Nitrogen protection device in protein ultrafiltration concentration

Abstract

The application discloses a nitrogen protection device in protein ultrafiltration concentration, which comprises a protein original solution container and further comprises a protein concentration unit, a buffer solution replacement unit and a nitrogen protection unit.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to a nitrogen protection device for protein ultrafiltration concentration. Background Technology

[0002] Ultrafiltration concentration is a crucial step in the preparation of high-concentration protein formulations. However, during ultrafiltration, increased pressure, viscosity, and flow rate can create excessively high shear forces near the membrane. Furthermore, proteins can accumulate in large quantities on the membrane surface, resulting in extremely high concentrations. In particular, as protein concentration increases, the risk of generating undesirably modified polymers due to weakened protein conformation and colloidal stability also increases. Simultaneously, the filter membrane itself interacts with the proteins, further exacerbating aggregate formation and protein heterogeneity. These degradation products become nucleation sites for protein aggregation, accelerating the aggregation of target proteins, manifesting as an increase in insoluble particles in the original solution, reduced protein purity, and even protein precipitation. These degradation products can alter the relative activity and immunogenicity of the drug, severely impacting product quality and increasing production costs for companies.

[0003] Currently, to address the problem of poor protein stability, some researchers have attempted to add surfactants, such as polysorbate 80 and poloxamer 188, during ultrafiltration. Studies have shown that surfactants can indeed reduce interactions between proteins and the filter membrane, as well as between proteins themselves, but the effects are minimal. Surfactants generate a large amount of foam in the protein solution under strong physical shear forces, causing numerous inconveniences in experiments and production. Furthermore, the surfactants introduced in this process increase the difficulty of controlling the amount of excipients used in later product formulations. In addition, some have tried to reduce protein precipitation by optimizing ultrafiltration parameters, but this still cannot fundamentally solve the problems of protein aggregation and poor stability during ultrafiltration. Summary of the Invention

[0004] To achieve the above objectives, the present invention discloses a nitrogen protection device for protein ultrafiltration concentration, comprising a protein stock solution container, and further comprising:

[0005] A protein concentration unit is connected to the protein stock solution container, and the protein concentration unit is used to circulate and concentrate the protein stock solution in the protein stock solution container;

[0006] A buffer replacement unit is connected to the protein stock solution container and is used to supply buffer solution to the protein stock solution container;

[0007] A nitrogen protection unit is connected to the protein stock solution container and the buffer replacement unit, respectively, and is used to fill the protein stock solution container and the buffer replacement unit with nitrogen.

[0008] Preferably, the protein concentration unit comprises:

[0009] An ultrafiltration membrane module, the ultrafiltration membrane module including a liquid inlet, a permeate outlet and a concentrate outlet;

[0010] A return line is provided, which connects the concentrate outlet and the protein stock container.

[0011] A pipeline with a first liquid supply pump is connected between the protein stock solution container and the liquid inlet;

[0012] The permeate outlet is connected to the permeate storage tank via a connecting pipe.

[0013] Preferably, the buffer replacement unit includes:

[0014] A buffer solution container, wherein the nitrogen protection unit is connected to the buffer solution container;

[0015] A second liquid supply pump line is provided, which is connected between the buffer solution container and the protein stock solution container.

[0016] Preferably, the ultrafiltration membrane module is a plate-type ultrafiltration membrane module.

[0017] Preferably, the device also includes the following usage method:

[0018] Step 1: After connecting the entire device, turn on the nitrogen protection unit. The nitrogen protection unit will fill the protein stock solution container and the buffer solution container with nitrogen to purge the air in the protein stock solution container and the buffer solution container and create an inert atmosphere.

[0019] Step 2: Turn on the first liquid supply pump line and concentrate the protein stock solution in an inert environment;

[0020] Step 3: Turn on the second supply pump line and replace the buffer solution of the protein stock solution in an inert environment;

[0021] Step 4: Collect the protein solution after ultrafiltration concentration and buffer replacement;

[0022] The first liquid supply pump has the same power as the second liquid supply pump, thereby achieving dynamic balance in the device.

[0023] Preferably, the nitrogen protection unit includes:

[0024] Nitrogen supply components;

[0025] A nitrogen supply pipeline, one end of which is connected to the nitrogen supply assembly, and the other end of which is connected to the buffer solution container and the protein stock solution container respectively.

[0026] Nitrogen outlets are installed on the buffer solution container and the protein stock solution container, respectively.

[0027] Preferably, the nitrogen supply assembly includes:

[0028] A placement shell, wherein a placement opening is provided on the side end of the placement shell;

[0029] A nitrogen cylinder placement slot is installed at the bottom of the placement shell;

[0030] A liftable nitrogen cylinder fixing assembly, wherein the liftable nitrogen cylinder fixing assembly moves within the nitrogen placement slot, and the nitrogen cylinder is placed on the liftable nitrogen cylinder fixing assembly;

[0031] A gas supply trough is provided inside the top of the housing. The nitrogen supply pipeline is connected to the gas supply trough. The liftable nitrogen cylinder fixing assembly raises the top of the nitrogen cylinder into the gas supply trough.

[0032] A camera is installed inside the housing at the top near the gas supply slot, and the camera is used to collect image information of the top of the nitrogen cylinder.

[0033] Preferably, the liftable nitrogen cylinder fixing assembly includes:

[0034] A mobile container, with electric rollers installed at the bottom, and the bottom of the nitrogen cylinder placed on top of the mobile container;

[0035] Lifting components, two of which are symmetrically installed on the mobile box with the nitrogen cylinder as the center, each lifting component includes a lifting rod that vertically passes through the top of the mobile box;

[0036] A fixing strap is installed on the two lifting rods and is used to fix a nitrogen cylinder located between the two lifting components.

[0037] A flipping assembly, two of which are symmetrically mounted on the top of the movable box, is used to flip the lifting rod;

[0038] A one-way drive assembly includes a supporting vertical rod, which is installed on the nitrogen cylinder placement slot. A strip-shaped flipping block is installed on the supporting vertical rod via a connecting shaft. A limiting locking rod is installed on the supporting vertical rod. The strip-shaped flipping block rests on the limiting locking rod, thereby limiting the strip-shaped flipping block to flip towards the flipping assembly.

[0039] Preferably, the lifting assembly includes:

[0040] A transverse sliding groove is provided at the top of the movable box, and the bottom end of the transverse sliding groove is connected to the inside of the movable box. A limit groove is provided in the transverse sliding groove, and the transverse slider is installed in the limit groove by a return spring.

[0041] A transverse sleeve is slidably connected in the transverse sliding groove. The transverse sleeve is rotatably mounted on the transverse sliding block via a mounting shaft. A return coil spring is mounted on the mounting shaft. The transverse sleeve is sleeved on the lifting rod.

[0042] A lifting rack, which is mounted on the lifting rod;

[0043] A lifting motor is installed on the top of the movable box, and a rotating gear that meshes with the lifting rack is installed at the output end of the lifting motor.

[0044] Preferably, the flipping component includes:

[0045] A flip sleeve is fitted onto the lifting rod;

[0046] A flip shaft is connected to a flip sleeve, and a limiting cross frame is installed on the top of the transverse box via a telescopic bracket. The flip shaft is slidably connected inside the limiting cross frame.

[0047] A limiting turntable and a flip turntable are coaxially mounted on the flip shaft. A limiting cross frame is located between the limiting turntable and the flip turntable. The limiting turntable is located close to the flip sleeve. A slot is provided on the outer circumference of the limiting turntable. An elastic rubber block that matches the slot is installed on the lifting rod through a supporting cross plate.

[0048] Rotating paddles, a plurality of rotating paddles are circumferentially mounted on the rotating turntable, and the strip-shaped rotating block extends into the space between two adjacent rotating paddles from the end away from the connecting shaft.

[0049] Preferably, a gas outlet seat is installed at the top of the nitrogen cylinder, and the gas outlet seat has vertically distributed gas outlet channels. The vertical section of the gas outlet channel is connected to the inside of the nitrogen cylinder, and the horizontal section of the gas outlet channel is connected to the side end of the gas outlet seat. A push rod extends from the top of the gas outlet seat into the vertical section of the gas outlet channel. A plug is installed in the vertical section of the gas outlet channel and connected to the push rod. A spring reset member is installed in the gas outlet channel and connected to the push rod. Attached Figure Description

[0050] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0051] Figure 1 This is a schematic diagram of the process of the present invention;

[0052] Figure 2 This is a schematic diagram of the protein concentration unit and buffer replacement unit in this invention;

[0053] Figure 3 This is a schematic diagram of the nitrogen supply component structure in this invention. Figure 1 ;

[0054] Figure 4 This is a schematic diagram of the nitrogen supply component structure in this invention. Figure 2 (Nitrogen cylinders are placed below the gas supply tank);

[0055] Figure 5 This is a schematic diagram of the structure of the liftable nitrogen cylinder fixing assembly in this invention. Figure 1 ;

[0056] Figure 6 This is a schematic diagram of the structure of the liftable nitrogen cylinder fixing assembly in this invention. Figure 2 (After the nitrogen cylinder is lifted);

[0057] Figure 7 for Figure 4 Enlarged view of number A;

[0058] Figure 8 This is a schematic diagram of the top structure of the nitrogen cylinder in this invention.

[0059] Figure 9 These are the detection results of insoluble particles before and after ultrafiltration in this invention.

[0060] In the diagram: 1. Protein stock solution container; 2. Protein concentration unit; 3. Buffer exchange unit; 4. Nitrogen protection unit; 21. Ultrafiltration membrane assembly; 22. Inlet; 23. Permeate outlet; 24. Concentrate outlet; 25. Return line; 26. Line with first supply pump; 27. Permeate storage tank; 31. Buffer solution container; 32. Line with second supply pump; 41. Nitrogen supply assembly; 42. Nitrogen supply line; 43. Nitrogen outlet; 44. Housing shell; 45. Placement port; 46. Nitrogen cylinder placement slot; 47. Liftable nitrogen cylinder fixing assembly; 48. Nitrogen cylinder; 49. Gas supply tank; 40. 51. Camera; 52. Moving box; 53. Electric roller; 54. Lifting assembly; 55. Lifting rod; 56. Fixing strap; 57. Tilting assembly; 58. One-way drive assembly; 59. Elastic rubber block; 50. Rotating lever; 61. Supporting vertical rod; 62. Horizontal sliding groove; 63. Horizontal sliding slider; 64. Horizontal sliding sleeve; 65. Lifting rack; 66. Rotating gear; 67. Tilting sleeve; 68. Tilting shaft; 69. Limiting horizontal frame; 60. Limiting turntable; 71. Tilting turntable; 72. Strip-shaped tilting block; 73. Limiting locking rod; 74. Air outlet seat; 75. Air outlet channel; 76. Top rod; 77. Block. Detailed Implementation

[0061] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] Example

[0063] The present invention will now be further described with reference to the accompanying drawings.

[0064] like Figures 1 to 9 As shown, this embodiment provides a nitrogen protection device for protein ultrafiltration concentration, including a protein stock solution container 1, and further comprising:

[0065] Protein concentration unit 2, which is connected to the protein stock solution container 1, is used to circulate and concentrate the protein stock solution in the protein stock solution container 1;

[0066] Buffer replacement unit 3, which is connected to the protein stock solution container 1, is used to supply buffer solution to the protein stock solution container 1;

[0067] Nitrogen protection unit 4 is connected to the protein stock solution container 1 and the buffer replacement unit 3 respectively, and is used to fill the protein stock solution container 1 and the buffer replacement unit 3 with nitrogen.

[0068] The working principle and beneficial effects of the above technical solution are as follows:

[0069] This invention discloses a nitrogen protection device for protein ultrafiltration concentration. By adding nitrogen protection during this ultrafiltration stage, the dissolved oxygen content in the ultrafiltration solution can be reduced, avoiding protein aggregation caused by exposure of hydrophobic domains due to oxidative modification, reducing the content of insoluble particles in the original solution, and fundamentally achieving the purpose of enhancing the stability of recombinant protein drugs, reducing aggregate formation, and ensuring product quality.

[0070] In one embodiment, the protein concentration unit 2 includes:

[0071] Ultrafiltration membrane module 21, the ultrafiltration membrane module 21 includes a liquid inlet 22, a permeate outlet 23 and a concentrate outlet 24;

[0072] Return line 25, the return line 25 is connected between the concentrate outlet 24 and the protein stock container 1;

[0073] A first liquid supply pump pipeline 26 is provided, which is connected between the protein stock solution container 1 and the inlet 22.

[0074] The permeate outlet 23 is connected to the permeate storage tank 27 via a connecting pipe.

[0075] The working principle of the above technical solution is as follows:

[0076] The protein stock solution in the protein stock solution container 1 is ultrafiltered by the ultrafiltration membrane assembly 21 through the first liquid supply pump pipeline 26, and the concentrate is returned to the protein stock solution container 1 from the concentrate outlet 24.

[0077] In one embodiment, the buffer replacement unit 3 includes:

[0078] Buffer container 31, the nitrogen protection unit 4 is connected to the buffer container 31;

[0079] A second liquid supply pump line 32 is provided, which is connected between the buffer container 31 and the protein stock container 1.

[0080] The working principle of the above technical solution is as follows:

[0081] The buffer solution in the buffer container 31 is delivered into the protein stock solution container 1 via the second liquid supply pump line 32.

[0082] In one embodiment, the ultrafiltration membrane module 21 is a plate-type ultrafiltration membrane module.

[0083] In one embodiment, the device further includes the following method of use:

[0084] Step 1: After connecting the entire device, turn on the nitrogen protection unit 4. The nitrogen protection unit 4 sends nitrogen gas into the protein stock solution container 1 and the buffer solution container 31 to purge the air in the protein stock solution container 1 and the buffer solution container 31 and create an inert atmosphere.

[0085] Step 2: Turn on the first liquid supply pump line 26 to concentrate the protein stock solution in an inert environment;

[0086] Step 3: Turn on the second supply pump line 32 to replace the buffer solution of the protein stock solution in an inert environment;

[0087] Step 4: Collect the protein solution after ultrafiltration concentration and buffer replacement;

[0088] The first liquid supply pump has the same power as the second liquid supply pump, thereby achieving dynamic balance in the device.

[0089] The working principle and beneficial effects of the above technical solution are as follows:

[0090] The principle behind this invention's ability to improve protein stability is as follows:

[0091] The inventors discovered that the instability of protein drugs is closely related to changes in their own structure, and that oxidative modification of amino acid residues is one of the important factors causing changes in protein structure.

[0092] During ultrafiltration concentration and buffer replacement, protein enrichment on the membrane surface and excessive shear forces near the membrane sac weaken protein conformation and colloidal stability. In this context, the presence of oxygen undoubtedly exacerbates protein-protein interactions. Oxidative modifications of amino acid residues can alter the secondary and tertiary structures of proteins, thereby changing hydrophobic domains on the protein surface. Hydrophobic interactions become the initial driving force for protein aggregate formation. The resulting degradation products further become nucleation sites for protein aggregation, accelerating the tendency of protein aggregation. For example, the oxidation of methionine at position 111 of interferon α2b alters its secondary structure, leading to decreased stability. Furthermore, many oxidized proteins exhibit high activity due to the introduction of free radicals, peroxides, or ketone groups. The activity of these groups leads to protein-protein interactions and the formation of insoluble protein aggregates. For instance, the oxidation products of Tyr and Phe can further participate in aggregate formation through 1,4 or 1,6 addition to the primary amine groups of proteins, resulting in non-reducing covalent cross-links between amino acid residues.

[0093] Therefore, by adding nitrogen protection during this ultrafiltration stage, the present invention can reduce the dissolved oxygen content in the ultrafiltration solution, avoid protein aggregation caused by exposure of hydrophobic domains due to oxidation modification, reduce the content of insoluble particles in the original solution, and fundamentally achieve the purpose of enhancing the stability of recombinant protein drugs, reducing aggregate formation, and ensuring product quality.

[0094] Recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) is a high-concentration protein drug approved for subcutaneous injection to treat rheumatoid arthritis. As a high-concentration protein formulation, it also faces problems such as protein aggregation and poor stability during ultrafiltration. Previous mass spectrometry peptide mapping analysis of samples revealed that rhIL-1Ra is easily oxidized, containing multiple easily oxidized sites. The M positions at positions 1, 66, 126, 137, and 143 of the amino acid sequence all show potential for oxidative modification. Component analysis of normal formulations and precipitated formulations determined that protein precipitation is mainly caused by the oxidation of methionine. This research provides a theoretical basis for improving the quality of rhIL-1Ra formulations.

[0095] Therefore, the applicant conducted experiments using rhIL-1Ra protein stock solution to investigate the ultrafiltration effect of the nitrogen-protected device used in the ultrafiltration concentration of this protein. The recombinant human interleukin-1 receptor antagonist was prepared by the applicant.

[0096] method:

[0097] ① Nitrogen protection is used in protein ultrafiltration concentration:

[0098] Connect the entire device, turn on the nitrogen protection unit 4, and purge the air (oxygen) from the device to create an inert atmosphere;

[0099] Ultrafiltration concentration stage: Turn on the first supply pump at 600 ml / min and transmembrane pressure of 1.5 bar to enter the concentration stage of the protein stock solution in an inert environment; the low concentration protein solution is introduced from the protein solution container into the ultrafiltration membrane module 21 through the pipeline, the permeate is sent from the permeate outlet 23 of the ultrafiltration membrane module 21 to the permeate storage tank 27, and the high concentration protein solution is sent from the concentrate outlet 24 of the ultrafiltration membrane module 21 back to the protein stock solution container 1;

[0100] Buffer replacement stage: After the protein concentration reaches 150 mg / mL, buffer replacement unit 3 is started. Under nitrogen protection throughout the process, the buffer is replaced by an equal volume. Finally, the protein solution with a relatively high concentration after ultrafiltration concentration and buffer replacement is collected.

[0101] ② Nitrogen protection without protein ultrafiltration concentration: Except for not activating nitrogen protection unit 4, the other experimental procedures are the same as those in “Nitrogen protection device in protein ultrafiltration concentration”.

[0102] (2) Evaluation Indicators

[0103] In response to the performance of recombinant human interleukin-1 receptor antagonists during experiments, and in accordance with the 2020 Pharmacopoeia standard, the detection of insoluble microparticle content (to evaluate protein aggregates and particle size) was selected as a method to evaluate the effectiveness of nitrogen-filled experiments.

[0104] (3) Results:

[0105] As shown in Table 1, Figure 9 As shown, following the above experimental method, four ultrafiltration experiments were conducted with nitrogen protection and two ultrafiltration experiments were conducted without nitrogen protection. The content of insoluble particles in the protein solution under nitrogen protection was much lower than that in the protein solution without nitrogen protection.

[0106] Table 1. Comparison of Ultrafiltration Effects of Different Ultrafiltration Methods

[0107]

[0108] Note: 4 ultrafiltration experiments with nitrogen protection - nitrogen ultrafiltration 1, nitrogen ultrafiltration 2, nitrogen ultrafiltration 3, nitrogen ultrafiltration 4; 2 ultrafiltration experiments without nitrogen protection - ultrafiltration without nitrogen 1, ultrafiltration without nitrogen 2.

[0109] (4) Conclusion: By designing and applying a nitrogen protection device for protein ultrafiltration concentration, the content of insoluble particles in rhIL-1Ra stock solution under different ultrafiltration devices was compared and investigated. The effect of nitrogen protection throughout the ultrafiltration stage was significantly better than that without nitrogen protection, and the stability of rhIL-1Ra was significantly improved. This indicates that a nitrogen protection device for protein ultrafiltration concentration plays an important role in improving the aggregation of easily oxidized proteins during ultrafiltration, and improving protein stability and product quality.

[0110] In one embodiment, the nitrogen protection unit 4 includes:

[0111] Nitrogen supply component 41;

[0112] Nitrogen supply line 42, one end of which is connected to nitrogen supply assembly 41, and the other end of which is connected to buffer container 31 and protein stock container 1 respectively.

[0113] Nitrogen outlet 43 is installed on the buffer container 31 and the protein stock container 1 respectively.

[0114] The working principle and beneficial effects of the above technical solution are as follows:

[0115] Nitrogen from nitrogen supply component 41 is supplied from nitrogen supply pipeline 42 into buffer container 31 and protein stock solution container 1 respectively, thereby purging the air (oxygen) in buffer container 31 and protein stock solution container 1 and creating an inert atmosphere.

[0116] In one example, the nitrogen supply assembly 41 includes:

[0117] A placement shell 44 is provided, and a placement opening 45 is provided on the side end of the placement shell 44;

[0118] Nitrogen cylinder placement slot 46, which is installed at the bottom of the placement shell 44;

[0119] A liftable nitrogen cylinder fixing assembly 47 moves within the nitrogen placement slot 46, and a nitrogen cylinder 48 is placed on the liftable nitrogen cylinder fixing assembly 47.

[0120] A gas supply trough 49 is provided inside the top of the housing 44. The nitrogen supply pipeline 42 is connected to the gas supply trough 49. The liftable nitrogen cylinder fixing assembly 47 raises the top of the nitrogen cylinder 48 into the gas supply trough 49.

[0121] Camera 40 is installed inside the housing 44 at the top near the gas supply slot 49. Camera 40 is used to collect image information of the top of the nitrogen cylinder 48.

[0122] The working principle and beneficial effects of the above technical solution are as follows:

[0123] Nitrogen cylinder 48 is placed into the liftable nitrogen cylinder fixing component 47 through the placement port 45. The liftable nitrogen cylinder fixing component 47 moves within the nitrogen cylinder placement slot 46 and moves the nitrogen cylinder 48 to below the gas supply slot 49. Then, the liftable nitrogen cylinder fixing component 47 lifts the nitrogen cylinder 48, thereby raising the top of the nitrogen cylinder 48 into the gas supply slot 49. The camera 40 is used to collect image information of the top of the nitrogen cylinder 48, thereby judging the integrity of the interface at the top of the nitrogen cylinder 48 through machine vision.

[0124] In one embodiment, the liftable nitrogen cylinder fixing assembly 47 includes:

[0125] The mobile box 51 is equipped with electric rollers 52 at its bottom end, and the nitrogen cylinder 48 is placed at the top of the mobile box 51 at its bottom end.

[0126] Lifting assembly 53, two lifting assemblies 53 are symmetrically installed on the mobile box 51 with nitrogen cylinder 48 as the center. The lifting assembly 53 includes a lifting rod 54, which is vertically inserted through the top of the mobile box 51.

[0127] A fixing strap 55 is installed on the two lifting rods 54 and is used to fix the nitrogen cylinder 48 located between the two lifting components 53.

[0128] Two flipping components 56 are symmetrically installed on the top of the movable box 51. The flipping components 56 are used to flip the lifting rod 54.

[0129] A one-way drive assembly 57 includes a support vertical rod 50, which is mounted on the nitrogen cylinder placement slot 46. A strip-shaped flipping block 71 is mounted on the support vertical rod 50 via a connecting shaft. A limiting locking rod 72 is mounted on the support vertical rod 50. The strip-shaped flipping block 71 rests on the limiting locking rod 72, thereby limiting the flipping of the strip-shaped flipping block 71 towards the flipping assembly 56.

[0130] The working principle and beneficial effects of the above technical solution are as follows:

[0131] The bottom of the nitrogen cylinder 48 is fixed to the fixed strap 55. The electric roller 52 drives the moving box 51 to move in the nitrogen cylinder placement slot 46. When the nitrogen cylinder 48 moves to below the gas supply slot 49, the lifting component 53 lifts the nitrogen cylinder 48 fixed to the fixed strap 55. When the lifting rod 54 lifts the flipping component 56 simultaneously, the one-way drive component 57 does not trigger the flipping component 56 to work. The top of the nitrogen cylinder 48 is sent into the gas supply slot 49, thereby triggering the gas supply of the nitrogen cylinder 48. When the gas supply of the nitrogen cylinder 48 ends, the lifting component 53 lowers the nitrogen cylinder 48 fixed to the fixed strap 55. The top of the nitrogen cylinder 48 is removed from the gas supply slot 49. When the lifting rod 54 lowers the flipping component 56 simultaneously, the one-way drive component 57 triggers the flipping component 56 to work, thereby causing the lifting rod 54 to flip the nitrogen cylinder 48 towards the camera 40. The top of the nitrogen cylinder 48 flips towards the camera 40 so that the camera 40 can capture the integrity of the interface at the top of the nitrogen cylinder 48.

[0132] In one embodiment, the lifting assembly 53 includes:

[0133] A transverse sliding groove 61 is provided at the top of the movable box 51, and the bottom end of the transverse sliding groove 61 is connected to the inside of the movable box 51. A limit sliding groove is provided inside the transverse sliding groove 61, and the transverse sliding block 62 is installed in the limit sliding groove by a return spring.

[0134] A transverse sleeve 63 is slidably connected to the transverse sliding groove 61. The transverse sleeve 63 is rotatably mounted on the transverse sliding block 62 via a mounting shaft. A reset coil spring is mounted on the mounting shaft. The transverse sleeve 63 is sleeved on the lifting rod 54.

[0135] A lifting rack 64 is mounted on the lifting rod 54;

[0136] A lifting motor is installed on the top of the movable box 51, and a rotating gear 65 that meshes with the lifting rack 64 is installed at the output end of the lifting motor.

[0137] The working principle and beneficial effects of the above technical solution are as follows:

[0138] When the lifting motor operates, it drives the rotating gear 65 installed at the output end of the lifting motor to rotate. With the cooperation of the rotating gear 65 and the lifting rack 64, the lifting rod 54 is driven to rise and fall within the transverse sleeve 63. When the flipping component 56 drives the lifting rod 54 to flip, the transverse sleeve 63 sleeved on the lifting rod 54 flips within the transverse slide groove 61. At this time, the transverse sleeve 63 flips on the transverse slider 62 with the mounting shaft as the center.

[0139] In one embodiment, the flipping component 56 includes:

[0140] A flip sleeve 66 is sleeved on the lifting rod 54;

[0141] A flip shaft 67 is connected to a flip sleeve 66. A limiting cross frame 68 is installed on the top of the transverse box 51 via a telescopic bracket. The flip shaft 67 is slidably connected inside the limiting cross frame 68.

[0142] A limiting turntable 69 and a flip turntable 60 are coaxially mounted on the flip shaft 67. A limiting cross frame 68 is located between the limiting turntable 69 and the flip turntable 60. The limiting turntable 69 is located close to the flip sleeve 66. A slot is provided on the outer circumference of the limiting turntable 69. An elastic rubber block 58 that matches the slot is installed on the lifting rod 54 through a supporting cross plate.

[0143] Rotate the lever 59. Multiple rotating levers 59 are circumferentially mounted on the rotating turntable 60. The strip-shaped rotating block 71 extends into the space between two adjacent rotating levers 59 from the end away from the connecting shaft.

[0144] In one embodiment, a gas outlet seat 73 is installed at the top of the nitrogen cylinder 48. The gas outlet seat 73 has vertically distributed gas outlet channels 74. The vertical section of the gas outlet channel 74 is connected to the inside of the nitrogen cylinder 48, and the horizontal section of the gas outlet channel 74 is connected to the side end of the gas outlet seat 73. A push rod 75 extends from the top of the gas outlet seat 73 into the vertical section of the gas outlet channel 74. A plug 76 is installed in the vertical section of the gas outlet channel 74 and connected to the push rod 75. A spring return member is installed in the gas outlet channel 74 and connected to the push rod 75.

[0145] The working principle and beneficial effects of the above technical solution are as follows:

[0146] When the lifting rod 54 is raised within the transverse sleeve 63, it drives the tilting sleeve 66 sleeved on the lifting rod 54 to rise. The tilting sleeve 66 drives the limiting turntable 69 and the tilting turntable 60 to rise synchronously via the tilting shaft 67. As the tilting turntable 60 drives the rotating paddle 59 to rise, it drives the strip-shaped tilting block 71 to tilt away from the tilting turntable 60 around the connecting shaft. The strip-shaped tilting block 71 does not interfere with the rise of the tilting turntable 60. The lifting rod 54 drives the nitrogen cylinder 48 to rise via the fixing strap 55. The gas outlet seat 73 located at the top of the nitrogen cylinder 48 is sent into the gas supply groove 49. The push rod 75 abuts against the bottom of the gas supply groove 49, thereby driving the block 76 connected to the push rod 75 to move within the gas outlet channel 74. As the outlet seat 73 is fully inserted into the supply trough 49, the block 76 releases its seal on the outlet channel 74, and the nitrogen in the nitrogen cylinder 48 is discharged from the supply trough 49 and from the nitrogen supply pipeline 42 connected to the supply trough 49. When it is necessary to periodically check the integrity of the interface at the top of the nitrogen cylinder 48, the lifting motor reverses its operation, thereby driving the rotating gear 65 installed at the output end of the lifting motor to rotate. With the cooperation of the rotating gear 65 and the lifting rack 64, the lifting rod 54 is driven to descend within the transverse sleeve 63. At this time, the outlet seat 73 disengages from the supply trough 49. In this position, the lifting rod 54 drives the tilting sleeve 66, the limiting turntable 69 installed on the tilting sleeve 66 via the tilting shaft 67, and the tilting turntable 6... As the lifting motor continues to operate in reverse, the rotating paddle 59 on the rotating turntable 60 contacts the strip-shaped rotating block 71. Due to the interference of the limit locking rod 72, the strip-shaped rotating block 71 cannot rotate downwards around the connecting shaft. This causes the rotating paddle 59 to rotate the rotating turntable 60 towards the placement opening 45. At the same time, to avoid the strip-shaped rotating block 71, the rotating paddle 59 also causes the rotating turntable 60 to move towards the placement opening 45. The rotating turntable 60, through the rotating shaft 67, drives the rotating sleeve 66 and the lifting rod 54 sleeved in the rotating sleeve 66 to rotate towards the placement opening 45 around the rotating shaft 67. At this time, the transverse sleeve 63 sleeved on the lifting rod 54 is transversely moving. The nitrogen cylinder 48, centered on the connecting shaft, rotates on the transverse slider 62 within the slide groove 61. This causes the top of the nitrogen cylinder 48, which is mounted on the lifting rod 64 via the fixing strap 55, to rotate towards the camera 40. Since the rotating turntable 60 needs to move towards the placement port 45, the transverse sleeve 63, which is sleeved on the lifting rod 54, drives the transverse slider 62 to slide within the limiting slide groove within the transverse slide groove 61. This causes the nitrogen cylinder 48, which is mounted on the lifting rod 64 via the fixing strap 55, to move horizontally towards the camera 40. This allows the outlet seat 73 to quickly detach from the gas supply groove 49 without colliding with the top rod 75, while also facilitating the camera 40 to capture the integrity of the top interface of the nitrogen cylinder 48.

[0147] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A nitrogen protection device for protein ultrafiltration concentration, comprising a protein stock solution container (1), characterized in that, It also includes: a protein concentration unit (2) connected to the protein stock solution container (1), the protein concentration unit (2) being used for circulating concentration of the protein stock solution in the protein stock solution container (1); a buffer replacement unit (3) connected to the protein stock solution container (1), the buffer replacement unit (3) being used to supply buffer to the protein stock solution container (1); and a nitrogen protection unit (4) connected to the protein stock solution container (1) and the buffer replacement unit (3) respectively, for filling the protein stock solution container (1) and the buffer replacement unit (3) with nitrogen. The protein concentration unit (2) includes: an ultrafiltration membrane assembly (21), which includes an inlet (22), a permeate outlet (23) and a concentrate outlet (24), a return line (25) connecting the concentrate outlet (24) and the protein stock container (1), a line with a first supply pump (26) connecting the protein stock container (1) and the inlet (22), and the permeate outlet (23) being connected to the permeate storage tank (27) via a connecting line; The buffer replacement unit (3) includes: The buffer container (31) is connected to the nitrogen protection unit (4); A second liquid supply pump line (32) is provided, which is connected between the buffer container (31) and the protein stock container (1); The nitrogen protection unit (4) includes: Nitrogen supply assembly (41); Nitrogen supply line (42), one end of which is connected to the nitrogen supply assembly (41), and the other end of which is connected to the buffer container (31) and the protein stock container (1). Nitrogen outlet (43), which is installed on the buffer container (31) and the protein stock container (1); The nitrogen supply assembly (41) includes: a placement shell (44), a placement port (45) on the side of the placement shell (44), a nitrogen cylinder placement slot (46) installed at the bottom of the placement shell (44), a liftable nitrogen cylinder fixing assembly (47) moving in the nitrogen placement slot (46), a nitrogen cylinder (48) placed on the liftable nitrogen cylinder fixing assembly (47), a gas supply slot (49) opened at the top of the placement shell (44), a nitrogen supply pipeline (42) connected to the gas supply slot (49), the liftable nitrogen cylinder fixing assembly (47) raising the top of the nitrogen cylinder (48) into the gas supply slot (49), and a camera (40) installed at the top of the placement shell (44) near the gas supply slot (49). The camera (40) is used to collect image information of the top of the nitrogen cylinder (48).

2. The nitrogen protection device for protein ultrafiltration concentration according to claim 1, characterized in that, The device also includes the following usage methods: Step 1: After connecting the entire device, turn on the nitrogen protection unit (4). The nitrogen protection unit (4) sends nitrogen into the protein stock solution container (1) and the buffer solution container (31) to purge the air in the protein stock solution container (1) and the buffer solution container (31) and create an inert atmosphere. Step 2: Turn on the first liquid supply pump line (26) and concentrate the protein stock solution in an inert environment; Step 3: Turn on the second supply pump line (32) and replace the buffer solution of the protein stock solution in an inert environment; Step 4: Collect the protein solution after ultrafiltration concentration and buffer replacement; The first liquid supply pump has the same power as the second liquid supply pump, thereby achieving dynamic balance in the device.

3. The nitrogen protection device for protein ultrafiltration concentration according to claim 1, characterized in that, The liftable nitrogen cylinder fixing assembly (47) includes: a movable box (51), with electric rollers (52) installed at the bottom of the movable box (51), the bottom of the nitrogen cylinder (48) placed at the top of the movable box (51), and two lifting assemblies (53) symmetrically installed on the movable box (51) with the nitrogen cylinder (48) as the center. Each lifting assembly (53) includes a lifting rod (54) that vertically passes through the top of the movable box (51). A fixing strap (55) is installed on the two lifting rods (54) and is used to fix the nitrogen cylinder located between the two lifting assemblies (53). Bottle (48), two flipping components (56) are symmetrically installed on the top of the mobile box (51). The flipping components (56) are used to flip the lifting rod (54). The one-way drive component (57) includes a support rod (50). The support rod (50) is installed on the nitrogen cylinder placement slot (46). The strip flipping block (71) is installed on the support rod (50) through a connecting shaft. The limiting lock rod (72) is installed on the support rod (50). The strip flipping block (71) is placed on the limiting lock rod (72), thereby limiting the strip flipping block (71) to flip towards the flipping component (56).

4. The nitrogen protection device for protein ultrafiltration concentration according to claim 3, characterized in that, The lifting assembly (53) includes: a transverse sliding groove (61), which is located at the top of the moving box (51). The bottom end of the transverse sliding groove (61) is connected to the inside of the moving box (51). A limit groove is provided inside the transverse sliding groove (61). The transverse sliding block (62) is installed in the limit groove by a return spring. The transverse sleeve (63) is slidably connected in the transverse sliding groove (61). The transverse sleeve (63) is rotatably installed on the transverse sliding block (62) by a mounting shaft. A return coil spring is installed on the mounting shaft. The transverse sleeve (63) is sleeved on the lifting rod (54). The lifting rack (64) is installed on the lifting rod (54). The lifting motor is installed at the top inside the moving box (51). A rotating gear (65) that meshes with the lifting rack (64) is installed at the output end of the lifting motor.

5. The nitrogen protection device for protein ultrafiltration concentration according to claim 3, characterized in that, The flipping assembly (56) includes: a flipping sleeve (66), which is sleeved on the lifting rod (54); a flipping shaft (67) connected to the flipping sleeve (66); a limiting cross frame (68) installed on the top of the movable box (51) via a telescopic bracket; the flipping shaft (67) slidably connected inside the limiting cross frame (68); a limiting turntable (69) and a flipping turntable (60) coaxially installed on the flipping shaft (67); and the limiting cross frame (68) is positioned... Between the limiting turntable (69) and the flipping turntable (60), the limiting turntable (69) is set close to the flipping sleeve (66). The outer ring of the limiting turntable (69) has a slot. The lifting rod (54) is equipped with an elastic rubber block (58) that matches the slot through the support plate. Multiple rotating paddles (59) are circumferentially installed on the flipping turntable (60). The strip-shaped flipping block (71) extends into the space between two adjacent rotating paddles (59) away from the connecting shaft end.

6. The nitrogen protection device for protein ultrafiltration concentration according to claim 1, characterized in that, The nitrogen cylinder (48) is equipped with an outlet seat (73) at the top. The outlet seat (73) is provided with vertically distributed outlet channels (74). The vertical section of the outlet channel (74) is connected to the nitrogen cylinder (48), and the horizontal section of the outlet channel (74) is connected to the side end of the outlet seat (73). The push rod (75) extends from the top of the outlet seat (73) into the vertical section of the outlet channel (74). The plug (76) is installed in the vertical section of the outlet channel (74) and connected to the push rod (75). The spring reset component is installed in the outlet channel (74) and connected to the push rod (75).

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