KR20230054247A - Method for purification of a protein - Google Patents

Abstract

One aspect of the technology disclosed by the present invention provides a method for purifying a cell culture medium expressing a target protein, which is a polypeptide represented by the amino acid sequence expressed in SEQ ID NO: 1 or a polypeptide having 75 % or more sequence homology thereto, comprising, after a step of culturing a cell line expressing the target protein, the steps of: (a) centrifuging the cell culture medium to recover the supernatant; and (b) filtering the recovered culture medium. In addition, the method for purifying the cell culture medium expressing the target protein according to the present invention can be used to mass-produce the protein while improving the protein production process and reducing protein production costs.

Description

Protein purification method {METHOD FOR PURIFICATION OF A PROTEIN}

The present invention is a method for purifying a cell culture medium expressing the protein, which can reduce production costs while mass-producing a target protein, and in particular, prevents SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2) infection and / It relates to a method for purifying a cell culture medium expressing an antigen protein of a vaccine used for the purpose of alleviating symptoms expressed during infection or infection.

The SARS-CoV-2 virus, which has been reported as a pandemic infection since December 2019, is a virus belonging to the Coronaviridae family, Betacoronavirus genus Sarbecovirus subgenus. It is known that the Receptor Binding Domain (RBD) of B. binds to the ACE2 receptor protein on the surface of human cells to cause infection. Spike glycoprotein monomers are separated into S1 subunits and S2 subunits by host cell proteases.

It is known that the main transmission route of the SARS-CoV-2 virus infection is close contact with droplets of an infected person. However, it is known that the virus may be transmitted by direct contact with an infected person or by touching a medium such as an item contaminated by droplets of an infected person and then touching eyes, nose, mouth, etc. without washing hands.

Meanwhile, as the social and economic damage caused by the SARS-CoV-2 virus infection intensified in 2020, emergency approval and approval for the use of vaccines that have the effect of alleviating the symptoms of SARS-CoV-2 virus infection in various countries such as the United States and Europe Together, vaccinations have been initiated in many countries around the world. However, even if the vaccine is vaccinated, SARS-CoV-2 virus infection in the vaccinated person cannot be prevented from the source.

Moreover, SARS-CoV-2 virus mutations such as alpha mutation, beta mutation, gamma mutation, epsilon mutation, delta mutation, kappa mutation, and eta mutation continue to be reported, and these mutated SARS-CoV-2 viruses in vaccinated persons Infections continue to occur. Therefore, for the purpose of maintaining an effective antibody level in the body, additional vaccination for vaccinated persons is recommended. Furthermore, governments of each country are expanding the target of SARS-CoV-2 vaccination to adolescents in consideration of the social and economic impact of SARS-CoV-2 infection. Demand for the production of antigenic proteins that can be used is rapidly increasing. A method for mass-producing a target protein in plants is already known (Patent Publication No. 10-2021-0117808), but research on a method for mass-producing a target protein in animal cells is still lacking. Therefore, it is required to develop a production method capable of mass-producing vaccine antigen proteins in animal cells in a short period of time.

(Patent Document 1) Patent Publication No. 10-2021-0117808

While conducting research on a method for reducing the production cost of vaccine antigen proteins, the present inventors recovered the cell culture medium expressing the target protein cultured in the bioreactor and supplied the culture medium to the centrifuge in the process of purifying the target protein. By changing the feed flow rate, which is the speed of centrifugation, the time required for the process can be shortened and the effect of reducing impurities in the centrifugation supernatant can be obtained. The present invention was completed by finding that the production process can be improved and the protein production cost can be reduced.

According to one aspect of the technology disclosed by the present application, as a method for purifying a cell culture medium expressing a target protein, after culturing a cell line expressing the target protein, (a) centrifuging the cell culture medium to obtain an upper layer recovering liquid; And (b) it provides a purification method comprising the step of filtering the recovered culture medium.

The method for purifying a cell culture solution expressing a target protein according to the present invention can be used to mass-produce the protein while improving the protein production process and reducing protein production costs.

1 shows the structure of a vector containing a nucleotide sequence encoding a target protein of SEQ ID NO: 1.
Figure 2 shows the content of the target protein in the supernatant obtained by setting the feed flow rate to 200 L/h, 300 L/h or 400 L/h in the process of centrifuging 2000 L of cell culture medium expressing the target protein and A1HC in the supernatant When filtering using a depth filter or a combination of filters having pore diameters of 0.5 μm/0.2 μm, the result of measuring the content of the target protein in the filtrate by Western blot is shown.
Figure 3 shows the content of the target protein in the supernatant obtained by centrifuging 2000 L of the cell culture medium expressing the target protein at a feed flow rate of 200 L/h, and filtration when the supernatant was filtered by applying a filter with a pore diameter of 0.45 μm. The result of measuring the target protein content in the liquid and the target protein content in the filtrate when the supernatant was filtered using a filter having a 0.2 μm pore diameter was measured by Western blotting.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a method for purifying a cell culture medium expressing a target protein, after culturing the cell line expressing the target protein, (a) centrifuging the cell culture medium to recover a supernatant; And (b) it provides a purification method comprising the step of filtering the recovered culture medium.

The "target protein" is any kind of exogenous product protein that can be expressed by cells. Typically, insulin, cytokine (interleukin, tumor necrosis factor, interferon, colony stimulating factor, chemokine, etc.), erythropoietin, antigen, antibody, antibody fragment, structural protein, regulatory protein, transcription factor, toxin protein, hormone, hormone Analogs, enzymes, enzyme inhibitors, transport proteins, receptors (e.g., tyrosine kinase receptors, etc.), fragments of receptors, biological defense inducers, storage proteins, movement proteins, exploitive proteins, reporter proteins, growth factors, etc., and such a target protein includes a "cloning site", which is a nucleic acid sequence into which a restriction enzyme recognition or cleavage site is introduced to allow insertion of the gene encoding the target protein into a vector for expression in a cell line. can do.

In the present invention, the target protein is one of the polypeptides described in claim 1 of International Patent Publication No. 2021-163438, International Patent Publication No. 2019-169120, US Patent Publication No. 9630994, International Patent Publication No. WO2021-163481, It may be one of the polypeptides disclosed in the specification of International Patent Publication No. WO2021-163438, or International Application No. PCT/US2021/037341. Preferably, the protein excluding the RBD and the linker portion from the target protein is a polypeptide having an amino acid sequence represented by SEQ ID NO: 7, 29, 30, 31, or 39 disclosed in International Patent Publication No. 2019-169120, or the above It may be a polypeptide having 85% or more sequence homology with the amino acid sequence of the polypeptide disclosed in International Patent Publication No. 2019-169120.

In one embodiment of the present invention, the target protein may be a protein including the Receptor Binding Domain of the SARS-CoV-2 spike protein and I53-50A, which is a nanoparticle structure. In addition, in one embodiment of the present invention, the target protein is the receptor binding domain of the SARS-CoV-2 spike protein, and I53-50A, a linker connecting the receptor binding domain of the SARS-CoV-2 spike protein and I53-50A. It may be a protein containing. Furthermore, in another embodiment of the present invention, the target protein is the receptor binding domain of the SARS-CoV-2 spike protein, I53-50A, a linker connecting the receptor binding domain of the SARS-CoV-2 spike protein and I53-50A, and It may be a protein comprising an N-terminal phosphatase signal peptide. At this time, the amino acid sequence of I53-50A, which is the structure of the nanoparticle, may be KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKATPGGVLAVGVREVGSVRGATE ring, The amino acid sequence of the Kerr may be GGSGGSGSGGSGGSGSEKAAKAEEAAR, and the amino acid sequence of the N-terminal phosphatase signal peptide may be MGILPSPGMPALLSLVSLLSVLLMGCVAETGT.

In another embodiment of the present invention, the target protein is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, or 70% or more, 75% or more, 80% or more, 85% or more of the amino acid sequence represented by SEQ ID NO: 1 At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology. there is. The target protein is encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO: 2, or 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the nucleotide sequence represented by SEQ ID NO: 2, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence homology can be encoded by a base sequence.

The cell line producing the target protein is a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92% , It may be a cell line having an expression vector containing a gene encoding a polypeptide having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology. . In one embodiment of the present invention, the expression vector may be a plasmid vector, but the polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or 70% or more, 75% or more, 80% or more, 85% or more , which encodes a polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology Any vector suitable for transfecting a gene may be used without limitation.

On the other hand, in the present invention, an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 1 may have a DNA sequence represented by SEQ ID NO: 3

As used herein, the term "vector" refers to a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and inserted into the genome of the host cell, or can be spontaneously replicated as an episome. Suitable expression vectors include expression control elements such as promoters, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. . When a genetic construct encoding a target protein is administered, the initiation codon and stop codon must be functional in the subject and must be in frame with the coding sequence.

The polynucleotide or vector according to one embodiment of the present invention is an independent molecule outside the genome, specifically a molecule capable of replicating, and may exist in a genetically modified host cell or non-human host organism, or may be present in a host cell or non-human host cell. -Can be stably integrated into the genome of human host organisms.

A host cell of a cell line expressing a target protein according to one embodiment of the present invention is a eukaryotic cell. The eukaryotic cells include fungus cells, plant cells or animal cells. Examples of the fungal cell may be yeast, specifically Saccharomyces genus ( Saccharomyces sp ) yeast, more specifically Saccharomyces cerevisiae ( S. cerevisiae ). In addition, examples of animal cells include insect cells or mammalian cells, and specific examples of animal cells include HEK293, 293T, NSO, Chinese hamster ovary cells (CHO), MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A, etc. In addition, suitable cell lines well known in the art can be obtained from cell line depositories such as the American Type Culture Collection (ATCC).

The host cell of the cell line expressing the target protein according to one embodiment of the present invention may be a Chinese hamster ovary (CHO) cell, preferably a recombinant adeno-associated virus (rAAV) by Glutamine Synthetase HD-BIOP3 cells in which exon 6 of the gene is knocked out can be used.

In order to introduce an expression vector having a genetic sequence encoding a target protein into the host cell, any method for introducing nucleic acid into the cell may be used, and techniques known in the art may be used depending on the host cell. For example, heat shock, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but are not limited thereto.

In one embodiment of the present invention, a cell line expressing a target protein may be cultured in a flask or bioreactor.

Unlike flasks, culture using a bioreactor can control and maintain culture conditions such as DO, glucose content, and pH, so that cell culture can be performed under favorable conditions and mass culture is possible. The types of flasks and bioreactors and culture conditions can be changed within a range commonly controllable by those skilled in the art.

In one embodiment of the present invention, a cell line expressing a target protein may be cultured in a flask or bioreactor. Unlike flasks, culture using a bioreactor can control and maintain culture conditions such as DO, glucose content, and pH, so that cell culture can be performed under favorable conditions and mass culture is possible.

The types of flasks and bioreactors and culture conditions can be changed within a range commonly controllable by those skilled in the art.

For example, a cell line expressing a target protein is inoculated into an Elenmeyer flask, cultured in suspension, subcultured to secure the minimum number of cells to be inoculated into a bioreactor, and the subcultured cell line is equipped with a disposable cell culture bag. The target protein can be produced by inoculating the prepared bioreactor and performing fed-batch culture.

In another embodiment of the invention, the culture period may be 6 days or more. Preferably, the culture period is 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days. can More preferably, the culture period may be 10 days.

The step (a) of centrifuging the cell culture medium to recover the supernatant may be performed using a general protein separation method.

In one embodiment of the present invention, in the step (a) of centrifuging the cell culture medium to recover the supernatant, a centrifugal separator commonly used in the art to which the present invention pertains may be used. For example, a centrifuge using a fixed-angle angle rotor, a centrifugal separator using a swing rotor, a continuous centrifuge, and the like may be used. Preferably, the step (a) of centrifuging the cell culture medium to recover the supernatant may be performed using a continuous centrifuge. The continuous centrifuge may be GEA's westfalia continuous centrifuge.

In another embodiment of the present invention, in the (a) step of centrifuging the cell culture medium to recover the supernatant, the g-force during centrifugation may be greater than 8000x g and less than 20000x g. In addition, the g-force during the centrifugation may be greater than 8000x g and less than 19000x g, preferably greater than 15000x g and less than 20000x g, greater than 16000x g and less than 20000x g, greater than 17000x g and less than 20000x g, greater than 18000x g and less than 20000x g, 19000x g or more and 2000x g or less, 15000x g or more and 19000x g or less, 16000x g or more and 19000x g or less, 17000x g or more and 19000x g or less, or 18000x g or more and 19000x g or less, but is not limited to these figures.

In another embodiment of the present invention, in the step (a) of centrifuging the cell culture medium and recovering the supernatant, the centrifugal vessel speed may be greater than 8000 rpm and less than 13000 rpm. In addition, the centrifugation vessel speed may be greater than 8000 rpm and less than 12000 rpm, preferably greater than 10000 rpm and less than 13000 rpm, greater than 11000 rpm and less than 13000 rpm, greater than 12000 rpm and less than 13000 rpm, greater than 10000 rpm and less than 12000 rpm, or 11000 rpm or more and 12000 rpm or less, but is not limited to these figures.

In another embodiment of the present invention, in the step of (a) centrifuging the cell culture medium to recover the supernatant, the feed flow rate at which the culture medium is supplied to the centrifuge is 200 L/h to 400 L /h, 200 L/h to 300 L/h, 210 L/h to 300 L/h, 220 L/h to 300 L/h, 230 L/h to 300 L/h, or 240 L/h to 300 It may be L/h. Preferably, the supply flow rate may be 250 L/h, but is not limited thereto.

In another embodiment of the present invention, in the step of (a) centrifuging the cell culture medium and recovering the supernatant, the ejection interval during centrifugation is 100 to 250 seconds, 120 to 240 seconds, 120 to 230 seconds , 140 to 230 seconds, 160 to 220 seconds, 180 to 210 seconds, or 190 to 210 seconds. Also, preferably, the discharge interval during the centrifugal separation may be 190 to 210 seconds or 201 seconds, but is not limited thereto.

In one embodiment of the present invention, it may be filtered using a filter to remove impurities in the culture medium recovered by the centrifugation. At this time, the pore diameter of the filter used for filtration may be 1 μm or less, 0.5 μm or less, 0.45 μm or less, 0.3 μm or less, 0.25 μm or less, 0.22 μm or less, or 0.2 μm or less, by combining filters having various pore diameters. can also be used In addition, the conductivity upon filtration may be 10 to 20 mS/cm, 10 to 19 mS/cm, 10 to 18 mS/cm, 10 to 17 mS/cm, or 10 to 16 mS/cm, but limited to these conditions. It doesn't work.

In another embodiment of the present invention, the (b) step of filtering the recovered culture medium may be performed using a microfiltration filter. The function of the microfiltration filter can be evaluated based on a pressure limit, eg, a feed load of <20 liters (l)/m 2 , or turbidity reduction. Further, non-limiting examples of the microfiltration filter include a microfiltration membrane, Millipore, COHC, A1HC, BIHC, XOHC depth filter, CUNO 60ZA, and CUNO 90ZA. Preferably, the microfiltration filter may be a depth filter, more preferably an A1HC depth filter, but is not limited to such a filter.

In another embodiment of the present invention, after filtering by applying a filter to the culture medium recovered by the centrifugation, it may be reacted by further treatment with a surfactant. The surfactant is sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-100 (trade name, manufactured by Rohm and Hass), Nodiet P-40 (trade name, manufactured by Shell), Tween-80, Tween-20 , CHAPS, Chapso, digitonin, urea, and mixtures thereof, but is not limited to these components. Preferably, the surfactant may be Triton X-100. In addition, the time for treating and reacting the surfactant may be 10 minutes or less, 20 minutes or less, 30 minutes or less, 30 minutes or less, 40 minutes or less, 50 minutes or less, or 60 minutes or less, preferably 10 minutes or less, but limited to these conditions It is not.

In one embodiment of the present invention, the filter filtrate treated with the surfactant may be refiltered. The pore diameter of the filter used for the re-filtration may be 1 μm or less, 0.5 μm or less, 0.45 μm or less, 0.3 μm or less, 0.25 μm or less, 0.22 μm or less, or 0.2 μm or less, and filters having various pore diameters may be combined to obtain can also be used A sterility test, a mycoplasma detection test, an exogenous factor identification test through a cell culture inoculation method, a mouse minute virus detection test, and a tuberculosis bacillus detection test may be performed on the recovered culture medium. There are no bacteria in the recovered culture medium, and no mycoplasma, exogenous factor, micromouse virus, or Mycobacterium tuberculosis are detected.

Example

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

Preparation Example 1. Preparation of cell lines expressing the target protein

To prepare a cell line expressing the polypeptide represented by the amino acid sequence of SEQ ID NO: 1, HD-BIOP3 cell line obtained from HORIZON Discovery, UK was used as a host cell. A recombinant expression vector plasmid (M-2560) was constructed by inserting the cDNA synthesized by Genscript into the Donor plasmid (pJV145) using the SalI/NotI restriction enzyme reaction (Fig. 1). The M-2560 vector has a DNA sequence represented by SEQ ID NO: 3.

A recombinant expression vector plasmid (M-2560) was transfected into the HD-BIOP3 cell line. Among the transfected cells, cells capable of stable single cell cloning were selected to prepare a research cell bank (RCB) having sufficient long-term passage stability. The RCB was transferred to the Andong plant of SK Bioscience, and a working cell bank (WCB) was manufactured in accordance with GMP standards.

One vial of WCB (Working Cell Bank) was dissolved in a water bath at 37 ° C., diluted with CD OptiCHO medium, centrifuged, and the pellet was resuspended in CD OptiCHO medium. 6 L culture at 5 x 10 ⁵ cells/mL in 10 L bioreactor, 30 L culture at 5 x 10 ⁵ cells/mL in 50 L bioreactor, 5 x 10 ⁵ cells/mL in 200 L bioreactor 200 L culture, 8 x 10 ⁵ cells / mL concentration in a 2000 L bioreactor was expanded to 1600 L culture was subcultured.

Production Example 2. Cultivation of a cell line expressing a target protein in a 2000 L bioreactor

After mounting the disposable cell culture bag in a 2000 L bioreactor and preparing for operation, 1400 L of Dynamis medium was injected, and WCB was inoculated at a concentration of 8.0 x 10 ⁵ cells/mL and cultured. During incubation, DO control proceeded through gas and oxygen, and pH control proceeded using 8% sodium bicarbonate and carbon dioxide gas.

Sampling was performed every day immediately after inoculation to measure the number of cells, check the presence of microbial contamination and the concentration of glucose in the culture medium, and when bubbles in the bioreactor reached the bottom of the air filter mounted on the condenser, ADCF antifoam was treated. This process was repeated until the end of the culture. Culture conditions used during culture are shown in Table 1 below.

Cell line culture conditions culture conditions set value Temperature (day 0-5 of incubation) 37℃ Temperature (after the 5th day of culture) 31℃ culture medium Dynamis medium for inoculation CD OptiCHO stirring speed 93 RPM culture volume 1600L initial inoculation concentration 8.0 x 10 ⁵ cells/mL amount of dissolved oxygen 40% Dissolved Oxygen Control gas, oxygen pH control 8% sodium bicarbonate, carbon dioxide

Experimental Example 1. Changes in Recovery Amount of Target Protein and Impurities According to Differences in Centrifugal Separation Feed Flow

In order to rapidly produce a large amount of the target protein, a large-scale bioreactor can be used or the volume of the culture medium can be increased. And there was a disadvantage that the production period increased because the waiting time in the centrifuge increased.

Therefore, in a bioreactor (2000L) used for large-scale protein production, 1400L of medium is filled to calibrate culture conditions such as dissolved oxygen content and pH, and then the culture of cells expressing the target protein is started with 1600L of medium. By adding culture additives for production during the culture process, the culture volume on the 10th day of culture was cultured to a culture volume of 2000 L. Centrifugation is performed using an industrial continuous centrifuge (2000L separable, westfalia continuous centrifuge, GEA), and at this time, centrifugation is performed by setting the condition of 100% output (full performance), and the supply flow rate at which the culture medium is supplied to the centrifuge Concentration of the target protein in the supernatant, yield, and The impurities and turbidity of the supernatant were measured. The 100% output (full performance) condition is a condition corresponding to a vessel speed of 11,800 rpm or 18,300 g-force.

Total protein concentration in the supernatant was determined via a BCA assay using the Pierce™ BCA Protein Assay Kit (Cat. No. 23225) provided by ThermoFisher Scientific. In addition, the concentration of the target protein in the supernatant was measured using HIC-HPLC.

The content of impurities in the supernatant was confirmed by measuring host cell proteins and host cell-derived DNA in the supernatant. At this time, the host cell protein concentration in the supernatant was determined by ELISA analysis using a Chinese hamster ovary cell (CHO) host cell protein (HCP) ELISA kit (catalog number: F550-1) from Cygnus Technologies. measured through Furthermore, the concentration of Host Cell Derived DNA (HCD) in the supernatant was measured by RT-PCR using Thermo Fisher Scientific's Applied Biosystems (trade name Applied Biosystems) resDNASEQ Quantitative CHO DNA Kit (Catalog Number: 4402085). analyzed through In addition, the turbidity of the supernatant was measured using a turbidimeter (Naphelometer).

Table 2 shows the total protein concentration in the supernatant, the target protein concentration, the yield of the target protein, and the measurement results of impurities in the supernatant and turbidity of the supernatant according to the change in the supply flow rate of the culture medium to the centrifuge.

Concentration of target protein, impurities and turbidity in the supernatant according to the difference in feed flow rate supply flow rate
(L/h) Total protein concentration in supernatant
(μg/ml) Target protein concentration (μg/ml) Host cell protein (HCP) (ng/ml host cell-derived DNA
(HCD) (ng/ml) turbidity
(NTU) 200 5639.778 1585 369140 26473.4 56.6 300 5794.096 1568.1 359130 27045.6 73.8 400 6081.003 1598.6 389700 32786 154

When the supply flow rate during centrifugation was 200 L/h, the centrifugation time of the 2000 L culture medium was 10 hours, whereas when the supply flow rate was increased to 300 L/h, it took about 6.7 hours to centrifuge the 2000 L culture medium. In addition, when the supply flow rate is 400 L / h, the time required for centrifugation of 2000 L culture medium is reduced to about 5 hours.

On the other hand, it was found that the content of the target protein in the recovered supernatant did not change significantly even when the feed flow rate during centrifugation increased from 200 L/h to 300 L/h or 400 L/h. In addition, it was found that host cell proteins and host cell-derived DNA, which are impurities in the supernatant, did not significantly increase or decrease even when the feed flow rate during centrifugation increased from 200 L/h to 300 L/h. Although the turbidity in the supernatant increased when the feed flow rate increased from 200 L/h to 300 L/h during centrifugation, this increase in turbidity was improved by applying a depth filter to the supernatant obtained by centrifugation. confirmed to be

Furthermore, when the supply flow rate increased from 200 L/h to 400 L/h during centrifugation, host cell proteins and host cell-derived DNA tended to increase. A significant number of was removed, and it was confirmed that the turbidity was significantly lowered.

On the other hand, if the supply flow rate during centrifugal separation is lower than 200 L / h, the centrifugal separation time increases to 10 hours or more, which inevitably causes a problem in that the production period is delayed. In addition, when the supply flow rate during centrifugation is higher than 400 L/h, the centrifugation time can be reduced by less than half compared to the case where the supply flow rate is 200 L/h. There was a problem that the number of filters to be used increased rapidly.

Therefore, when the feed flow rate is 200 L / h to 400 L / h when centrifuging the culture medium of the cell line expressing the target protein, shortening the production period and reducing production cost without affecting the concentration of the target protein in the supernatant confirmed to be

Experimental Example 2. Turbidity change according to centrifugation feed flow difference

Initiate the culture of cells expressing the target protein in a 1600 L medium in a 2000 L bioreactor, and add culture additives for the production of the target protein during the culture process, so that the culture volume on the 10th day of culture becomes 2000 L, and then industrial continuous The supernatant was recovered using a centrifuge (westfalia continuous centrifuge, GEA). When the supernatant was recovered, centrifugation was performed under the condition of 100% output (full performance), and the 100% output (full performance) condition corresponds to a container speed of 11,800 rpm or 18,300x g. The turbidity of the supernatant obtained by centrifugation at a supply flow rate of 200 L/h, 250 L/h or 300 L/h and a discharge interval of 252 seconds, 201 seconds, or 168 seconds, respectively, was measured using a turbidimeter, and the measurement results are shown in the table. 3.

Change in turbidity in the supernatant according to the difference in feed flow rate Supply flow rate (L/h) Discharge interval during centrifugation Turbidity (NTU) 200 252 seconds 134 250 201 seconds 135 300 168 seconds 166

Even when the feed flow rate increased from 200 L/h to 250 L/h during centrifugation, the turbidity in the supernatant did not change much. In Experimental Example 1, it was confirmed that there was no significant difference from the change width (about 20) shown when the supply flow rate was increased from 200 L / h to 300 L / h.

Although the turbidity was not measured when the feed flow rate was 400 L/h during centrifugation, considering the turbidity at 200 L/h and 300 L/h, turbidity similar to that measured in Experimental Example 1 would be obtained. it was expected Therefore, even if the culture volume increases during commercial culture of protein-expressing cells, it was confirmed that using a supply flow rate of 200 L/h to 400 L/h shortens the production period and obtains a supernatant with low turbidity due to low impurities. It became.

Experimental Example 3. Purification of target protein and removal of impurities according to the use of a filter used for filtration of the centrifugal supernatant

When the filter was applied to the centrifuged supernatant obtained in Experimental Example 1 and filtered, the change in the content of the target protein in the filtrate and the effect of reducing impurities were confirmed.

The centrifugal supernatant obtained in Experimental Example 1 was filtered using a combination of an A1HC depth filter, a filter having a pore diameter of 0.5 μm and a filter having a pore diameter of 0.2 μm, a filter having a pore diameter of 0.45 μm, or a filter having a pore diameter of 0.45 μm When the filtrate was prepared, the change in the content of the target protein in the filtrate and the effect of removing impurities were measured through Western blotting.

When a filter is applied to the centrifugation supernatant, the content of the target protein in the filtrate is similar to that of the supernatant of centrifugation without a filter applied, and the band sizes of the target protein monomer and target protein dimer are similar, so the target protein content appeared to be maintained (see Figures 2 and 3).

On the other hand, when a filter was applied to the centrifugation supernatant, impurities were reduced, so that the target protein monomer and target protein dimer bands were more distinct than the target protein monomer and target protein dimer bands observed in the centrifugation supernatant to which no filter was applied, It was found that the other bands (ie, impurities other than the target protein) were observed relatively blurry as the sharpness of the other bands decreased (see FIGS. 2 and 3).

<110> SK bioscience Co., Ltd. <120> METHOD FOR PURIFICATION OF A PROTEIN <130> FPD/202205-0055 <150> KR 10-2021-0137835 <151> 2021-10-15 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val 1 5 10 15 Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Thr 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Gly Ser Gly 225 230 235 240 Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Glu Lys Ala Ala 245 250 255 Lys Ala Glu Glu Ala Ala Arg Lys Met Glu Leu Phe Lys Lys His 260 265 270 Lys Ile Val Ala Val Leu Arg Ala Asn Ser Val Glu Glu Ala Ile Glu 275 280 285 Lys Ala Val Ala Val Phe Ala Gly Gly Val His Leu Ile Glu Ile Thr 290 295 300 Phe Thr Val Pro Asp Ala Asp Thr Val Ile Lys Ala Leu Ser Val Leu 305 310 315 320 Lys Glu Lys Gly Ala Ile Ile Gly Ala Gly Thr Val Thr Ser Val Glu 325 330 335 Gln Ala Arg Lys Ala Val Glu Ser Gly Ala Glu Phe Ile Val Ser Pro 340 345 350 His Leu Asp Glu Glu Ile Ser Gln Phe Ala Lys Glu Lys Gly Val Phe 355 360 365 Tyr Met Pro Gly Val Met Thr Pro Thr Glu Leu Val Lys Ala Met Lys 370 375 380 Leu Gly His Thr Ile Leu Lys Leu Phe Pro Gly Glu Val Val Gly Pro 385 390 395 400 Gln Phe Val Lys Ala Met Lys Gly Pro Phe Pro Asn Val Lys Phe Val 405 410 415 Pro Thr Gly Gly Val Asn Leu Asp Asn Val Ala Glu Trp Phe Lys Ala 420 425 430 Gly Val Leu Ala Val Gly Val Gly Ser Ala Leu Val Lys Gly Thr Pro 435 440 445 Asp Glu Val Arg Glu Lys Ala Lys Ala Phe Val Glu Lys Ile Arg Gly 450 455 460 Ala Thr Glu 465 <210> 2 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 atgggaatcc tgccaagccc tggaatgcca gccctgctgt ccctggtgtc tctgctgagc 60 gtgctgctga tgggatgcgt ggcagagacc ggaacaaggt tccctaacat caccaacctg 120 tgcccattcg gcgatcatccgttg cctgccatccgttg taactgctgatccgttgt ctggaaccgg 180 aagagaatct ctaattgcgt ggccgactat agcgtgctgt acaatagcgc ctccttctct 240 acctttaagt gctatggcgt gtctcccacc aagctgaacg acctgtgctt cacaaacgtg 300 tacgccgaca gctttgtgat ccggggcgat gaggtgagac agatcgcacc aggacagacc 360 ggcaagatcg cagactacaa ctataagctg cctgacgatt tcacaggctg cgtgatcgcc 420 tggaatagca acaatctgga ttccaaagtg ggcggcaact acaattatct gtacaggctg 480 tcaacgagcat gccgaacctgaa gccgaggcatcag 540 ggctccacac catgcaacgg agtggagggc ttcaattgtt attttcccct gcagagctac 600 ggcttccagc ctaccaatgg cgtgggctat cagccataca gagtggtggt gctgtccttt 660 gagctgctgc acgcaccagc aaccgtgtgc ggacctaaga agtccacaggg cggctctgga 720 ct ggaagcgaga aggcagcaaa ggcagaggag 780 gcagcaagga agatggagga gctgttcaag aagcacaaga tcgtggccgt gctgagagcc 840 aactctgtgg aggaggccat cgagaaggca gtggccgtgt tcgcaggagg agtgcacctg 900 atcgagatca cctttacagt gcccgtcact6gcc aggacctga tcaagct9 agg gagcaatcat cggagcagga accgtgacat ctgtggagca ggcaaggaag 1020 gcagtggagt ccggagccga gtttatcgtg tctcctcacc tggatgagga gatctcccag 1080 ttcgccaagg agaagggcgt gttttacatg cctggcgtga tgaccccaac agagcatgag cggact aggac cagac ctgttcc caggagaggt ggtgggacca 1200 cagtttgtga aggccatgaa gggcccattc cccaatgtga agtttgtgcc tacaggcggc 1260 gtgaacctgg acaatgtggc agagtggttc aaggcaggcg tgctggcagt gggagtggga 1320 tctgccctgg tgaagggaac cccagatgag gtgatgggaggaag 1 ctgagggaggaag 0 aagatcaggg gagcaacaga gtga 1404 <210> 3 <211> 10704 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtggt tcggcagctaga8 cttaactatg cgggattgc 0 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcggtc aagttgggt aagttgggt 300 acgttgggta acgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgtttaaact cccgccccta actccgccca gttccgccca ttctccgccc 480 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 540 ttcctaggaggggt agttgatgatgagggt ct tttgcaaaaa gctagctggt 600 tctttccgcc tcagaaggta cctaaccaag ttcctctttc agaggttatt tcaggccacc 660 ttccaccatg gccacctcag caagttccca cttgaacaaa aacatcaagc aaatgtactt 720 gtgcctgccc cagggtgagac tagaggtccgagac gtactggaga 780 aggactgcgc tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag aagagttacc 840 tgagtggaat tttgatggct ctagtacctt tcagtctgag ggctccaaca gtgacatgta 900 tctcagccct gttgccatgt ttcgggaccc cttccgcaga gatcccaaca agctggtgtt 960 ctgtcagaagtcc ttcagaagggcc ttcagaagtac attgac acc cgtgtaa 1020 acggataatg gacatggtga gcaaccagca cccctggttt ggaatggaac aggagtatac 1080 tctgatggga acagatgggc acccttttgg ttggccttcc aatggctttc ctgggcccca 1140 aggtccgtat tactgtggtg tgggcgcaga t1 caaagggcctaggat tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat 1260 gcctgcccag tgggaatttc aaataggacc ctgtgaagga atccgcatgg gagatcatct 1320 ctgggtggcc cgtttcatct tgcatcgagt atgtgaagac tttggggtaa tagcaacctt 1380 tgaccccaag cccattcctg ggaactggaa tggt4cggcaca 4tagtgcaggcca caaggccatg cgggaggaga atggtctgaa gcacatcgag gaggccatcg agaaactaag 1500 caagcggcac cggtaccaca ttcgagccta cgatcccaag gggggcctgg acaatgcccg 1560 tcgtctgact gggttccacg aaacgtccaa catcaacgac ttttctg6cat cc agtcgcca ttttctg6cat cc agtcgcca ccgca ttccccggac tgtcggccag gagaagaaag gttactttga 1680 agaccgccgc ccctctgcca attgtgaccc ctttgcagtg acagaagcca tcgtccgcac 1740 atgccttctc aatgagactg gcgacgagcc cttccaatac aaaaactaac gcccgcccca 1800 cgacccgcag cgcccgaccg aaaggacgacc1cacacat8 catagacat gcata tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1920 gaaatttgg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1980 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtagtaa accatta 204 attctacgta gataaaagtt ttgttacttt 2100 atagaagaaa ttttgagttt ttgttttttt taataaataa ataaacataa ataaattgtt 2160 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat 2220 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaag tacattgatt 2acaca8 tacatgatt atttaca8 ttatc tttctagggt taatctagct gcgtgttctg 2340 cagcgtgtcg agcatcttca tctgctccat cacgctgtaa aacacatttg caccgcgagt 2400 ctgcccgtcc tccacgggtt caaaaacgtg aatgaacgag gcgcgctcat atcatgatta 2460 cgccaaggggccggg gccaactgggccggc tccatttctg cggcatccag 2520 ccaggatacc cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc 2580 acaccggcac ggcagttcca tttaaatggc tgtcgccggt attgttcggg ttgctgatgc 2640 gcttcgggct gaccatccgg aactgt0ga gggaaaagccg cgacgaactg gggtatcc tca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca 2760 tggtaaacgt gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt 2820 tccatgccgc ttcaacctcg cgggaaaagg cacgggcttc ttccagcagca gtcatcctcg atgccaggact ag ctgagccgga aaaaagaccc gacgatatga tcctgatgca 2940 gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt 3000 gctctctctt tctaaatagc gcgaatccgt cgctggcat ttaggacatc tcagtcgccg 3060 cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaaggtt gtcactgatt ac ttgaactgaacgtata 3g1 gcatgat tatcttttac gtgactttta agatttaact 3180 catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata 3240 ttttcttgtt atagatatca agcttataga tctggggaca gccccccccc aaagccccca 3300 gggatgtaat tacgtgccctc gccagggcccgcc ct 3360 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 3420 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag 3480 acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag aatgacgtgg cacaggacagaa 3540 tacagggaacgg gcc cat ccagatccaa ccccctgcta 3600 tgtgcagggt catcaaccag cagcccaggc tgcccagagc cacatccagc ctggccttga 3660 atgcctgcag ggatggggca tccacagcct ccttgggcaa cctgttcagt gcgtcaccac 3720 cctctggggg aaaaactccatac tcctcccatatc tcctcccatgt gtgtaaa 3780 gccattcccc cttgtcctat caagggggag tttgctgtga cattgttggt ctggggtgac 3840 acatgtttgc caattcagtg catcacggag aggcagattt ggggataagg aagtgcagga 3900 cagcatggac gtgggacatg caggtgttga gggctctggg acactctcca agtcacagcg 3960 ttcagaacag gattagagacagaccttaaggagacc 4020 agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc tgagcctgca 4080 tgtttgatgg tgtctggatg caagcagaag gggtggaaga gcttgcctgg agagatacag 4140 ctgggtcagt aggactggga caggcagctg gagaattgcat0gt gaa2catgcc atgtagatgt 4020 aaagccctcc aagatcccca agaccaaccc caacccaccc 4260 accgtgccca ctggccatgt ccctcagtgc cacatcccca cagttcttca tcacctccag 4320 ggacggtgac ccccccacct ccgtgggcag ctgtgccact gcagcaccgc tctttggaga 4380 aggtaaatct tgctaaatcc agccctgcctaggacca ctc 4440 tcatccaact ccaggacgga gtcagtgagg atggggctct agagtcaaca ggaaagttcc 4500 attggagcca agtacattga gtcaataggg actttccaat gggttttgcc cagtacataa 4560 ggtcaatggg aggtaagcca atgggttttt cccattactg gcacgtccgt0 gcctgagtcatgta gcctgagtccgtta gcctgatccgtta cagtaca taaggtcaat aggggtgaat caacaggaaa 4680 gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt ttgcccagta 4740 caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcacg tacataaggt 4800 caataggggt gagtcattgg gtttttccag ccaatt8ttaat taagtacg0cca attgac gtcaatgggc tattgaaact aatgcaacgt gacctttaaa cggtactttc 4920 ccatagctga ttaatgggaa agtaccgttc tcgagccaat acacgtcaat gggaagtgaa 4980 agggcagcca aaacgtaaca ccgccccggt tttcccctggct aaattccata ttgg4ggctgc t5 ct0gattacggc t5 tctacgtggg tatataagca gagctctccc tatcagtgat 5100 agagatctcc ctatcagtga tagagatcga gctcagcgtc ggtaccgtac ctcttccgca 5160 tcgctgtctg cgagggccag ctgttggggt gagtggcggg tgtggcttcc gcgggccccg 5220 gagctggagc cctgctctga gcgggccggt 5 ctgatatgcg 2 ctggtcagtctta g gagggtcgtct ttcccacttc gagtggcggg cggtgcgggg gtgagagtgc gaggcctagc 5340 ggcaaccccg tagcctcgcc tcgtgtccgg cttgaggcct agcgtggtgt ccgccgccgc 5400 gtgccactcc ggccgcacta tgcgtttttt gtccttgctg gca6 cctagcc agcgattg cct4 ggag ggtgtggggc tcactcttaa ggagcccatg aagcttacgt 5520 tggataggaa tggaagggca ggaggggcga ctggggcccg cccgccttcg gagcacatgt 5580 ccgacgccac ctggatgggg cgaggcctgt ggctttccga agcaatcggg cgtgagttta 5640 gcctacctgg gccatgtggc cctagcactg ggcacggtct ggcctggcg0 ccctcat5cccacagt agggt gaggccgtcc cgcccggcac cagttgcttg cgcggaaaga 5760 tggccgctcc cggggccctg ttgcaaggag ctcaaaatgg aggacgcggc agcccggtgg 5820 agcgggcggg tgagtcaccc acacaaagga agagggcctt gcccctcgcc ccgt8tgacctaggt 5820 agcgggcggg cggccgcat agtcacctcg ggcttctctt gagcaccgct 5940 cgtcgcggcg gggggagggg atctaatggc gttggagttt gttcacattt ggtgggtgga 6000 gactagtcag gccagcctgg cgctggaagt cattcttgga atttgcccct ttgagtttgg 6060 agcgaggcta attctcaagc ctcttagcgg ttcaaaggta ttttctaaac ct gggtt2cctag c ggggt2cctag c aact cttcgcggtc tttccagtac tcttggatcg gaaacccgtc 6180 ggcctccgaa cggtactccg ccaccgaggg acctgagcga gtccgcatcg accggatcgg 6240 aaaacctcgt cgacgccgcc accatgggaa tcctgccaag ccgtggaatg ccagcccttgc 6240 agct30ctgc gctgc tgatggggatg cgtggcagag accggaacaa 6360 ggttccctaa catcaccaac ctgtgcccat tcggcgaggt gtttaacgcc acacgctttg 6420 cctccgtgta tgcctggaac cggaagagaa tctctaattg cgtggccgac tatagcgtgc 6480 tgtacaatag cgcctccttc tctaccttta agtgctatgg cgtgtctccc accaagctga 6540 acgacctaggtg gttagcaagctg gttagcaacctggtg gatccggggc gatgaggtga 6600 gacagatcgc accaggacag accggcaaga tcgcagacta caactataag ctgcctgacg 6660 atttcacagg ctgcgtgatc gcctggaata gcaacaatct ggattccaaa gtgggcggca 6720 actacaatta tctgtacagg ctggttccgca 7gagggccactag cagcaccga gatctaccag gcaggctcca caccatgcaa cggagtggag ggcttcaatt 6840 gttattttcc cctgcagagc tacggcttcc agcctaccaa tggcgtgggc tatcagccat 6900 acagagtggt ggtgctgtcc tttgagctgc tgcacgcacc agcaaccgtg tgcggaccta 6960 agaagcagcggctgg at gagcagcggctgg at ccggagga tctggaagcg 7020 agaaggcagc aaaggcagag gaggcagcaa ggaagatgga ggagctgttc aagaagcaca 7080 agatcgtggc cgtgctgaga gccaactctg tggaggaggc catcgagaag gcagtggccg 7140 tgttcgcagg aggagtgcacac gccgtcgtagac ctgatcgccagag 7200 tgatcaaggc cctgtccgtg ctgaaggaga aggggagcaat catcggagca ggaaccgtga 7260 catctgtgga gcaggcaagg aaggcagtgg agtccggagc cgagtttatc gtgtctcctc 7320 acctggatga ggagatctcc cagttcgcca aggagaaggg cgtgttttac atgcctggcg 7380 tgatgacccc aacagagctg gtgaagct0gggc 7 cc4agct0ggg tcccaggaga ggtggtggga ccacagtttg tgaaggccat gaagggccca ttccccaatg 7500 tgaagtttgt gcctacaggc ggcgtgaacc tggacaatgt ggcagagtgg ttcaaggcag 7560 gcgtgctggc agtgggagtg ggatctgccc tggtgaaggg gaacccaggaggga 7acccaggaggga ggcctttgtg gagaagatca ggggagcaac agagtgagcg gccgccacac 7680 atcataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 7740 tttatttgg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 7800 caagttaaca acaacaattg cattcattttt atgttcagggtcagg ttt86gggtga 7800 caagttaaca aa gcaagtaaaa cctctacaaa tgtggtacac aaagtgggga gctctagagg 7920 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag 7980 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggggggcgcc cgagggggggcgcgccgagggggggcgca 8040 gccgagggggcgtcgcc cacggg atcgctttcc tctgaacgct 8100 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag gggagctcta 8160 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 8220 gcagcagcga gccgcccggg gctccgctcc ggtcccccc2g0 caggtcccccc2g cc tccccccccgc cgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 8340 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaaggatcca 8400 tggccggcca tctgcagatc atatgatcgg atgccgggac cgacgagtc4gc aggag8cgt gtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8520 caattccaca cacacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8580 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8640 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggggag aggcggtttg cgtattgggc gct8cctgggc gctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8760 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 8820 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttcctgcca tggcttcctgcca tggcttcctgcca 8820 cgt acg agcatcacaa aaatcgacgc tcaagtcaga 8940 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 9000 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 9060 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg tagagct2ggtt gacct 91 acccc ccgttcagcc cgaccgctgc gccttatccg 9180 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 9240 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 9300 ggcctaacta cgggctacctt agac g ctgaagccag 9360 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9420 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9480 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9540 tggtcatgag attatcaaaa aggatcttca cctagatcct ttaatagaattataa ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9660 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9720 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatcaccact gcaatgactgatac 9780 cgctgactgatacg at aaaccagcca gccggaaggg 9840 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9900 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9960 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc gccatt2cag0 gccgtcaggaggt cccatgt tgtgcaaaaa agcggttagc tccttcggtc 10080 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 10140 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtcagagtc ggtacagagt0 ggtacagagt0 ggtacaggtact gaccgag ttgctcttgc ccggcgtcaa 10260 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 10320 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10380 ctcggcacc caactgatct tcagcatctt ttactttcac cagcgtttct ggggaacgagcaa 10440 aggaacagagcaa gaataagggc gacacggaaa tgttgaatac 10500 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 10560 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10620 gaacatatagtgcc acctgacgttaac tatta ata 10680ggcgtatcac gaggcccttt cgtc 10704

Claims (13)

A method for purifying a cell culture solution expressing a target protein, which is a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or a polypeptide having 75% or more sequence homology thereto, after the step of culturing a cell line expressing the target protein to,
(a) recovering a supernatant by centrifuging the cell culture medium; and
(b) filtering the recovered culture medium
Including, purification method. The method of claim 1, wherein the cell line is derived from Chinese Hamster Ovary (CHO) cells. The method of claim 1, wherein the step (a) of centrifuging the cell culture medium to recover the supernatant is performed using a continuous centrifuge. The purification method according to claim 1, wherein in the step (a) of centrifuging the cell culture medium and recovering the supernatant, the feed flow during centrifugation is 200 L/h to 400 L/h. The purification method according to claim 4, wherein the supply flow rate is 200 to 300 L/h. The purification method according to claim 5, wherein the feed flow rate is 250 L/h. The method of claim 1, wherein in the step (a) of centrifuging the cell culture medium to recover the supernatant, the g-force during centrifugation is greater than 8000x g and less than 20000x g. The purification method according to claim 1, wherein in the step (a) of centrifuging the cell culture medium and recovering the supernatant, the speed of the centrifugation vessel is greater than 8000 rpm and less than 13000 rpm. The method of claim 1, wherein in the step (a) of centrifuging the cell culture medium to recover the supernatant, an ejection interval during centrifugation is 100 to 250 seconds. The purification method according to claim 9, wherein the discharge interval during the centrifugation is 190 to 210 seconds. The purification method according to claim 1, wherein step (b) filtering the recovered culture medium is performed using a microfiltration filter. The purification method according to claim 11, wherein the microfiltration filter is a depth filter. The purification method according to claim 11, wherein the microfiltration filter filtrate is further treated with a surfactant.

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