised between about 5.0 and about 6.0 preferably of about 5.5 and consisting of 3M of acetate and 15% (v:v) of acetic acid, preferably used at a temperature comprised between 2? C. and 8? C., preferably of about 4? C.

(22) This neutralization tank 31 is in fluid connection, via a fifth tubing 32 and via a pump 33, to an inlet or opening 34 of a third static mixer 35. The neutralization medium can be cooled before use by storage of the neutralization tank 31 in a cold room for a sufficient amount of time, or cooled down in a continuous manner from room temperature (comprised between 20? C. and 25? C.) to a lower temperature comprised between 2? C. and 8? C., preferably of about 4? C., by a cryostat 36 and a heat exchanger 39 surrounding a section of the fifth tubing 32.

(23) The inlet or opening 34 of this third static mixer 35 is also I fluid connection to the fourth tubing 27 providing the cells lysate. Preferably, this third static mixer 35 comprises between 6 and 16 mixing elements, preferably 10 mixing elements.

(24) The inlet 34 of this third static mixer 35 is preferably in fluid connection by a Y-type connection to the two tubing elements 32 and 27, so that both fluids flows are sent to the static mixer 35 to obtain a rapid and efficient neutralization of the cells lysate to stop the lysis reaction and to form a neutralized cells lysate.

(25) The inventors have further optimized the tubing diameter and length, and the pump output in order to assess the optimal mean contact time of the lysed cells under neutralization. The apparatus developed allows short mean contact time, such as less than 3 minutes, preferably of (about) 0.5 minute to (about) 3 minutes, more preferably (about) 1 minute, which advantageously leads to an efficient neutralization of the lysed cells before the addition of the precipitation agent.

(26) The outlet or exit 37 of this third static mixer 35 is in fluid connection to a sixth tubing 38 collecting the obtained neutralized lysate mixture. The sixth tubing 38 can be directed and is in fluid connection to the inlet or opening 45 of a fourth static mixer 41 forming part of the precipitation unit 40.

(27) The inlet or opening 45 of the fourth static mixer 41, is also in fluid connection to a seventh tubing 42, via a pump 43, to a divalent salt(s) solution tank 44 comprising a solution of divalent ions salt(s) (RM4), preferably hydrated calcium chloride.

(28) Again, a Y-type connection is preferably used between both tubings 38 and 42, so that both fluids flows are sent to the fourth static mixer 41 to obtain a rapid and efficient mixing. A solution of divalent salt(s) pumped from the tank 44, via the seventh tubing 42 to the inlet 45 of the fourth static mixer 41, is added continuously to the neutralized cells lysate added into the fourth static mixer 41 from the sixth tubing 38 to obtain a cell lysate mixture. This fourth static mixer 41 insures a thorough mixing of the dense and viscous solutions and improves the precipitation reaction. However, others means than this static mixers, including means for generating a Venturi effect, can be used for the efficient mixing of fluids, including the dense and viscous divalent salt(s) solution obtained from the tank 44.

(29) This fourth static mixer 41 comprises between 4 mixing elements and 18 mixing elements, preferably only 4 mixing elements. An optimum number of 10 mixing elements could be selected for an efficient mixing of the salts and the neutralized lysed cells to obtain the cells lysate mixture. However, as a higher number of mixing elements will produce smaller particles and precipitate, which are less easily eliminated from the soluble fraction during separation (decantation or clarification) and would reduce the production and purification yield and increase purification time.

(30) The outlet or exit 46 of the fourth static mixer 41 is in fluid connection, via an eighth tubing 47 to a separation tank 51 of the separation unit 50. The cells lysate mixture 52 is supplied to this separation tank 51 from the fourth static mixer 41. It will be convenient to note that the fourth static mixer 41 is optional, and tubing 38 can be directly fed to separation tank 51 of the separation (or decantation or clarification) unit 50.

(31) In the apparatus according to the invention, the separation unit 50 is used for separation, decantation or clarification of a cells lysate mixture 52 from the sequence(s) of interest from impurities (or contaminants) and is improving the yield, especially the delay and the efficiency of separation, decantation or clarification and improving recovery of the extra-chromosomal nucleic acids sequence(s) of interest from this cells lysate mixture 52.

(32) The separation unit 50 represented in FIGS. 2 and 3 is connected to an injection (floatation) unit 70 represented in FIG. 4 and allowing injection inside the separation tank 51, of a liquid aqueous medium more preferably water, comprising large amounts of a dissolved gas being preferably air, nitrogen, CO.sub.2, oxygen or ozone, more preferably air into the separation tank 51. This liquid aqueous medium is maintained at room temperature with the gas, preferably air, and introduced at a suitable pressure, preferably a pressure of at least 2 barg.

(33) The separation, decantation or clarification is efficiently improved by means of an injection of this liquid aqueous medium, preferably water comprising saturated or near-saturated levels of the dissolved gas, preferably selected from the group consisting of air, CO.sub.2 or nitrogen, or a mixture thereof, via one or more injection pipe(s) 53 having an exit 54, possibly comprising a nozzle into the cells lysate mixture 52 present in the separation tank 51. The exit 54 of this injection pipe 53 is disposed in the direction opposite to the surface 55 of the cells lysate mixture 52 present in the separation tank 51 and towards or in the direction of the bottom surface 58 of the separation tank 51.

(34) As represented in FIG. 3, the configuration of the decantation unit 50 may include a separation tank 51 wherein more than one injection pipe(s) 53 are introduced, preferably two, three or four injection pipes 53, possibly arranged at diagonally opposite positions in the separation tank 51, preferably with an exit 54 of each injection pipe 53 being disposed in the same plane parallel to the plane formed by the bottom of the separation tank 51 and advantageously at equivalent distance from each other and from the sides and bottom of the decantation tank. In FIG. 3 four injection pipes 53 are represented at diametrically or diagonally opposite positions.

(35) As represented in the FIG. 3, the exit 54 of each injection pipe 53 is disposed facing away from the top surface 55 of the cells lysate mixture 52 present in the separation tank 51 and this exit 54 is facing the tank bottom surface 58, but oriented to inject the liquid aqueous medium vertically downwards the direction of this bottom surface 58. This configuration avoids the formation of important convection movement in the cells lysate and avoids the dispersion of the precipitates or flocs from the top precipitate layer.

(36) In the separation unit 50 of the apparatus according to the invention, the cells lysate mixture 52 is introduced by the eighth tubing 47 disposed along the side wall of the separation tank 51 and fills partially the separation tank 51. There should remain a free volume in this separation tank 51 sufficient to handle the addition of the liquid aqueous medium saturated with gas under pressure.

(37) The exit 54 of the injection pipe(s) 53 is advantageously positioned at a height 57 from the bottom of the separation tank 51 between ? and ? of the height 56 of this separation tank 51, more advantageously positioned at a height 57 from the bottom of the separation tank 51 between ? and ?.

(38) The injection pipe 53 may present a nozzle exit 54, with a narrow exit. Advantageously the injection pipe 53 presents a ratio between its internal diameter and the diameter of its nozzle exit 54, which is higher than or equal to (about) 2, more preferably higher than or equal to (about) 3, and may be lower than or equal to 50. This configuration and ratio between the internal diameter pipe size and its nozzle exit ensure that the depressurization of the liquid aqueous medium only starts at the nozzle itself, and not within the injection tubing 53. By so doing, the liquid aqueous medium with the dissolved gas is kept at a suitable elevated pressure up to the exit nozzle, so that pressure drops at the nozzle exit only. However, in specific configuration of the apparatus of the invention allowing production of lower amounts of extra-chromosomal nucleic acids sequence(s) of interest, this ratio is of (about) 1 or near the value of 1 with an injection pipe 53 that neither include a nozzle and nor a reduction of the exit 54 diameter. As a result, a rapid and efficient separation, clarification or decantation of the cells lysate mixture 52 from its contaminants or impurities, which are dispersed in the separation tank 51 can be obtained. The contaminants or impurities will float at the top surface 55 of the cells lysate mixture 52. Preferably, this separation duration is comprised between (about) 5 minutes and (about) 3 hours, but could be applied for longer periods of up to 1 or 2 days.

(39) As represented in FIG. 4, the added liquid aqueous medium containing the dissolved gas is obtained from the injection pipe 53 which is connected, preferably through a valve 71 to the injection or floatation unit 70.

(40) The injection (or floatation) unit 70 comprises a tank 72 containing a liquid aqueous medium, preferably water. This liquid aqueous medium tank 72 comprises an inlet, advantageously arranged in a lower part of the tank 72, which is connected, preferably via a valve 73 to a tubing element 74 of a bubbling device, supplying in the liquid aqueous medium tank 72 a gas, preferably air. Additional tubing elements (75 and 76) and valves (77 and 78) for a gas (air) introduction and expulsion can be connected to the aqueous medium tank 72.

(41) The liquid aqueous medium tank 72 is advantageously a closed vessel arranged to hold a liquid under suitable elevated pressure. The separation (or decantation) tank 51 is advantageously configured to hold the cells lysate mixture 52 at substantially atmospheric pressure.

(42) The liquid aqueous medium is enriched and advantageously saturated with the dissolved gas under pressure, by a bubbling of the pressurized gas obtained from the bubbling device into the liquid aqueous medium. By so doing, this liquid aqueous medium is enriched in the dissolved gas (Henry's law). Advantageously, the bubbling is performed for a sufficient amount of time to reach the complete saturation. The pressure in the liquid aqueous medium tank 72 is advantageously kept constant during the bubbling, at a value of 2 barg or more, preferably between 2 barg and 50 barg, more preferably between 3 barg and 25 barg.

(43) The liquid aqueous medium enriched in dissolved gas is injected in the separation tank 51 advantageously during a period comprised between a few seconds and a few minutes, preferably between 5 seconds and minutes. Preferably, in the method and apparatus according to the invention, the volume % of the injected liquid aqueous medium is comprised between 0.2% and 25% (v/v), preferably comprised between 0.5% and 15%, more preferably comprised between 1% and 10%, compared to the volume of the (decanted) cells lysate mixture.

(44) Referring again to FIGS. 2 and 3, the separation tank 51, further includes a collecting tubing 61 for recovering the soluble fraction or clarified phase containing the extra-chromosomal nucleic acids sequence(s) of interest, from the cells lysate mixture 52 present into the separation tank 51.

(45) The inlet of the collecting tubing 61 is advantageously disposed at the bottom of the separation tank 51 for obtaining an efficient pumping of the soluble fraction or clarified phase with minimising liquid movements in the separation tank 51 to avoid a re-suspension of the contaminants or impurities precipitates. Tubing 61 advantageously removes the soluble fraction from separation tank 51 in the bottom part 57, below the level of exit 54 of injection pipe 53. Tubing 61 can be connected to a filtration unit.

(46) A further purification with known means (such as filters, chromatographic columns and collecting tanks) and method steps well known by the skilled person can also be performed.

(47) As represented in FIG. 5, the flow of this soluble fraction or clarified phase of the cells lysate mixture, outside the separation tank 51 is advantageously obtained through the opening of a valve 62 and a pump 63 connected to another section of a tubing element 64 allowing the flow of the soluble fraction or clarified phase of the cells lysate mixture towards another filtration unit 60 for its further purification with methods steps well known by the skilled person. This filtration unit can comprise one or more filters 65 and 66 and/or one or more chromatographic columns with suitable elution material. Furthermore during filtration, ultrafiltration and/or diafiltration, one or more tubing section(s), especially section(s) of the tubing element 64 could be surrounded by a heat exchange device 67 connected to a cryostat 68 for a cooling of the filtrated and clarified lysate to a temperature as closed to 4? C. as possible (in the rage of 2? C. to 8? C.) preferably a temperature that can be maintained for an adequate time (overnight) in suitable (and possibly refrigerated) tank 69.

(48) In the apparatus and method according to the invention, the tubing elements may include flow meters or other devices for controlling the introduction of suitable volumes (per unit of time) of the different fluids necessary for the lysis system, the tanks or reservoirs. The apparatus may also include weight monitoring elements, such as weight balances of the different tanks or reservoirs to measure the amount of introduced solution or active compounds (lysis solution, neutralization solution, cells in suspension, liquid aqueous medium saturated in gas under pressure) at each step of the method according to the invention. Conditions and efficiency of purification obtained by the apparatus and the method of the invention are summarized in the following table 1 which presents an overview of possible process conditions.

(49) TABLE-US-00001 TABLE 1 Liquid aqueous 4? C. to room temperature medium temperature (the air dissolves much slower at 4? C.) Air pressure 6 barg Compressed gas >=1 minutes bubbling in the aqueous medium duration Lysate volume From almost nothing (less than 1 liter) up treated to cubic meters (3 ? 1500 L planned for the Step2 capacity expansion at KEGT), depending on the injection system. Liquid aqueous From 0.5 to 20% per injection, successive medium volume (% injections improving the floatation effect v/v) Liquid aqueous From 5 seconds to 5 minutes depending on medium injection the injection flow rate and injected volume. duration Floatation Between 5 min and 3 hours, typically 15-30 duration min between each injection or before collection. Full process From cells lysis to final 0.2 ?m duration filtration: less than around 8 hours at any scale. separation Volume of clear lysate recovered/total efficiency volume (lysate + precipitates layer): By gravimetric decanting: 70% to 75% recovery With floatation: 85% to 90% recovery .Math. 10 to 15% volume recovery gain Turbidimetry By gravimetric decanting: about 50 NTU reduction With floatation: <20 NTU .Math. More than 2x turbidimetry reduction .Math. Depth filtration membrane area needed decreased by about the same factor.

(50) The advantages brought by the new process and apparatus of the invention are numerous. It is a simple and robust, efficient, reproducible and automatable process and apparatus (or plant) can be used.

(51) A goal of the process and apparatus according to the invention is to keep separation duration very low, preferably at a maximum of two hours. This goal was reached and even exceeded, the full separation being obtained after only about 1 hour or even lower time.

(52) Another goal is to obtain a robust separation (decantation or clarification) where, for each run, almost no particles were left on the bottom of the separation tank (51) or dispersed in the cells lysate mixture, as they quickly clog the clarification depth filters and should not be pumped. This goal is reached as almost all particles are floating on the top of the separation (decantation or clarification) tank 51 after the floatation, and even exceeded as the clarified fraction of the lysate is much clearer (decreased turbidity) than with a classical gravimetric separation step. As a consequence, less depth filters surface is needed for separation, which means lower costs, wastes, space needed in the zone, handling, etc.

(53) An additional goal is to enhance the recovery of the (total) soluble fraction or clarified phase, and is also reached as the soluble fraction or clarified phase lost in the precipitates layer is now replaced by a gas, such as air, this precipitates layer floating above this soluble fraction or clarified phase instead of being immersed in it. The volume of recovered soluble fraction or clarified phase has therefore been increased by 10% to 15%.

(54) A further goal of the new process and apparatus of the invention is the treatment of large amounts of cells pellets, preferably by increasing the flow rates, while keeping at least the same process duration time (lower than about 4 hours) for every scale and to obtain the recovery of large amounts (kgs) of purified extra-chromosomal nucleic acids sequence(s) of interest.

(55) The inventors have also tested the direct injection of gas bubbles, instead of the liquid aqueous medium comprising the dissolved gas into the cells lysate mixture contained in the separation tank 51. However, the injection of gas bubbles generates down to up movements due to bubble rising to the top, and therefore a mixing of the soluble fraction with the precipitate layer, containing impurities and contaminants of the extra-chromosomal nucleic acids sequence(s) to be recovered from the cells lysate mixture. This mixing and introduction of flocs and others solid particles, including contaminants of the extra-chromosomal nucleic acids sequence(s) to recovered in the soluble fraction or clarified phase, will reduce the efficiency of this soluble fraction recovery and also the extra-chromosomal nucleic acids sequence(s) purification.

(56) On the other side, in the method and apparatus according to the invention, the injection of the liquid aqueous medium comprising dissolved gas into the cells lysate mixture, allows advantageously a complete or almost complete recovery of the soluble fraction or clarified phase that is not, or almost not contaminated by the precipitate layer made of flocs floating above the soluble fraction of the cells lysate mixture.

(57) In addition, the method and apparatus according to the invention does not contain gas injection frit or other micro-porous material that would be necessary to reduce the bubbles sizes, such devices being very prone to be clogged by the precipitated particles and also being an important source of cross-contaminations from batch to batch due to their poor cleanability. The design of the method and apparatus according to the invention allows after the soluble fraction (or clarified phase) of the cells lysate recovery, a rapid and efficient subsequent washing of the separation (decantation or clarification) means, especially the separation tank 51 for its further use, possibly through injection of washing liquid(s) from the injection pipe 53 or from others tubing elements into this separation tank 51.

(58) After lysis, the precipitates have a density similar to the liquid phase (soluble fraction or clarified phase) of the cells lysate. Still, they often tend to rise very slowly to the top of the separation tank due to a few microbubbles sticking to them, which are coming from small amounts of gas dissolved in the starting reagents. The addition of gas bubbles as proposed in the state of the art publication, in continuous during the lysis process is a way to increase the amount of bubbles to help this floatation effect. On the other hand, in the method of the state of the art, the binding strength of the bubbles to the precipitates is very low, so mixing movements or agitation easily unsticks the bubbles from the precipitates, breaking the floatation effect and making them sink again.

(59) At the exit of a continuous lysis system, the lysate must be sent to a separation tank, where the precipitate made of flocs 101 or particles 100 (made of the impurities of the sequences(s) of interest to be recovered) are separated from the soluble fraction. If the cells lysate is sent to the bottom (exit immersed into the cells lysate) and if gas bubbles are added in continuous during the lysis, as proposed in the state of the art, those gas bubbles will quickly go up right above the exit (like an air pump in an aquarium) and will produce an upward stream within the cells lysate, leading to mixing movements preventing the precipitates to settle at the top of the separation tank correctly. If the cell lysate is sent to the top of the separation tank, it will also create mixing movements within the top layer of particles 100 that have already started to float and will push them back into the solution, breaking part of the floatation effect. These mixing movements are one of the drawbacks of the state of the art requiring continuous addition of bubbles during the lysis process. Another drawback comes from the continuous nature of the treatment. New cells lysate with precipitates and bubbles are added continuously to the separation tank that already contains the cells lysate previously generated and which has already started to settle. This leads to treatment heterogeneity in the separation tank.

(60) The present invention brings a solution to avoid these mixing movements and treatment heterogeneity by performing the floatation in a static manner, when the lysis is finished and the separation tank 51 is filled. Injecting the liquid aqueous medium with the dissolved gas under pressure allows the treatment of the whole cells lysate volume at the same time. As a liquid is injected instead of a gas, it quickly mixes to the whole volume to treat. Right after injection, due to the fact that the separation tank is at atmospheric pressure, the gas that was dissolved in the liquid medium under pressure (i.e. in a liquid phase) turns back into the gaseous phase, mainly in contact with the particles playing the role of nucleation centers. Therefore, the microbubbles stick to the precipitates and all particles rise at the same time in a linear way to the top of the separation tank 51, pushing each other in the same direction. This creates a strong plunger effect: the particles being above are pushed further out of the soluble fraction of the cells lysate by the ones being below. This plunger effect leads to much higher separation efficiency. After treatment, most of the particles have not only been separated at the top of the separation tank 51, but also pushed out of the liquid phase or Gas coming from the injection of the liquid medium saturated with a dissolved gas, will replace the liquid cell lysate that was previous entrapped in the spaces between the particles and push the precipitate layer (101), thus significantly increasing the recovery yield, as shown in the following table 2 and within the comparative FIGS. 6 and 7 (see volume gain 102 with the applied floatation method of the invention and remaining particles 100 obtained with the applied gravimetric method of the state of the art).

(61) Finally, the present invention is designed in such a way that succeeding injections of the aqueous liquid medium with the dissolved gas under pressure is performed without dispersing again the particles that have been separated. Each succeeding injection decrease the amount of micro-particles left in cell lysate and eases clarification

(62) TABLE-US-00002 TABLE 2 Gravimetric Floatation Volume recovery 70-75% 85-90%