Method for removing thalli in fermentation liquor and application thereof
Through the combined method of multiple membrane stack filtration, filter element filtration, anion exchange chromatography and hydrophobic chromatography, the problems of low efficiency and high cost of bacterial removal in fermentation broth in the existing technology are solved, and efficient and low-cost bacterial removal and protein preparation effects are achieved.
Patent Information
- Application Number
- CN202510878333.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-19
AI Technical Summary
The existing technology has problems such as expensive equipment, low efficiency, high cost and poor effect in removing bacteria from fermentation broth. Especially when treating Staphylococcus epidermidis fermentation broth, the existing method is difficult to achieve efficient and low-cost bacteria removal.
A combination of multiple membrane stack filtration, filter cartridge filtration, anion exchange chromatography and hydrophobic chromatography was used to achieve efficient removal of bacteria from the fermentation broth by optimizing filtration conditions and buffer gradient elution.
The efficiency and effect of bacterial removal were significantly improved, the purification cost was reduced, the purification rate and the preparation effect of the target protein were increased, and the safety of the feed solution was enhanced.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, in particular to a method for removing bacteria from fermentation liquid and application thereof. Background Art
[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.
[0003] At present, for the fermentation broth of bacteria, such as Escherichia coli, Bacillus subtilis, yeast, etc., centrifugation, diatomaceous earth filtration, membrane stack filtration, hollow fiber column ultrafiltration, membrane package ultrafiltration, filter cartridge filtration and other methods are usually used to remove bacteria. However, these methods generally have the following defects: (1) Centrifugation: The continuous flow tubular centrifuge used in the factory is expensive and has low processing efficiency. Generally, 50L / h is considered a large scale, and the processing effect is poor. Generally, the turbidity of the supernatant after treatment can only reach about 100NTU. (2) Diatomaceous earth filtration: The processing effect is poor and can only be used as a rough treatment. Generally, the turbidity of the supernatant after treatment exceeds 1000NTU, and impurities will be mixed into the supernatant after treatment. (3) Membrane stack filtration: There are many types of membrane stacks. The most suitable model needs to be selected for different bacteria, different growth characteristics, different collection times, and different fermentation processes. (4) Hollow fiber column: The equipment price is extremely high, the processing efficiency is low, and generally does not exceed 10L / h / m 2 (5) Membrane ultrafiltration: The equipment is expensive and the treatment efficiency is low, generally not exceeding 10L / h / m 2 (6) Filter element filtration: It cannot process liquid with too high turbidity. As the turbidity of the liquid increases, the filter element load will decrease exponentially.
[0004] Staphylococcus epidermidis ( Staphylococcus epidermidis Staphylococcus epidermidis is a Gram-positive coccus widely found in human skin, mucous membranes, and the external auditory canal. It belongs to the coagulase-negative staphylococcus (CoNS) group and is less pathogenic. Staphylococcus epidermidis is spherical or slightly elliptical, approximately 1.0 micron in diameter, often clustering in grape-like clusters. It lacks flagella and is nonmotile. It grows well on standard culture media, either aerobically or facultatively anaerobically, with an optimal growth temperature of 37°C.
[0005] Recombinant Staphylococcus epidermidis is a subunit engineered bacterium characterized by high solubility of the target protein, high expression levels, and minimal interference from native proteins. The target protein produced by fermentation with recombinant Staphylococcus epidermidis is secreted into the fermentation supernatant, necessitating post-fermentation processing to remove the bacteria and retain the supernatant. The more thoroughly the bacteria are removed during this step, the lower the turbidity of the supernatant, which significantly reduces the difficulty of subsequent concentration processing, improves protein inactivation stability, and reduces vaccine immune side effects.
[0006] Given the aforementioned shortcomings of existing methods for removing bacteria from fermentation broth, and the fact that methods for removing bacteria from fermentation broths of Staphylococcus epidermidis have not yet been fully developed, there is currently an unresolved issue regarding how to improve these methods and make them applicable to the removal of bacteria from fermentation broths of Staphylococcus epidermidis.
[0007] In view of this, the present invention is proposed. Summary of the Invention
[0008] The object of the present invention is to provide a method for removing bacteria from fermentation broth and its application, so as to improve the effect of removing bacteria from fermentation broth.
[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, a method for removing bacteria from a fermentation broth is provided, comprising sequentially subjecting the fermentation broth to at least two membrane stack filtrations, filter element filtrations, anion exchange chromatography, and hydrophobic chromatography.
[0010] In an optional embodiment, the at least two membrane stack filtrations include initially filtering the fermentation broth using a membrane stack with a pore size of 0.04 to 9 μm, and then performing a secondary filtration using a membrane stack with a pore size of 0.1 to 9 μm.
[0011] In an optional embodiment, the inlet hydraulic pressure of the primary filtration is 0.4~0.6 bar, the outlet hydraulic pressure is 0.1~0.3 bar, and the flow rate is 3~5 L / min; and / or, the inlet hydraulic pressure of the secondary filtration is 0.3~0.5 bar, the outlet hydraulic pressure is 0.1~0.3 bar, and the flow rate is 7~9 L / min.
[0012] In an optional embodiment, in the preliminary filtration, the filtration area used is 1.7 to 2.3 m per 1000 L of fermentation liquid. 2 Membrane stack; and / or, in the secondary filtration, the filtration area used per 1000L fermentation liquid is 1.7~2.3m 2 Membrane stack.
[0013] In an optional embodiment, the stack membrane for the primary filtration and the stack membrane for the secondary filtration are independently selected from the cobetter® CSD-C series stacked filter elements.
[0014] In an optional embodiment, the filter element filtration uses a filter element with a pore size of 0.8~0.2μm.
[0015] In an optional embodiment, the anion exchange chromatography includes loading the filtrate obtained after filtering the filter element onto an anion exchange chromatography column equilibrated with a first buffer, and washing with the first buffer after loading; then gradient eluting the anion exchange chromatography column with a mixture of the first buffer and the second buffer and collecting the eluate.
[0016] The first buffer solution is 48-52 mM Tris solution with a pH of 7.8-8.2; the second buffer solution is a solution containing 48-52 mM Tris and 0.8-1.2 M NaCl with a pH of 7.8-8.2.
[0017] In an optional embodiment, during the gradient elution process, the volume percentage of the second buffer solution is increased from 55% to 70%.
[0018] In an optional embodiment, the gradient elution program, measured in percentage by volume of the second buffer, is as follows: from 0 to 10 min, from 55% to 60%, from 10 to 60 min, from 60% to 65%, from 60 to 150 min, from 65% to 70%, and maintained at 70% from 150 to 240 min.
[0019] In an optional embodiment, the anion exchange chromatography uses an anion exchange chromatography column Cellufine MAXQ-H.
[0020] In an optional embodiment, the hydrophobic chromatography includes loading the filtrate obtained after anion exchange chromatography onto a hydrophobic chromatography column equilibrated with a fourth buffer, and then gradient eluting the anion exchange chromatography column with a mixture of a third buffer and a fourth buffer and collecting the eluate.
[0021] The third buffer solution is a 48-52 mM Tris solution with a pH of 7.8-8.2, and the fourth buffer solution is a solution containing 48-52 mM Tris and 0.8-1.2 M NaCl with a pH of 7.8-8.2.
[0022] In an optional embodiment, during the gradient elution process, the volume percentage of the third buffer is increased from 40% to 65%.
[0023] In an optional embodiment, the gradient elution program, measured in percentage by volume of the third buffer, is as follows: from 0 to 20 min, from 40% to 45%, from 20 to 80 min, from 45% to 50%, from 80 to 140 min, from 50% to 55%, from 140 to 200 min, from 55% to 60%, and from 200 to 240 min, from 60% to 65%.
[0024] In an optional embodiment, the hydrophobic chromatography uses a hydrophobic chromatography column MAX pheny 1.
[0025] In an optional embodiment, the method further comprises the step of filtering the eluate obtained through the hydrophobic chromatography using a membrane bag.
[0026] In an optional embodiment, the fermentation broth is Staphylococcus epidermidis fermentation broth.
[0027] In a second aspect, the invention provides the application of the method for removing bacteria from fermentation broth described in the first aspect in preparing a target protein.
[0028] In an optional embodiment, the target protein is expressed by Staphylococcus epidermidis.
[0029] In a third aspect, a protein preparation method is provided, which comprises fermenting a strain expressing a target protein, and then removing bacteria from the fermentation broth using the method described in the first aspect.
[0030] In an optional embodiment, the target protein is expressed by Staphylococcus epidermidis.
[0031] Compared with the prior art, the present invention uses a method combining membrane filtration, filter element filtration, anion exchange chromatography and hydrophobic chromatography to greatly improve the efficiency and effect of removing host bacteria, significantly reducing the purification cost. This method not only shortens the time required for filter element filtration, anion exchange chromatography and hydrophobic chromatography, but also reduces the loss of consumables required in the above steps, thereby reducing the cost of consumables. The present invention rationally arranges the purification means at each stage, so that the filtrate obtained in the previous step can be fully purified while ensuring the efficiency and low cost of the current purification step; through overall process improvement, the impurity removal effect is better than most conventional methods, a better target protein preparation effect is obtained, and the safety of the feed liquid is greatly improved. DETAILED DESCRIPTION
[0032] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0033] It should be noted that: Unless otherwise specified, all embodiments and preferred implementation methods mentioned herein can be combined with each other to form new technical solutions. All technical features and preferred features mentioned herein can be combined with each other to form new technical solutions. The components or preferred components involved can be combined with each other to form new technical solutions.
[0034]
[0046] "Range" disclosed herein in the form of lower limits and upper limits can have one or more lower limits, and one or more upper limits, respectively.
[0035] Herein, unless otherwise indicated, the various reactions or process steps may be performed sequentially or in any non-sequential manner. Preferably, the reaction methods herein are performed sequentially.
[0036] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual relationship, order, or importance between these entities or actions, such as numbering first, second, etc.
[0037] As used herein, unless otherwise stated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
[0038] Herein, the terms "comprise" or "comprising" are intended to imply the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
[0039] Unless otherwise indicated, the professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to this article.
[0040] In a first aspect, a method for removing bacteria from a fermentation broth is provided, the method comprising sequentially subjecting the fermentation broth to at least two membrane stack filtrations, filter element filtrations, anion exchange chromatography, and hydrophobic chromatography.
[0041] Membrane stack filtration is a fluid filtration technology that utilizes a multi-layer membrane structure. Its core component is a filter element composed of stacked layers of membranes. This membrane stack is composed of porous materials with a specific pore size distribution, which can selectively intercept impurities in the fluid based on their size. When the fluid passes through the membrane stack, impurities larger than the membrane pore size are retained on the membrane surface, while smaller molecules or pure fluids pass through the membrane layer and flow out. By optimizing the arrangement and combination of multiple membrane stack filtration processes and combining them with filter element treatment, the subsequent purification rate can be accelerated. Through membrane stack optimization, the cost of the required equipment and consumables is lower than most conventional methods. The preferred implementation is as follows: In an optional embodiment, the at least two membrane stack filtrations include initially filtering the fermentation broth using a membrane stack with a pore size of 0.04 to 9 μm, and then performing a secondary filtration using a membrane stack with a pore size of 0.1 to 9 μm.
[0042] In an optional embodiment, the stack membrane for the primary filtration and the stack membrane for the secondary filtration are independently selected from the cobetter® CSD-C series stacked filter elements.
[0043] In an optional embodiment, a cobetter® CSD-C series product model C16D0140PC23DOES stacked filter element stack is used for preliminary filtration.
[0044] In an optional embodiment, the inlet pressure of the preliminary filtration is 0.4~0.6 bar, for example but not limited to 0.4, 0.5 or 0.6 bar; the outlet pressure is 0.1~0.3 bar, for example but not limited to 0.1, 0.2, or 0.3 bar; the flow rate is 3~5 L / min, for example but not limited to 3, 4 or 5 L / min.
[0045] In an optional embodiment, in the preliminary filtration, the filtration area used is 1.7 to 2.3 m per 1000 L of fermentation liquid. 2 Membrane stack, preferably with a filtration area of 2.0m 2 Membrane stack.
[0046] In an optional embodiment, a cobetter® CSD-C series product model C16DS840PC23DOES stacked filter element is used for secondary filtration.
[0047] In an optional embodiment, the inlet pressure of the secondary filtration is 0.3~0.5 bar, for example but not limited to 0.3, 0.4 or 0.5 bar; the outlet pressure is 0.1~0.3 bar, for example but not limited to 0.1, 0.2, or 0.3 bar; the flow rate is 7~9 L / min, for example but not limited to 7, 8 or 9 L / min.
[0048] In an optional embodiment, in the secondary filtration, the filtration area used for each 1000L of fermentation liquid is 1.7~2.3m 2 Membrane stack, preferably with a filtration area of 2.0m 2 Membrane stack.
[0049] Cartridge filtration involves filtering the filtrate obtained through a cartridge-based membrane stack. A filter element, also known as a filter core, is a filter element that removes microorganisms from liquids or gases through physical retention to achieve sterility. In an optional embodiment, the filter core is a sterilizing-grade filter with a pore size of 0.8-0.2 μm.
[0050] In an optional embodiment, a 5-inch 0.8-0.2 μm sterilization grade filter element is used for filtering every 1000 L of fermentation broth.
[0051] In an optional embodiment, the anion exchange chromatography includes loading the filtrate obtained after filtering the filter element onto an anion exchange chromatography column equilibrated with a first buffer, washing with the first buffer after loading to remove unadsorbed substances in the anion exchange chromatography column; then gradient eluting the anion exchange chromatography column with a mixture of the first buffer and the second buffer and collecting the eluate. The first buffer is a 48-52 mM Tris solution with a pH of 7.8-8.2. The pH is, for example, but not limited to, 7.8, 8.0, or 8.2, and the Tris concentration is, for example, but not limited to, 48, 50, or 52 mM. The second buffer is a solution containing 48-52 mM Tris and 0.8-1.2 M NaCl, with a pH of 7.8-8.2. The Tris concentration is, for example, but not limited to, 48, 50, or 52 mM, the NaCl concentration is, for example, but not limited to, 0.8, 1.0, or 1.2 M, and the pH is, for example, but not limited to, 7.8, 8.0, or 8.2.
[0052] In an optional embodiment, the loading flow rate is 1.8 to 2.2 mL / min, for example but not limited to 1.8, 2.0 or 2.2 mL / min.
[0053] In an optional embodiment, during the gradient elution process, the volume percentage of the second buffer solution is increased from 55% to 70%.
[0054] In an optional embodiment, the gradient elution program, measured in percentage by volume of the second buffer, is as follows: from 0 to 10 min, from 55% to 60%, from 10 to 60 min, from 60% to 65%, from 60 to 150 min, from 65% to 70%, and maintained at 70% from 150 to 240 min.
[0055] In an optional embodiment, the elution flow rate is 1.3-1.7 mL / min, for example but not limited to 1.3, 1.5 or 1.7 mL / min.
[0056] In an optional embodiment, anion exchange chromatography is performed using an anion exchange chromatography column Cellufine MAX QH.
[0057] In an optional embodiment, the hydrophobic chromatography includes loading the filtrate obtained after the anion exchange chromatography onto a hydrophobic chromatography column equilibrated with a fourth buffer, and then gradient eluting the anion exchange chromatography column with a mixture of a third buffer and a fourth buffer and collecting the eluate. The third buffer is a 48-52 mM Tris solution with a pH of 7.8-8.2. The pH is, for example, but not limited to, 7.8, 8.0, or 8.2, and the Tris concentration is, for example, but not limited to, 48, 50, or 52 mM. The fourth buffer is a solution containing 48-52 mMTris and 0.8-1.2 M NaCl, with a pH of 7.8-8.2. The Tris concentration is, for example, but not limited to, 48, 50, or 52 mM, the NaCl concentration is, for example, but not limited to, 0.8, 1.0, or 1.2 M, and the pH is, for example, but not limited to, 7.8, 8.0, or 8.2.
[0058] In an optional embodiment, the loading flow rate is 1.8 to 2.2 mL / min, for example but not limited to 1.8, 2.0 or 2.2 mL / min.
[0059] In an optional embodiment, during the gradient elution process, the volume percentage of the third buffer is increased from 40% to 65%.
[0060] In an optional embodiment, the gradient elution program, measured in percentage by volume of the third buffer, is as follows: from 0 to 20 min, from 40% to 45%, from 20 to 80 min, from 45% to 50%, from 80 to 140 min, from 50% to 55%, from 140 to 200 min, from 55% to 60%, and from 200 to 240 min, from 60% to 65%.
[0061] In an optional embodiment, the elution flow rate is 0.8-1.2 mL / min, for example but not limited to 0.8, 1.0 or 1.2 mL / min.
[0062] In an optional embodiment, hydrophobic chromatography is performed using a hydrophobic chromatography column MAX pheny 1.
[0063] In an optional embodiment, the method further includes the step of filtering the eluate obtained through the hydrophobic chromatography using a membrane bag. Membrane bag filtration is a filtration method based on membrane separation technology, which mainly achieves the separation and concentration of substances through tangential flow filtration. The molecular weight cutoff of the membrane bag can be selected according to the molecular weight of the target protein.
[0064] In an optional embodiment, the molecular weight cut-off of the membrane package is 100 kD.
[0065] In an optional embodiment, the flow rate during membrane filtration is 9-11 mL / min, for example but not limited to 9, 10 or 11 mL / min.
[0066] In an optional embodiment, the method for removing bacteria from the fermentation broth comprises the following steps: (1) Membrane pile treatment: The harvested 1000 L bacterial solution is first filtered through a 2m 2 The 0140 membrane stack with a pore size of 0.04~9μm was used for preliminary filtration under the conditions of inlet pressure of 0.4~0.6 bar, outlet pressure of 0.1~0.3 bar, and flow rate of 3~5 L / min; then the filter area was 2 m 2 The S840 membrane stack with a pore size of 0.1~9μm was used for secondary filtration under the conditions of an inlet hydraulic pressure of 0.3~0.5 bar, an outlet hydraulic pressure of 0.1~0.3 bar, and a flow rate of 7~9 L / min to obtain a supernatant with a turbidity value lower than 50 NTU.
[0067] (2) Filter treatment: The supernatant after the membrane stack treatment is filtered through a 5-inch 0.8~0.2μm sterilization grade filter to obtain a supernatant with a turbidity value lower than 10 NTU.
[0068] (3) Anion exchange chromatography was performed using an anion exchange chromatography column Cellufine MAX QH: the anion exchange chromatography column was equilibrated with the first buffer, and then the supernatant after the filter element treatment was loaded, and the loading speed was 1.8~2.2 mL / min; the anion exchange chromatography column was washed with the first buffer until the absorbance value at a wavelength of 280 nm began to rise significantly and stabilize; the washing speed was 2 mL / min; and then the anion exchange chromatography column was gradient eluted with a mixture of the first buffer and the second buffer, the volume percentage of the second buffer was 55%~70%, and the gradient elution program calculated by the volume percentage of the second buffer was: 0 to 10 min, from 55% to 60%, 10 to 60 min, from 60% to 65%, 60 to 150 min, from 65% to 70%, 150 to 240 min, maintained at 70%, and the elution flow rate was 1.3~1.7 mL / min. The eluate is collected as the first eluate, and the volume ratio of the obtained first eluate to the sample to be purified is 1:(8-10). Optional chromatography column specifications are: filler particle size 90 μm, chromatography column diameter 80 cm, and height 60 cm.
[0069] The first buffer solution is 48-52 mM Tris solution with a pH of 7.8-8.2; the second buffer solution is a solution containing 48-52 mM Tris and 0.8-1.2 M NaCl with a pH of 7.8-8.2.
[0070] (4) Using a hydrophobic chromatography column MAX pheny 1, hydrophobic chromatography was performed: the first eluate was loaded onto the hydrophobic chromatography column pretreated with the fourth buffer, and the hydrophobic chromatography column was eluted using a gradient solution of a mixture of the third buffer and the fourth buffer. The volume percentage of the third buffer was 40% to 65%. The gradient elution program calculated based on the volume percentage of the third buffer was as follows: from 0 to 20 min, from 40% to 45%, from 20 to 80 min, from 45% to 50%, from 80 to 140 min, from 50% to 55%, from 140 to 200 min, from 55% to 60%, from 200 to 240 min, from 60% to 65%, and the elution rate was 0.8 to 1.2 mL / min. The eluate was collected as the second eluate, and the volume ratio of the first eluate to the second eluate was 1:(0.8 to 1.2). The optional specifications of the chromatography column are: filler particle size is 90 μm, chromatography column diameter is 80 cm, and height is 40 cm.
[0071] The third buffer solution is a 48-52 mM Tris solution with a pH of 7.8-8.2; the fourth buffer solution is a solution containing 48-52 mM Tris and 0.8-1.2 M NaCl with a pH of 7.8-8.2.
[0072] (5) The eluate from step (4) was subjected to membrane filtration, and the obtained filtrate was the target purified liquid. The molecular weight cutoff of the membrane used was 100 kD, and the flow rate during the membrane filtration was 9-11 mL / min. During the membrane filtration, the flow-through valve was closed and only the filtrate was collected.
[0073] In an optional embodiment, the fermentation broth is Staphylococcus epidermidis fermentation broth.
[0074] In an optional embodiment, the concentration of the bacteria in the fermentation broth is 10 9 ~10 12 CFU / mL.
[0075] The method for removing bacteria from fermentation broth provided in the first aspect has the following beneficial effects: (1) Time efficiency is improved. In the preferred scheme, by optimizing processes including inlet hydraulic pressure, outlet hydraulic pressure, flow rate, etc., the crude treatment effect of the feed liquid is greater than most conventional methods, and is consistent with the most efficient diatomaceous earth and membrane stack; after efficient crude treatment, the supernatant is better than the supernatant provided by other crude treatment methods, and the purification rate of the two-stage ion chromatography is faster.
[0076] (2) Improved effect. In the preferred scheme, by optimizing the type, model, specification and arrangement of the membrane stack, the turbidity of the treated liquid is lower than that of most conventional methods, and is similar to the filter element with the lowest turbidity. In the preferred scheme, the turbidity of the treated supernatant is lower than 10NTU, which is consistent with the existing best process effect, and the viscosity is lower than the minimum detection limit of the viscometer. Using this highly clarified liquid for chromatographic purification can effectively improve the chromatographic effect, and the purity of the target protein obtained exceeds 95%.
[0077] (3) Cost reduction: through membrane stack area testing and optimization, the cost of equipment and consumables is lower than most conventional methods; after the efficient crude treatment of the liquid for chromatography purification, the required consumables are greatly reduced. In the preferred solution, only 2 2m3 per 1000L of fermentation liquid are required. 2 The cost of a membrane stack and a 5-inch filter element is lower than all existing processes.
[0078] (4) Safety improvement: Through overall process improvement, the impurity removal effect is better than most conventional methods, achieving better protein preparation results and greatly improving the safety of the feed liquid. The treated supernatant has been verified by mice to have no harmful substances remaining.
[0079] In a second aspect, the invention also provides the use of the method for removing bacteria from fermentation broth described in the first aspect in preparing protein.
[0080] In an optional embodiment, the target protein is expressed by Staphylococcus epidermidis.
[0081] In a third aspect, a protein preparation method is also provided, which comprises fermenting a strain expressing a target protein, and then removing bacteria from the fermentation broth using the method described in the first aspect.
[0082] In an optional embodiment, the target protein is expressed by Staphylococcus epidermidis.
[0083] In an optional embodiment, the Staphylococcus epidermidis secretes and expresses the target protein.
[0084] In an optional embodiment, the preparation method further comprises subjecting the fermentation broth from which the bacteria have been removed to one or more treatments selected from the group consisting of purification, enrichment, concentration, drying, and sterilization.
[0085] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.
[0086] The device information used in the following examples is as follows: 0.04~9μm 0140 membrane stack: cobetter® CSD-C series, model: C16D0140PC23DOES stacked filter element, number of layers is 10: 0.1~9μm S840 membrane stack: cobetter® CSD-C series, model: C16DS840PC23DOES stacked filter element, number of layers is 10; 0.04~18.0µm S840 membrane stack: cobetter® CSD-C series, model: C16DS840PC22DOES stacked filter element, number of layers is 10; 0.1~9μm 4070 membrane stack: Model: C16D4070PC23DOES stacked filter element, number of layers is 10; The filter element is Sartopore® 2 filter element: material is PES, length is 5 inches, membrane area is 0.3m 2 , pore size is 0.2μm.
[0087] The anion exchange chromatography column is Cellufine MAX QH, with a filler particle size of 90 μm, a chromatography column diameter of 80 cm, and a height of 60 cm.
[0088] The hydrophobic chromatography column is MAX pheny 1, the filler particle size is 90 μm, the chromatography column diameter is 80 cm, and the height is 40 cm.
[0089] Example 1 This example aims to remove bacteria produced by fermentation of recombinant Staphylococcus epidermidis in large quantities exceeding 1000L of bacterial liquid by combining membrane stack treatment, filter element treatment, anion chromatography, and hydrophobic chromatography. The specific method is as follows: (1) Membrane pile treatment: The harvested 1000 L bacterial solution is first filtered through a 2m 2 The 0140 membrane stack with a pore size of 0.04~9μm was used for preliminary filtration under the conditions of inlet pressure of 0.5 bar, outlet pressure of 0.2 bar, and flow rate of 4 L / min; then the filter area was 2 m 2 The S840 membrane stack with a pore size of 0.1~9μm was subjected to secondary filtration under the conditions of an inlet hydraulic pressure of 0.4 bar, an outlet hydraulic pressure of 0.2 bar, and a flow rate of 8 L / min to obtain a supernatant with a turbidity value of 45NTU.
[0090] (2) Filter treatment: The supernatant after the membrane stack treatment was filtered through a Sartojet® 36FF filter cartridge to obtain a supernatant with a turbidity value of 8 NTU, which was used as the sample to be purified in step (3). The concentration of the sample to be purified was 150-250 μg / mL and the volume was 5 L.
[0091] (3) Anion exchange chromatography was performed using an anion exchange chromatography column Cellufine MAX QH: the anion exchange chromatography column was equilibrated with the first buffer, and then the supernatant after the filter element treatment was loaded at a loading rate of 2 mL / min; the anion exchange chromatography column was washed with the first buffer until the absorbance value at a wavelength of 280 nm began to rise significantly and then stabilized; the washing rate was 2 mL / min; the anion exchange chromatography column was then gradiently eluted with a mixture of the first buffer and the second buffer, and the elution procedure was as follows based on the volume percentage of the second buffer: from 0 to 10 min, from 55% to 60%, from 10 to 60 min, from 60% to 65%, from 60 to 150 min, from 65% to 70%, and from 150 to 240 min, maintained at 70%, and the elution flow rate was 1.5 mL / min. The eluate was collected as the first eluate, and the volume ratio of the obtained first eluate to the sample to be purified was 1: (8~10).
[0092] The turbidity of the first eluate was 2 NTU and the protein purity was 87%.
[0093] (4) Hydrophobic chromatography was performed using a hydrophobic chromatography column MAX pheny 1: the first eluate was loaded onto the hydrophobic chromatography column pretreated with the third buffer at a loading rate of 2 mL / min; the hydrophobic chromatography column was eluted using a gradient of a mixture of the third buffer and the fourth buffer. The elution procedure, based on the volume percentage of the third buffer, was as follows: from 0 to 20 min, from 40% to 45%, from 20 to 80 min, from 45% to 50%, from 80 to 140 min, from 50% to 55%, from 140 to 200 min, from 55% to 60%, and from 200 to 240 min, from 60% to 65%, with an elution rate of 1 mL / min. The eluate was collected as the second eluate, and the volume ratio of the first eluate to the second eluate was 1:(0.8-1.2). The turbidity of the second eluate was 1 NTU, and the protein purity was 96%.
[0094] (5) The eluate from step (4) was filtered through a membrane filter. The resulting filtrate was the purified target solution. The membrane filter had a molecular weight cutoff of 100 kD and a flow rate of 10 mL / min. The flow-through valve was closed during the membrane filter, and only the filtrate was collected. The turbidity of the filtrate was 1 NTU, and the protein purity was 96%.
[0095] In this embodiment, the first buffer solution and the third buffer solution are 50 mM Tris solutions with a pH of 8; the second buffer solution and the fourth buffer solution are solutions containing 50 mM Tris and 1 M NaCl with a pH of 8.
[0096] The filtrate obtained in step (5) was injected intravenously into 18-22 g Kunming white mice at a dose of 0.4 mL, and no abnormal reaction occurred, indicating that it had no toxic effect on mice.
[0097] Example 2 Selection of different types of membrane stacks: Based on factors such as the turbidity, viscosity, and protein adsorption characteristics of the feed solution, various types of membrane stacks such as 0140, S840, and 4070 were selected, with various pore size specifications such as 0.04-9.0µm, 0.1-9.0µm, and 0.04-18.0µm for experiments. The preliminary filtration conditions and secondary filtration conditions of each experimental group were the same as those in Example 1. The experimental groups were divided into the following groups: Table 1
[0098] The above experimental results show that the optimal combination for recombinant Staphylococcus epidermidis is to first use the 0140 membrane stack with a specification of 0.04-9.0 µm for coarse filtration to remove bacteria, and then use the S840 membrane stack with a specification of 0.1-9.0 µm for filtration to remove nucleic acids and other viscous substances. This combination results in the lowest turbidity in the filtrate.
[0099] Example 3 The membrane stack treatment process was selected based on Example 2. A 0140 membrane stack with a specification of 0.04-9.0 µm was used for primary filtration, followed by a S840 membrane stack with a specification of 0.1-9.0 µm for secondary filtration. The inlet and outlet pressures and flow rates of the two steps were adjusted. The experimental groups were as follows: Experimental group 1: primary filtration at an inlet pressure of 0.5 bar, an outlet pressure of 0.2 bar, and a flow rate of 4 L / min; secondary filtration at an inlet pressure of 0.4 bar, an outlet pressure of 0.2 bar, and a flow rate of 8 L / min, with a turbidity of 45; Experimental group 2: primary filtration at an inlet pressure of 0.4 bar, an outlet pressure of 0.3 bar, and a flow rate of 3 L / min; secondary filtration at an inlet pressure of 0.3 bar, an outlet pressure of 0.3 bar, and a flow rate of 7 L / min; turbidity of 43; Experimental group 3: Primary filtration was performed at an inlet pressure of 0.6 bar, an outlet pressure of 0.1 bar, and a flow rate of 5 L / min. Secondary filtration was performed at an inlet pressure of 0.5 bar, an outlet pressure of 0.1 bar, and a flow rate of 9 L / min. The turbidity was 45. Experimental group 4: primary filtration was performed at an inlet pressure of 0.2 bar, an outlet pressure of 0.5 bar, and a flow rate of 1 L / min. Secondary filtration was performed at an inlet pressure of 0.6 bar, an outlet pressure of 0.1 bar, and a flow rate of 5 L / min. The turbidity was 51. Experimental group 5: Primary filtration was performed at an inlet pressure of 1 bar, an outlet pressure of 0.1 bar, and a flow rate of 7 L / min. Secondary filtration was performed at an inlet pressure of 1 bar, an outlet pressure of 0.1 bar, and a flow rate of 10 L / min. The turbidity was 55.
[0100] From the above experimental results, it can be seen that the turbidity of experimental groups 1-3 is below 50, indicating that their process conditions can achieve better filtration effects and have better filtration flux.
[0101] Example 4 Table 2 compares the effects of removing bacteria from Staphylococcus epidermidis fermentation broth using different process flows.
[0102] Table 2
[0103] Example 5 The effects of different process flows on removing bacteria from Staphylococcus epidermidis fermentation broth were compared. The experimental groups were as follows: Experimental Group 1: The only difference from Example 1 is that the supernatant obtained from the membrane stack treatment in step (1) is directly subjected to anion exchange chromatography in step (3) without the filter element treatment in step (2).
[0104] Experimental Group 2: The only difference from Example 1 is that the supernatant after the filter element treatment is directly subjected to step (4) hydrophobic chromatography without step (3) anion exchange chromatography.
[0105] Experimental Group 3: The only difference from Example 1 is that the filtrate from the anion exchange chromatography was directly subjected to membrane filtration without undergoing the hydrophobic chromatography in step (4).
[0106] Experimental Group 4: The only difference from Example 1 is that step (4) hydrophobic chromatography is performed first, and then step (3) anion exchange chromatography is performed.
[0107] Experimental Group 5: The only difference from Example 1 is that step (3) was replaced with gel filtration chromatography, using Superdex 200 chromatography packing for purification. The chromatography column size was 16 mm × 100 cm, requiring approximately 200 ml of chromatography packing, allowing a 10 ml sample to be loaded, with a protein concentration of approximately 2 mg / ml. If 1000 L of feed solution was processed with a protein concentration of 0.5 mg / ml, ultrafiltration and concentration would need to be 4-fold, i.e., to 250 L. Based on a sample loading of 5-10% of the column volume, 1250-2500 L of packing would be required, resulting in high costs and impractical for industrial scale-up.
[0108] Experimental Group 6: The only difference from Example 1 is that step (4) was replaced with gel filtration chromatography, and Superdex 200 chromatography packing was used for purification. The chromatography column size was 16 mm × 100 cm, requiring approximately 200 ml of chromatography packing, and a 10 ml sample load was possible, with a protein concentration of approximately 2 mg / ml. If 1000 L of feed solution was processed with a protein concentration of 0.5 mg / ml, ultrafiltration and concentration would need to be 4-fold, i.e., to 250 L. Based on a sample load of 5-10% of the column volume, 1250-2500 L of packing would be required, resulting in high costs and impractical for industrial scale-up.
[0109] Experimental Group 7: The only difference from Example 1 is that step (2) filter element treatment is performed first, and then step (1) membrane stack treatment is performed. Because the filter element is in direct contact with the high-turbidity fermentation liquid and is easily clogged by impurities, the replacement frequency increases by 3-5 times and the cost of consumables increases by 200%-300%, so industrial scale-up cannot be achieved.
[0110] Experimental Group 8: The only difference from Example 1 is that step (1) is replaced by first filtering through diatomaceous earth, then centrifuging, and then filtering the centrifuged supernatant through a filter element in step (2). The diatomaceous earth filtration parameters are: using 1000 pore size diatomaceous earth, at a flow rate of 500 L / h; the centrifugation parameters are: using a continuous flow centrifuge, at a centrifugal force of 14800 g, and a centrifugal speed of 50 L / min.
[0111] Experimental Group 9: The only difference from Example 1 is that the membrane stack treatment step in step (1) is replaced by: the harvested 1000 L scale bacterial liquid is filtered through a 2m 2 The 0140 membrane stack with a pore size of 0.04~9μm was used for preliminary filtration under the conditions of an inlet hydraulic pressure of 0.5 bar, an outlet hydraulic pressure of 0.2 bar, and a flow rate of 4 L / min.
[0112] Experimental Group 10: The only difference from Example 1 is that the membrane stack treatment step in step (1) was replaced by the harvested 1000 L scale bacterial liquid, which was filtered over a 2m 2 The S840 membrane stack with a pore size of 0.1~9μm was subjected to secondary filtration under the conditions of an inlet hydraulic pressure of 0.4 bar, an outlet hydraulic pressure of 0.2 bar, and a flow rate of 8 L / min to obtain a supernatant with a turbidity value of 45NTU.
[0113] Control group: The method of combined treatment of membrane stack treatment, filter element treatment, anion chromatography and hydrophobic chromatography treatment in Example 1 was adopted.
[0114] Table 3
[0115] Example 6 Comparative Example 1 was used to remove bacteria from different bacterial fermentation broths using the method provided in Example 1: the method provided in Example 1 was used to remove bacteria from the fermentation broth of recombinant Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis, and yeast, respectively.
[0116] Table 4
[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for removing bacteria from a fermentation broth, characterized in that: The method comprises sequentially subjecting the fermentation liquid to at least two membrane pile filtrations, filter element filtration, anion exchange chromatography and hydrophobic chromatography.
2. The method according to claim 1, characterized in that The at least two membrane stack filtrations include firstly filtering the fermentation broth using a membrane stack with a pore size of 0.04-9 μm, and then filtering it twice using a membrane stack with a pore size of 0.1-9 μm.
3. The method according to claim 2, wherein The inlet pressure of the primary filtration is 0.4-0.6 bar, the outlet pressure is 0.1-0.3 bar, and the flow rate is 3-5 L / min; and / or the inlet pressure of the secondary filtration is 0.3-0.5 bar, the outlet pressure is 0.1-0.3 bar, and the flow rate is 7-9 L / min; Optionally, in the preliminary filtration, a filtration area of 1.7 to 2.3 m2 is used per 1000 L of fermentation liquid. 2 Membrane stack; and / or, in the secondary filtration, the filtration area used per 1000L fermentation liquid is 1.7~2.3m 2 Membrane stack; Optionally, the stack membrane for the primary filtration and the stack membrane for the secondary filtration are independently selected from cobetter® CSD-C series laminated filter elements.
4. The method according to claim 1, wherein The filter element filtration uses a filter element with a pore size of 0.8~0.2μm.
5. The method according to claim 1, characterized in that The anion exchange chromatography comprises loading the filtrate obtained after filtering the filter element onto an anion exchange chromatography column equilibrated with a first buffer, washing with the first buffer after loading; then gradient eluting the anion exchange chromatography column with a mixture of the first buffer and the second buffer and collecting the eluate; The first buffer is a 48-52 mM Tris solution with a pH of 7.8-8.2; The second buffer is a solution containing 48–52 mM Tris and 0.8–1.2 M NaCl, with a pH of 7.8–8.2; Optionally, during the gradient elution process, the volume percentage of the second buffer is increased from 55% to 70%; Optionally, the gradient elution program, calculated as a percentage by volume of the second buffer, is as follows: from 0 to 10 min, increasing from 55% to 60%, from 10 to 60 min, increasing from 60% to 65%, from 60 to 150 min, increasing from 65% to 70%, and maintaining at 70% from 150 to 240 min; Optionally, the anion exchange chromatography uses an anion exchange chromatography column Cellufine MAX QH.
6. The method according to claim 1, characterized in that The hydrophobic chromatography comprises loading the filtrate obtained after the anion exchange chromatography onto a hydrophobic chromatography column equilibrated with a fourth buffer, then gradient eluting the anion exchange chromatography column with a mixture of the third buffer and the fourth buffer and collecting the eluate; The third buffer is a 48-52 mM Tris solution with a pH of 7.8-8.2, and the fourth buffer is a solution containing 48-52 mM Tris and 0.8-1.2 M NaCl with a pH of 7.8-8.2; Optionally, during the gradient elution process, the volume percentage of the third buffer is increased from 40% to 65%; Optionally, the gradient elution program, calculated as a percentage by volume of the third buffer, is as follows: from 0 to 20 min, increasing from 40% to 45%, from 20 to 80 min, increasing from 45% to 50%, from 80 to 140 min, increasing from 50% to 55%, from 140 to 200 min, increasing from 55% to 60%, and from 200 to 240 min, increasing from 60% to 65%; Optionally, the hydrophobic chromatography uses a hydrophobic chromatography column MAX pheny 1; Optionally, the method further comprises the step of filtering the eluate obtained through the hydrophobic chromatography using a membrane bag.
7. The method according to any one of claims 1 to 6, characterized in that The fermentation broth is Staphylococcus epidermidis fermentation broth.
8. Use of the method for removing bacteria from fermentation broth according to any one of claims 1 to 7 in the preparation of a target protein.
9. The use according to claim 8, characterized in that The target protein is expressed by Staphylococcus epidermidis.
10. A method for preparing a protein, characterized in that: The method comprises fermenting a strain to express a target protein, and then removing the bacterial cells in the fermentation broth using the method according to any one of claims 1 to 7; Optionally, the target protein is expressed by Staphylococcus epidermidis.