CN115430198A - Protein purification method and protein purification system - Google Patents

Protein purification method and protein purification system Download PDF

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CN115430198A
CN115430198A CN202211154006.5A CN202211154006A CN115430198A CN 115430198 A CN115430198 A CN 115430198A CN 202211154006 A CN202211154006 A CN 202211154006A CN 115430198 A CN115430198 A CN 115430198A
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ultrafiltration
unit
diafiltration
tangential flow
protein purification
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方峰
郭静
岳慧敏
徐志豪
樊鹏程
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Yingmai Biological Medicine Hangzhou Co ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D36/00—Filter circuits or combinations of filters with other separating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/08—Polysaccharides
    • B01D71/10—Cellulose; Modified cellulose
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/66—Polymers having sulfur in the main chain, with or without nitrogen, oxygen or carbon only
    • B01D71/68—Polysulfones; Polyethersulfones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

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  • Organic Chemistry (AREA)
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  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Water Supply & Treatment (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to the technical field of protein purification, and particularly relates to a protein purification method and a protein purification system. The protein purification method comprises the following steps: and (3) performing virus removal filtration on the treated substance after the fine purification chromatography after first ultrafiltration and/or first diafiltration. According to the invention, the first ultrafiltration and/or the first diafiltration are carried out on the treated substance after the fine purification chromatography before virus removal filtration, so that the process performance of virus removal filtration can be optimized, the process time of virus removal filtration is shortened, the loading capacity is increased, the required membrane area is reduced, and the cost is reduced.

Description

Protein purification method and protein purification system
Technical Field
The invention relates to the technical field of protein purification, and particularly relates to a protein purification method and a protein purification system.
Background
The prior protein purification technical method is generally carried out by a virus removal filtration unit, an ultrafiltration percolation unit and a final preparation filtration canning unit in sequence after fine purification chromatography. Wherein, the virus-removing filter unit is connected with the fine purification chromatography unit in the last step and the ultrafiltration diafiltration unit in the next step, namely, the sample solution of the virus-removing filter unit comes from the product of the fine purification chromatography in the last step, and the product is used as the sample solution of the ultrafiltration diafiltration in the next step.
Therefore, the process performance of virus removal and filtration is greatly related to the state of the sample loading product (namely the purified chromatography product in the previous step), and factors such as the solution system of the sample loading product, pH, conductivity value, protein concentration and the like directly influence the process performance of virus removal and filtration. Therefore, the process performance of virus removal filtration is often limited by the state of the purified chromatography product of the previous step. Since the solution state of the purified chromatography product may cause the virus removal filtration to have a low loading capacity (membrane area requirement is increased, cost is increased), and flux is reduced (process time is prolonged), further adjustments are often required to be made to the virus removal filtration sample loading product, such as changing the pH, conductivity value, protein concentration, and the like of the sample loading product. The adjusting operation steps are complicated, the adjustable limit of the product is limited, and the product cannot be adjusted too much and greatly.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a protein purification method, which can optimize the process performance of virus removal filtration by performing first ultrafiltration and/or first diafiltration on a treated product after fine purification chromatography before virus removal filtration, thereby shortening the process time of virus removal filtration, increasing the loading capacity, reducing the required membrane area, and reducing the cost.
The second purpose of the invention is to provide a protein purification system, which has the advantages of simple equipment, high production efficiency, good controllability and the like.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides a protein purification method, which comprises the following steps:
and (3) performing virus removal filtration on the treated product after the fine purification chromatography after first ultrafiltration and/or first diafiltration.
Preferably, a step of performing a second ultrafiltration and/or a second diafiltration is included after the virus removal filtration.
Preferably, the first ultrafiltration, the first diafiltration, the second ultrafiltration and the second diafiltration comprise tangential flow filtration and/or unidirectional tangential flow filtration.
Preferably, the flux of the tangential flow filtration is 100-400 LMH, the transmembrane pressure is 0.5-1.5 bar, and the loading capacity is less than or equal to 500g/m 2 ；
Preferably, the flux of the unidirectional tangential flow filtration is 10-90 LMH, the transmembrane pressure is 1-2 bar, and the loading capacity is less than or equal to 100g/m 2 。
The invention also provides a protein purification system which is suitable for the protein purification method and comprises a fine purification chromatography unit, a first ultrafiltration and diafiltration unit and a virus removal and filtration unit which are connected in sequence.
Preferably, the virus removal filtration unit is further connected to a second ultrafiltration diafiltration unit.
Preferably, the first ultrafiltration diafiltration unit comprises a tangential flow filtration device and/or a unidirectional tangential flow filtration device;
preferably, the second ultrafiltration diafiltration unit comprises a tangential flow filtration device and/or a unidirectional tangential flow filtration device.
Preferably, the first ultrafiltration diafiltration unit comprises a tangential flow filtration device and the second ultrafiltration diafiltration unit comprises a tangential flow filtration device;
alternatively, the first ultrafiltration diafiltration unit comprises a tangential flow filtration device and the second ultrafiltration diafiltration unit comprises a unidirectional tangential flow filtration device;
alternatively, the first ultrafiltration diafiltration unit comprises a one-way tangential flow filtration device and the second ultrafiltration diafiltration unit comprises a tangential flow filtration device.
Preferably, the material of the ultrafiltration membrane of the first ultrafiltration diafiltration unit and/or the second ultrafiltration diafiltration unit comprises polyethersulfone and/or regenerated cellulose.
Preferably, the protein purification system further comprises a capture chromatography unit and a virus inactivation unit connected together; wherein the virus inactivation unit is connected with the fine purification chromatography unit;
preferably, the protein purification system further comprises a preparation unit connected to the virus removal filtration unit or the second ultrafiltration diafiltration unit.
Compared with the prior art, the invention has the beneficial effects that:
(1) The protein purification method provided by the invention can optimize the process performance of virus removal filtration by performing the first ultrafiltration and/or the first diafiltration on the treated substance after the fine purification chromatography before the virus removal filtration, thereby shortening the process time of the virus removal filtration, increasing the loading capacity, reducing the required membrane area and reducing the cost.
(2) The protein purification method provided by the invention has various treatment schemes, can flexibly change the state of a sample, and has the advantages of good process controllability, simple operation and high production efficiency.
(3) The protein purification system provided by the invention can utilize the existing traditional unit operation system equipment (virus removal filtration system equipment and ultrafiltration percolation system equipment), does not need to make great modification on the equipment, and can simply modify and integrate the existing equipment, thereby simplifying the process flow and improving the production efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the structure of a protein purification system provided by the present invention;
FIG. 2 is a schematic diagram of the structure of a protein purification system according to scheme A provided herein;
FIG. 3 is a schematic diagram of the structure of the protein purification system of scheme B provided in the present invention;
FIG. 4 is a schematic diagram of the structure of the protein purification system of scheme C provided herein;
FIG. 5 is a schematic diagram of the structure of the protein purification system of scheme D provided herein;
FIG. 6 is a schematic view of another embodiment of the protein purification system provided by the present invention;
FIG. 7 is a schematic view of another structure of the protein purification system provided by the present invention.
Reference numerals:
1-a fine purification chromatography unit; 2-a first ultrafiltration diafiltration unit; 21-a first recycle tank; 22-a first ultrafiltration membrane package; 23-a first dialysate reservoir; 24-a first stirring device; 25-a second SPTFF film package; 3-a virus removal filtration unit; 31-a pre-filter; 32-a virus removal filter; 33-a water for injection storage tank; 34-an equilibrium buffer storage tank; 4-a second ultrafiltration diafiltration unit; 41-a second recycle tank; 42-a second ultrafiltration membrane package; 43-a second dialysate reservoir; 44-a second stirring device; 45-a first storage device; 46-a buffer tank; 47-first SPTFF film package; 48-a third stirring device; 49-a second storage device; 5-a cache tank; 6-a capture chromatography unit; 7-a virus inactivation unit; 8-formulation unit.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are a part of the embodiments of the present invention, rather than all of the embodiments, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
In a first aspect, the present invention provides a protein purification method comprising the steps of:
and (3) performing virus removal filtration on the treated product after the fine purification chromatography after first ultrafiltration and/or first diafiltration.
Wherein, the treated matter after the fine purification chromatography can be subjected to first ultrafiltration, and the treated matter is concentrated to the specific protein concentration; the processed matter after the fine purification chromatography can also be subjected to first percolation, and the dialysis liquid change of the processed matter is carried out to a target buffer solution system or is diluted to a specific target concentration; ultrafiltration diafiltration may also be performed, where the treatment is concentrated to a specific protein concentration and the dialysis fluid is exchanged into the target buffer solution system.
After the treated matter after the fine purification chromatography is subjected to the first ultrafiltration and/or the first diafiltration, the sample subjected to the virus removal filtration can reach a better condition state, such as a specific protein concentration, a buffer solution system, pH and a conductivity value, and the better loading product state enables the virus removal filtration unit 3 to have better process performance. Therefore, the protein purification method provided by the invention has higher process loading capacity and shorter process time, can reduce the membrane area of virus removal filtration, shortens the process time and obviously reduces the cost.
Preferably, a step of performing a second ultrafiltration and/or a second diafiltration is included after the virus removal filtration.
In some embodiments of the invention, the product after the virus removal filtration may be subjected to a second ultrafiltration and/or a second diafiltration to meet the product requirements of subsequent formulation operations. The second ultrafiltration and/or second diafiltration may allow for a variety of adjustments and treatments to the sample, including but not limited to: (1) ultrafiltration, concentrating the product to a specific protein concentration; (2) Ultrafiltration and diafiltration, namely, after the product is concentrated to specific protein concentration, dialyzing and changing the solution to a target buffer solution system, or further concentrating the product dialyzed and changed solution to specific protein concentration after being transferred to the target buffer solution system; (3) And (4) performing diafiltration, and performing dialysis liquid change on the product to a target buffer solution system or diluting the product to a specific target concentration.
In some embodiments of the present invention, if the product after the virus removal filtration reaches or slightly exceeds the protein concentration range required by the preparation, and the dialysis solution is changed to the target buffer solution system meeting the preparation requirement, and the protein concentration further meets the protein concentration range required by the subsequent preparation stage after the virus removal filtration, the product can directly enter the subsequent preparation process without performing the second ultrafiltration and/or the second diafiltration.
Preferably, the first ultrafiltration, the first diafiltration, the second ultrafiltration and the second diafiltration comprise Tangential Flow Filtration (TFF) and/or unidirectional tangential flow filtration (SPTFF).
Wherein tangential flow filtration, i.e. recirculation TFF, the product can be recirculated through the recirculation loop.
Unidirectional tangential flow filtration refers to single pass TFF, i.e., single channel TFF, which allows direct flow-through concentration without product recirculation (SPTFF).
In some embodiments of the invention, the protein purification method specifically comprises the following steps: and (3) filtering the treated substance after the fine purification chromatography by using a first tangential flow, then performing virus removal filtration, then performing second tangential flow filtration, and preparing the preparation. Wherein the first tangential flow and the second tangential flow filtration can effect a variety of treatment modalities on the product, including but not limited to ultrafiltration, ultrafiltration/diafiltration/ultrafiltration, diafiltration, and the like.
Or, the treated matter after the fine purification chromatography is subjected to tangential flow filtration, virus removal filtration is carried out, and then the preparation is carried out. If the tangential flow filtration can realize the ultrafiltration concentration of the product to a specific concentration range, and the dialysis solution is changed to a target buffer solution system meeting the preparation requirement, after the virus removal filtration is carried out, the product reaches the protein concentration range required by the preparation, and the target buffer solution system is not changed, the second ultrafiltration and/or the second diafiltration can not be carried out, so that the process is simplified, the production time is shortened, and the production efficiency is improved.
Or, after tangential flow filtration, the treated matter after the fine purification chromatography is subjected to virus removal filtration, then unidirectional tangential flow filtration and then preparation. If one step ultrafiltration concentration of the product is required after virus removal filtration without diafiltration operation, one-way tangential flow filtration (SPTFF) is performed after virus removal filtration to simplify the process.
Or, after the treated matter after the fine purification chromatography is subjected to one-way tangential flow filtration, virus removal filtration is carried out, then tangential flow filtration is carried out, and then the preparation is carried out. If the product before virus removal filtration needs to be concentrated without diafiltration, one-way tangential flow filtration (SPTFF) can be performed before virus removal filtration to simplify the process.
Preferably, the tangential flow filtration has a flux of 100 to 400LMH (relative to the area of the membrane package used), including but not limited to the point of any one of 150LMH, 200LMH, 250LMH, 300LMH, 350LMH, or a range of values between any two. Wherein LMH is L/(m) 2 ·h)。
The transmembrane pressure (TMP) of the tangential flow filtration is between 0.5 and 1.5bar, including but not limited to a point value of any one of 0.7bar, 0.9bar, 1.0bar, 1.2bar, 1.4bar, or a range value therebetween.
The loading capacity of the tangential flow filtration is less than or equal to 500g/m 2 (relative to the area of the membrane package used); including but not limited to 450g/m 2 、400g/m 2 、350g/m 2 、300g/m 2 、250g/m 2 、200g/m 2 、150g/m 2 、100g/m 2 、50g/m 2 A point value of any one of them, or a range value between any two.
Preferably, the flux of the unidirectional tangential flow filtration is in the range of 10 to 90LMH (relative to the area of the membrane package used), including but not limited to the point value of any one of 20LMH, 30LMH, 40LMH, 50LMH, 60LMH, 70LMH, 80LMH or a range of values between any two.
The transmembrane pressure (TMP) of the unidirectional tangential flow filtration is 1 to 2bar, including but not limited to a point value of any one of 1.2bar, 1.4bar, 1.5bar, 1.7bar, 1.9bar, or a range value between any two.
Said unidirectional tangential flow throughThe filter capacity is less than or equal to 100g/m 2 (relative to the area of the membrane used), including but not limited to 90g/m 2 、80g/m 2 、70g/m 2 、60g/m 2 、50g/m 2 、40g/m 2 、30g/m 2 、20g/m 2 、10g/m 2 A point value of any one of them, or a range value between any two.
In some specific embodiments of the present invention, the purification chromatography further comprises a step of capture chromatography and virus inactivation. Preferably, a step of formulation is included after the virus removal filtration, the second ultrafiltration or the second diafiltration.
In some specific embodiments of the present invention, the pore size of the membrane used for the tangential flow filtration and/or the unidirectional tangential flow filtration can be any, conventional pore size, and can be selected according to the protein molecular weight, such as, but not limited to, 10kd,30kd,50kd, etc.
In some embodiments of the present invention, the protein concentration of the tangential flow filtration and/or the unidirectional tangential flow filtration may be any, conventional concentration, and may be set according to the requirements of the formulation. Preferably 5g/L to 50g/L, including but not limited to any one of 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or a range of values between any two.
In a second aspect, the present invention provides a protein purification system suitable for use in a protein purification method as described above. As shown in FIG. 1, the protein purification system comprises a fine purification chromatography unit 1, a first ultrafiltration and diafiltration unit 2 and a virus removal and filtration unit 3 which are connected in sequence.
Wherein the first ultrafiltration diafiltration unit 2 is adapted to perform a first ultrafiltration and/or a first diafiltration.
According to the invention, the first ultrafiltration and diafiltration unit 2 is arranged in front of the virus removal and filtration unit 3, so that various flexible adjustments can be made on the sample loading of the virus removal and filtration unit 3, the state of the sample loading can be flexibly changed, and the process performance of virus removal and filtration is optimized, thereby shortening the process time of virus removal and filtration, increasing the loading capacity, reducing the required membrane area and reducing the cost.
In addition, the protein purification system provided by the invention can utilize the existing traditional unit operation system equipment (virus removal filtration system equipment and ultrafiltration filtration system equipment) without great modification on the equipment, and the existing equipment is simply modified and integrated, so that the process flow can be simplified, the production efficiency can be improved, and the process performance and controllability can be improved.
Preferably, referring to fig. 1, the virus-removing filtration unit 3 is further connected to a second ultrafiltration diafiltration unit 4. The second ultrafiltration diafiltration unit 4 is used to perform a second ultrafiltration and/or a second diafiltration.
In some embodiments of the invention, the second ultrafiltration diafiltration unit 4 is optional, and may or may not be provided according to particular needs.
Preferably, said first ultrafiltration diafiltration unit 2 comprises a tangential flow filtration unit (TFF unit) and/or a unidirectional tangential flow filtration unit (SPTFF unit).
Preferably, said second ultrafiltration diafiltration unit 4 comprises a tangential flow filtration unit (TFF unit) and/or a unidirectional tangential flow filtration unit (SPTFF unit).
Preferably, first ultrafiltration diafiltration unit 2 comprises a tangential flow filtration device and second ultrafiltration diafiltration unit 4 comprises a tangential flow filtration device (denoted as version a). That is, both first ultrafiltration diafiltration unit 2 and second ultrafiltration diafiltration unit 4 employ TFF devices. That is, the protein purification system includes a fine purification chromatography unit 1, a first ultrafiltration diafiltration unit 2 (first TFF device), a virus removal filtration unit 3, and a second ultrafiltration diafiltration unit 4 (second TFF device) connected in series, see fig. 2.
In some particular embodiments of the invention, as shown in fig. 2, the first ultrafiltration diafiltration unit 2 comprises a first recycle tank 21 and a first ultrafiltration membrane module 22 connected; wherein the first circulation tank 21 is connected with the virus removing and filtering unit 3; the first circulation tank 21 is also connected to the fine chromatography unit 1. Preferably, a first dialysate storage tank 23 is also connected to the first circulation tank 21. Preferably, a first stirring device 24 is arranged in the first circulation tank 21.
As shown in fig. 2, the virus-removing filtration unit 3 comprises a pre-filter 31 and a virus-removing filter 32 which are connected, wherein the pre-filter 31 is connected with the first circulation tank 21, and the virus-removing filter 32 is connected with the second ultrafiltration and diafiltration unit 4. A water for injection storage tank 33 and/or an equilibration buffer storage tank 34 are also provided between the pre-filter 31 and the first circulation tank 21. The water for injection storage tank 33 and/or the equilibration buffer storage tank 34 are connected to the pre-filter 31 and the virus removal filter 32, respectively.
The material of the pre-filter 31 includes Polyethersulfone (PES), and the pore size of the pre-filter 31 may be any conventional pore size, such as 0.22 μm,0.1 μm, but is not limited thereto. The virus removing filter 32 may be any conventional virus removing filter 32 available by purchase, for example, planova of Asahi Kasei corporation TM Series of filters, merckmix
Figure BDA0003857637790000091
Series of filters, pelasus by Perl TM Series of filters, or of Sadoris
Figure BDA0003857637790000092
Series of filters, but not limited thereto. The pressure difference of the virus-removing filtering selection filter is different according to the product characteristics, and a proper specific pressure range is selected, for example, the control operation pressure of the Planova series filter is in the range of 0.7-1.0 bar.
As shown in fig. 2, said second ultrafiltration diafiltration unit 4 comprises a second recycle tank 41 and a second ultrafiltration membrane pack 42 connected; preferably, a second dialysate storage tank 43 is also connected to the second circulation tank 41. Preferably, a second stirring device 44 is arranged in the second circulation tank 41. Preferably, a first storage device 45 is further connected to the second circulation tank 41, said first storage device 45 being used for storing or temporarily storing the product that has undergone the second ultrafiltration and/or second diafiltration.
The first ultrafiltration diafiltration unit 2 (first TFF unit) and the second ultrafiltration diafiltration unit 4 (second TFF unit) may be used to perform a variety of processes including, but not limited to, ultrafiltration/diafiltration, etc., and also to achieve efficient recovery of the product in the ultrafiltration membrane module and the piping.
The working principle of the first ultrafiltration by the first ultrafiltration and diafiltration unit 2 is as follows: referring to fig. 2, the treated material after the fine purification chromatography enters the first circulation tank 21 from the fine purification chromatography unit 1, and the first stirring device 24 is turned on. Subsequently, the treated matter in the first circulation tank 21 passes through an ultrafiltration membrane pack, and protein molecules are trapped on the inner side of the membrane pack; the solution micromolecules permeate the outer side of the membrane under the action of transmembrane pressure (TMP) to be discharged. The protein molecules trapped inside the membrane are returned to the first circulation tank 21, while the feed flow rate and transmembrane pressure (TMP) are controlled, and the circulation is repeated, and the treated product in the first circulation tank 21 is continuously concentrated to reach the target concentration, and then stopped.
The operating principle of the first diafiltration with the first ultrafiltration diafiltration unit 2 is as follows: starting the first stirring device 24, leading the dialysate to enter the first circulation tank 21 from the first dialysate storage tank 23 and then enter the first ultrafiltration membrane package 22, and trapping protein molecules on the inner side of the membrane; the solution micromolecules permeate the outer side of the membrane under the action of transmembrane pressure (TMP) to be discharged. The volume of the first circulation tank 21 is maintained constant, the feed flow rate and the TMP are controlled, and the product dialysis solution exchange of the diafiltration process is completed after the dialysate amount which is more than or equal to 6 times the volume of the first circulation tank 21.
And (3) recovering the product in the ultrafiltration membrane package after the treatment is finished, conveying the treated matters in the dialysate top washing-ultrafiltration membrane package-loop to the first circulating tank 21, and uniformly stirring.
The working principle of virus removal and filtration by adopting the virus removal and filtration unit 3 is as follows: 1) Water for injection (WFI) is introduced from the water for injection storage tank 33 into the pre-filter 31 and the virus removal filter 32, and is flushed while controlling the flow path pressure to be lower than the maximum operating pressure of the filter; 2) The balance buffer solution enters the pre-filter 31 and the virus removing filter 32 from the balance buffer solution storage tank 34 to be pre-balanced, and the pressure of the flow path is controlled within the process requirement pressure range; 3) Loading and filtering, wherein the treated substance in the first circulating tank 21 enters the pre-filter 31 and the virus removing filter 32, and after virus removing and filtering treatment, the treated substance is collected and conveyed to the second circulating tank 41, and the flow rate of the raw materials is controlled so that the pressure of the filter is constant within the process requirement range; 4) The product is recovered by top washing with the equilibration buffer, which enters the prefilter 31 and the virus-removing filter 32, and the treated matter in the pipeline and the filter is recovered to the second circulation tank 41, while the pressure of the flow path is controlled within the pressure range required by the process.
The working principle of the second ultrafiltration using second ultrafiltration diafiltration unit 4 is as follows: the treated material in the second circulation tank 41 enters the second ultrafiltration membrane 42, protein molecules are trapped on the inner side of the membrane, and solution small molecules permeate the outer side of the membrane under the action of transmembrane pressure (TMP) to be discharged. The protein molecules trapped inside the membrane are returned to the second circulation tank 41 through a loop during which the feed flow rate and transmembrane pressure (TMP) are controlled by a pump and a valve, and the circulation is repeated, and the treated product in the second circulation tank 41 is continuously concentrated to reach the target concentration, and then is stopped.
The operating principle of the second diafiltration with the second ultrafiltration diafiltration unit 4 is as follows: the second stirring device 44 is turned on, the dialysate flows from the second dialysate storage tank 43 into the second circulation tank 41, and then into the second ultrafiltration membrane 42, the protein molecules are trapped inside the membrane, and the small molecules of the solution permeate through the outside of the membrane under the action of transmembrane pressure (TMP) to perform waste discharge. The volume of the second circulation tank 41 is kept constant, the feed flow rate and TMP are controlled, and the product dialysis exchange fluid of the diafiltration process is completed after a dialysis fluid volume of not less than 6 times the volume of the second circulation tank 41.
And (3) recovering the product in the ultrafiltration membrane package after the treatment is finished, conveying the treated product to a first storage device 45 for storage or temporary storage, conveying the pipeline and the residual product in the ultrafiltration membrane package to a second circulation tank 41 through a loop, and then conveying the product to the first storage device 45.
Alternatively, first ultrafiltration diafiltration unit 2 comprises a tangential flow filtration device and second ultrafiltration diafiltration unit 4 comprises a unidirectional tangential flow filtration device (denoted as variant B). That is, the protein purification system includes a purification chromatography unit 1, a first ultrafiltration and diafiltration unit 2 (TFF device), a virus removal filtration unit 3, and a second ultrafiltration and diafiltration unit 4 (SPTFF device) connected in series, see fig. 3.
Wherein, the tangential flow filtration device is adopted for tangential flow filtration, and the purpose is as follows: and adjusting the sample product subjected to virus removal and filtration to a better protein concentration and a solution system state, wherein the solution system is a solution system meeting the preparation requirement.
The one-way tangential flow filter device realizes one-step concentration of the treated substances. The ultrafiltration membrane of the unidirectional tangential flow filtration device may be of any conventional type, such as Cadence from Perl TM The merckmicrobio corporation modifies the common ultrafiltration membrane module in series by clamps, but is not limited thereto.
As shown in fig. 3, the protein purification system corresponding to scheme B has substantially the same structure as the protein purification system corresponding to scheme a except that the second ultrafiltration diafiltration unit 4, a tangential flow filtration device, is replaced with a unidirectional tangential flow filtration device. In particular, the second ultrafiltration diafiltration unit 4-one-way tangential flow filtration unit in protocol B comprises a connected buffer tank 46 and a first SPTFF membrane module 47; wherein, the buffer tank 46 is connected with the virus removal filtering unit 3. Preferably, a third stirring device 48 is arranged in the buffer tank 46; the first SPTFF membrane package 47 is further connected to a second reservoir 49.
The structure and the working principle of the tangential flow filtration device, and the structure and the working principle of the virus removing filtration unit 3 are the same as those of the scheme A.
The working principle of the second ultrafiltration by using the unidirectional tangential flow filtration device is as follows: the treated substance treated by the virus removing and filtering unit 3 enters a buffer tank 46 and then enters a first SPTFF membrane 47, protein molecules are intercepted at the inner side of the membrane, solution micromolecules are discharged from the outer side of the membrane under the action of TMP for waste discharge, and products are directly recovered after reaching the target protein concentration. During the period, the flow rate of the sample injection is regulated and controlled by a pump, and the TMP is controlled by a valve.
Alternatively, first ultrafiltration diafiltration unit 2 comprises a one-way tangential flow filtration device and second ultrafiltration diafiltration unit 4 comprises a tangential flow filtration device (denoted as version C). That is, the protein purification system includes a purification chromatography unit 1, a first ultrafiltration and diafiltration unit 2 (SPTFF device), a virus removal filtration unit 3, and a second ultrafiltration and diafiltration unit 4 (TFF device) connected in series, see fig. 4.
Wherein, the purpose of adopting the unidirectional tangential flow filter device to carry out unidirectional tangential flow filtration is as follows: the sample product after virus removal and filtration (namely the treated matter before virus removal and filtration) is concentrated to a better protein concentration, and one-step concentration is realized. The tangential flow filtration using the tangential flow filtration device is aimed at: and (4) ultrafiltering and percolating the virus-removed and filtered product into a protein concentration and solution system meeting the preparation requirements.
As shown in fig. 4, the protein purification system corresponding to scheme C has substantially the same structure as the protein purification system corresponding to scheme a except that the first ultrafiltration diafiltration unit 2, a tangential flow filtration device, is replaced with a unidirectional tangential flow filtration device. Specifically, referring to fig. 4, the first ultrafiltration diafiltration unit 2-one-way tangential flow filtration device in scheme C comprises a second SPTFF membrane package 25, said second SPTFF membrane package 25 being connected to said polishing chromatography unit 1, said second SPTFF membrane package 25 being further connected to said virus removal filtration unit 3. Preferably, a buffer tank 5 is arranged between the second SPTFF membrane package 25 and the virus removal and filtration unit 3.
Wherein, the working principle of the first ultrafiltration by adopting the first ultrafiltration percolation unit 2-one-way tangential flow filtration device is as follows: the treated matter after the fine purification chromatography enters a second SPTFF membrane pack 25, protein molecules are intercepted at the inner side of the membrane, solution micromolecules are discharged from the outer side of the membrane under the action of TMP for waste discharge, and the product is recovered to a buffer tank 5 after reaching the target protein concentration. The period is adjusted to control the flow rate of sample injection and control TMP.
The structure of the virus-removing filtration unit 3 and the second ultrafiltration diafiltration unit 4 (TFF unit) and their working principle are the same as in the above-described embodiment a.
Alternatively, the second ultrafiltration diafiltration unit 4 is not provided. That is, the protein purification system comprises a fine purification chromatography unit 1, a first ultrafiltration diafiltration unit 2 (TFF unit) and a virus removal filtration unit 3 (denoted as scheme D) connected in series, see fig. 5.
The structures of the first ultrafiltration and diafiltration unit 2 (TFF unit) and the virus removal and filtration unit 3 and their working principles are the same as those of the above-mentioned embodiment a.
Preferably, the material of the ultrafiltration membrane of said first ultrafiltration and diafiltration unit 2 and/or said second ultrafiltration and diafiltration unit 4 comprises Polyethersulfone (PES) and/or regenerated cellulose.
Preferably, as shown in fig. 6, the protein purification system further comprises a capture chromatography unit 6 and a virus inactivation unit 7 connected; wherein the virus inactivation unit 7 is connected with the fine purification chromatography unit 1.
Preferably, as shown in fig. 6 and 7, the protein purification system further comprises a preparation unit 8, the preparation unit 8 being connected to the virus removal filtration unit 3 (fig. 7) or the second ultrafiltration diafiltration unit 4 (fig. 6).
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
The present embodiment provides a protein purification system, as shown in fig. 2, which includes a fine purification chromatography unit 1, a first ultrafiltration and diafiltration unit 2, a virus removal and filtration unit 3, and a second ultrafiltration and diafiltration unit 4, which are connected in sequence.
Wherein the first ultrafiltration diafiltration unit 2 is a tangential flow filtration device (first TFF device) and the second ultrafiltration diafiltration unit 4 is a tangential flow filtration device (second TFF device).
The first uf unit 2 includes a first circulation tank 21 and a first uf membrane module 22 connected. The first circulating tank 21 is connected with the virus removing and filtering unit 3; the other end of the first circulation tank 21 is also connected to the fine purification chromatography unit 1. The first circulation tank 21 is also connected to a first dialysate storage tank 23. A first stirring device 24 is arranged in the first circulation tank 21.
The virus-removing filter unit 3 comprises a pre-filter 31 and a virus-removing filter 32 which are connected, wherein the pre-filter 31 is connected with the first circulation tank 21, and the virus-removing filter 32 is connected with the second ultrafiltration and diafiltration unit 4. An injection water storage tank 33 and a balance buffer storage tank 34 are further provided between the pre-filter 31 and the first circulation tank 21.
The second ultrafiltration diafiltration unit 4 comprises a second recycle tank 41 and a second ultrafiltration membrane pack 42 connected. A second dialysate storage tank 43 is also connected to the second circulation tank 41. A second stirring device 44 is arranged in the second circulation tank 41. The second circulation tank 41 is also connected to a first storage device 45.
The present invention also provides a protein purification method using the protein purification system, comprising the following steps:
the product Mab1, after fine purification chromatography, the protein concentration of the product is 11.2g/L, the volume is 220mL, the pH is 5.53, the conductivity value is 19.32mS/cm, and the buffer solution system is 50mM NaAc-HAc, naCl system.
The treated material after the fine purification chromatography was introduced into the first ultrafiltration/diafiltration unit 2, and ultrafiltration/diafiltration was carried out so that the protein concentration reached 20g/L, and the buffer system was replaced with 20mM Histindine, pH 6.2. Wherein the ultrafiltration membrane of the first ultrafiltration and diafiltration unit 2 is made of Mercmcrobo Pellicon 3PES material with 30KD and the membrane area is 0.0088m 2 Film load of 280g/m 2 Firstly, the product is ultrafiltered and concentrated to 18-22 g/L, then the product is diafiltered and replaced by diafiltration buffer solution 20mM Histidine with 6 times of the product, and the pH value is 6.2. 123mL of product is recovered, and then virus removal filtration is carried out, wherein the membrane area required by virus removal filtration is 0.001m 2 . The product after virus removal and filtration enters a second ultrafiltration and diafiltration unit 4 for concentration, the ultrafiltration membrane used by the second ultrafiltration and diafiltration unit 4 is made of Pellicon 3PES material, 30KD, and the membrane area is 0.0088m 2 Film loading 270g/m 2 And concentrating the product to a concentration range of 75-85 g/L, and then carrying out subsequent preparation operation.
Wherein the process parameters of the ultrafiltration/diafiltration in the first ultrafiltration/diafiltration unit 2 are as shown in table 1 below.
The membrane used for virus removal filtration was Asahi-converted Planova 20N, and the membrane surface wasProduct of 0.001m 2 The process parameters for virus removal filtration are shown in Table 2 below.
TABLE 1 Process parameters of the ultrafiltration/diafiltration in the first ultrafiltration/diafiltration unit 2
Figure BDA0003857637790000151
TABLE 2 Process parameters for virus removal filtration
Figure BDA0003857637790000161
Wherein the filtration flux is 27.3LMH, and the process time for filtering the required sample after finishing 123mL is 4.5 hours. The yield of ultrafiltration/diafiltration in the first ultrafiltration diafiltration unit 2 was 99.1% and the yield of virus removal filtration was 100.2%, which met the requirements of conventional unit step operation. According to the process, under the condition of increasing the sample amount, the virus removal membrane loading capacity can be still further improved, the membrane area used is further reduced, and the process time is shortened.
Example 2
The protein purification system provided in this example is essentially the same as that of example 1, except that the second ultrafiltration diafiltration unit 4-tangential flow filtration device (TFF device) is replaced with a unidirectional tangential flow filtration device (SPTFF device). Referring to fig. 3, the second ultrafiltration diafiltration unit 4-one-way tangential flow filtration unit comprises a connected buffer tank 46 and a first SPTFF membrane module 47. The buffer tank 46 is connected with the virus removing filter unit 3. A third stirring device 48 is arranged in the buffer tank 46. A second storage means 49 is further connected to the first SPTFF membrane package 47.
Wherein the unidirectional tangential flow filtration device is a Peltier Cadence TM One-way flow on-line concentration filter, 30KD pore size, regenerated cellulose material, and selected membrane area of 0.065m 2 The flow rate is controlled to be 0.05-0.1L/min, and the TMP is controlled to be 1.2-2.0 bar.
The protein purification method provided in this example uses the protein purification system provided in this example, and the protein purification method is substantially the same as in example 1. Specifically, in this example, the same product source and volume as in example 1 were used, the product was fed into a first ultrafiltration diafiltration unit 2-TFF unit, protein concentration was ultrafiltered and concentrated to 18-22 g/L using the same tangential flow filtration process as in example 1, the product dialysis fluid was diafiltered to a buffer solution system of 20mM histidine, ph 6.2, the recovered product was subjected to virus-removal filtration, the process parameters were the same as in example 1, the recovered virus-removal filtration product was fed into an SPTFF unit, one-step concentration to 75-85 g/L was completed, and then the subsequent formulation operation was performed.
Compared with the embodiment 1, the embodiment simplifies the protein purification system, has simpler and more convenient operation, shortens the process time, improves the membrane loading capacity of virus removal filtration, reduces the membrane consumption and reduces the production cost on the premise of ensuring the product yield and quality.
Example 3
The protein purification system provided in this example is essentially the same as that of example 1, except that the first ultrafiltration diafiltration unit 2-tangential flow filtration device (TFF device) is replaced with a unidirectional tangential flow filtration device (SPTFF device). Referring to fig. 4, the first ultrafiltration diafiltration unit 2-one-way tangential flow filtration device comprises a second SPTFF membrane module 25, the second SPTFF membrane module 25 being connected to the polishing chromatography unit 1, the second SPTFF membrane module 25 being further connected to the virus removal filtration unit 3. A buffer tank 5 is arranged between the second SPTFF membrane package 25 and the virus removing and filtering unit 3.
Wherein the first ultrafiltration diafiltration unit 2-unidirectional tangential flow filtration device is a peltier Cadence TM One-way flow on-line concentration filter with 30KD pore diameter and regenerated cellulose material, and the selected membrane area is 0.065m 2 The flow rate is controlled to be 0.05-0.1L/min, and the TMP is controlled to be 1.2-2.0 bar.
The protein purification method provided in this embodiment uses the protein purification system provided in this embodiment, and includes the following steps:
the product Mab2, after fine purification chromatography, the protein concentration of the product is 13.5g/L, the volume is 250mL, the pH value is 5.47, the conductivity value is 12.13mS/cm, and the buffer solution system is 50mM NaAc-HAc, naCl system.
The treated matter after the fine purification chromatography enters a first ultrafiltration and diafiltration unit 2-one-way tangential flow filtration device, the concentration of the protein of the product is concentrated to 20-25 g/L, the product after the one-step concentration enters a buffer tank 5, and the residual product of the system is recovered. Then, the product collected in the buffer tank 5 enters the virus removing and filtering unit 3 for virus removing and filtering. And (3) feeding the product subjected to virus removal and filtration into a second ultrafiltration and diafiltration unit 4-TFF device, performing ultrafiltration/diafiltration/ultrafiltration, further performing ultrafiltration and concentration on the product to 30-40 g/L, performing diafiltration to change the liquid to 20mM Glutamic Acid/Histidine, pH 5.0, and 6 times of the volume of the product, performing ultrafiltration and concentration on the product to 83-90 g/L after the liquid change is finished, and performing subsequent preparation operation.
Wherein the process parameters for ultrafiltration concentration using the first ultrafiltration diafiltration unit 2-SPTFF unit are shown in table 3 below.
The process parameters for ultrafiltration/diafiltration/ultrafiltration using the second ultrafiltration diafiltration unit 4-TFF unit were essentially the same as in example 1 (see table 1). The ultrafiltration membrane of the second ultrafiltration diafiltration unit 4-TFF unit is made of Pellicon 3PES material of Mercury, 30KD, and the membrane area is 0.0088m 2 Film load of 372g/m 2 . The buffer used for equilibration/diafiltration/rinse was 20mM Glutaminc Acid/Histidine, pH 5.0.
The filter used for virus removal filtration was a Merck Millico ViResolve Micro 40 filter with a membrane area of 3.4cm 2 The material is PES. The process parameters for virus removal filtration are shown in Table 4 below.
TABLE 3 Process parameters for ultrafiltration concentration using SPTFF unit
Figure BDA0003857637790000181
Figure BDA0003857637790000191
The concentration of the SPTFF product obtained after one-step concentration is 23.3g/L, the volume is 142mL, and the yield of the step is 98.3%.
TABLE 4 Process parameters for virus removal filtration
Figure BDA0003857637790000192
The filtration flux was 48LMH, and the process time for filtration of the desired sample was 8.7 hours after 142mL had been completed. The results show that the membrane loading of the virus removal step is higher, less membrane area is required and the cost is lower during normal process time.
The product after virus removal filtration enters the second recycle tank 41 of the TFF unit, and the yield of the step is 99.0%.
Example 4
The protein purification system provided in this example is essentially the same as that of example 1, except that the second ultrafiltration diafiltration unit 4 is not provided. See fig. 5.
The protein purification method provided in this embodiment uses the protein purification system provided in this embodiment, and includes the following steps:
the product Mab3, after fine purification chromatography, has a protein concentration of 10.2g/L, a volume of 300mL, a pH of 5.55, a conductivity value of 21.31mS/cm, and a buffer solution system of 50mM NaAc-HAc, naCl system.
And (3) allowing the treated product after the fine purification chromatography to enter a first ultrafiltration and diafiltration unit 2-TFF device for ultrafiltration/diafiltration/ultrafiltration, performing ultrafiltration and concentration on the product to 20-30 g/L, performing diafiltration and dialysis on the product to 20mM NaAc-HAc solution with pH of 5.3, performing ultrafiltration and concentration on the product again to 30-40 g/L, collecting the product to a first circulation tank 21, and recovering the residual product in the system. The product collected in the first circulation tank 21 is introduced into the virus-removing filtration unit 3 to be virus-removed and filtered. The protein concentration of the product after virus removal and filtration is 31.3g/L, the solution system is 20mM NaAc-HAc, the pH is 5.3, the product meets the product requirements of subsequent preparation steps, and the product is directly subjected to subsequent preparation operation.
Wherein the process parameters for ultrafiltration/diafiltration/ultrafiltration were essentially the same as in example 1 using the first ultrafiltration diafiltration unit 2-TFF unit (see table 1). The ultrafiltration membrane of the second ultrafiltration diafiltration unit 4-TFF unit is made of Pellicon 3PES material of Mercury, 30KD, and the membrane area is 0.0088m 2 The film loading was 348g/m 2 . The buffer used for equilibration/diafiltration/rinse was 20mM NaAc-HAc, pH 5.3.
The ultrafiltration membrane used in the TFF device is made of Pellicon 3PES material of Merck Raobo, 30KD and the membrane area is 0.0088m 2 . The yield of this step was 96.3%.
The virus-removing filtration membrane used for virus-removing filtration was Asahi platova 20N, and the membrane area was 0.001m 2 . The process time was 6 hours and the yield of this step was 98.9%.
Comparative example 1
The present comparative example provides a protein purification system substantially identical to that of example 1, except that the first ultrafiltration diafiltration unit 2 is not provided. That is, the protein purification system of this comparative example comprises a purification chromatography unit 1, a virus removal filtration unit 3, and a second ultrafiltration-diafiltration unit 4, which are connected in this order.
The protein purification method of the present comparative example used the protein purification system of the present comparative example, comprising the steps of: the product after the fine purification chromatography (using the same product Mab1 as in example 1 and using the same initial conditions for the sample) was subjected to a virus removal filtration followed by an ultrafiltration/diafiltration, a protein concentrate exchange to 20mm histidine, ph 6.2 and then to the formulation step. Wherein, the membrane used for virus removal filtration and the process parameters of the virus removal filtration are the same as those of the embodiment 1. The process parameters of ultrafiltration/diafiltration were the same as in example 1.
The filtration flux is 28.8LMH, the process time for filtering the sample after the completion of 220mL is 7.6 hours, and the actual membrane loading is 2464g/m 2 。
As can be seen by comparing the process times of example 1 and comparative example 1, the process time of example 1 was shortened by 3.1 hours compared to comparative example 1.
In conclusion, the protein purification system and method provided by the invention can obtain higher process loading capacity and shorter process time, can reduce the membrane area of virus removal filtration, and can obviously reduce the cost.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit it; those of ordinary skill in the art will understand that: modifications may be made to the above-described embodiments, or equivalents may be substituted for some or all of the features thereof without departing from the spirit and scope of the present invention; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention; it is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. A protein purification method is characterized by comprising the following steps:
and (3) performing virus removal filtration on the treated product after the fine purification chromatography after first ultrafiltration and/or first diafiltration.
2. The protein purification process according to claim 1, further comprising a step of performing a second ultrafiltration and/or a second diafiltration after said virus removal filtration.
3. The protein purification process according to claim 2, wherein the first ultrafiltration, the first diafiltration, the second ultrafiltration and the second diafiltration comprise tangential flow filtration and/or unidirectional tangential flow filtration.
4. The protein purification process according to claim 3, wherein the tangential flow filtration has a flux of 100 to 400LMH, a transmembrane pressure of 0.5 to 1.5bar and a loading of 500g/m or less 2 ；
Preferably, the flux of the unidirectional tangential flow filtration is 10-90 LMH, the transmembrane pressure is 1-2 bar, and the loading capacity is less than or equal to 100g/m 2 。
5. A protein purification system suitable for the protein purification method according to any one of claims 1 to 4, comprising a fine purification chromatography unit, a first ultrafiltration and diafiltration unit, and a virus removal and filtration unit, which are connected in this order.
6. The protein purification system according to claim 5, wherein a second ultrafiltration diafiltration unit is connected to the virus removal filtration unit.
7. The protein purification system according to claim 6, wherein the first ultrafiltration diafiltration unit comprises a tangential flow filtration device and/or a unidirectional tangential flow filtration device;
preferably, the second ultrafiltration diafiltration unit comprises a tangential flow filtration device and/or a unidirectional tangential flow filtration device.
8. The protein purification system according to claim 6, wherein the first ultrafiltration diafiltration unit comprises a tangential flow filtration device and the second ultrafiltration diafiltration unit comprises a tangential flow filtration device;
alternatively, the first ultrafiltration diafiltration unit comprises a tangential flow filtration device and the second ultrafiltration diafiltration unit comprises a unidirectional tangential flow filtration device;
alternatively, the first ultrafiltration diafiltration unit comprises a unidirectional tangential flow filtration device and the second ultrafiltration diafiltration unit comprises a tangential flow filtration device.
9. The protein purification system according to claim 6, wherein the ultrafiltration membrane of the first and/or second ultrafiltration diafiltration unit comprises polyethersulfone and/or regenerated cellulose.
10. The protein purification system of claim 6, further comprising a capture chromatography unit and a virus inactivation unit connected; wherein the virus inactivation unit is connected with the fine purification chromatography unit;
preferably, the protein purification system further comprises a formulation unit connected to the virus removal filtration unit or the second ultrafiltration diafiltration unit.
CN202211154006.5A 2022-09-21 2022-09-21 Protein purification method and protein purification system Pending CN115430198A (en)

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CN106794424A (en) * 2014-05-13 2017-05-31 美国安进公司 For filter and the Process Control System and method of filter process
US20200190137A1 (en) * 2018-12-18 2020-06-18 Amgen Inc. Methods for purifying proteins
CN112279919A (en) * 2020-10-02 2021-01-29 朱吉安 Preparation method of anti-PD-1 antibody
CN113710685A (en) * 2019-04-04 2021-11-26 吉瑞工厂 Improved affinity chromatography of immunoglobulins by using flocculation prior to capture
CN114829371A (en) * 2019-12-12 2022-07-29 Emd密理博公司 Enhanced virus filtration using diafiltration buffer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106794424A (en) * 2014-05-13 2017-05-31 美国安进公司 For filter and the Process Control System and method of filter process
US20200190137A1 (en) * 2018-12-18 2020-06-18 Amgen Inc. Methods for purifying proteins
CN113710685A (en) * 2019-04-04 2021-11-26 吉瑞工厂 Improved affinity chromatography of immunoglobulins by using flocculation prior to capture
CN114829371A (en) * 2019-12-12 2022-07-29 Emd密理博公司 Enhanced virus filtration using diafiltration buffer
CN112279919A (en) * 2020-10-02 2021-01-29 朱吉安 Preparation method of anti-PD-1 antibody

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