A method for solid-liquid separation after cell lysis by using micro-nano bubble technology

Abstract

The application is suitable for the field of biopharmaceuticals, and provides a method for solid-liquid separation after cell lysis by using micro-nano bubble technology, which comprises the following steps: resuspending collected cells with P1 solution to obtain a cell resuspension; mixing the cell resuspension with P2 solution for alkaline lysis reaction; mixing P3 solution with P2 solution for acid neutralization after the alkaline lysis reaction is completed; mixing P4 solution with the cell resuspension for flocculation reaction after the acid neutralization is completed; introducing micro-nano bubbles into the flocculated feed liquid and standing, so that the micro-nano bubbles adsorb the flocculation and drive it to float, thereby forming a cake-shaped solid impurity layer above the feed liquid; and performing clarification filtration from the relatively clear feed liquid below. The application uses micro-nano bubbles to replace the traditional NaHCO3 or NH4HCO3 floatation liquid chemical gas production mode, has high floatation efficiency and good solid-liquid separation effect, avoids the introduction of process impurities and health and safety hazards, shortens the production waiting time, and reduces the degradation risk of the target product.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, and in particular relates to a method for solid-liquid separation after cell lysis using micro-nano bubble technology. Background Technology

[0002] In the biopharmaceutical field, the feed solution after cell lysis contains a large amount of cell debris and impurities, requiring solid-liquid separation to provide a clean feed solution for subsequent purification. In plasmid production, cell lysis often employs an alkaline lysis method, where host cell proteins, RNA, and DNA are released and denatured under alkaline conditions. The addition of an acidic potassium acetate high-salt buffer causes the denatured host DNA, proteins, and ruptured cell walls to entangle and form a precipitate. After adding a neutralizing solution, the plasmid DNA renatures and remains soluble. Further flocculation of impurities is achieved by adding a flocculant, and finally, the addition of NaHCO3 or NH4HCO3 solution generates bubbles that carry the precipitate to the surface, achieving preliminary solid-liquid separation.

[0003] However, in the above-mentioned solid-liquid separation scheme that uses NaHCO3 or NH4HCO3 flotation liquid to generate bubbles, the bubble generation rate is too fast and the size is too large, resulting in weak adsorption and carrying capacity. Furthermore, the continuous gas generation during the clarification and filtration process affects the filtration operation and recovery rate. The long settling time leads to the risk of degradation of the target product, and the need for refrigeration facilities increases energy consumption. The introduction of flotation liquid introduces new process impurities and increases the volume of the feed liquid, increasing the pressure on downstream processing, and making the production process complex and costly. The use of NH4HCO3 generates NH3 gas, which affects the environment and the health and safety of operators. Summary of the Invention

[0004] The purpose of this invention is to provide a method for solid-liquid separation after cell lysis using micro-nano bubble technology, aiming to solve the problems existing in the background technology.

[0005] This invention is achieved by using micro / nano bubble technology for solid-liquid separation after cell lysis, comprising the following steps:

[0006] Step 1: Resuspend the collected cells in P1 solution to obtain a cell resuspension.

[0007] The P1 solution is a solution containing 25 mmol / L tris(hydroxymethyl)aminomethane (Tris), 7.9 mmol / L ethylenediaminetetraacetic acid (EDTA), and 55 mmol / L glucose, with a pH of 7.5 ± 0.2; the cell resuspension contains 5% to 25% (w / v) of cells.

[0008] Step 2: Mix the cell resuspension with P2 solution and carry out an alkaline lysis reaction.

[0009] The P2 solution is a lysis buffer containing 0.2 mol / L sodium hydroxide (NaOH) and 1% sodium dodecyl sulfate (SDS); the mixing ratio of the cell resuspension to the P2 solution is 0.8:1.2 to 1.2:0.8; the alkaline lysis reaction time is 10 seconds to 300 seconds. Mixing can be performed using static mixing, stirring, or shaking.

[0010] Step 3: After the alkaline pyrolysis reaction is completed, the P3 solution is mixed with the P2 solution for acid neutralization to terminate the alkaline pyrolysis reaction and flocculate some impurities.

[0011] The P3 solution is a 3 mol / L potassium acetate solution; the mixing ratio of the P3 solution to the P2 solution is 0.8:1.2 to 1.2:0.8; the acid neutralization time is 10 seconds to 300 seconds. The mixing method can be static mixing, stirring, or shaking.

[0012] Step 4: After acid neutralization, mix the P4 solution with the cell resuspension to carry out the flocculation reaction.

[0013] The P4 solution is a 1-5 mol / L calcium chloride (CaCl2) solution or zinc sulfate (ZnSO4) solution; the mixing ratio of the P4 solution to the cell resuspension is 0.5:1.0 to 1.5:1.0; the flocculation reaction time is 0.5 minutes to 60 minutes. Mixing methods can include stirring, orbital oscillation, or wave-like oscillation.

[0014] Step 5: Introduce micro-nano bubbles into the liquid treated in Step 4, let it stand, and allow the micro-nano bubbles to adsorb the flocculents and carry them to the surface, thereby forming a cake-shaped solid impurity layer above the liquid.

[0015] Micro- and nano-bubbles can be introduced in any of the following ways:

[0016] Method 1: Add micro-nano bubbles to the P4 solution using a micro-nano bubble generator, then mix the P4 solution containing micro-nano bubbles with the cell resuspension to carry out a flocculation reaction;

[0017] Method 2: Use a micro-nano bubble generator to add micro-nano bubbles to other buffer solutions or water, and then add the other buffer solutions or water containing micro-nano bubbles to the solution treated in step 4.

[0018] The content of micro-nano bubbles, measured by turbidity, is 1000–20000 NTU; the settling time is 1 minute–120 minutes.

[0019] Step 6: Clarify and filter the liquid obtained in Step 5 from below relative to the clarified liquid, and collect the filtered clarified liquid.

[0020] The clarification filtration employs a deep membrane stack and / or a capsule filter; the filtration pressure is less than 2 bar; the pore size of the deep membrane stack is 0.1 μm to 100 μm, and the pore size of the capsule filter is 0.1 μm to 0.65 μm. After filtration, 0 to 5 times the dead volume of the filter (purified water, water for injection, or buffer solution) can be used as a top wash solution to wash the filter, and the top wash solution is combined with the collected clarified liquid.

[0021] The present invention provides a method for solid-liquid separation after cell lysis using micro-nano bubble technology, which has the following beneficial effects:

[0022] This invention replaces the traditional chemical gas generation method of NaHCO3 or NH4HCO3 flotation liquid by using micro-nano bubble technology. It utilizes the characteristics of micro-nano bubbles, such as small particle size, large specific surface area, slow rising speed and high interfacial potential, so that the bubbles can efficiently adsorb and carry flocculents to float to the surface, forming a compact cake-shaped solid impurity layer above the liquid.

[0023] This invention eliminates the need for adding a flotation solution, avoiding the introduction of process impurities such as NaHCO3 and NH4HCO3. It also reduces the number of steps involved in preparing and adding the flotation solution, as well as production waiting time, significantly improving production efficiency. Bacteria and their lysed impurities carry a positive charge in an acidic environment, while micro-nano bubbles carry a negative charge. These bubbles more easily adsorb flocculent precipitates during their ascent, promoting solid-liquid separation. The negative charge of the target product, DNA particles, remains unaffected. The micro-nano bubbles exhibit stronger flotation capabilities and better solid-liquid separation, significantly reducing the difficulty and processing pressure of clarification and filtration, and saving on the use of related membrane materials. Eliminating the need for a flotation solution results in a smaller process volume, reducing downstream processing pressure. By saving materials, reducing investment in facilities and equipment, and shortening production waiting time, production costs are significantly reduced. Simultaneously, the production waiting time from the alkaline pyrolysis step to the clarification and filtration step is shortened, reducing the risk of target product degradation and changes in quality properties, increasing the yield and quality of the target product, and completely avoiding the health and safety hazards associated with the potential use of NH4HCO3. Attached Figure Description

[0024] Figure 1 A comparison of the process flow diagrams for plasmid alkaline pyrolysis and flocculation flotation before and after optimization;

[0025] Figure 2 State diagrams of water and 5 mol / L CaCl2 solution incorporating micro / nano bubbles.

[0026] Figure 3 The graph shows the change in turbidity of a 5 mol / L CaCl2 solution over time after the addition of micro / nano bubbles.

[0027] Figure 4The images show the actual results of the alkaline pyrolysis and flocculation flotation experiment (12.5g of bacterial sludge / batch);

[0028] Figure 5 Comparison of turbidity of the clarified and filtered liquid after alkaline pyrolysis and flocculation flotation experiments (12.5g of bacterial sludge / batch);

[0029] Figure 6 A comparison chart of recovery rates between alkaline pyrolysis and flocculation flotation experiments (12.5g of bacterial sludge / batch);

[0030] Figure 7 Agarose gel electrophoresis image for alkaline lysis and flocculation flotation experiments (12.5g bacterial sludge / batch);

[0031] Figure 8 A comparison of the supercoil ratio of plasmid monomers in alkaline lysis and flocculation flotation experiments (12.5g of bacterial sludge / batch);

[0032] Figure 9 Comparison of endotoxin content after clarification and filtration in alkaline pyrolysis and flocculation flotation experiments (12.5g of bacterial sludge / batch);

[0033] Figure 10 The images show the actual results of the alkaline pyrolysis and flocculation flotation experiment (50g of mycelium sludge / batch).

[0034] Figure 11 Agarose gel electrophoresis image for alkaline lysis and flocculation flotation experiments (50g bacterial sludge / batch). Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0037] Micro- and nanobubbles generally refer to tiny bubbles with a diameter of less than 100 μm. They can be classified into microbubbles (1 μm to 100 μm) and nanobubbles (less than 1 μm) based on their diameter. Compared to ordinary bubbles, micro- and nanobubbles have many advantages, including slower ascent speed, larger specific surface area, ability to generate hydroxyl radicals, higher interfacial potential, and higher gas solubility. Air flotation involves introducing or generating a large number of microbubbles into water, causing them to adhere to impurity flocs and float to the surface due to buoyancy, thus achieving efficient solid-liquid separation. The high potential and longer contact time between micro- and nanobubbles and suspended solids improve the adhesion efficiency between bubbles and suspended solids, significantly enhancing air flotation efficiency. Bacteria and their lysed impurity flocs typically carry a positive charge in acidic environments, while the generated micro- and nanobubbles carry a negative charge. Therefore, micro- and nanobubbles more easily adsorb flocs during their ascent, promoting solid-liquid separation and ultimately forming a relatively compact, cake-like solid impurity layer on top of the feed liquid. The relatively clear feed liquid below can then be further clarified using membrane filtration. The target product, DNA particles, carries a negative charge and is unaffected by it. In addition, the small particle size of micro- and nano-bubbles means that the "explosion" effect during their breakage is not significant, and they will not cause secondary damage to the stratification of flocculents and cake-like solid impurities.

[0038] Example 1

[0039] The plasmids and host cell information used in this embodiment are shown in the table below:

[0040] plasmid name plasmid A plasmid size 0.8kbp resistance gene KanR host cells E.coli Stbl3 <![CDATA[Bacterial concentration (OD 600 )]]> 86 Plasmid volume yield 498mg / L

[0041] This embodiment performs alkaline pyrolysis and flocculation flotation operations according to the following steps:

[0042] Step 1: Cell resuspension

[0043] The collected cells were resuspended in solution P1 to obtain a cell suspension with a cell concentration of 10% (w / v). Solution P1 was a solution containing 25 mmol / L tris(hydroxymethyl)aminomethane (Tris), 7.9 mmol / L ethylenediaminetetraacetic acid (EDTA), and 55 mmol / L glucose, with a pH of 7.5 ± 0.2.

[0044] Step 2: Alkali pyrolysis

[0045] The cell resuspension and P2 solution were mixed at a ratio of 1.00:1.00 (using static mixing) and subjected to alkaline lysis. The P2 solution was a lysis buffer containing 0.2 mol / L sodium hydroxide (NaOH) and 1% sodium dodecyl sulfate (SDS). The alkaline lysis reaction time was controlled within the range of 10 to 300 seconds based on actual operation.

[0046] Step 3: Acid neutralization

[0047] After the alkaline pyrolysis reaction is completed, solution P3 and solution P2 are mixed at a ratio of 1.00:1.00 (using static mixing) for acid neutralization to terminate the alkaline pyrolysis reaction and flocculate some impurities. Solution P3 is a 3 mol / L potassium acetate solution. The acid neutralization time is controlled within the range of 10 to 300 seconds based on actual operation.

[0048] Step 4: Precipitation / flocculation reaction

[0049] After acid neutralization, the P4 solution and cell resuspension were mixed at a ratio of 0.75:1.00 (using stirring) for flocculation. The P4 solution was a 1–5 mol / L calcium chloride (CaCl2) solution or zinc sulfate (ZnSO4) solution. The flocculation reaction time was controlled between 0.5 and 60 minutes. Furthermore, micro-nano bubbles were added to the P4 solution using a micro-nano bubble generator, allowing the P4 solution containing micro-nano bubbles to mix with the cell resuspension for flocculation (i.e., introducing micro-nano bubbles using method one). The micro-nano bubble content, measured by turbidity, was controlled within the range of 1000–20000 NTU.

[0050] Step 5: Let it float to the surface

[0051] After the flocculation reaction is completed, the mixture is allowed to stand for a period of 1 to 120 minutes. This allows the micro-nano bubbles to adsorb the flocculents and carry them to the surface, thus forming a cake-like solid impurity layer above the liquid.

[0052] Step 6: Clarification and Filtration

[0053] The feed solution obtained in step 5 is filtered relative to the clarified feed solution from below. Filtration is performed using a deep-layer membrane stack (pore size 0.1 μm–100 μm) and / or a capsule filter (pore size 0.1 μm–0.65 μm), with the filtration pressure controlled at less than 2 bar. The filtered clarified feed solution is collected. After filtration, the filter is washed with 0–5 times the filter dead volume of purified water, water for injection, or a buffer solution as the wash solution, and the wash solution is combined with the collected clarified feed solution.

[0054] Flocculation and flotation effect of the feed liquid after alkaline pyrolysis

[0055] Figure 1 The process flow diagrams of plasmid alkaline pyrolysis and flocculation flotation before and after optimization are shown.

[0056] Figure 2 The liquid states and their changes over time after micro- and nano-bubbles were loaded into water and 5 mol / L CaCl2 solution, respectively. Figure 2 (1) shows the change of liquid state over time after micro-nano bubbles are loaded with water as a medium; Figure 2(2) shows the change of liquid state over time after micro-nano bubbles are loaded with 5 mol / L CaCl2 solution.

[0057] Figure 3 The turbidity of a 5 mol / L CaCl2 solution after incorporating micro / nano bubbles is shown (taking...). Figure 2 The solution circled in red (measured using a Hach TL23 turbidimeter within 10 seconds) changes over time.

[0058] Figure 4 The results of the alkaline pyrolysis and flocculation flotation experiments (12.5g of bacterial sludge / batch) in this embodiment are shown. Figure 4 In the middle, beakers A through F contain the same neutralized alkaline lysis solution; Figure 4 (2) is a side view of the beaker after 15 minutes of floating. A is the negative control, B is NaHCO3, C is NH4HCO3, and D to F are micro-nano bubbles. Figure 4 (3) is a side view of the beaker 60 minutes after it started to float. Figure 4 (4) shows a comparison of the beakers after 60 minutes of initial floating (top view). The experimental results show that the solid-liquid separation effect of the micro-nano bubble treatment group is better than that of the NaHCO3 group and the NH4HCO3 group, and the solid-liquid stratification is more obvious.

[0059] Figure 5 The results show a comparison of turbidity in the clarified and filtered liquid. The test results indicate that the turbidity of the micro / nano bubble treatment group was significantly lower than that of the NaHCO3 and NH4HCO3 groups.

[0060] Recovery rate determination

[0061] After flotation and clarification filtration, the alkaline pyrolysis feed solution was concentrated 5 to 15 times and washed 20 to 50 times using 100KD ultrafiltration centrifuge tubes. The plasmid concentration was detected and the recovery rate was calculated.

[0062] Figure 6 The recovery rate comparison results are shown. The results show that the plasmid recovery rate of the micro / nano bubble treatment group is significantly higher than that of the NaHCO3 group and the NH4HCO3 group.

[0063] Agarose gel electrophoresis

[0064] After clarifying and filtering, the liquid was washed with 100 kDa ultrafiltration and then subjected to agarose gel electrophoresis (1.0% agarose gel, 150 V, 30 min). Figure 7 The results are shown in the agarose gel electrophoresis chart, where M is the marker (DNA bar), A is NC (negative control), B is NaHCO3, C is NH4HCO3, and D is MNB (micro / nanobubbles). The electrophoresis results show that the plasmid bands in the micro / nanobubble treated group were clear and showed no significant difference compared to the negative control.

[0065] Monomer superspiral ratio analysis

[0066] analyze Figure 7 The content of different plasmid morphologies was determined, and the ratio of monomer supercoiled was calculated. Figure 8 The results show a comparison of the supercoil ratio of plasmid monomers. The results indicate that the supercoil ratio of monomers in the micro / nano bubble treatment group is higher than that in the NaHCO3 and NH4HCO3 groups.

[0067] Endotoxin concentration detection

[0068] Endotoxins, also known as lipopolysaccharides, are components of the outer wall of Gram-negative bacterial cell walls, composed of lipids and polysaccharides. Endotoxins are highly hazardous, causing cell damage, inflammation, shock, organ dysfunction, and even death. Humans are extremely sensitive to them; even trace amounts can cause toxic reactions such as fever. During the alkaline lysis of *E. coli* fermentation broth, endotoxins are released into the solution. Because endotoxins typically form vesicle-like structures, their molecular weight, charge, and hydrophobicity are very similar to plasmid DNA. Although endotoxins can be removed by precipitation, filtration, and chromatography, the combined removal capacity of these methods is relatively limited in actual production. Therefore, the amount of endotoxins released during the alkaline lysis and solid-liquid separation stages should be minimized to reduce the pressure on downstream processing. Figure 9 The results show a comparison of endotoxin content after clarification and filtration. The results indicate that the endotoxin content in the micro / nanobubble treatment group was significantly lower than that in the NaHCO3 and NH4HCO3 groups.

[0069] Example 2

[0070] The plasmids and host cell information used in this embodiment are shown in the table below:

[0071] plasmid name plasmid B plasmid size 1.2kbp resistance gene KanR host cells E. coli DH5α <![CDATA[Bacterial concentration (OD 600 )]]> 57 Plasmid volume yield 579mg / L

[0072] In this embodiment, alkaline pyrolysis and flocculation flotation operations are performed according to steps 1 to 6 above (using method one to introduce micro-nano bubbles). The operating parameters are the same as in Example 1, and will not be repeated here.

[0073] Figure 10 The results of the alkaline pyrolysis and flocculation flotation experiments (50g of bacterial sludge / batch) in this embodiment are shown, where A is before flotation, B is after flotation for 30 minutes with micro-nano bubbles, and C is after flotation for 90 minutes with micro-nano bubbles. The results show that after micro-nano bubble treatment, the solid-liquid separation effect of the material liquid is good, and the cake-shaped solid impurities are clearly layered and compact.

[0074] After the clarified and filtered solution was washed with 100KD ultrafiltration, it was subjected to agarose gel electrophoresis. Figure 11The image shows the results of agarose gel electrophoresis, where M stands for Marker (DNA bar), 1 for MiniPrep (plasmid mini-prep), and 2 for MNB (micro / nanobubbles). The electrophoresis results show that the plasmid bands treated with micro / nanobubbles are clear and of good quality.

[0075] The data from this embodiment are summarized in the table below:

[0076] plasmid recovery rate 84.1% Monomer superspiral ratio 99.8% endotoxin residue <20 EU / mg

[0077] The results of the above embodiments show that the solid-liquid separation operation after plasmid alkaline lysis using micro-nano bubble technology has a better solid-liquid separation effect than the flotation effect using NaHCO3 and NH4HCO3, and performs better in key indicators such as plasmid recovery rate, plasmid monomer supercoil content, and endotoxin residue. At the same time, the solid-liquid stratification is more obvious, significantly reducing the difficulty of clarification and filtration.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for solid-liquid separation after cell lysis using micro / nano bubble technology, characterized in that, The method for solid-liquid separation after cell lysis using micro / nano bubble technology includes: Step 1: Resuspend the collected cells in P1 solution to obtain a cell resuspension with a cell content of 10%. Step 2: Mix the cell resuspension with P2 solution and carry out an alkaline lysis reaction; Step 3: After the alkaline pyrolysis reaction is completed, the P3 solution is mixed with the P2 solution for acid neutralization to terminate the alkaline pyrolysis reaction and flocculate impurities; Step 4: After acid neutralization, the P4 solution with added micro-nano bubbles is mixed with the cell resuspension to carry out a flocculation reaction. After the flocculation reaction is completed, the mixture is allowed to stand and separate into layers to achieve preliminary solid-liquid separation. Step 5: Clarify and filter the solid-liquid separated liquid portion, and collect the clarified liquid after filtration.

2. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The P1 solution is a solution containing 25 mmol / L Tris, 7.9 mmol / L EDTA, and 55 mmol / L glucose, with a pH of 7.5 ± 0.

2. The cell resuspension contains 5%–25% cells. The P2 solution is a lysis buffer containing 0.2 mol / L NaOH and 1% SDS. The mixing ratio of the cell resuspension to the P2 solution is 0.8:1.2 to 1.2:0.8, and the alkaline lysis reaction time is 10 seconds to 300 seconds. The P3 solution is a 3 mol / L potassium acetate solution, and the mixing ratio of the P3 solution to the P2 solution is 0.8:1.2 to 1.2:0.8, with an acid neutralization time of 10 seconds to 300 seconds; The P4 solution is one of a 1-5 mol / L CaCl2 solution or a ZnSO4 solution, and the mixing ratio of the P4 solution to the cell resuspension is 0.5:1.0 to 1.5:1.

0. The flocculation reaction time is 0.5 minutes to 60 minutes.

3. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The method for introducing micro / nano bubbles in step 4 is as follows: Micro-nano bubbles were added to the P4 solution using a micro-nano bubble generator, and then the P4 solution containing micro-nano bubbles was mixed with the cell resuspension to carry out a flocculation reaction.

4. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The method for introducing micro / nano bubbles in step 4 is as follows: Micro-nano bubbles are added to other buffer solutions using a micro-nano bubble generator, and then the other buffer solutions containing micro-nano bubbles are added to the feed solution after the flocculation reaction is completed.

5. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The micro-nano bubble content mentioned in step 4 is 1000-20000 NTU in terms of turbidity, and the settling time is 1 minute to 120 minutes.

6. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The clarification filtration uses a deep membrane stack with a filtration pressure of less than 2 bar, and the pore size of the deep membrane stack is 0.1 μm to 100 μm.

7. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The clarification filtration is performed using a capsule filter with a filtration pressure of less than 2 bar, and the pore size of the capsule filter is 0.1 μm to 0.65 μm.

8. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, The clarification filtration employs a deep membrane stack and a capsule filter for filtration at a pressure of less than 2 bar. The pore size of the deep membrane stack is 0.1 μm to 100 μm, and the pore size of the capsule filter is 0.1 μm to 0.65 μm.

9. The method for solid-liquid separation after cell lysis using micro / nano bubble technology according to claim 1, characterized in that, After the clarification and filtration are completed, the filter is washed with a top wash solution consisting of 0 to 5 times the dead volume of the filter, purified water, water for injection, or a buffer solution, and the top wash solution is then combined with the collected clarified liquid.

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