CN111072773A - Antibody purification method, antibody and application - Google Patents
Antibody purification method, antibody and application Download PDFInfo
- Publication number
- CN111072773A CN111072773A CN201811226606.1A CN201811226606A CN111072773A CN 111072773 A CN111072773 A CN 111072773A CN 201811226606 A CN201811226606 A CN 201811226606A CN 111072773 A CN111072773 A CN 111072773A
- Authority
- CN
- China
- Prior art keywords
- antibody
- chromatography
- buffer
- monitor
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a purification method of an antibody, the antibody and application, and relates to the technical field of protein purification. The purification method comprises the following steps: providing a cell fermentation broth containing a crude antibody; providing a buffer for chromatography; then using AKTA Avant150 to carry out combined chromatography on the antibody crude product; wherein the combinatorial chromatography comprises in sequence (a) affinity chromatography to capture the antibody; (b) gel filtration chromatography displacement Buffer for desalting; (c) the antibody was chromatographed again to purify the antibody. The antibody purification method has the advantages of simple operation, high purification efficiency, less sample loss and high purity of the purified antibody.
Description
Technical Field
The invention relates to the technical field of protein purification, in particular to a purification method of an antibody, the antibody and application.
Background
After the first approval of therapeutic monoclonal antibodies (mAbs) in 1986, the need for therapeutic mAbs has increased significantly. By 11 months at 2015, 53 novel therapeutic monoclonal antibodies entered phase 3 clinical studies with a 104% increase over 2009, six of which were first approved in the eu or us in 2016. There are currently about 4 new therapeutic mab products available to market each year, and with this increasing rate, the market is expected to have 70 mabs by 2020, with a global sales of $ 1250 billion.
The purification preparation of therapeutic monoclonal antibodies is mostly based on Protein a affinity chromatography as the first step, but usually, the purity of one-step Protein a affinity chromatography and the like do not meet the requirements of therapeutic antibodies (generally, the purity requirement is more than 95%), and when a CHO cell line is used as a host to express antibodies, cell culture supernatant contains not only target antibodies but also host proteins (HCPs), host dna (hcd) and other impurities, and the single-step Protein a chromatography does not remove all impurities, so that an additional step or a plurality of steps of chromatography processes are needed to meet the requirements.
The most common chromatographic procedures for the purification of therapeutic monoclonal antibodies are Protein a (affinity chromatography, AC), desalting (BEX), Cation Exchange (CEX), desalting (BEX), Anion Exchange (AEX). The desalting process may use desalting columns or ultrafiltration desalting. At present, almost most of purification workers adopt a manual one-step chromatography method, each step of chromatography needs separate sampling, elution, sample treatment and next step of chromatography operation, and few manual two-step chromatography methods are used for purification. Manual chromatography not only can cause the loss of samples, but also has the disadvantages of much labor consumption and large equipment investment.
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 method for purifying an antibody, which has the advantages of simple operation, high purification efficiency, less sample loss, and high purity of the purified antibody.
The second object of the present invention is to provide an antibody purified by the above-mentioned antibody purification method.
The third purpose of the invention is to provide an application of the purification method of the antibody in preparing antibody medicines.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a method for purifying an antibody, which comprises the following steps: providing a cell fermentation broth containing a crude antibody; providing a buffer for chromatography; then using AKTA Avant150 to carry out combined chromatography on the antibody crude product; the combined chromatography comprises the following steps in sequence:
(a) affinity chromatography to capture the antibody;
(b) gel filtration chromatography displacement Buffer for desalting;
(c) carrying out chromatographic separation on the antibody again to carry out fine purification treatment on the antibody;
wherein, the connecting mode of pipeline and chromatographic column among AKTA Avant150 is: the Out1 outlet of the device is connected to the loading port of the Loop, and the affinity chromatography column used in the step (a) is connected to the Position 3; replacing the Loop with the gel filtration chromatography column used in step (b); the chromatography column used in step (c) is attached at Position 1.
Preferably, the packing used in the affinity chromatography column of step (a) comprises at least one of Protein a, Protein G or Protein L; protein A is preferred.
Preferably, the manner of chromatographically separating the antibody again in step (c) comprises: ion exchange chromatography, affinity chromatography, hydroxylapatite chromatography or hydrophobic interaction chromatography.
Preferably, the mode of chromatographically separating the antibody again in step (c) is ion exchange chromatography, preferably cation exchange chromatography.
Preferably, the mode of chromatographically separating the antibody again in step (c) is hydrophobic interaction chromatography.
Preferably, the purification method further comprises setting a loading inlet of a sample pump of AKTA Avant150 as a variable for sequentially purifying a plurality of antibody samples.
Preferably, the flow path of the combined chromatography comprises:
(x1) affinity loading: injecting the antibody crude product into a pipeline from Injection valve through a Sample pump, flowing through an affinity chromatography column to be specifically combined with filler in the chromatography column, then sequentially flowing the filtrate to a Fraction collection through a UV monitor, a Conductivity monitor and a pH valve, and collecting the flow liquid;
(x2) affinity Wash impurity: buffer A1 flows through System pump A from Injection valve Injection pipeline, passes through affinity chromatography column to elute impurities, then the filtrate flows to Waste through UV monitor, reduction monitor and pH valve in sequence, and flows out to Waste liquid;
(x3) affinity eluting antibody and gel filtration chromatography loading: buffer B1 flows through the affinity chromatography column after being injected into the pipeline from Injection value through System pump B for affinity elution of the antibody captured by the affinity chromatography column, then the filtrate with the antibody sample flows to Outlet value through UV monitor, reduction monitor and pH value in sequence, and flows back to Injection value without draining after peak collection and is injected into the gel filtration chromatography column;
(x4) chromatographic loading: buffer A2 is injected into the pipeline from Injection valve through System pump A, the antibody sample is eluted from the gel filtration chromatographic column and simultaneously loaded into the chromatographic column used in the step (c), the filtrate flows to the Outlet valve through UVmonitor, reduction monitor and pH valve in sequence, and the filtrate is collected after flowing out;
(x5) chromatographic elution: and (2) injecting Buffer A2 from the Injection valve into the pipeline through System pump A, simultaneously flowing Buffer B2 from the Injection valve into the pipeline through System pump B, then flowing through the chromatographic column for gradient elution to elute the antibody combined with the chromatographic column, then flowing the filtrate to the separation Collection after passing through UV monitor, reduction monitor and pHvalve in sequence, and collecting the filtrate after flowing out to obtain the purified antibody.
Preferably, the flow path of the combined chromatography further comprises a step for regeneration of the chromatography column:
(x6) regeneration of the affinity chromatography column used in step (a): buffer B3 was passed through System pump B from Injection valve Injection line, through affinity chromatography column to wash out impurities for regeneration treatment, the filtrate was passed through UVmonitor, reduction monitor and pH valve in sequence to Waste, then Buffer A1 was passed through System pump A from Injection valve Injection line, through affinity chromatography column to wash out the remaining alkali solution in the column, the filtrate was passed through UVmonitor, reduction monitor and pH valve in sequence to Waste.
(X7) regeneration of the chromatography columns used in step (b) and step (c): and (3) injecting Buffer B3 from the Injection valve through the System pump B, firstly passing through the gel filtration chromatographic column used in the step (B), then passing through the chromatographic column used in the step (c), sequentially passing the filtrate through the UV monitor, the Conductivity monitor and the pH valve to flow to the step, then sequentially passing Buffer A2 from the Injection valve through the System pump A, firstly passing through the gel filtration chromatographic column used in the step (B), then passing through the chromatographic column used in the step (c), and sequentially passing the filtrate through the UV monitor, the Conductivity monitor and the pH valve to flow to the step.
Preferably, the antibody desalted by the gel filtration chromatography column is mixed and then loaded to the cation exchange chromatography column;
the buffer used in the channel includes:
BufferA1：PBS，pH7.2～7.4；
buffer a 2: a NaAC solution having a pH of 5.0 to 5.5 and a concentration of 20 to 50 mM;
buffer b 1: a NaAC solution having a pH of 3.2 to 3.4 and a concentration of 20 to 50 mM;
buffer b2: the pH value is 5.0-5.5, and the NaAC solution contains NaAC with the concentration of 20-50 mM and NaCl solution with the concentration of 1-2M;
buffer B3 0.1-0.2M NaOH solution.
Preferably, the antibody desalted by the gel filtration chromatography column is mixed and then loaded to the hydrophobic interaction chromatography column;
the buffer used in the channel includes:
BufferA1：PBS，pH7.2～7.4；
buffer a 2: a pH of 7.5 to 8.0, a concentration of PB of 20 to 50mM and (NH) of 1.5M2SO4A solution;
buffer b 1: a NaAC solution having a pH of 3.2 to 3.4 and a concentration of 20 to 50mM
BufferB2, pH 7.5-8.0, containing PB at a concentration of 20-50 mM,
buffer b3: containing a 0.1M NaOH solution.
The invention also provides an antibody purified by using the antibody purification method.
The invention also provides application of the purification method of the antibody in preparation of antibody drugs.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a method for purifying an antibody, which comprises the following steps: providing a cell fermentation broth containing a crude antibody; providing a buffer for chromatography; the crude antibody was then subjected to combinatorial chromatography using AKTA Avant 150. The combined chromatography process does not need manual participation, the instrument can run overnight, the night time is fully utilized, and the utilization rate of equipment is high.
(2) The sample treated by the method has high flux, and the labor cost investment is greatly reduced; the operation is simple, the program operation can be completed only by inputting the sample name and the sample inlet in the edited program, and errors caused by manual operation are not easy to occur; and in the whole chromatography process, the sample stays in the instrument pipeline or the column all the time, so that the loss in the middle treatment process of the sample is reduced.
(3) The antibody purified by the full-automatic three-step chromatography has higher purity than the antibody purified by the one-step Protein A, and has less impurity content.
(4) The method for purifying the antibody by full-automatic three-step chromatography reduces about half of time compared with manual three-step chromatography for processing the same number of samples, and greatly improves the purification speed of the antibody drug in the early stage of research and development of small samples.
(5) The full-automatic three-step chromatography purification antibody sample is completely established based on AKTA Avant150, and purification accessories or equipment such as a pump, an ultraviolet detector and the like are not required to be additionally arranged.
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 flow path for affinity loading provided in example 1 of the present invention;
FIG. 2 is a schematic flow diagram of an affinity elution impurity provided in example 1 of the present invention;
FIG. 3 is a schematic flow diagram of affinity elution and gel filtration chromatography loading provided in example 1 of the present invention;
FIG. 4 is a schematic flow diagram of a chromatographic loading provided in example 1 of the present invention;
FIG. 5 is a schematic flow diagram of chromatographic elution provided in example 1 of the present invention;
FIG. 6 and FIG. 7 are schematic views of the flow path for affinity regeneration provided in example 1 of the present invention;
FIG. 8 and FIG. 9 are schematic views of the flow path for affinity regeneration provided in example 1 of the present invention;
fig. 10 is a flowchart of a main procedure for operating the AKTA Avant150 according to embodiment 1 of the present invention;
FIG. 11 is a chromatogram of a purified antibody provided in Effect example 1 of the present invention;
FIG. 12-A is the SEC (TSK G3000) results for the antibody after purification using ProteinA affinity chromatography alone;
FIG. 12-B is the SEC (TSK G3000) results for antibodies purified using the purification methods provided in the examples;
FIG. 13 is a comparison of HCP removal;
FIG. 14 is a graph showing the comparison between the time-consuming purification method and the artificial purification method provided in example 1.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.
The invention provides a method for purifying an antibody, which is based on AKTA Avant150 to complete three-step purification of the antibody automatically, and comprises the following steps: providing a cell fermentation broth containing a crude antibody; providing a buffer for chromatography; then using AKTA Avant150 to carry out combined chromatography on the antibody crude product; the combined chromatography comprises the following steps in sequence:
(a) affinity chromatography to capture the antibody;
(b) gel filtration chromatography displacement Buffer for desalting;
(c) carrying out chromatographic separation on the antibody again to carry out fine purification treatment on the antibody;
wherein, the connecting mode of pipeline and chromatographic column among AKTA Avant150 is: the Out1 outlet of the device is connected to the loading port of the Loop, and the affinity chromatography column used in the step (a) is connected to the Position 3; replacing the Loop with the gel filtration chromatography column used in step (b); the chromatography column used in step (c) is attached at Position 1.
The AKTA protein purification system is a liquid chromatography system used in the purification process of the U.S. general electric production, and can be used in combination with a chromatography column and a method module to perform various chromatographic techniques. Under the control of software, the AKTA protein purification system can quickly complete the purification work of the protein.
The invention uses AKTA Avant150 to complete three-step chromatography of the antibody in full automation, and the three-step chromatography sequentially comprises affinity chromatography, gel filtration chromatography and chromatography for fine purification treatment of the antibody. The antibody purification treatment of the invention means that the antibody is purified again by using a chromatography method after affinity chromatography and gel filtration chromatography so as to further improve the purity of the antibody. The present invention is not limited to the mode of the last step of the three-step chromatography, as long as the antibody can be further purified.
Affinity chromatography, which is the most effective method for separating and purifying proteins at present, is a technique for achieving the separation purpose based on the specific affinity between a bioactive substance and a specific ligand. Affinity chromatography is designed according to the specificity of target proteins, such as enzymes and substrates, antigens and antibodies, and the like. Affinity chromatography has the advantages of high purity, high yield, capability of maintaining the natural activity of biomacromolecules and the like, and has good selectivity. Affinity chromatography media for the separation of antibody fragments are primarily protein ligands that interact with immunoglobulins.
Gel filtration chromatography, also known as Size Exclusion Chromatography (SEC), uses the molecular weight difference between proteins to be separated to separate target proteins, and allows small molecular weight proteins to enter small channels of the medium during gel filtration chromatography, thereby prolonging the retention time. The smaller the molecular weight of the protein, the more internal channels are accessible and the longer the residence time in the medium, thus allowing the separation of different proteins according to molecular weight. The GFC separation mechanism is clear, the applicability is wide, and after the GFC separation is applied to affinity chromatography separation, the antibody fragment can be further refined.
According to the antibody purification method provided by the invention, manual participation is not needed in the combined chromatography process, the instrument can run overnight, the night time is fully utilized, and the equipment utilization rate is high. The sample treated by the method has high flux, and the labor cost investment is greatly reduced; the operation is simple, the program operation can be completed only by inputting the sample name and the sample inlet in the edited program, and errors caused by manual operation are not easy to occur; and in the whole chromatography process, the sample stays in the instrument pipeline or the column all the time, so that the loss in the middle treatment process of the sample is reduced. The antibody purified by the full-automatic three-step chromatography has higher purity than the antibody purified by the one-step Protein A, and has less impurity content. The method for purifying the antibody by full-automatic three-step chromatography reduces about half of time compared with manual three-step chromatography for processing the same number of samples, and greatly improves the purification speed of the antibody drug in the early stage of research and development of small samples. The full-automatic three-step chromatography purification antibody sample is completely established based on AKTA avant, and purification accessories or equipment such as a pump, an ultraviolet detector and the like are not required to be additionally arranged.
In some alternative embodiments, the packing used in the affinity chromatography column of step (a) comprises at least one of Protein a derived from staphylococcus aureus, Protein G derived from streptococcus or Protein L derived from streptococcus magnus; protein A is preferred.
Protein a is a Protein on the cell wall of staphylococcus aureus that selectively binds to the Fc domain of immunoglobulins. The Protein A ligand can be coupled to different matrixes, such as cross-linked agarose, surface modified porous glass, polystyrene, ceramic shells or other organic high molecular materials to form different Protein A fillers, and the Protein A fillers have higher loading capacity, selectivity and stability.
In some alternative embodiments, the means for re-chromatographically separating the antibody in step (c) comprises: ion exchange chromatography, affinity chromatography, hydroxylapatite chromatography or hydrophobic interaction chromatography, preferably using ion exchange chromatography, more preferably cation exchange chromatography; or preferably hydrophobic interaction chromatography.
Ion Exchange Chromatography (IEC): the separation is carried out by utilizing the difference of the acting force between the ion exchange medium and different protein molecules, and the difference of the surface static charge of the protein molecules is mainly relied on. The proteins all have specific isoelectric points (pI), when the environment is higher than the pI, the net charge presented on the protein surface is negative, when the environment is lower than the pI, the net charge presented on the protein surface is positive, the electric charge carried by the proteins is adjusted by adjusting the mobile phase according to the pI of each protein, wherein the proteins with the responsible (positive) charges are adsorbed by an anion (cation) ion exchange medium, the unbound proteins directly pass out, and the adsorbed proteins are eluted by changing the concentration of the mobile phase or salt, thereby achieving the purpose of separation.
Hydrophobic Interaction Chromatography (HIC) relies on the difference of hydrophobic interaction between biomacromolecules and hydrophobic ligands to realize the separation and purification of protein, and under the condition of high salt, hydrophobic groups on the surface of the protein are exposed and adsorbed by combining with the hydrophobic interaction of the ligands; when the salt concentration is decreased, the hydration layer on the surface of the protein increases, so that hydrophobic interaction is weakened, thereby realizing protein desorption in which the hydrophobicity of the antibody is stronger than that of most impurity components in most cases, and thus HIC can be used as a purification step for antibody purification.
Hydroxyapatite chromatography (HAC) is a chromatographic separation technique using hydroxyapatite as a medium. Hydroxyapatite (Ca)10(PO4)6(OH)2) The difference from other chromatography media is that the hydroxyapatite is not only a functional ligand, but also two action sites on the chromatography matrix hydroxyapatite, wherein the PO with negative electricity4 3-Can be ionically bound to positively charged proteins, has ion exchange properties, and can be eluted with a salt concentration gradient while positively charged Ca is present2+The site can be bound to the free carboxyl group of the negatively charged protein in a metal-chelating manner.
In some optional embodiments, the purification method further comprises setting a sample inlet (SampL inlet) of a sample pump of the AKTA Avant150 as a variable, so that a plurality of antibody samples can be purified in three steps in sequence, and at most seven antibody samples can be purified, thereby saving a lot of experimental time.
In some alternative embodiments, when performing three-step chromatography using AKTA Avant150, the flow path of the crude protein to each buffer comprises the following process:
(x1) affinity loading: injecting the antibody crude product into a pipeline from Injection valve through a Sample pump, flowing through an affinity chromatography column to be specifically combined with filler in the chromatography column, then sequentially flowing the filtrate to a Fraction collection through a UV monitor, a Conductivity monitor and a pH valve, and collecting the flow liquid;
(x2) affinity Wash impurity: buffer A1 flows through System pump A from Injection valve Injection pipeline, passes through affinity chromatography column to elute impurities, then the filtrate flows to Waste through UV monitor, reduction monitor and pH valve in sequence, and flows out to Waste liquid;
(x3) affinity eluting antibody and gel filtration chromatography loading: buffer B1 flows through the affinity chromatography column after being injected into the pipeline from Injection value through System pump B for affinity elution of the antibody captured by the affinity chromatography column, then the filtrate with the antibody sample flows to Outlet value through UV monitor, reduction monitor and pH value in sequence, and flows back to Injection value without draining after peak collection and is injected into the gel filtration chromatography column;
(x4) chromatographic loading: buffer A2 is injected into the pipeline from Injection valve through System pump A, the antibody sample is eluted from the gel filtration chromatographic column and simultaneously loaded into the chromatographic column used in the step (c), the filtrate flows to the Outlet valve through UVmonitor, reduction monitor and pH valve in sequence, and the filtrate is collected after flowing out;
(x5) chromatographic elution: and (2) injecting Buffer A2 from the Injection valve into the pipeline through System pump A, simultaneously flowing Buffer B2 from the Injection valve into the pipeline through System pump B, then flowing through the chromatographic column for gradient elution to elute the antibody combined with the chromatographic column, then flowing the filtrate to the separation Collection after passing through UV monitor, reduction monitor and pHvalve in sequence, and collecting the filtrate after flowing out to obtain the purified antibody.
In some alternative embodiments, when performing three-step chromatography using AKTA Avant150, a plurality of antibody samples are purified sequentially, and therefore, after purifying each antibody sample, the chromatography column needs to be regenerated, and the flow path between the crude protein and each buffer solution when regenerating the chromatography column comprises the following processes:
(x6) regeneration of the affinity chromatography column used in step (a): buffer B3 was passed through System pump B from Injection valve Injection line, through affinity chromatography column to wash out impurities for regeneration treatment, the filtrate was passed through UVmonitor, reduction monitor and pH valve in sequence to Waste, then Buffer A1 was passed through System pump A from Injection valve Injection line, through affinity chromatography column to wash out the remaining alkali solution in the column, the filtrate was passed through UVmonitor, reduction monitor and pH valve in sequence to Waste.
(X7) regeneration of the chromatography columns used in step (b) and step (c): and (3) injecting Buffer B3 from the Injection valve through the System pump B, firstly passing through the gel filtration chromatographic column used in the step (B), then passing through the chromatographic column used in the step (c), sequentially passing the filtrate through the UV monitor, the Conductivity monitor and the pH valve to flow to the step, then sequentially passing Buffer A2 from the Injection valve through the System pump A, firstly passing through the gel filtration chromatographic column used in the step (B), then passing through the chromatographic column used in the step (c), and sequentially passing the filtrate through the UV monitor, the Conductivity monitor and the pH valve to flow to the step.
Wherein, the System pump A and the System pump B are high-end precision pumps for providing buffer solution during purification operation. Sample pump is a high-end precision pump used to provide samples or buffers during purification runs. Injection valve is a valve that directs the sample onto the column. UV monitor is a monitor, while measuring the absorbance of UV/Vis at three wavelengths in the range of 190 to 700 nm. Conductivity monitor is a monitor that continuously measures the conductance of buffer and sample solutions. pH value is a valve that allows a pH electrode to be included in the flow path or to bypass the electrode during operation. The pH electrode can be calibrated when installed in the pH Valve, which also allows for the inclusion of a flow restrictor in the flow path or the bypassing of the flow restrictor during operation. The Fraction collection is a circular Fraction collector that can perform up to 175 fractionations.
In some alternative embodiments, the last step of the three-step chromatography uses cation exchange chromatography, whereby the antibody desalted through the gel filtration chromatography column is mixed and loaded onto the cation exchange chromatography column; in this purification system, the buffers used in the flow path preferably include the following:
buffer a 1: PBS, pH7.2 ~ 7.4, for example but not limited to 7.2, 7.3 or 7.4.
Buffer a 2: a pH of 5.0 to 5.5, such as but not limited to 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5; the NaAC solution having a concentration of 20 to 50mM may be, for example, but not limited to, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM.
Buffer b 1: a pH of 3.2 to 3.4, such as but not limited to 3.2, 3.3 or 3.4; the NaAC solution having a concentration of 20 to 50mM may be, for example, but not limited to, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM.
Buffer b2: a pH of 5.0 to 5.5, such as but not limited to 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5; contains NaAC with a concentration of 20-50 mM, such as but not limited to 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50 mM; and a NaCl solution with a concentration of 1-2M, such as but not limited to 1M, 1.2M, 1.4M, 1.6M, 1.8M or 2M.
The Buffer B3 is 0.1-0.2M NaOH solution, for example, but not limited to, 0.1M, 0.12M, 0.15M, 0.18M or 0.2M.
In other alternative embodiments, where the final step of the three-step chromatography uses hydrophobic interaction chromatography, the buffers used in the flow path in the purification system preferably include the following:
buffer a 1: PBS, pH7.2 ~ 7.4, for example but not limited to 7.2, 7.3 or 7.4.
Buffer a 2: a pH of 7.5 to 8.0, such as but not limited to 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0; contains PB at a concentration of 20-50 mM, which may be, but not limited to, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM; and 1.5M of (NH)2SO4A solution.
Buffer b 1: a pH of 3.2 to 3.4, such as but not limited to 3.2, 3.3 or 3.4; the NaAC solution having a concentration of 20 to 50mM may be, for example, but not limited to, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM.
Buffer b2: a pH of 7.5 to 8.0, such as but not limited to 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0; the concentration of PB contained in the composition is 20-50 mM, and may be, for example, but not limited to, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM.
Buffer b3: containing a 0.1M NaOH solution.
The invention also provides an antibody purified by using the method for purifying the antibody, and the antibody and the method for preparing the antibody are based on the same inventive concept, so that all the beneficial effects of the method for purifying the antibody are achieved, and the details are not repeated.
The invention also provides an application of the antibody purification method in preparing antibody drugs, and the antibody purification method can purify antibodies with higher purity, reduce the possibility of introducing other impurities in the purification process, and shorten the purification time, so that the antibody purification method is applied to preparing antibody drugs, and the quality and the production efficiency of the antibody drugs can be better improved.
The advantageous effects of the present invention are further illustrated below with reference to preferred embodiments:
this example provides a method for the automated purification of monoclonal antibodies by AKTA avant-based three-step chromatography (Protein A-BEX-CEX), comprising the following steps:
1.1 connect the Out1 outlet of the device to the loading port of the Loop, replace the Loop Loop with a BEX column (50mL desaling, GEHealthcare), attach an affinity column (5mL Bogelong, AT ProteinA Dimond) AT Position3 and attach an ion column (5mL Capto S GE Healthcare) AT Position 1.
1.2 automated flow Path for three-step chromatography
(x1) affinity loading: the crude antibody is injected into the pipeline from Injection valve through Sample pump, flows through the affinity chromatography column to be specifically combined with the filler in the chromatography column, and then the filtrate flows to the separation collection through UV monitor, reduction monitor and pH valve in sequence, and the flow liquid is collected, which is shown in reference figure 1.
(x2) affinity Wash impurity: buffer A1 was injected from Injection valve via System pump A, passed through an affinity chromatography column to elute impurities, and then the filtrate was passed through UV monitor, Conductivity monitor and pH valve in sequence to Waste, and flowed out into Waste as shown in FIG. 2.
(x3) affinity eluting antibody and gel filtration chromatography loading: buffer B1 flows through the affinity chromatography column after being injected into the line from Injection value via System pump B for affinity elution of the antibody captured by the affinity chromatography column, then the filtrate with the antibody sample flows to Outlet value via UV monitor, reduction monitor and pH value in sequence, and flows back to Injection value without draining after peak collection and is injected into the gel filtration chromatography column, as shown in FIG. 3.
(x4) chromatographic loading: buffer a2 was loaded onto the chromatography column used in step (c) while eluting the antibody sample from the Injection valve Injection line via System pump a, and the filtrate was flowed to the Outlet valve via the UVmonitor, Conductivity monitor and pH valve in this order, and the filtrate was collected after flowing out, as shown in fig. 4.
(x5) chromatographic elution: buffer A2 is injected into the pipeline from the Injection valve through the System pump A, and simultaneously Buffer B2 is injected into the pipeline from the Injection valve through the System pump B and then flows through the chromatographic column for gradient elution so as to elute the antibody combined with the chromatographic column, then the filtrate flows to the separation Collection after passing through the UV monitor, the Conductivity monitor and the pHvalve in sequence, and the purified antibody is obtained after the filtrate flows out, and the method is shown in figure 5.
(x6) regeneration of the affinity chromatography column used in step (a): buffer B3 was regenerated by passing through System pump B from Injection valve through the affinity chromatography column to wash out impurities, the filtrate was passed through UVmonitor, Conductivity monitor and pH valve in this order to flow to Waste, then Buffer A1 was passed through System pump A from Injection valve through the affinity chromatography column to wash out the remaining alkali solution in the column, and the filtrate was passed through UVmonitor, Conductivity monitor and pH valve in this order to flow to Waste, as shown in FIGS. 6 and 7.
(X7) regeneration of the chromatography columns used in step (b) and step (c): the Buffer B3 is injected into the pipeline from the Injection valve through the System pump B, passes through the gel filtration chromatography column used in the step (B), then passes through the chromatography column used in the step (c), the filtrate sequentially passes through the UV monitor, the Conductivity monitor and the pH valve to flow to the step, then the Buffer A2 is injected into the pipeline from the Injection valve through the System pump A, passes through the gel filtration chromatography column used in the step (B), then passes through the chromatography column used in the step (c), and the filtrate sequentially passes through the UV monitor, the Conductivity monitor and the pH valve to flow to the step, which is shown in reference to FIG. 8 and FIG. 9.
Wherein the bold lines in fig. 1-9 indicate the path through which the sample and/or buffer flows.
Wherein, BufferA 1: PBS pH 7.2; buffer a 2: a NaAC solution at pH 5.0 with a concentration of 20 mM; buffer b 1: a NaAC solution at pH 3.4 with a concentration of 20 mM; buffer b2: pH 5.0, containing NaAC at a concentration of 20mM and NaCl at a concentration of 1M; buffer B3 0.1-0.2M NaOH solution.
3 editing of program method
Textinstruction in the editor of AKTAAvant150 results in the main program Phase shown in FIG. 10.
Based on the above procedure, only the sample name (SampleID) and the sample inlet (Sampleinlet) need to be input into the Scouting, and the corresponding pipeline is filled with the buffer solution.
Wherein, the Buffer solution corresponding to the A1& Buffer pipeline is Buffer A1; the buffer solution corresponding to the B1 pipeline is BufferB 1; the buffer solution corresponding to the B2 pipeline is 0.1M NaOH; the buffer solution corresponding to the A2 pipeline is BufferA 2; the buffer solution corresponding to the B3 pipeline is BufferB 2.
And 3, putting a 50ml collecting pipe (collecting flow) and a 15ml collecting pipe (collecting sample) into the collector, wherein S1-S7 are corresponding antibody fermentation supernatant samples, and the three-step chromatography process from affinity, desalting and liquid changing to ion exchange of 7 antibodies can be automatically completed.
Effect example 1
Three-step full-automatic purification preparation of 7 monoclonal antibodies
50ml CHO cell fermentation (expression amount about 0.5mg/ml) samples were filtered with 0.22um needle filter for future use, all buffer solutions were filled into the corresponding lines according to the invention method, PBS was pre-filled in S1-S7, and the filtered fermentation supernatant was inserted.
The columns and corresponding tubing were connected as in example 1, the sample name (SampleID) and sample inlet (Sampleinlet) were entered in Scouting according to the procedure of example 1, and the program was run in Systemcontrol.
The chromatogram for one of the samples is shown in FIG. 11; the fractions eluted from each tube after the full-automatic three-step chromatography are identified, the samples with better purity are combined, the SEC (TSK G3000) results are shown in figure 12-A and figure 12-B, and the purity of the three-step chromatography is higher than that of the one-step Protein A as can be seen from figure 12-A and figure 12-B.
The comparison of HCP removal is shown in FIG. 13, where A- (ProteinA + BEX + CEX) represents fully automated three-step chromatography and M- (ProteinA + BEX + CEX) represents manual three-step chromatography, and it can be seen from the figure that fully automated three-step chromatography can significantly remove HCP compared to one-step ProteinA affinity chromatography and has the same decontamination effect as manual three-step chromatography.
The time consumption comparison result is shown in FIG. 14, and the total time of the single-step chromatography is more than 32h under the condition that the total time of the single-step chromatography is uninterrupted by manpower and machine; the full-automatic three-step chromatography only needs to manually prepare a sample and set a program, and the machine is used for overnight operation, so that the total time consumption is reduced by about half compared with the single-step chromatography, the actual working time of manual participation is about 4 hours, and the labor cost is greatly reduced.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and 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.
Claims (10)
1. A method for purifying an antibody, comprising: providing a cell fermentation broth containing a crude antibody; providing a buffer for chromatography; then using AKTA Avant150 to carry out combined chromatography on the antibody crude product; the combined chromatography comprises the following steps in sequence:
(a) affinity chromatography to capture the antibody;
(b) gel filtration chromatography displacement Buffer for desalting;
(c) carrying out chromatographic separation on the antibody again to carry out fine purification treatment on the antibody;
wherein, the connecting mode of pipeline and chromatographic column among AKTA Avant150 is: the Out1 outlet of the device is connected to the loading port of the Loop, and the affinity chromatography column used in step (a) is connected to the Position 3; replacing the Loop with the gel filtration chromatography column used in step (b); the chromatography column used in step (c) is attached at Position 1.
2. The method of claim 1, wherein the packing used in the affinity column of step (a) comprises at least one of Protein a, Protein G or Protein L; protein A is preferred.
3. The method for purifying an antibody according to claim 1, wherein the step (c) of separating the antibody again by chromatography comprises: ion exchange chromatography, affinity chromatography, hydroxyapatite chromatography or hydrophobic interaction chromatography;
preferably, the mode of chromatographically separating the antibody again in step (c) is ion exchange chromatography, preferably cation exchange chromatography;
preferably, the mode of chromatographically separating the antibody again in step (c) is hydrophobic interaction chromatography.
4. The method for purifying antibody according to claim 1, wherein the method further comprises setting a loading inlet of a sample pump of AKTAAvant150 as a variable for sequentially purifying a plurality of antibody samples.
5. The method for purifying an antibody according to claim 1, wherein the flow path of the combined chromatography comprises:
(x1) affinity loading: injecting the antibody crude product into a pipeline from Injection valve through a Sample pump, flowing through an affinity chromatography column to be specifically combined with a filler in the chromatography column, then sequentially flowing the filtrate to a Fraction collection through a UV monitor, a Conductivity monitor and a pHvalve, and collecting the flow liquid;
(x2) affinity Wash impurity: buffer A1 flows through System pump A from Injection valve Injection pipeline, passes through affinity chromatography column to elute impurities, then the filtrate flows to Waste through UV monitor, reduction monitor and pH valve in sequence, and flows out to Waste liquid;
(x3) affinity eluting antibody and gel filtration chromatography loading: buffer B1 flows through the affinity chromatography column after passing through the System pump B from the Injection valve Injection line for affinity elution of the antibody captured by the affinity chromatography column, then the filtrate with the antibody sample flows to the Outlet valve through the UV monitor, the reduction monitor and the pH valve in sequence, and flows back to the Injection valve without draining after peak collection and is injected into the gel filtration chromatography column;
(x4) chromatographic loading: buffer A2 is injected into the pipeline from Injection valve through System pump A, the antibody sample is eluted from the gel filtration chromatographic column and simultaneously loaded into the chromatographic column used in the step (c), the filtrate flows to the Outlet valve through UV monitor, reduction monitor and pH valve in sequence, and the filtrate is collected after flowing out;
(x5) chromatographic elution: and (2) injecting Buffer A2 from the Injection valve into the pipeline through System pump A, simultaneously flowing Buffer B2 from the Injection valve into the pipeline through System pump B, then flowing through the chromatographic column for gradient elution to elute the antibody combined with the chromatographic column, then flowing the filtrate to the separation Collection after passing through UV monitor, reduction monitor and pHvalve in sequence, and collecting the filtrate after flowing out to obtain the purified antibody.
6. The method for purifying an antibody according to claim 5, wherein the flow path of the combined chromatography further comprises a step for regenerating a chromatography column:
(x6) regeneration of the affinity chromatography column used in step (a): injecting Buffer B3 from the Injection valve through System pump B, regenerating through an affinity chromatography column to wash off impurities, flowing filtrate sequentially through UV monitor, reduction monitor and pH valve to water, then injecting Buffer A1 from the Injection valve through the System pump A, washing off residual alkali solution in the column through the affinity chromatography column, and flowing filtrate sequentially through UVmonitor, reduction monitor and pH valve to water;
(X7) regeneration of the chromatography columns used in step (b) and step (c): and (3) injecting Buffer B3 from the Injection valve through the System pump B, firstly passing through the gel filtration chromatographic column used in the step (B), then passing through the chromatographic column used in the step (c), sequentially passing the filtrate through the UV monitor, the Conductivity monitor and the pH valve to flow to the step, then sequentially passing Buffer A2 from the Injection valve through the System pump A, firstly passing through the gel filtration chromatographic column used in the step (B), then passing through the chromatographic column used in the step (c), and sequentially passing the filtrate through the UV monitor, the Conductivity monitor and the pH valve to flow to the step.
7. The method for purifying an antibody according to claim 6, wherein the antibody desalted by the gel filtration chromatography column is mixed and then applied to a cation exchange chromatography column;
the buffer used in the channel includes:
BufferA1：PBS，pH7.2～7.4；
buffer a 2: a NaAC solution having a pH of 5.0 to 5.5 and a concentration of 20 to 50 mM;
buffer b 1: a NaAC solution having a pH of 3.2 to 3.4 and a concentration of 20 to 50 mM;
buffer b2: the pH value is 5.0-5.5, and the NaAC solution contains NaAC with the concentration of 20-50 mM and NaCl solution with the concentration of 1-2M;
buffer B3: 0.1-0.2M NaOH solution.
8. The method for purifying an antibody according to claim 6, wherein the antibody desalted by the gel filtration chromatography column is mixed and then applied to the hydrophobic interaction chromatography column;
the buffer used in the channel includes:
BufferA1：PBS，pH7.2～7.4；
buffer a 2: a pH of 7.5 to 8.0, a concentration of PB of 20 to 50mM and (NH) of 1.5M2SO4A solution;
buffer b 1: a NaAC solution having a pH of 3.2 to 3.4 and a concentration of 20 to 50 mM;
buffer b2: a pH of 7.5 to 8.0, containing PB at a concentration of 20 to 50 mM;
buffer b3: containing a 0.1M NaOH solution.
9. An antibody purified by the method for purifying an antibody according to any one of claims 1 to 8.
10. Use of a method of purifying an antibody according to any one of claims 1 to 8 in the manufacture of a medicament for the antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811226606.1A CN111072773A (en) | 2018-10-19 | 2018-10-19 | Antibody purification method, antibody and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811226606.1A CN111072773A (en) | 2018-10-19 | 2018-10-19 | Antibody purification method, antibody and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111072773A true CN111072773A (en) | 2020-04-28 |
Family
ID=70309508
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811226606.1A Pending CN111072773A (en) | 2018-10-19 | 2018-10-19 | Antibody purification method, antibody and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111072773A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311498A (en) * | 2011-08-19 | 2012-01-11 | 中国科学院过程工程研究所 | Method for separation of recombinant human antibody from transgenic mammal milk |
| CN102321150A (en) * | 2011-08-19 | 2012-01-18 | 中国科学院过程工程研究所 | Method for separating recombinant human antibody from breast bioreactor expression product |
| CN105017418A (en) * | 2014-03-27 | 2015-11-04 | 上海药明康德新药开发有限公司 | Monoclonal antibody purification process |
| CN105111309A (en) * | 2008-10-20 | 2015-12-02 | Abbvie公司 | Isolation and purification of antibodies using protein an appinity chromatography |
| CN105263947A (en) * | 2013-06-25 | 2016-01-20 | 卡迪拉保健有限公司 | Purification process for monoclonal antibodies |
| CN106146660A (en) * | 2015-04-16 | 2016-11-23 | 湖北生物医药产业技术研究院有限公司 | The method of isolated and purified monoclonal antibody |
| CN107964044A (en) * | 2016-10-19 | 2018-04-27 | 无锡科捷诺生物科技有限责任公司 | The method that anti-CD-20 monoclonal antibody is purified from milk sample |
-
2018
- 2018-10-19 CN CN201811226606.1A patent/CN111072773A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105111309A (en) * | 2008-10-20 | 2015-12-02 | Abbvie公司 | Isolation and purification of antibodies using protein an appinity chromatography |
| CN102311498A (en) * | 2011-08-19 | 2012-01-11 | 中国科学院过程工程研究所 | Method for separation of recombinant human antibody from transgenic mammal milk |
| CN102321150A (en) * | 2011-08-19 | 2012-01-18 | 中国科学院过程工程研究所 | Method for separating recombinant human antibody from breast bioreactor expression product |
| CN105263947A (en) * | 2013-06-25 | 2016-01-20 | 卡迪拉保健有限公司 | Purification process for monoclonal antibodies |
| CN105017418A (en) * | 2014-03-27 | 2015-11-04 | 上海药明康德新药开发有限公司 | Monoclonal antibody purification process |
| CN106146660A (en) * | 2015-04-16 | 2016-11-23 | 湖北生物医药产业技术研究院有限公司 | The method of isolated and purified monoclonal antibody |
| CN107964044A (en) * | 2016-10-19 | 2018-04-27 | 无锡科捷诺生物科技有限责任公司 | The method that anti-CD-20 monoclonal antibody is purified from milk sample |
Non-Patent Citations (1)
| Title |
|---|
| 王宁等: "陶瓷羟基磷灰石去除单抗聚集体的工艺研究", 《中国生化药物杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2017245478B2 (en) | Protein purification using Bis-Tris buffer | |
| CN102762585B (en) | Single unit antibody purification | |
| JP6092892B2 (en) | Antibody purification method | |
| JP6463734B2 (en) | Continuous multi-step method for purifying antibodies | |
| AU2009200842B2 (en) | Method of purifying polypeptides by simulated moving bed chromatography | |
| Hochuli | Large-scale chromatography of recombinant proteins | |
| KR101569783B1 (en) | A Method of Antibody Purification | |
| CN105073769B (en) | Increase the method for purity of protein using the chromatography based on A albumen | |
| CN103038247A (en) | Apparatus and process of purification of proteins | |
| CN112409477B (en) | IgM purification method | |
| CN109030686A (en) | A kind of three-dimensional liquid chromatogram protein purification device and its application method | |
| CN103497248B (en) | A kind of method of isolated and purified antibody from cells and supernatant | |
| CN111072773A (en) | Antibody purification method, antibody and application | |
| US10717023B1 (en) | Method for continuous purification | |
| Chen et al. | Comparison of mono Q, superose-6, and ABx fast protein liquid chromatograhy for the purification of IgM Monoclonal antibodies | |
| CN115353561A (en) | Antibody purification method | |
| CN103619868A (en) | Single unit chromatography antibody purification | |
| WO2011015919A1 (en) | A highly efficient process of purification and production of recombinant infliximab | |
| EP2782925B1 (en) | Protein purification using bis-tris buffer | |
| CN111153993A (en) | Preparation method of anti-TNF- α monoclonal antibody | |
| CN115819608A (en) | Method for purifying fusion protein | |
| Schwartz | protein A to purify immunoglobulins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200428 |
|
| RJ01 | Rejection of invention patent application after publication |