Alkali cracking system and equipment
By using an alkaline lysis chip with S-shaped segment small units connected in series in the alkaline lysis system, the problems of alkaline solution uniformity and time control were solved, achieving efficient extraction of plasmid DNA, which is suitable for large-scale mass production of mRNA vaccines.
Patent Information
- Application Number
- CN202422803884.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-11-07
- Estimated Expiration
- 2034-11-18
AI Technical Summary
In large-scale industrial production, existing technologies cannot maintain the uniformity of the alkaline solution during alkaline lysis, leading to plasmid DNA degradation. Furthermore, it is difficult to precisely control the alkaline lysis time and acid neutralization process, which limits the large-scale mass production of mRNA vaccines.
A chip for alkaline lysis with multiple S-shaped segments connected in series is used. By mixing alkaline solution and bacterial solution, and by precisely controlling the position of the acid inlet and the fluid velocity, the alkaline lysis time and mixing uniformity are achieved, thus avoiding the degradation of plasmid DNA.
It achieves uniformity and precise control of the alkaline lysis process, improves plasmid yield, is suitable for industrial-scale production, and reduces plasmid DNA loss.
Smart Images
Figure CN223522498U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model belongs to the field of biological medicine, and relates to an alkali lysis system and equipment. BACKGROUND
[0002] An mRNA vaccine is a nucleic acid vaccine, which produces an antigen by injecting a partial mRNA fragment of a virus into a human cell, and then excites a specific immune response to achieve the effect of forming an immune memory. Plasmid production is the first step of the mRNA vaccine, and usually uses recombinant E. coli containing a nucleotide sequence of interest to ferment and culture, so as to amplify and produce a sufficient amount of plasmid in the bacterial body. The bacterial body is collected, resuspended with a resuspension solution, and then lysed with a strong alkali, and after extraction and purification, the mRNA is obtained through subsequent enzyme cutting, transcription, and further purification.
[0003] The alkali lysis method is a commonly used method for extracting plasmid, and has been widely used in the field of gene therapy for DNA extraction and purification. The principle is that the bacterial suspension is exposed to a strong anion detergent with a high pH value, so that the cell wall is broken, the chromosomal DNA and the protein are denatured, and the plasmid DNA is released into the supernatant. During the lysis process, the bacterial protein, the broken cell wall, and the denatured chromosomal DNA will intertwine into a large complex, and the latter is covered with dodecyl sulfate and precipitated, and through a certain clarification process, the supernatant containing the plasmid DNA is obtained for subsequent purification of the plasmid DNA
[0004] In the prior art, under the laboratory scale, alkali lysis generally uses a beaker or a container with a volume of less than 50L to prepare alkali and acid solutions, and then manually pours the alkali solution into the bacterial suspension, so that the alkali lysis time can be accurately controlled. This operation cannot be applied to large-scale high-intensity batch operations, and cannot meet the needs of large-scale industrial production. In large-scale industrial production, how to maintain the uniformity of the alkali solution and avoid local over-strength alkali to cause degradation of the plasmid DNA is a major technical difficulty at present; how to accurately control the alkali lysis time and add acid for neutralization in time is also another major technical difficulty, which also limits the large-scale batch production of the corresponding mRNA vaccine.
[0005] Therefore, how to quickly and efficiently implement the process steps of alkali lysis to meet the needs of large-scale batch production is an important technical problem that needs to be solved by those skilled in the art at present. UTILITY MODEL CONTENTS
[0006] Based on the above-mentioned defects of the prior art that the alkali lysis operation is not suitable for scale-up production and the local solubility of the alkali solution is too high, which easily leads to the breakage and loss of the plasmid DNA.
[0007] The utility model discloses a first aspect provides a kind of alkali lysis system;Including lysis core component, conveying component, power component;The lysis core component is alkali lysis with chip, and when the main passage in the alkali lysis with chip is mixed lysis with lye by the bacteria liquid to be lysed, and after mixed neutralization with acid solution, alkali lysis is completed;The conveying component is connected with alkali lysis with chip, power component respectively, so that the bacteria liquid to be lysed reaches each component;The power component is connected with conveying component, and power is provided for the liquid flow in alkali lysis system;
[0008] Further, the alkali lysis with chip includes a substrate and a channel located inside the substrate. The channel includes a main channel, an alkali inlet, a bacteria liquid inlet located at one end of the main channel, a mixed outlet located at the other end of the main channel, and an acid inlet located in the middle of the main channel. The alkali inlet, the bacteria liquid inlet, and the acid inlet are connected to the main channel through a sample inlet channel.
[0009] Further, the main channel includes a plurality of S-shaped segments connected in series.
[0010] Further, the main channel includes 2-300 S-shaped segments connected in series.
[0011] Further, the main channel has 1-200 S-shaped segments between the acid inlet and the alkali inlet or the bacteria liquid inlet, and 1-300 S-shaped segments between the acid inlet and the mixed outlet.
[0012] Further, the main channel has an inner diameter of 0.1-100 mm.
[0013] Further, the S-shaped segment has a length of 0.5-50 mm.
[0014] Further, the acid inlet, the alkali inlet, and the bacteria liquid inlet have a diameter of 0.5-20 mm.
[0015] Further, the main channel has an inner bending radius of 0.3-30 mm for the S-shaped segment, and an outer bending radius of 0.4-40 mm.
[0016] Further, the angle between the alkali inlet and the bacteria liquid inlet is 10-180 degrees.
[0017] Further, the angle between the acid inlet and the S-shaped segment is 10-170 degrees.
[0018] Further, the substrate is one of circular, oval, square, trapezoidal column, hexagonal, pentagonal, triangular, and star-shaped.
[0019] Further, the conveying component is a pipe, preferably a circular pipe.
[0020] Further, the power component is a device for providing flow power to the liquid in the system;
[0021] Further, the power component is a pump, compressed gas, preferably a plunger pump;
[0022] The alkali lysis device comprises the alkali lysis system of the first aspect of the alkali lysis system.
[0023] Further, the alkali lysis device comprises a chip for alkali lysis, a conveying pipeline, a plunger pump and an interface, wherein the chip for alkali lysis and the conveying pipeline are defined as above.
[0024] Further, the interface comprises a bacterial liquid interface, an alkali liquid interface, an acid liquid interface and a sample outlet interface.
[0025] Further, the alkali lysis device further comprises a control system; and the control system is a terminal control system.
[0026] Further, the terminal is an intelligent interactive terminal.
[0027] Further, the intelligent interactive terminal comprises a computer terminal, a mobile phone terminal and a PLC terminal.
[0028] In the embodiment of the utility model, the alkali lysis device comprises an alkali lysis system for alkali lysis of fermentation bacterial liquid.
[0029] In the embodiment of the utility model, the terminal refers to a device or program for interacting with a computer system or network.
[0030] In the embodiment of the utility model, the PC terminal is a device for providing input and output functions for connecting to a computer system.
[0031] In the embodiment of the utility model, the multiple channels in the substrate can be connected in parallel to realize higher flux alkali lysis.
[0032] The utility model has the advantages of:
[0033] The alkali lysis system of the utility model, adopt the alkali lysis chip with multiple S shape section small units in series, carry out the mixed lysis of bacteria liquid and lye, can realize the uniformity of mixing quickly, effectively reduce the degradation of plasmid DNA caused by local lye over-concentration, and introduce acid liquor in the suitable position of the main channel formed by S shape section small units to neutralize the alkalinity in the mixed liquid after alkali lysis, adjust the position of acid liquor sampling port, cooperate with the speed of fluid in each sampling channel, accurately control the time of alkali lysis, make the plasmid release not incomplete due to insufficient alkali lysis time caused by acid liquor adding too early, and not cause excessive loss of plasmid due to acid liquor adding too late, relative to prior art alkali lysis, it is favorable to accurately control the alkali lysis operation, improve the plasmid yield, and is favorable to linear amplification to have the excellent effect of industrial practicability. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 It is the process schematic view of the alkali lysis system of the utility model;
[0035] Figure 2 It is the structure schematic view of the alkali lysis chip;
[0036] Figure 3 It is the local schematic view of the alkali lysis chip of the utility model;
[0037] Figure 4 It is the structure schematic view of the alkali lysis equipment of the utility model;
[0038] Figure 5 It is the structure schematic view of the alkali lysis chip of another embodiment of the utility model;
[0039] In the drawing, 1, base plate; 2, main channel; 3, lye sampling port; 4, bacteria liquid sampling port; 5, S shape section; 6, acid liquor sampling port; 7, mixed sampling outlet; 8, sampling channel; 9, conveying pipeline; 10, acid liquor pump; 11, bacteria liquid pump; 12, lye pump; 13, acid liquor interface; 14, bacteria liquid interface; 15, lye interface; 16, sampling interface. DETAILED DESCRIPTION
[0040] The utility model will be further explained in detail in combination with the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the related content, and not limited to the utility model. In addition, it should be noted that, for the convenience of description, only the parts related to the utility model are shown in the drawings.
[0041] It should be noted that the embodiments in the utility model and the features in the embodiments can be combined with each other without conflict. The technical scheme of the utility model will be described in detail below with reference to the drawings and in combination with the embodiments.
[0042] Unless otherwise indicated, the exemplary implementations / examples will be understood to provide exemplary features of various details of some ways in which the inventive concept can be implemented in practice. Thus, unless otherwise indicated, the features of the various implementations / examples can be combined, separated, interchanged, and / or rearranged, additionally, without departing from the inventive concept.
[0043] The use of cross-hatching and / or shading in the drawings is generally used to illustrate the boundaries, of adjacent components. As such, unless specified, the presence of cross-hatching or shading in a drawing is not intended to connote or dictate a particular material, material property, dimension, ratio, etc., to a component, but rather is generally used to illustrate boundaries between adjacent components. Additionally, in the drawings, the size and relative sizes of components can be exaggerated for clarity and / or descriptive purposes. When exemplary embodiments can be practiced differently, a specific sequence of processes can be performed in an order other than described. For example, two processes described in succession can be performed at substantially the same time or in the reverse order as described. Further, like reference numerals denote like components.
[0044] When a component is referred to as being "on" or "over", "connected to", or "coupled to" another component, it can be directly on, connected, or coupled to the other component, or intervening components can be present. When a component is referred to as being "directly on", "directly connected to", or "directly coupled to" another component, there are no intervening components present. As such, the term "connected" can refer to physical or electrical connection, with or without intervening components.
[0045] For descriptive purposes, the inventive concept can use spatial or relative terms, such as "below", "lower", "lower", "down", "downward", "above", "upper" and "higher", "side" (e.g., as in "sidewall"), to describe the relationship between one component and another component as shown in the drawings. In addition to the orientation depicted in the drawings, spatially relative terms can also encompass different orientations of the device in use, operation, and / or manufacture. For example, if the device in the drawings were turned over, a component described as "below" or "under" another component would then be oriented "above" the other component. Therefore, the exemplary term "below" can encompass both an orientation of above and below. Moreover, the device can be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
[0046] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "containing," "contains," or "containing," "contains," or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising" as an open transition term without precluding any additional or other elements. It is also noted that, as used herein, the terms "substantially," "approximately," and other similar terms are used as terms of approximation and not as terms of degree, unless the context clearly indicates otherwise.
[0047] The detection method used in the utility model, if no special description, adopts the general method operation disclosed in prior art.
[0048] The specific embodiment of the utility model is as follows:
[0049] As Figure 1 shown, Figure 1 is the base lysis system process schematic diagram of the utility model; the lysis bacteria liquid and the lysis alkali respectively obtain power through power component, and after being transported into lysis core component through the transport component connected between components, the lysis bacteria liquid and the lysis alkali are transported, and the lysis bacteria liquid and the lysis alkali are mixed and contacted to carry out lysis bacteria liquid cell lysis under alkaline condition, then the acid liquid is transported to lysis core component through transport component after obtaining power through power component to carry out alkaline neutralization, and the final lysis solution after lysis is obtained;
[0050] The base lysis equipment of the specific embodiment of the utility model adopts Figure 1 The base lysis system shown in the utility model, the lysis core component is a base lysis chip, as Figure 2 shown, the base lysis chip includes square substrate 1 and channel in substrate 1 interior;The channel includes main channel 2 and lysis alkali sample inlet 3 and bacteria liquid sample inlet 4 at one end of main channel 2, mixed sample outlet 7 at the other end of main channel 2, acid liquid sample inlet 6 in the middle of main channel 2;The lysis alkali sample inlet 3, the bacteria liquid sample inlet 4, the acid liquid sample inlet 6 are all connected with main channel 2 through sample inlet channel 8;The main channel 2 includes 12 S-shaped segments 5 connected in series, and the S-shaped segments between the acid liquid sample inlet and the lysis alkali sample inlet or the bacteria liquid sample inlet are 6, and the S-shaped segments between the acid liquid sample inlet and the mixed sample outlet are 6;
[0051] As Figure 3As shown in the numerical units of μm, the inner diameter of the main channel 2 is 450 μm; the inner bending radius of the S-shaped section is 450 μm; the outer bending radius is 900 μm; the diameter of the sample inlet is 600 μm;
[0052] In the embodiment, the length of the S-shaped section is 5 mm;
[0053] The included angle between the alkali sample inlet and the bacterial liquid sample inlet is 60 degrees;
[0054] The included angle between the acid sample inlet and the S-shaped section is 30 degrees;
[0055] In actual use, the bacterial liquid to be lysed flows in through the bacterial liquid sample inlet 4, the NaOH alkali liquid flows in through the alkali sample inlet 3, and the two liquids enter the main channel 2 and are mixed in the inner cavity of the S-shaped section to perform lysis of the E. coli at a PH value of 12. After the mixed liquid passes through the S-shaped section for 6 sections, the mixed liquid is mixed and neutralized with the acid liquid entering from the acid sample inlet 6 to neutralize the alkalinity in the main channel 2. After the mixed liquid passes through the S-shaped section for 6 sections, the mixed liquid flows out from the mixed sample outlet 7, and the lysed liquid after alkali lysis enters the next process;
[0056] As shown in the numerical units of μm, the inner diameter of the main channel 2 is 450 μm; the inner bending radius of the S-shaped section is 450 μm; the outer bending radius is 900 μm; the diameter of the sample inlet is 600 μm; Figure 4 As shown in the numerical units of μm, the inner diameter of the main channel 2 is 450 μm; the inner bending radius of the S-shaped section is 450 μm; the outer bending radius is 900 μm; the diameter of the sample inlet is 600 μm;
[0057] In other embodiments of the utility model, similar operations are adopted, and the alkali lysis chip contains two channels in the substrate as shown in the numerical units of μm; Figure 5
[0058] By using the alkali lysis system and the equipment, the uniformity of mixing can be quickly realized, the defect that the plasmid DNA is degraded due to the local alkali solution being too thick is effectively reduced, the acid solution is introduced at a suitable position of a main channel composed of S-shaped small units to neutralize the alkalinity in the mixed solution, the position of the acid solution sampling port is adjusted, the speed of the fluid in each sampling channel is matched, the alkali lysis time is accurately controlled, the plasmid is not completely released due to insufficient alkali lysis time caused by the acid solution being added too early, and the plasmid is not excessively lost due to the acid solution being added too late, compared with the prior art alkali lysis, the alkali lysis operation can be accurately controlled, the plasmid quality and the yield are improved, and the application of workshop industrialization amplification production can be realized.
Claims
1. An alkaline lysis system characterized by: The device comprises a lysis core component, a conveying component and a power component; the lysis core component is an alkali lysis chip; when the bacteria liquid to be lysed passes through the main channel in the alkali lysis chip, the bacteria liquid is mixed with alkali liquid for lysis, and after mixed with acid liquid for neutralization, the alkali lysis is completed; the conveying component is connected with the alkali lysis chip and the power component respectively, so that the bacteria liquid to be lysed reaches each component; the power component is connected with the conveying component, and provides power for the liquid flow in the alkali lysis system.
2. The system of claim 1, wherein: The alkali lysis chip comprises a substrate (1) and a channel in the substrate (1); wherein the channel comprises a main channel (2), an alkali liquid inlet (3) and a bacteria liquid inlet (4) at one end of the main channel (2), a mixed outlet (7) at the other end of the main channel (2), and an acid liquid inlet (6) in the middle of the main channel (2); the alkali liquid inlet (3), the bacteria liquid inlet (4) and the acid liquid inlet (6) are connected with the main channel (2) through an inlet channel (8).
3. The system of claim 2, wherein: The main channel (2) comprises 2-300 S-shaped sections (5) connected in series; the length of the S-shaped section (5) is 0.5-50 mm.
4. The system of claim 2, wherein: The S-shaped section (5) between the acid liquid inlet (6) and the alkali liquid inlet (3) or the bacteria liquid inlet (4) is 1-200; the S-shaped section (5) between the acid liquid inlet (6) and the mixed outlet (7) is 1-300.
5. The system of claim 3, wherein: The inner diameter of the main channel (2) is 0.1-100 mm; the diameter of the acid liquid inlet (6), the alkali liquid inlet (3) and the bacteria liquid inlet (4) is 0.5-20 mm.
6. The system of claim 3, wherein: The inner bending radius of the S-shaped section (5) is 0.3-30 mm; the outer bending radius is 0.4-40 mm.
7. The system of claim 2, wherein: The included angle between the alkali liquid inlet (3) and the bacteria liquid inlet (4) is 10-180 degrees; the included angle between the acid liquid inlet (6) and the S-shaped section (5) is 10-170 degrees.
8. The system of claim 2, wherein: The alkali lysis chip, the substrate (1) comprises 1-20 channels in the substrate (1).
9. The system of claim 8, wherein: The multiple channels in the substrate (1) can be connected in parallel to realize higher throughput alkali lysis.
10. An alkaline lysis apparatus comprising or using the system of any one of claims 1 to 9; characterized in that: The alkali lysis device comprises an alkali lysis chip, a conveying pipeline, a plunger pump and an interface.