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• • 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
• • 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

Definitions

• the present invention relates to the automated extraction of a plasmid of interest produced by a bacterium.
• a plasmid of interest in a bacterium such as Escherichia coli
• alkaline lysis followed by a neutralization and precipitation step eg Birnboim and Dolly, 1979, Nucleic Acids Research, 7, pages 1513-1523.
• This method is simple to put into practice and works well in discontinuous mode (“batch”); it is suitable for the production of small quantities of plasmids, such as the order of a gram, at most.
• Patent application WO 2010/136503 (also published, among others under EP2435569B1, US8,822,672 e ⁇ US 9,416,400) describes a process for the continuous extraction of plasmids, based on a pipe arrangement system for the different solutions and ⁇ their mixtures e ⁇ flow control by pumps.
• this method provides for the addition of a concentrated precipitation solution.
• the neutralization and precipitation solutions are ⁇ homogeneously mixed by adapting the internal diameter of the piping, so as to cause a Venturi effect locally.
• this system is advantageously disposable, so that it does not require cleaning.
• this system is ⁇ very effective for very large quantities of plasmids to be purified, for example 100 g of plasmid, it entails fixed costs (disposable device, losses of plasmid) which become significant if the quantities of plasmids to be purify his weakest ⁇ .
• discontinuous mode the efficiency of systems in discontinuous mode is limited by physical parameters such as the size of the containers, the time for bringing the solutions into contact, or even the need for rapid homogenization.
• an ideal purification is achieved from low volumes, which allows good control of the mixtures, but greatly complicates production on a larger scale.
• the present invention relates to a method for extracting a plasmid synthesized by a microorganism comprising the successive steps of: obtaining a receptacle 1 coupled to a withdrawal pump 8, said receptacle 1 being provided with a means of mechanical stirring 9, obtaining a cell suspension 2 of said microorganism comprising said plasmid to be extracted; adding to said cell suspension 2 an alkaline lysis solution 3 at a predetermined rate (Q 1 ) so as to form a homogeneous mixture; placing at a predetermined rate (Q2) said homogeneous mixture in said receptacle 1; after a predetermined time, preferably with gentle agitation, adding thereto at a predetermined rate (Q3) a neutralization solution 5 comprising acetic acid in said receptacle, via a plurality of orifices 7; after a predetermined time, adding thereto at a predetermined rate (Q4) a precipitation solution 6 in said receptacle, via a
• This extracted and purified plasmid can advantageously be used directly, or after sterilizing filtration, ultrafiltration and/or polishing by chromatography.
• Figure 1 shows semi-schematically a container according to the invention.
• Figure 2 shows an HPLC analysis of plasmid exfrai ⁇ by the automated method according to the invention
• Figure 3 shows the effect of variations #1; #2; #3 e ⁇ #4 made to the preferred automated process.
• the inventors have succeeded in developing a process for extracting plasmids which retains the flexibility of a process in batch mode, for ⁇ by making it possible to process large quantities of plasmid e ⁇ with an extraction yield e ⁇ a level of purity very high.
• a first aspect of the present invention is ⁇ a method for extracting a synthesized plasmid comprising the successive steps of: obtaining a receptacle 1 coupled to a withdrawal pump 8, said receptacle 1 being provided with a means of mechanical agitation 9, obtaining a cell suspension 2 comprising said plasmid to be extracted; adding to said cell suspension 2 an alkaline lysis solution 3 at a predetermined rate Q1 so as to form a homogeneous mixture; placing at a predetermined rate Q2 said homogeneous mixture in said receptacle 1; after a predetermined time, preferably with gentle stirring, add thereto at a predetermined rate Q3 a neutralization solution 5 comprising acetic acid in said receptacle, via a plurality of orifices 7; after a predetermined time, adding thereto at a predetermined rate Q4 a precipitation solution 6 in said receptacle, via a plurality of orifices 7; after a predetermined time with gentle stirring, withdraw
• the cells synthesizing the plasmid of interest are typically Gram-negative bacteria, for example Escherichia coli. However, other Gram-negative or even Gram-positive bacteria, or even other microorganisms, such as Saccharomyces Sp. or Pichia Sp. may also be suitable.
• the plasmid is advantageously in so-called “super coiled” form.
• the size of the plasmid is not limiting because the present method also works with large plasmids.
• larger plasmids eg > 10 kb, e.g. between 10 and 20 kb
• plasmids encoding viral vectors and/or comprising repeated and/or inverted sequences impose a more precise control of the times, in particular of the contact time with the alkaline lysis medium e ⁇ of the addition time e ⁇ of contact with the neutralization solution.
• the size of the receptacle 1 is not particularly limited. Receptacles 1 that are too small do not allow the treatment of sufficient quantities of plasmids, whereas receptacles 1 filled with too large quantities of solution risk requiring too much pumping time, which is penalizing, since this complicates, even prevents the control of different times. Indeed, the solutions being pumped one-by-one, there is a heterogeneity at the beginning of each step: too long a pumping time will therefore cause a double heterogeneity at the level of the compositions e ⁇ of the contact times.
• receptacles 1 filled in the end (after incorporation of the 2+3+5+6 solutions) with 5 to 10 liters of the solutions were very simple to use.
• receptacles 1 filled in the end (after incorporation of the solutions 2+3+5+6) per 100 liters become too complex to control.
• a preferred size of usable volumes of receptacles are between 1 liter and 50 liters, preferably between 2.5 liters and 20 liters, even more preferably between 5 liters and 10 liters.
• receptacles 1 of larger, or even much larger, capacity can be used, even if they are only intended to be filled with a maximum of 2.5 to 10 liters.
• the inventors have determined that the contact between the cell suspension and the lysis solution is advantageously between 2 and 5 minutes.
• the inventors have deduced that the addition of the solutions 2 e ⁇ 3 can be done in, for example, one minute, without this causing any problem.
• the cell suspension 2 and the lysis solution 3 are ⁇ homogenized upstream of the receptacle 1, for example via a static mixing system 4.
• the term preferably means the term “static mixer” fo ⁇ device which causes turbulence to the combined flow of the cell suspension 2 e ⁇ of the lysis solution 3, resulting in a rapid homogenization of this combined flow.
• This type of mixture is advantageous in that it is not associated with excessive shearing forces.
• the cell suspension is ⁇ preferably a cell culture pellet which has been taken up in a TRIS-EDTA buffer; suspension es ⁇ at a concentration of 10 to 300, preferably 50 to 200, even more preferably 75 to 150, such as about 100 g/L (cell weight:total suspension volume).
• the lysis solution 3 is ⁇ at a pH of between 12.0 and ⁇ 12.5, preferably the pH of the lysis solution is ⁇ fixed by a hydroxide of an alkali.
• the lysis solution 3 additionally comprises a detergent, preferably 0.1% by weight of sodium dodecyl sulphate.
• This ensures the neutralized solution (2, 3 e ⁇ 5 solutions combined) has a pH below 7.0, preferably below 6.0 e ⁇ , preferably (logically) above 4.5, preferably above at 5.0.
• the suspension comprising the plasmid remains in the presence of the neutralization solution for at least 1 minute, for example between 2 and 3 minutes. Excessive times allow the genomic DNA to renature, which is detrimental. Thus, in the present batch process it is preferable that the neutralization solutions 5 e ⁇ of precipitation 6 are added quickly. Thus, for plasmids smaller than 10 kb, a contact time with the neutralization solution between 20 and 80 seconds is advantageous.
• a contact time with the neutralization solution of 1 to 3 minutes is preferred.
• the addition time of the 5 e ⁇ 6 solutions becomes a parameter that should be controlled very finely, ideally 30 seconds each, at most.
• the precipitation solution 6 comprises a water-soluble calcium salt (such as CaCh) at a concentration of between 3.5 and 6 M, preferably about 5 M. Thanks to this concentrated calcium solution, the calcium concentration in the (suspension) solution after addition of the precipitation solution (solutions 2, 3, 5 and 6 combined) is at least 0.8 M, for example at least 1.0 M, or even at least 1.2 M.
• the inventors prefer to use a highly concentrated precipitation solution, despite its viscosity, so as to keep volumes reasonable, while ensuring a final calcium concentration which is ⁇ sufficient.
• Such a final calcium concentration (> 0.8 M, or even > IM) allows impurities to precipitate, in particular e ⁇ RNA from genomic DNA which has not had time to renature, but also proteins and endotoxins (if the starting cell synthesizes them), or at least a significant part of the endotoxins.
• the plurality of orifices 7 are ⁇ a pierced piping system.
• This system can be a simple drilled pipe, a coil, or even a ring.
• the advantage of the plurality of orifices is to inject the neutralization solution 5 and/or the precipitation solution 6 at a plurality of places in the receptacle, which results in a plurality of micro heterogeneities, rather than to massive heterogeneity.
• the plurality of orifices 7 used for the addition of the neutralization solution 5 e ⁇ the plurality of orifices 7 used for the addition of the precipitation solution 6 are ⁇ the same plurality of orifices 7: the containers containing the precipitation solutions e ⁇ of neutralization being connected upstream of the receptacle 1. This makes it possible not to multiply the devices, e ⁇ also makes it possible to purge all of the neutralization solution.
• the neutralization 5 and/or precipitation 6 solutions are substantially added via the bottom of the receptacle 1, preferably at least 30, 40, or even (at least or exactly) 50% by weight and/or volume of said solutions of neutralization and/or precipitation is ⁇ added to the bottom 20% of said receptacle. This allows a controlled injection of the solutions and increases their diffusion.
• the neutralization solution 5 is ⁇ added substantially via the bottom of the receptacle 1, preferably at least 30, 40, or even (at least or exactly) 50% by weight and/or volume of the neutralization solution 5 ⁇ added to the bottom 20% of said receptacle e ⁇
• the precipitating solution 6 is ⁇ substantially added to the top of receptacle 1, preferably at least 30, 40, or even (at least or exactly) 50% by weight of the solution precipitation 6 bran ⁇ added to the top 20% of said receptacle.
• This can be done by varying the height of the system comprising the plurality of orifices 7, or by using two systems comprising the plurality of distinct orifices 7.
• the neutralization solution 5 is ⁇ substantially added to the top of the receptacle 1, preferably at least 30, 40, or even (at least or exactly) 50% by weight of the neutralization solution 5 is ⁇ added in the top 20% of said receptacle e ⁇ the precipitation solution 6 is ⁇ added substantially via the bottom of the receptacle 1, preferably at least 30, 40, or even (at least or exactly) 50% by weight and/or volume of the 6 bran ⁇ precipitation solution added to bottom 20% of said receptacle.
• This can be done by varying the height of the system comprising the plurality of orifices 7, or by using two systems comprising the plurality of separate orifices 7.
• the flow rates Q1, Q2, Q3, Q4 and Q5 are independently between 0.5 L/min and 25 L/min, more preferably between 1 L/min and 10 L/min.
• the flow rates Q1, Q2, Q3, Q4 and/or Q5 can be independently constant; or the flow rates Q1, Q2, Q3, Q4 and/or Q5 can be independently variable.
• the flow rates Q1, Q2, Q3, Q4 and/or Q5, preferably Q3 and/or Q4 can be increased over time (lower at the start of pumping, maximum at the end) so as to limit the heterogeneities when adding these viscous solutions.
• An advantageous way to proceed with the increase in flow rates is ⁇ to keep a constant, or substantially constant, flow/volume ratio.
• the flow rate Q5 is preferably constant.
• the flow rate Q5 is ⁇ , preferably, constant e ⁇ maximum.
• the flow rates Q1 e ⁇ Q2 are paired (determined together) so that (i) all of the cell suspension 2 e ⁇ of the lysis solution 3 are pumped at the same time e ⁇ (ii) so that the concentration of the mixture (cell content, pH), for example at the outlet of the static mixer 4, constant self ⁇ .
• the gentle agitation 9 generates insufficient shearing stresses to shear the plasmid DNA or the genomic DNA of the host.
• a possible preliminary step consists of a test ⁇ of the acceptability of the shear stresses, according to the type of microorganism, the size of the plasmid to be purified and the chosen concentrations of the solutions.
• a related aspect of the present invention is ⁇ a method for purifying the plasmid of interest from the supernatant clarified according to the above, comprising the steps of: harvesting the clarified supernatant comprising said plasmid, filtering the clarified supernatant on a filter with a porosity of between 0.1 and 0.4 miti, preferably between 0.15 and 0.3 miti, favorably around 0.2 miti, to give a filtered solution comprising said plasmid, optionally ⁇ , ultrafiltration of the filtered solution, polishing by chromatography on an anion exchange resin, preferably said polishing comprising a step of washing the plasmid attached to the resin by means of a solution comprising polyoxyethylene (10) isooctylcyclohexyl ether; a formulation of the plasmid in a final solution.
• the inventors only recovered 200 mg of the plasmid per litre, and this plasmid contained measurable quantities of genomic DNA and RNA, which requires, in practice, to add a chromatography step, which means additional costs, e ⁇ loss of yield.
• yields which varied from simple to double, but also variable contents of contaminating RNA and in plasmid in "Open circular” form, the best yield being associated with an increased content of contaminants.
• the ratio of plasmid DNA in unrolled form (open circular) varied from 7 to 12%; values above 10% are ⁇ considered high.
• the desired shape is ⁇ the so-called “super coiled” one, and it is ⁇ difficult to achieve a separation of these two forms by HPLC or by fou ⁇ other means, the two peaks tending to overlap.
• 1.2 liters of a concentrated cell suspension 2 (100 g of cells/lifre in the same Tris-EDTA medium) containing the plasmid was inserted into container 1 with a capacity of 10 liters, via a peristaltic pump at the same time as 1.2 liters of the alkaline lysis solution 3 (NaOH; pH 12.5; SDS 0.1% by weight), these two solutions passing through a static mixer 4.
• the injection time was of 30 seconds.
• the vessel was under slow (non-shearing) mechanical agitation for exactly 90 seconds.
• this device can be easily adapted (volumes, flow rates) by slightly modifying the duration of the agitation.
• the inventors then analyzed by HPLC the plasmid obtained by the process according to the invention.
• the main advantages of the process according to the invention relate to the reproducibility and the possibility of production on a larger scale: a plurality of reactors according to the invention can be managed in parallel by a single operator, whereas the Manual stirring requires one operator per bottle, and cannot be performed at too high a rate, otherwise there is a risk of abusing the physical capacities of the operator, which will have an impact on the quality of the plasmid.
• Example 3 comparative example The inventors compared 4 conditions with respect to the condition according to the invention (see the HPLC profiles in Figure 3).
• the diffusion ring is placed in the middle of the bottle, not at the bottom; 2. Diffusion ring omitted;
• Condition #1 shows heterogeneities at an intermediate level between the process where the diffusion ring is ⁇ at the bottom, and the process where the diffusion ring is ⁇ moved towards the middle of the container.
• the filtration parameters are also degraded, where the filters had to be changed twice for conditions #1 and #4 and once for condition #2.
• RNA (RNA:plasmid) contamination also increases, except for condition #3.

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• 2021
  • 2021-07-02 BE BE20215517A patent/BE1029550B1/fr active IP Right Grant
• 2022
  • 2022-07-04 US US18/572,642 patent/US20240287496A1/en active Pending
  • 2022-07-04 EP EP22744169.8A patent/EP4363571A1/de active Pending
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