Automated Plasmid Extraction

Abstract

A method for discontinuously extracting plasmids from microorganisms and purifying a plasmid of interest, the method comprising adding a neutralizing solution under gentle mechanical agitation, followed by adding a precipitation solution through multiple orifices.

Description

[Technical field]

[0001] The present invention relates to the automated extraction of plasmids of interest produced by bacteria. [Background technology]

[0002] Production of a plasmid of interest in bacteria such as E. coli is known, as is alkaline lysis followed by neutralization and precipitation steps (see, for example, Birnboim and Dolly, 1979, Nucleic Acids Research, 7, pp. 1513-1523).

[0003] The method is easy to perform, works well in batch mode, and is suitable for producing small amounts of plasmid, up to the order of grams.

[0004] In patent application WO2010 / 136503 (also published inter alia in EP2435569B1, US8,822,672 and US9,416,400) a method for continuous plasmid extraction is described, based on a system of pipe arrangements for different solutions and their mixtures and flow control by pumps. In addition to a neutralizing solution, typically consisting of acetic acid / acetate buffer, a concentrated precipitation solution is added in this method. The neutralizing and precipitation solutions are homogeneously mixed by adapting the internal diameter of the pipes to create a Venturi effect locally. In fact, the inventors of this patent application point out that for such amounts of viscous solutions, mechanical stirring is not possible and risks shearing the genomic DNA and even the plasmids, which is unacceptable, unlike stirring by the Venturi effect. Moreover, advantageously, the system is single-use, so no cleaning is required. However, while the system is very effective for very large amounts of plasmid to be purified, for example 100 g of plasmid, the fixed costs (single-use equipment, losses of plasmid) are high when small amounts of plasmid are to be purified.

[0005] Thus, there is a range of quantities that are difficult to process in a batch process and cannot be processed very efficiently in the continuous processes described above.

[0006] In fact, the efficiency of batch-mode systems is limited by physical parameters such as the size of the vessel, the time it takes to contact the solutions, or the need for rapid homogenization. Thus, while batch mode achieves ideal purification in small quantities and allows good control over the mixture, it significantly complicates large-scale production. Summary of the Invention

[0007] The present invention relates to a method for extracting plasmids synthesized by microorganisms, The method comprises the following successive steps: - obtaining a vessel 1 coupled to an extraction pump 8, the vessel 1 being equipped with mechanical stirring means 9; obtaining a cell suspension 2 of the microorganism containing the plasmid to be extracted; adding an alkaline lysis solution 3 to the cell suspension 2 at a predetermined flow rate Q1 to form a homogenous mixture; Putting the homogenous mixture into a container 1 at a predetermined flow rate Q2; After a predetermined time, preferably with gentle stirring, a neutralizing solution 5 of acetic acid from a container is added through a plurality of orifices 7 at a predetermined flow rate Q3; After a predetermined time, adding the precipitation solution 6 from the vessel at a predetermined flow rate Q4 via a plurality of orifices 7; Extracting the formed suspension after a certain time under gentle stirring via an extraction pump 8 at a certain flow rate Q5; Clarifying the extracted mixture, preferably by centrifugation, and recovering the clarified supernatant containing the plasmid of interest.

[0008] This extracted and purified plasmid can be advantageously used as is or after sterile filtration, ultrafiltration, and / or polishing chromatography. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 is a semi-schematic view of a container according to the invention. [Diagram 2] FIG. 2 shows the HPLC analysis of plasmids extracted by the automated method according to the invention. [Diagram 3] FIG. 3 shows the effects of variations #1, #2, #3, and #4 performed in a preferred automated method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present inventors have succeeded in developing a plasmid extraction method that can process large amounts of plasmid while maintaining the flexibility of the batch mode method, and that achieves extremely high extraction yields and purity.

[0011] A first aspect of the invention is a method for extracting a synthesized plasmid, comprising the successive steps of: The successive steps include: - obtaining a vessel 1 coupled to an extraction pump 8, the vessel 1 being equipped with mechanical stirring means 9; Obtaining a cell suspension 2 containing the plasmid to be extracted; adding an alkaline lysis solution 3 to the cell suspension 2 at a predetermined flow rate Q1 to form a homogenous mixture; Putting the homogenous mixture into a container 1 at a predetermined flow rate Q2; After a predetermined time, adding a neutralizing solution 5 of acetic acid from a container at a predetermined flow rate Q3 through a number of orifices 7, preferably with gentle stirring; After a predetermined time, adding the precipitation solution 6 from the vessel at a predetermined flow rate Q4 via a plurality of orifices 7; After a predetermined time under gentle stirring, the formed suspension is extracted via said extraction pump 8 at a predetermined flow rate Q5, the extracted mixture is clarified, preferably centrifuged, and the clarified supernatant containing the plasmid of interest is recovered.

[0012] The cells in which the desired plasmid is synthesized are generally gram-negative bacteria, such as E. coli, but other gram-negative or gram-positive bacteria or other microorganisms, such as Saccharomyces sp. or Pichia sp., are also suitable.

[0013] The plasmid is advantageously so-called "supercoiled".

[0014] The size of the plasmid is not a limiting factor, as the method also works with large plasmids. However, as described below, larger size plasmids (e.g., greater than 10 kb, e.g., between 10 and 20 kb, including plasmids encoding viral vectors, and / or plasmids consisting of repeat and / or inverted sequences) require more precise timing control, especially with regard to contact time with alkaline lysis medium, addition of neutralization solution and contact time.

[0015] The size of the vessel 1 is not particularly limited. If the vessel 1 is too small, a sufficient amount of plasmid cannot be processed. On the other hand, if the vessel 1 is filled with an excessively large amount of solution, there is a risk that the pumping time will be too long, which is disadvantageous because it complicates or prevents the control of different times. In fact, since the solutions are pumped one by one, there is non-uniformity at the beginning of each step.

[0016] The inventors have found that a container 1 with 5-10 liters of solution at the end (after adding solutions 2+3+5+6) is very easy to use. On the other hand, a container 1 with 100 liters of solution at the end (after adding solutions 2+3+5+6) becomes too difficult to control. Therefore, the preferred size of the usable volume of the container is between 1 liter and 50 liters, preferably between 2.5 liters and 20 liters, more preferably between 5 liters and 10 liters.

[0017] Obviously, containers 1 with a larger or even significantly larger volume (than 50 or 100 liters) can be used, even if they are only intended to be filled with a maximum of 2.5 to 10 liters.

[0018] Surprisingly, the inventors have discovered that this heterogeneity, a potential double heterogeneity, is not lethal as long as it is controlled and / or not in excess.

[0019] For example, the inventors have determined that the contact time between the cell suspension and the lysis solution is advantageously between 2 and 5 minutes, and therefore the inventors have reasoned that solutions 2 and 3 can be added in, for example, 1 minute without any problems.

[0020] More preferably, the cell suspension 2 and the lysis solution 3 are homogenized upstream of the vessel 1, for example via a static mixing system 4.

[0021] In the context of the present invention, the term "static mixer" is understood to mean any device that preferably induces turbulence in the confluent flow of the cell suspension 2 and the lysis solution 3, resulting in rapid homogenization of said confluent flows. The inventors have noted that this type of mixing is advantageous in that it does not involve excessively high shear forces.

[0022] The cell suspension is preferably a cell culture pellet taken up in TRIS-EDTA buffer, and the suspension has a concentration of 10-300, preferably 50-200, more preferably 75-150, for example about 100 g / L (cell weight:total volume of suspension).

[0023] This allows for initial homogenization without the involvement of shear forces, and since the two solutions are combined at this level, potential inhomogeneities are greatly reduced.

[0024] Preferably, the pH of the dissolution solution 3 is between 12.0 and 12.5, and the pH of the dissolution solution is preferably fixed by an alkaline hydroxide.

[0025] This allows for rapid dissolution and denaturation of genomic DNA. The use of alkaline hydroxides such as NaOH allows for a very basic pH with low buffering capacity, which makes subsequent neutralization easy. However, unless the pH is carefully fixed, sufficient dissolution and denaturation cannot be achieved, and there is a risk of irreversibly denaturing the target plasmid.

[0026] Preferably, the dissolution solution 3 further comprises a detergent, preferably 0.1% by weight sodium dodecyl sulfate.

[0027] Advantageously, the neutralization solution 5 has a pH between 4.5 and 5.7. This solution preferably consists of acetic acid at a concentration of more than 1 M, more preferably the neutralization solution is fixed at a pH of about 5.5 by an acetic acid / acetic acid mixture, which results in a neutralization solution (solutions 2, 3 and 5 combined) with a pH below 7.0, preferably below 6.0, and preferably (logically) a pH above 4.5, preferably above 5.0.

[0028] Preferably, the suspension containing the plasmid remains in the presence of the neutralizing solution for at least 1 minute, for example 2-3 minutes. Too long a contact time would re-denaturate the genomic DNA, which would be detrimental. Therefore, in this batch process, it is preferred to add the neutralizing solution 5 and the precipitation solution 6 quickly.

[0029] Therefore, for plasmids less than 10 kb in size, a contact time with the neutralizing solution of 20 to 80 seconds is advantageous.

[0030] Conversely, for plasmids larger than 10 kb, especially those containing repeats and / or inverted sequences, a contact time with the neutralizing solution of 1 to 3 minutes is preferred, in which case the addition times of solutions 5 and 6 become parameters that must be very carefully controlled, ideally around 30 seconds each.

[0031] However, flow rates cannot be adopted completely freely. Physical flow and therefore pressure constraints must be respected, especially since (i) the mixture containing solutions 2+3+5 and (ii) solution 6 are very viscous. In addition, it is advisable to use robust and / or commonly used pumps with efficient maintenance. If necessary, especially for large plasmids, reduce the volumes of the different solutions to be able to add them quickly, especially solutions 5 and 6 (see paragraph above).

[0032] Peristaltic pumps are advantageous because they provide a well-controlled flow rate and are shear-free.

[0033] Advantageously, the precipitation solution 6 contains a water-soluble calcium salt (CaCl) at a concentration between 3.5 and 6 M, preferably about 5 M. 2 etc.)

[0034] Thanks to this concentrated calcium solution, the calcium concentration in the (suspended) 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. We prefer to use a very concentrated precipitation solution, despite its viscosity, in order to maintain a reasonable volume while ensuring a sufficient final calcium concentration. Such a final calcium concentration (0.8 M or more, or even 1 M or more) allows to precipitate impurities, especially RNA and genomic DNA that have not had time to denature, as well as proteins and endotoxins (if the starting cells synthesize them), or at least a significant portion of the endotoxins.

[0035] Advantageously, the multiple orifices 7 are a perforated piping system, which may be a simple perforated pipe, a coil or a ring.

[0036] The advantage of having multiple orifices is that the neutralization solution 5 and / or precipitation solution 6 are injected into multiple locations within the vessel, resulting in multiple micro-inhomogeneities rather than mass inhomogeneities. This, combined with gentle mechanical agitation 9, allows for non-shearing, rapid homogenization, which addresses the dual problem of inhomogeneity discussed above.

[0037] The inventors were surprised that this approach could homogenize the solution sufficiently quickly without causing the shear problems described above.

[0038] Advantageously, the orifices 7 used for adding the neutralizing solution 5 and the orifices 7 used for adding the precipitation solution 6 are the same orifices 7. The vessels containing the precipitation solution and the neutralizing solution are connected upstream of the vessel 1. This avoids the need for multiple devices and also makes it possible to purge all of the neutralizing solution.

[0039] Advantageously, the neutralizing solution 5 and / or the precipitation solution 6 are added substantially from the bottom of the vessel 1, preferably at least 30, 40 or even (at least or exactly) 50% by weight and / or volume of said neutralizing solution and / or precipitation solution being added in the bottom 20% of the vessel, which allows a controlled injection of the solutions and increases their diffusion.

[0040] According to a variant, the neutralizing solution 5 is added substantially from the bottom of the vessel 1, preferably at least 30, 40 or (at least or exactly) 50% by weight and / or volume of the neutralizing solution 5 is added to the bottom 20% of the vessel, and the precipitation solution 6 is added substantially from the top of the vessel 1, preferably at least 30, 40 or (at least or exactly) 50% by weight of the precipitation solution 6 is added to the top 20% of said vessel. This can be done by varying the height of the system of multiple orifices 7 or by using two systems of multiple separate orifices 7.

[0041] According to a second variant, the neutralization solution 5 is added substantially to the top of the vessel 1, preferably at least 30, 40 or (at least or exactly) 50% by weight of the neutralization solution 5 being added to the top 20% of the vessel, and the precipitation solution 6 is added substantially via the bottom of the vessel 1, preferably at least 30, 40 or (at least or exactly) 50% by weight and / or volume of the precipitation solution 6 being added to the bottom 20% of the vessel. This can be done by varying the height of the system of multiple orifices 7 or by using two systems of multiple separate orifices 7.

[0042] Preferably, the flow rates Q1, Q2, Q3, Q4 and Q5 are each independently 0.5 L / min to 25 L / min, more preferably 1 L / min to 10 L / min.

[0043] 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. For example, the flow rates Q1, Q2, Q3, Q4 and / or Q5, preferably Q3 and / or Q4, can be increased over time (lowest at the beginning of pumping and highest at the end) to limit non-uniformity in dosing these viscous solutions. An advantageous way to increase the flow rate is to keep the flow rate / volume ratio constant or substantially constant. The flow rates Q1, Q2, Q3 and / or Q4 may be variable (increasing over time), but regardless, the flow rate Q5 is preferably constant. The extraction 8 is preferably very rapid so that agglomerates do not get in the way. Thus, the flow rate Q5 is preferably constant and fastest. The flow rates Q1 and Q2 are preferably matched (determined together) so that (i) all of the cell suspension 2 and lysis solution 3 are pumped simultaneously, and (ii) the concentration of the mixed solution (content in cells, pH) at, for example, the outlet of the static mixer 4 is constant.

[0044] Advantageously, gentle agitation 9 generates insufficient shear stress to shear plasmid or genomic DNA from the host.

[0045] Therefore, a preliminary step would be to test the tolerance of shear stress depending on the type of microorganism, the size of the plasmid to be purified, and the concentration of the solution.

[0046] A related aspect of the invention is a method for purifying a plasmid of interest from a clarified supernatant according to the above, comprising the steps of: harvesting the clarified supernatant containing the plasmid; filtering the clarified supernatant through a filter having a porosity of 0.1 to 0.4 μm, preferably 0.15 to 0.3 μm, and advantageously around 0.2 μm, to obtain a filtered solution containing the plasmid; Optionally, ultrafiltering the filtered solution; a chromatographic polishing step on an anion exchange resin, preferably comprising washing the plasmid immobilized on the resin with a solution containing polyoxyethylene(10) isooctylcyclohexyl ether; and formulating the plasmid into a final solution.

[0047] (Example) It will be understood that the invention is in no way limited to the embodiments described above, but many variations are possible without departing from the scope of the appended claims.

[0048] (Comparative Example) We have attempted to develop an "in-volt" batch method. For ease of reference, we refer to FIG. 1, which does not have elements 4 and 7, as well as piping and a pump system upstream of the vessel 1. To do this, 0.3 liters of concentrated cell suspension 2 producing plasmids (100 g cells / liter Tris-EDTA medium) were inserted into the vessel 1 of volume 2 liters, then 0.3 liters of alkaline lysis solution 3 (NaOH; pH 12.5; SDS 0.1% by weight) were quickly added and the vessel 1 was manually stirred for exactly 90 seconds by the operator. Then, 0.6 liters of neutralization solution 5 (AcOH 15% v:v / 3M potassium acetate, pH 4.5-5.7) were quickly added and the contents of the vessel 1 were manually stirred for exactly 90 seconds. Finally, 0.23 liters of precipitation solution 6 (CaCl 2 , 5M) was quickly added and the contents of vessel 1 were manually mixed for exactly 90 seconds. The mixture was then extracted for clarification by centrifugation.

[0049] We were only able to recover 200 mg of plasmid per liter, which contained measurable amounts of genomic DNA and RNA. By repeating this manual method with the aim of improving it, we obtained yields that varied from single to double, but also with different contents of contaminating RNA and of plasmid in "open circular" form, the highest yields being associated with an increased content of contaminants. Thus, the proportion of plasmid DNA in open circular form varied between 7 and 12%. In fact, the desired form is the so-called "supercoiled form", and it is difficult to separate these two forms by HPLC or other means, since the two cells tend to overlap.

[0050] Faced with these results, the inventors believe that the increased contaminant levels are due to the higher shear forces applied during this manual extraction, which vary depending on the batch (identity of the operator, potential fatigue of the operator).

[0051] Example 1 The inventors had the idea to develop this system, which they considered ineffective. To do so, 1.2 liters of concentrated cell suspension 2 containing plasmids (100 g / liter of cells in the same Tris-EDTA medium) were pumped by a peristaltic pump into a vessel 1 with a volume of 10 liters, simultaneously with 1.2 liters of alkaline lysis solution 3 (NaOH; pH 12.5; SDS 0.1% by weight), and these two solutions were passed through a static mixer 4. The injection time was 30 seconds. The vessel was subjected to slow mechanical stirring 9 (non-sheared) for 90 seconds. Then, 2.4 liters of neutralization solution 5, the same as in the comparative example, were quickly added to the bottom (flow rate 6 L / min) through a peristaltic pump and a diffusion ring perforated with a number of orifices 7, and the contents of vessel 1 were mechanically stirred 9, but non-sheared, for exactly 90 seconds. Finally, 0.92 L of precipitation solution 6 (CaCl2, 5 M) was quickly added (flow rate 2.3 L / min) via the same device drilled with a peristaltic pump and an orifice 7, for exactly 90 seconds under slow mechanical stirring 9. The mixture was then extracted 8 for clarification by centrifugation.

[0052] We recovered approximately 400 mg / L of plasmid, which contained no measurable amounts of genomic DNA and very little RNA.

[0053] Stirring is performed for 90 seconds and we conclude that this device can be easily adapted by slightly modifying the stirring time (volume, flow rate).

[0054] Example 2 The inventors analyzed the plasmids obtained by the method according to the invention by HPLC. The results showed that the extraction yield was 84%, with only 7.2% "open circular" plasmid content (and therefore approximately 93% in supercoiled form). See Figure 2, the first peak on the left represents residual salts, the second peak represents RNA, the double peak represents plasmid, and the peak on the right is the largest and is in supercoiled form, where the HPLC signal is saturated.

[0055] In addition, the main advantage of the method according to the invention is related to reproducibility and the possibility of larger scale production: multiple reactors according to the invention can be managed in parallel by one operator, whereas manual stirring requires one operator per bottle and cannot be performed very quickly.

[0056] Example 3 - Comparative Example We compared four conditions with the conditions according to the invention (see HPLC profiles in FIG. 3). 1. Place the diffusion ring in the middle of the bottle, not at the bottom; 2. The diffusion ring is omitted, 3. The static mixer is omitted, 4. Both the diffusion ring and the static mixer are omitted.

[0057] Visual inspection shows strong inhomogeneity at the top of the bottle for conditions #2 and #3. Condition #4 is less affected by inhomogeneity based on simple visual analysis. Condition #1 shows an intermediate level of inhomogeneity between the method with the diffusion ring at the bottom and the method where the diffusion ring was moved to the center of the container.

[0058] Filtration parameters also deteriorated, requiring two filter changes in conditions #1 and #4 and one in condition #2.

[0059] However, the most significant difference was seen in the yield, which dropped to 67% in condition #1, but only 22.6, 14, and 30% in conditions #2, #3, and #4. The proportion of "open circular" plasmids also increased: 16% in condition #1, 9.9% in condition #3, and 13.7% in condition #4. RNA contamination (RNA:plasmid) also increased, except for condition #3.

Claims

1. A method for extracting a plasmid synthesized by a microorganism, comprising the following consecutive steps: Said consecutive steps are: Obtaining a container (1) coupled to an extraction pump (8), said container (1) comprising mechanical stirring means (9); Obtaining a cell suspension (2) of said microorganism containing the plasmid to be extracted; Adding an alkaline lysis solution (3) to said cell suspension (2) at a predetermined flow rate Q1 to form a homogeneous mixture; Introducing said homogeneous mixture into said container (1) at a predetermined flow rate Q2; After a predetermined time, preferably while gently stirring, adding a neutralization solution (5) consisting of acetic acid of said container at a predetermined flow rate Q3 through a plurality of orifices (7); After a predetermined time, adding a precipitation solution (6) of said container at a predetermined flow rate Q4 through a plurality of orifices (7), said precipitation solution (6) consisting of a water-soluble calcium salt having a concentration of 3.5 to 6M, and the calcium concentration in said solution after adding said precipitation solution being at least 0.8M; After a predetermined time while gently stirring, extracting the formed suspension at a predetermined flow rate Q5 through said extraction pump (8); Clarifying the extracted mixture, preferably by centrifugation, and recovering the clarified supernatant containing said plasmid of interest.

2. The method according to claim 1, wherein said cell suspension (2) and said lysis solution (3) are homogenized upstream of said container via a static mixing system (4).

3. The method according to claim 1 or 2, wherein said lysis solution (3) has a pH between 12.0 and 12.5, and preferably, the pH is fixed by an alkali hydroxide.

4. The method according to claim 3, wherein said lysis solution (2) further contains a detergent, preferably 0.1 wt% sodium dodecyl sulfate.

5. The method according to any one of claims 1 to 4, wherein said neutralization solution (5) has a pH between 4.5 and 5.

7.

6. The method according to claim 1, wherein said neutralization solution (5) consists of acetic acid having a concentration exceeding 1M, and preferably, said neutralization solution has a pH fixed by an acetic acid / acetic acid mixture.

7. The method according to claim 6, wherein said solution is neutralized to a pH less than 7.0, preferably less than 6.0, preferably pH 4.5 or more, preferably 5.0 or more.

8. The precipitation solution (6) consists of a water-soluble calcium salt with a concentration of approximately 5 M, according to the method of claim 1.

9. The calcium concentration of the solution after adding the precipitation solution is at least 1.0 M, according to the method of claim 8.

10. The plurality of orifices (7) is a perforated piping system, according to the method of claim 1.

11. The plurality of orifices (7) used for adding the neutralization solution and the plurality of orifices (7) used for adding the precipitation solution are the same plurality of orifices (7), according to the method of claim 1.

12. The neutralization liquid (5) and / or the precipitation liquid (6) are added substantially at the bottom of the container (1), preferably, at least 50% by weight of the neutralization liquid and / or the precipitation liquid is added to the bottom 20% of the container, according to the method of claim 1.

13. 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, and even more preferably between 2 L / min and 8 L / min, according to the method of claim 1.

14. Gentle stirring generates shear stress insufficient to shear plasmid DNA or genomic DNA from the host, according to the method of claim 1.

15. The solution is added and / or extracted by a peristaltic pump, according to the method of claim 1.

16. A process for purifying the plasmid of interest from the clarified supernatant according to claim 1, comprising the step of collecting the clarified supernatant containing the plasmid, filtering the clarified supernatant through a filter having a porosity of 0.1 - 0.4 μm, preferably 0.15 - 0.3 μm, and advantageously around 0.2 μm to obtain a filtered solution containing the plasmid, optionally, ultrafiltrating the filtered solution, a chromatographic polishing step on an anion exchange resin, preferably including the step of washing the plasmid immobilized on the resin with a solution containing polyoxyethylene (10) isooctyl cyclohexyl ether, formulating the plasmid into a final solution, and a process comprising the above steps.

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