Non-protein a purification method for adalimumab

Abstract

The present application relates to a method for preparing an antibody group and an antibody group prepared by the method, which can prepare a high-purity and high-quality desired antibody group by removing impurities without using an expensive protein A column, and in particular, can achieve process automation and greatly reduce production costs.

Description

Technical Field

[0001] This invention relates to a method for preparing high-purity and high-yield antibody populations at low cost without using a protein A column for affinity chromatography. Background Technology

[0002] The latest trend in biopharmaceutical research and development focuses on the development of antibody fragments. Compared to the past, more biosimilar therapeutic proteins have been developed. However, major challenges in developing biosimilar products include the high cost of downstream processing, non-selective removal of impurities associated with the process and products, and protein degradation of the products. Compared to full-size monoclonal antibody (mAb) therapeutics, antibody fragments offer specific advantages, such as enhanced penetration into tumors and specific epitope binding that is inaccessible to full-size mAbs. Advances in high-valent cloning and continuous bio-manufacturing fermentation processes have shifted the focus of biopharmaceutical products towards improving the economics of overall downstream processing. Downstream processing accounts for approximately 60% to 70% of the overall cost of preparing monoclonal antibody therapeutics. This process includes capture, intermediate, and polishing procedures, involving the use of various expensive chromatographic operations.

[0003] Therefore, more research and development is needed on purification processes for antibody drugs.

[0004] Specifically, the purification process used for producing antibody drugs mainly utilizes protein A columns. While this method offers the advantage of producing high-purity results in the initial steps, it is more than 30 times more expensive than conventional ion exchange resins, resulting in high production costs.

[0005] According to previous reports, protein A resin accounts for a high proportion, approximately 35%, of the raw material cost for antibody drug production. Even trace amounts of protein A eluted from the column can trigger immune or physiological responses in the human body. Therefore, purification processes using protein A columns present challenges in monitoring and removing residual protein A according to the process. Furthermore, protein A, as a biocompatible group, suffers from weak chemical stability. During column regeneration, regeneration must be performed while maintaining the activity of protein A. Consequently, the 1M NaOH solution required in the washing process cannot be used, making it impossible to completely remove impurities adhering to the column. This results in a significantly reduced number of column regeneration cycles compared to other conventional chemical methods.

[0006] To address this issue, numerous efforts have been made to remove impurities and develop high-purity antibodies using cation exchange columns, hydrophobic columns, anion exchange columns, and other methods.

[0007] However, when preparing antibodies using columns other than those using protein A as described above, numerous problems were discovered until high yields and high purity of antibodies were achieved.

[0008] For example, in the case of cation exchange columns, various buffers are mixed in the purification process. This complexity due to the use of multiple buffers makes it difficult to collect the eluted protein. In particular, when using cation exchange columns, the washing process requires the use of buffer components with a large number of different compositions, thus posing significant challenges to automation and process simplification.

[0009] Due to the problems mentioned above, antibody production costs and time are greatly increased, making it difficult to achieve sufficient quality assurance and automating the process.

[0010] In this context, providing a convenient method for obtaining biosimilar products, and especially for the isolation and purification of purified antibody fragments, is crucial. Addressing the problem of adhering to existing isolation and purification procedures, the inventors aim to provide a method for purifying antibody populations. The purified antibody populations obtained through this invention meet excellent purity and activity standards. Summary of the Invention

[0011] Technical problems to be solved

[0012] The inventors, without using the expensive Protein A column typically used for antibody purification, sequentially utilized a cation exchange column, a hydrophobic interaction column, and an anion exchange column under suitable conditions after a pretreatment step that reduced the pH of the culture supernatant used to remove precipitates. In this process, the method was modified to perform virus filtration and filtration processes at appropriate time points, thereby confirming the ability to prepare high-quality antibody populations. Therefore, the present invention provides an invention with the following objectives.

[0013] One object of the present invention is to provide a method for preparing an antibody population, comprising: step (a), loading a sample containing an antibody mixture onto an equilibrated cation exchange column, washing the cation exchange column, and eluting the antibody bound to the column using an elution buffer, thereby removing host cell protein (HCP) and heteroantibodies from the sample containing the antibody mixture.

[0014] Step (b) involves loading the sample containing the mixed salts of the antibody eluent eluted in step (a) above onto a hydrophobic interaction column and eluting the antibody bound to the column using an elution buffer to remove host cell protein (HCP) and residual DNA from the antibody eluent.

[0015] Step (c) involves passing the antibody elution buffer from step (b) that has removed host cell protein (HCP) and residual DNA through an anion exchange column to collect the flow-through, thereby removing host cell protein (HCP) and residual DNA.

[0016] Step (d) involves passing the flow-through liquid from step (c) through a virus filter to remove the virus; and

[0017] Step (e) involves concentrating and buffer exchanging the antibody eluent eluted in step (d) above to prepare antibody populations containing residual DNA and host cell proteins at concentrations of less than 10 ppb and 10 ppm, respectively.

[0018] Another object of the present invention is to provide an antibody population comprising more than 60% of the major active antibodies prepared by the above method.

[0019] means for solving problems

[0020] As one embodiment for achieving the above objectives, the present invention provides a method for preparing an antibody population, comprising: step (a), loading a sample containing an antibody mixture onto an equilibrated cation exchange column, washing the cation exchange column, and eluting the antibody bound to the column using an elution buffer, thereby removing host cell protein (HCP) and heterologous antibodies from the sample containing the antibody mixture.

[0021] Step (b) involves loading the sample containing the mixed salts of the antibody eluent eluted in step (a) above onto a hydrophobic interaction column and eluting the antibody bound to the column using an elution buffer to remove host cell protein (HCP) and residual DNA from the antibody eluent.

[0022] Step (c) involves passing the antibody elution buffer from step (b) that has removed host cell protein (HCP) and residual DNA through an anion exchange column to collect the flow-through, thereby removing host cell protein (HCP) and residual DNA.

[0023] Step (d) involves passing the flow-through liquid from step (c) through a virus filter to remove the virus; and

[0024] Step (e) involves concentrating and buffer exchanging the antibody eluent eluted in step (d) above to prepare antibody populations containing residual DNA and host cell proteins at concentrations of less than 10 ppb and 10 ppm, respectively.

[0025] In addition to the primary active antibody, antibody products prepared from host cells also contain various isomeric antibodies, host cell proteins (HCPs), host cell-derived DNA, and factors for cell growth. These isomeric antibodies are antibodies in a form modified by deamination or oxidation of some amino acids in the antibody, and each isomeric antibody has different biological activities. In antibody products expressed through host cells, the proportion of these isomeric antibodies is high. Especially in the case of antibody biosimilars, it is crucial to prepare products with quality similar to the control drug. Therefore, after producing antibodies using host cells, a process is needed to adjust the content of isomeric antibodies. Therefore, this invention provides a method that improves purity by removing host cell proteins and host cell-derived DNA, increasing the proportion of the primary active antibody compared to the host cell culture medium, and effectively preparing a high-purity and high-quality antibody population containing the desired proportions of acidic isomeric antibodies, the primary active antibody, and basic isomeric antibodies.

[0026] In particular, the method of the present invention provides a method with the following excellent advantages: while providing high-purity and high-quality antibody populations as described above, it also automates the process and greatly reduces production costs.

[0027] In this invention, the term "antibody population" refers to an antibody group comprising a major active antibody and isomeric antibodies. For the purposes of this invention, the aforementioned antibody population refers to an antibody group comprising a major active antibody and isomeric antibodies in a desired proportion. The aforementioned antibody population may contain only one antibody, or it may contain all antibody groups comprising both a major active antibody and an isomeric antibody. More specifically, for the purposes of this invention, the aforementioned antibody population refers to an antibody group that increases the proportion of the major active antibody by removing impurities such as host cell proteins and isomeric antibodies from antibody products prepared from host cells.

[0028] In particular, when the method of the present invention is used in the preparation of antibody biosimilars, antibody groups containing major active antibodies and isomeric antibodies with the same or corresponding components as the control drug can be prepared.

[0029] The desired antibody group can be prepared by purification using a cation exchange resin column, comprising isomeric antibodies of the desired range and the major active antibody. Specifically, the proportion of the major active antibody is 50% or more, more specifically, 60% or more, the proportion of basic isomeric antibodies is 20% or less, and the proportion of acidic isomeric antibodies is 20% or less. Specifically, the antibody group may contain 60% or more of the major active antibody (specifically, 65% or more of the major active substance), 20% or less of the acidic isomeric antibody, and 20% or less of the basic isomeric antibody.

[0030] In embodiments of the present invention, by utilizing Fractogel COO - A cation exchange column was used to prepare an antibody group containing a similar amount to the Humira reference drug—less than 20% of acidic isomeric antibodies, more than 60% of major active antibodies, and less than 20% of basic isomeric antibodies.

[0031] In this invention, the term "antibody" refers to a substance produced by stimulating antigens in the immune system, specifically a substance that circulates in the lymph and blood by specifically binding to a particular antigen to elicit an antigen-antibody reaction. For the purposes of this invention, the aforementioned antibody is one of the proteins used for high-quality purification, and can be effectively purified by the methods of this invention.

[0032] Typically, the isoelectric point of the aforementioned antibodies is higher than that of other proteins. Therefore, by adsorbing the culture supernatant onto the column using an initial cation exchange resin, a first purification with high purity can be achieved during elution. The isoelectric point (pI) is the average effective charge on the surface of a protein molecule; that is, the pH at which the potential of the protein's electric double layer becomes 0, meaning that the protein's groups dissociate, making the number of cationic and anionic groups equal, thus resulting in an effective charge of 0. The antibodies used for purification in this invention are not limited to this; specifically, the isoelectric point can be 7 to 10, and more specifically, it can be antibodies with an isoelectric point of 7 to 9. Furthermore, the antibodies of this invention are not limited to this; specifically, they can include all therapeutic antibodies commonly used in the art. More specifically, it can be adalimumab, a TNF-α inhibitor. The aforementioned adalimumab, also known as Humira, is a TNF-α inhibitor antibody developed by AbbVie, a US company known for its use in the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, and psoriasis.

[0033] In this invention, the term "major active antibody" refers to the main component included in the antibody group of this invention. It means an antibody in which a portion of its amino acids are modified through deamination or oxidation without reducing its biological activity; that is, it is not an acidic or basic isomeric antibody. The aforementioned major active antibody is the most important component for regulating the quality of the desired antibody group and is the antibody with the highest biological activity among its components.

[0034] In this invention, the term "isomeric antibody" refers to an antibody in which a portion of the amino acids of the main active antibody is modified by deamination or oxidation, including acidic isomers and basic isomers. Examples include isomers where asparagine in the amino acid is deaminated to become aspartate, and isomers where methionine in the amino acid is oxidized to become methionine sulfate. Furthermore, isomers modified to pyruglutamate by forming a pentagonal ring structure containing glutamate at the N-terminus of the heavy chain are also included. When antibodies are generated in host cells such as CHO cells, these isomers are contained in a high proportion in the host cell culture medium; therefore, they should be removed by a process such as chromatography and contained in the antibody population at the desired proportion.

[0035] Therefore, in host cells in which a vector containing a polynucleotide encoding an antibody is introduced, to prepare a high-quality antibody population, it is necessary to appropriately remove the isomeric antibodies as described above and include the main active antibody and the isomeric antibody at the desired levels. Furthermore, to prepare a high-purity antibody population, impurities such as host cell proteins (HCPs), host cell-derived DNA (HCDs), and factors used for cell growth must be removed. Therefore, this invention develops a method for preparing an antibody population that not only regulates the content of the aforementioned isomeric antibodies but also effectively removes impurities such as host cell proteins.

[0036] In particular, the method of the present invention provides a method with the following excellent advantages: it automates the process while providing high-purity and high-quality antibody populations, and also significantly reduces production costs and preparation time. Furthermore, regarding virus inactivation and removal capabilities, when using the purification process of the present invention, even if unexpected virus expression or contamination occurs, it can be removed.

[0037] The method for preparing the antibody population of the present invention includes step (a), which involves loading a sample containing an antibody mixture onto an equilibrated cation exchange column, washing the cation exchange column, and eluting the antibody bound to the column using an elution buffer, thereby removing host cell protein (HCP) and heteroantibodies from the sample containing the antibody mixture.

[0038] Step (a) is a method of collecting an antibody eluent containing antibodies, namely, removing host cell proteins and isomeric antibodies from a sample containing an antibody mixture.

[0039] In this invention, the term "sample containing an antibody mixture" refers to a partially purified sample from the culture supernatant of cells producing antibodies or from fragments of such cells. Specifically, it refers to a partially purified sample containing a mixture of antibodies, including both the major active antibody and the isomeric antibody. This partial purification involves filtration, but other proteins besides the antibodies may still be present.

[0040] The antibody-containing mixture sample is prepared by sequentially performing the following steps: culturing host cells to produce the target antibody; removing the host cells to prepare a culture supernatant; and adjusting the pH of the culture supernatant to a pH below the isoelectric point of the target antibody, specifically removing precipitates formed by adjusting the pH to 4 to 6. That is, the antibody-containing mixture sample can be prepared by a method including the step of adjusting the pH of the culture supernatant to 4 to 6 to remove precipitates.

[0041] In preparing samples by removing cells after reducing the pH, there is a drawback: it can promote cell apoptosis and increase the host cell protein content by lowering the pH. Therefore, a sample preparation method that adjusts the pH of the culture supernatant after cell removal is more advantageous for reducing the host cell protein content. Therefore, specifically, in this invention, samples prepared by adjusting the pH of the culture supernatant after cell removal are used.

[0042] The methods for removing the cells described above can be those commonly used in the art, but are not limited thereto. In particular, filters can be used, and more specifically, barrier filters can be used.

[0043] The antibody-containing mixture described above can be conditioned to have a conductivity of 5 mS / cm to 7 mS / cm before injection into the cation exchange column, but is not limited to this. In embodiments of the present invention, purified water can be added to the pretreated supernatant to adjust the conductivity before injection into the column.

[0044] According to the present invention, in the process of re-filtering the culture supernatant after removing cells by a filter once in the culture medium, the content of host cell protein is significantly reduced, and excellent results are shown in turbidity analysis.

[0045] Furthermore, the precipitate formed by reducing pH contains a large amount of host cell protein, and removing it can reduce the content of host cell protein. The removal of this precipitate is a step before adsorbing antibodies onto the cation exchange column. In the case of antibodies, the isoelectric point is high; therefore, aggregation does not occur under acidic conditions of reduced pH. Conversely, typically, in the case of host cell proteins (HCPs) with an isoelectric point lower than that of antibodies, the charge is removed under acidic conditions of reduced pH, and a large amount precipitates due to van der Waals aggregation. Therefore, the pH range of the culture supernatant to be reduced can be lower than the following values: values ​​close to the isoelectric point of the antibody to be purified in this invention while increasing the precipitation of the host cell protein to be removed. That is, the pH can be 1 to 6 lower than the isoelectric point of the antibody to be purified, more specifically, it can be 2 to 5 lower. Therefore, the pH can ultimately be 3 to 7, more specifically, it can be 4 to 6, and even more specifically, it can be 4.5 to 5.5. At this point, the precipitate can be removed using filters commonly used in the art, such as sterilizing filters. In embodiments of the invention, when the pH is reduced to 5 to remove the precipitate, it has been confirmed that a pretreatment step with high purity can be performed. When the precipitate is removed by the pretreatment step described above, the antibody population can be purified more effectively in subsequent steps using cation exchange columns, hydrophobic interaction columns, or anion exchange columns.

[0046] In step (a), the sample containing the antibody mixture prepared as described above is injected into a cation exchange column. After washing the cation exchange column to remove the heterologous antibody and host cell protein, the antibody bound to the column is eluted.

[0047] In this invention, the term "cation exchange column" refers to a column filled with cation exchange resin. In the above steps, impurities can be removed by performing cation exchange chromatography, specifically, host cell proteins and isomeric antibodies can be removed. The aforementioned cation exchange resin is a synthetic resin that functions to exchange cations in an aqueous solution with its own cations. In the case of antibodies, it has a high isoelectric point, and therefore carries cations in pH buffers below the isoelectric point value. Therefore, the quality of antibody populations can be improved by using cation exchange resins capable of adsorbing antibodies carrying the aforementioned cations. The aforementioned cation exchange resin can be any resin commonly used in the art, although not limited thereto; specifically, a resin with COO2 can be used. - Or, for columns with functional groups of SO3, more specifically, carboxymethyl (CM), fractogel, sulfoethyl (SE), sulfopropyl (SP), phosphate (P), or sulfonate (S), etc., can be used; more specifically, fractogel SO3 can be used. -(s), POROS XS, POROS HS, carboxymethyl agarose (CM sepharose) or Fractogel COO - .

[0048] Step (a) above removes both host cell proteins and isomeric antibodies of the target antibody.

[0049] At this point, the aforementioned host cell proteins may contain all impurities except for the antibody to be purified. These impurities may include not only host cell proteins but also DNA derived from the host cell and factors used for cell growth. Therefore, by removing these host cell proteins, the antibody to be purified can be purified to a high purity.

[0050] Furthermore, the above steps are steps to remove isomeric antibodies in order to adjust the quality. These isomeric antibodies can be acidic isomeric antibodies and / or basic isomeric antibodies. Various isomeric antibodies exist in antibody products expressed through host cells; therefore, in order to prepare antibody biosimilars, it is important to make the quality as similar as possible to the control drug in terms of demonstrating homogeneity. The aforementioned isomeric antibodies are modified forms of a few amino acids of the major active antibody, and the major active antibody, acidic isomeric antibody, and basic isomeric antibody have slightly different charges. Therefore, this charge difference can be used to separate isomeric antibodies. However, the charge difference is only a small difference caused by a few amino acids; therefore, very detailed conditions need to be set for separation. Therefore, in this invention, a cation exchange column is used to effectively remove the aforementioned acidic and basic isomeric antibodies. That is, the method of this invention also has excellent advantages in adjusting the charge variants to the desired ratio by using a cation exchange column.

[0051] Furthermore, the aforementioned isomeric antibodies are modified by deamination or oxidation of a portion of the amino acids in the antibody. It is well known that each isomeric antibody has different biological activities, and maintaining a constant distribution of these isomeric antibodies is an important factor in maintaining consistent quality. However, typically, the content of acidic and basic isomeric antibodies in the host cell culture supernatant used to produce antibodies is relatively high compared to the major active antibody. Therefore, it is necessary to remove a portion of these isomeric antibodies to adjust the content of the three antibody forms. In this invention, the antibody group is prepared such that it contains the major active antibody at a proportion of 50%, specifically 60% or more. Moreover, in the method of this invention, it is possible to prepare the antibody group in a manner where the proportion of the major active antibody is 60% or more, the proportion of basic isomeric antibodies is 20% or less, and the proportion of acidic isomeric antibodies is 20% or less.

[0052] In step (a) above, in the step of loading the sample containing the antibody mixture into the equilibrated cation exchange column, an equilibration buffer containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride can be used.

[0053] Specifically, it can be Fractogel COO2 equilibrated with an equilibration buffer containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride. - The step involves loading the sample containing the antibody mixture onto the column. With the aforementioned equilibration buffer, a conductivity of 5 mS / cm to 7 mS / cm is possible, more specifically, a conductivity of 5.5 mS / cm to 6.5 mS / cm is possible, and even more specifically, a conductivity of approximately 6.0 mS / cm is possible. In this equilibration process, it is preferable to use the aforementioned buffer at a volume of approximately 11 column volumes or more, more specifically, 14 column volumes.

[0054] According to an embodiment of the present invention, Fractogel COO2 is equilibrated with an equilibration buffer containing 20 mM acetate at pH 5.0 and 40 mM sodium chloride, having a conductivity of 6.0 mS / cm. - The sample containing the antibody mixture is loaded onto the column, and the purification process begins using a cation exchange column.

[0055] In detail, the washing step in step (a) above may include the following two steps: a first washing step to attach antibodies that are not attached to the column; and a second washing step to remove acidic isomeric antibodies.

[0056] According to previously known methods of utilizing cation exchange columns, there are multiple washing steps, and the composition, pH, and conductivity of the buffer solutions used in each step are significantly different.

[0057] In contrast, the present invention uses a washing step of only two steps and a buffer solution with simple composition, thereby achieving cost reduction and automation in the preparation process.

[0058] Specifically, the washing steps of the present invention may include: 1) a first wash using a buffer solution containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride; and 2) a second wash using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 55 mM to 59 mM, specifically 57 mM sodium chloride.

[0059] In the first washing process of this invention, the first wash can be performed using a buffer containing 15 mM to 30 mM acetate and 35 mM to 45 mM sodium chloride at pH 4.5 to 5.5, which contains the same components as the buffer used in the loading step. This has the advantage that no additional buffer preparation process is required, and antibodies not attached to the column can be added via a continuous process. In this first wash, the aforementioned buffer can be used in quantities of approximately 3 to 7 column volumes, specifically approximately 5 column volumes.

[0060] In the second washing process of this invention, the second washing can be performed using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 55 mM to 59 mM, specifically 57 mM, sodium chloride.

[0061] The second washing process and the subsequent elution (desorption) process of the present invention allow for different treatment settings of the two buffer solutions to perform purification in a cation exchange column. Unlike conventional techniques that involve repeatedly performing washing and equilibration steps or performing a continuous process more than four times using buffers with different compositions, in the present invention, the purification process of step (a) can be simply performed by adjusting only the ratio of the two buffer solutions. Therefore, in terms of the preparation process, it offers the advantages of cost reduction and process simplification.

[0062] That is, in this invention, the washing and elution process in step (a) can be performed by increasing the molar concentration of sodium chloride in a 25mM to 35mM acetate buffer with a specified pH of 5.5 to 6.5. Specifically, for a 25mM to 35mM acetate buffer with a pH of 5.5 to 6.5, the mixing ratio of the 25mM to 35mM acetate buffer with a pH of 5.5 to 6.5 and the buffer solution containing sodium chloride at a preset molar concentration is changed (increased), and the molar concentration of sodium chloride is increased to perform the washing and desorption process.

[0063] For example, according to an embodiment of the invention, in the second washing process, a first buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and a second buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 100 mM sodium chloride can be used. Specifically, the buffer solution composition required for the second washing process can be prepared by mixing 41 to 45 wt% of the first buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 55 to 59 wt% of the second buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 100 mM sodium chloride. In this mixing case, appropriate volumes of each buffer solution can be supplied from the two buffer storage spaces according to the mixing ratios of each step set by the machine.

[0064] In the second washing step of a specific embodiment of the present invention, the first buffer solution and the second buffer solution are treated together at 43% and 57% by weight, respectively, and the second washing process is performed using a buffer solution containing 30 mM acetate and 57 mM sodium chloride at pH 5.5 to 6.5. In this second washing, preferably, the buffer solution is used up to approximately 3 to 17 column volumes, specifically approximately 15 column volumes or 0.1 AU.

[0065] The above step (a) may include an elution (desorption) step, specifically, it may include three steps: a first elution step, a second washing step, and a third elution step.

[0066] Specifically, this can be achieved by the following steps: Step 1), eluting the antibody for the first time using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 57 mM to 63 mM, specifically, 60 mM sodium chloride; Step 2), eluting the antibody for the second time using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 67 mM to 73 mM, specifically, 70 mM sodium chloride; and Step 3), eluting the antibody for the third time using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 77 mM to 83 mM, specifically, 80 mM sodium chloride.

[0067] In this invention, as confirmed in the previous second washing process, it can be easily carried out by simply adjusting the ratio of the two buffer solutions used in the elution process. This results in cost reduction and automation of the preparation process.

[0068] The elution process in step (a) can be performed using a 25mM to 35mM acetate buffer at a specified pH of 5.5 to 6.5, with an increased molar concentration of sodium chloride. Specifically, the desorption process is performed by changing (increasing) the mixing ratio of the 25mM to 35mM acetate buffer at pH 5.5 to 6.5 and the sodium chloride buffer at a predetermined molar concentration, and by increasing the molar concentration of sodium chloride.

[0069] For example, according to an embodiment of the invention, the buffer solution used for the elution process can be a first buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and a second buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 100 mM sodium chloride.

[0070] Specifically, in the first elution process, a first buffer may be included, comprising 37 to 43 wt% of a first buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5, and a second buffer comprising 57 to 63 wt% of a second buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 100 mM sodium chloride.

[0071] Specifically, in the second elution process, a first buffer may be included in the form of 27% to 33% by weight of a first buffer containing 25mM to 35mM acetate at pH 5.5 to 6.5 and 67% to 73% by weight of a second buffer containing 25mM to 35mM acetate at pH 5.5 to 6.5 and 100mM sodium chloride.

[0072] Specifically, in the third elution process, a first buffer may be included at 17% to 23% by weight of a first buffer containing 25mM to 35mM acetate at pH 5.5 to 6.5 and 77% to 83% by weight of a second buffer containing 25mM to 35mM acetate at pH 5.5 to 6.5 and 100mM sodium chloride.

[0073] In the first elution step of a specific embodiment of the present invention, the first buffer solution and the second buffer solution are treated together at 40% by weight and 60% by weight, respectively, and the first elution process is performed using a buffer solution containing 30 mM acetate and 60 mM sodium chloride at pH 5.5 to 6.5. Preferably, in this first elution case, the buffer solution is used at a volume of about 5 to 20 column volumes, more specifically, about 7 to 17 column volumes, and more specifically, about 8 to 15 column volumes.

[0074] In the second elution step of a specific embodiment of the present invention, the first buffer solution and the second buffer solution are processed together at 30% and 70% by weight, respectively, and the second elution process is performed using a buffer solution containing 30 mM acetate and 70 mM sodium chloride at pH 5.5 to 6.5. Preferably, in this second elution case, the buffer solution is used at a volume of about 7 to 20 column volumes, more specifically, about 8 to 17 column volumes, and more specifically, 9 to 15 column volumes.

[0075] In the third elution step of a specific embodiment of the present invention, the first buffer solution and the second buffer solution are treated together at 20% and 80% by weight, respectively, and the third elution process is performed using a buffer solution containing 30 mM acetate and 80 mM sodium chloride at pH 5.5 to 6.5. Preferably, in this third elution, the buffer solution is used up to about 6 to 18 column volumes, more specifically, about 8 to 16 column volumes, and more specifically, 9 to 14 column volumes or 0.1 AU.

[0076] That is, in step (a) of the present invention, the sample containing the antibody mixture is loaded onto an equilibrated cation exchange column, the cation exchange column is washed, and the antibody bound to the column is eluted using an elution buffer, thereby removing host cell proteins and isomeric antibodies from the sample containing the antibody mixture. Specifically, the loading, washing, and elution steps may include:

[0077] Step 1) Equivalent Fractogel COO2 to a buffer solution containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride. - The column is loaded with a sample containing a mixture of antibodies;

[0078] Step 2), perform the first wash using a buffer solution containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride.

[0079] Step 3), a second wash is performed using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 55 mM to 59 mM, specifically 57 mM sodium chloride.

[0080] Step 4) The antibody is first eluted using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 57 mM to 63 mM, specifically, 60 mM sodium chloride.

[0081] Step 5), the antibody is eluted a second time using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5, and specifically, 67 mM to 73 mM sodium chloride, specifically 70 mM sodium chloride; and

[0082] Step 6) The antibody is eluted a third time using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 77 mM to 83 mM, specifically, 80 mM sodium chloride.

[0083] In embodiments of the present invention, the antibody is purified by a process including equilibration and loading, first washing, second washing, first elution, second elution, and third elution, thereby significantly reducing the amount of protein and DNA derived from host cells to purify the antibody with a high content of major active antibody.

[0084] In the above-described method for removing isomeric antibodies, to simultaneously remove both acidic and basic isomeric antibodies, specifically, Fractogel COO2 can be used. - .

[0085] When mixing large amounts of acidic and basic isoantibodies, a cation exchange column with separation capabilities is required to remove the basic isoantibody. In this case, Fractogel COO2, which uses methacrylate polymer resin as a carrier, is preferably preferred. - Simultaneously removes both acidic and basic isomeric antibodies.

[0086] In the cation exchange chromatography of this embodiment, the flow rate is also 180 cm / hr, which has the advantage of being more than 1.5 times faster (e.g., more than 1.55 to 2.24 times faster) than conventionally known methods. At this high flow rate, higher productivity relative to time is achieved compared to existing methods.

[0087] In this invention, the step of inactivating the virus may be included after step (a) and before step (b) described above.

[0088] Specifically, virus inactivation includes rendering the virus contained in the above-mentioned eluent inactive or removing the virus from the above-mentioned eluent. Methods for rendering or removing the virus inactive include heat inactivation, pH inactivation, or chemical inactivation methods, etc. Specifically, pH inactivation methods can be used, but are not limited to this. The above-mentioned pH inactivation method is a method that utilizes pH treatment to a degree sufficient to render the virus inactive. Such pH inactivation methods include low-pH virus inactivation methods. These methods can be performed in the pH range of 3.0 to 4.0, specifically, by titrating the antibody eluent eluted in step (a) at pH 3.8, but are not limited to this.

[0089] Specifically, the antibody eluent eluted in step (a) may contain more than 60% of the major active antibody, less than 20% of the acidic isomeric antibody and less than 20% of the basic isomeric antibody.

[0090] The method for preparing the antibody population of the present invention includes step (b), loading the sample containing the mixed salt of the antibody eluent eluted in step (a) onto a hydrophobic interaction column, and eluting the antibody bound to the column using an elution buffer, thereby removing host cell protein (HCP) and residual DNA from the antibody eluent.

[0091] In the method of the present invention, step (b) is a step of collecting antibody eluent from which host cell protein (HCP) and residual DNA are also removed from the antibody eluent collected in step (a).

[0092] The antibody eluent collected in step (a) above can be the collected eluent itself or in a form diluted with other buffers. Furthermore, as mentioned earlier, it can also be the eluent sample after a step of inactivating the virus. In this invention, the sample loaded onto the hydrophobic interaction column can also be prepared by adding salt to the antibody eluent eluted in step (a). The type of salt contained in the sample loaded onto the hydrophobic interaction column is not particularly limited; in embodiments of this invention, citrate is used. Furthermore, the sample can be a sample adjusted to have a salt concentration of 0.8 to 1.2 times that of the equilibration buffer of the hydrophobic interaction column in step (b), specifically, it can be a sample adjusted to have the same salt concentration as the equilibration buffer as the antibody eluent in step (a), but is not limited thereto.

[0093] Furthermore, in the elution step of step (b) above, the antibody attached to the column is eluted using a concentration gradient. In embodiments of the present invention, it has been confirmed that the concentration gradient method is more advantageous in terms of yield and elution volume compared to a step-by-step method.

[0094] Step (b) above aims to improve purity by removing impurities such as host cell proteins and residual DNA that were not removed in step (a), and provides a purification step that utilizes the hydrophobicity difference between the separation mechanism and the cation exchange column in purification step (a) to remove host cell proteins from the hydrophobic interaction column.

[0095] In this invention, the term "hydrophobic interaction column" refers to a column filled with a hydrophobic interaction resin. The steps described above refer to a column capable of performing hydrophobic interaction chromatography to remove impurities, specifically, a column capable of removing host cell proteins. Proteins are generally hydrophilic, but include regions that are both hydrophilic and hydrophobic. The water-transporting properties of these regions are not expressed under conditions of strong electrostatic interactions, but are relatively strongly expressed when the electrostatic interactions are weakened by increasing the ionic strength or dielectric constant of the solvent. Specifically, if a hydrophobic ligand (long hydrocarbon chain or aromatic ring) is introduced into a substrate (agarose gel, organic polymer carrier, etc.) for hydrophilic chromatography and equilibrated with a concentrated salt concentration, various proteins can be adsorbed. Then, if the salt concentration is reduced, elution is performed according to the characteristics of the proteins, thereby achieving separation. That is, when a hydrophobic environment is provided using salt, differences in the strength of adsorption on a specific column occur due to the individual differences in hydrophobicity of the proteins. Using this principle, the steps described above (b) for removing host cell proteins and residual DNA using a hydrophobic interaction column can be performed.

[0096] The aforementioned hydrophobic interaction resins may be of those commonly known in the art, but are not limited thereto. Specifically, phenyl columns, butyl columns, phenyl sepharose gel, or Fractogel EMA phenyl columns may be used, and more specifically, phenyl agarose gel may be used.

[0097] Furthermore, step (b) above may include the following steps: loading a hydrophobic interaction column equilibrated with an equilibration buffer containing 25 mM to 35 mM acetate (pH 5.5 to 6.5) and 0.3 M to 1.0 M citrate, loading a sample with the same citrate concentration as the antibody elution buffer in step (a), and eluting the antibody by applying an elution buffer containing 25 mM to 35 mM acetate (pH 5.5 to 6.5) in a concentration gradient manner.

[0098] In this invention, it was confirmed that the yield was high when using acetate buffer at pH 6.0 as the buffer solution, and the yield was also excellent when using a concentration gradient of the buffer solution in the elution method. Thus, it was confirmed that the above steps of this preparation method using a concentration gradient can effectively prepare high-purity antibody populations.

[0099] The method for preparing the antibody population of the present invention may include a step of filtering the antibody eluent from step (b) using a filter after step (b) and before step (c).

[0100] For example, the filtration in this invention can perform ultrafiltration and / or percolation.

[0101] In detail, ultrafiltration can be performed. As used in this application, the term "ultrafiltration" or "UF" refers to any technique that treats a solution or suspension using a semi-permeable membrane that allows solvent or solute molecules to pass through while retaining macromolecules. Ultrafiltration can be used to increase the concentration of macromolecules in a solution or suspension.

[0102] Furthermore, in detail, perfiltration can be performed. The terms "diafiltration" or "DF" as used in this application refer to a specialized filtration method that uses a solvent to dilute the residue and then filters it again to reduce the soluble permeate content. For example, perfiltration may or may not induce an increase in the concentration of retained components containing proteins. For example, in continuous perfiltration, the solvent is continuously added to the residue at the same rate as the filtrate is generated. In this case, the volume of the residue and the concentration of the retained components do not change during the process. Alternatively, in discontinuous or sequential dilution perfiltration, the ultrafiltration step is accompanied by the addition of solvent to the residue side; when the volume of solvent added to the residue is greater than the volume of the generated filtrate, the retained components may have a high concentration. Perfiltration can be used to change other properties of solutions or suspensions such as pH, ionic strength, salt composition, buffer composition, or macromolecules.

[0103] This single-stage filtration process can further reduce the content of host cell proteins.

[0104] The method for preparing the antibody population of the present invention includes step (c), in which the antibody eluent from step (b) which removes host cell protein (HCP) and residual DNA is passed through an anion exchange column to collect the flow-through, thereby removing host cell protein (HCP) and residual DNA.

[0105] In the method of the present invention, step (c) is a step of collecting the desired antibody group after removing impurities from the antibody eluent collected in step (b). Specifically, step (c) is a step of collecting the flow-through by passing the antibody eluent from step (b) after removing host cell proteins and residual DNA through an anion exchange column. Furthermore, the antibody eluent collected in step (b) can be the collected eluent itself, or in a form diluted with other buffers, or in a form that has undergone filtration, etc., but is not limited to these forms.

[0106] In this invention, the term "anion exchange column" refers to a column filled with anion exchange resin. In the above steps, it refers to a column capable of performing anion exchange chromatography to remove impurities, specifically a column capable of removing host cell proteins, but not limited thereto. The aforementioned anion exchange resin is a synthetic resin that performs the action of exchanging its own anions with specific anions in an aqueous solution. The anion exchange column can adsorb anion-containing proteins above its isoelectric point. For antibodies, the isoelectric point is high; therefore, when using a neutral pH buffer, antibodies do not adhere to the anion exchange resin and overflow. However, impurities containing host cell proteins have a low isoelectric point and can be adsorbed onto the anion exchange resin and removed. Thus, the above principle can be used to prepare high-purity antibody populations.

[0107] The anion exchange resins described above may be those commonly used in the art, though not limited thereto. Specifically, Q sepharose, quaternary aminoethyl or quaternary amino(Q) resins may be used, and more specifically, Q Fast Flow resins may be used.

[0108] Furthermore, the above method can utilize an equilibration buffer with a pH lower than that of the target antibody, specifically, an equilibration buffer with a pH of 7.0 to 8.0, and more specifically, an equilibration buffer containing Tris hydrogen chloride (pH 7.0 to 8.0).

[0109] The host cell proteins removed in the above steps encompass all impurities other than the antibodies to be purified mentioned above. This includes not only the host cell proteins themselves but also DNA derived from the host cell and factors used for cell growth. Therefore, removing host cell proteins in this step allows for the purification of only the desired antibody at high purity. Furthermore, the anion exchange column effectively removes not only host cell proteins but also endotoxins. Thus, even when endotoxins are removed along with host cell proteins in the final purification step, a high-purity target antibody group can still be purified.

[0110] The method for preparing the antibody population of the present invention includes step (d), in which the flow-through liquid in step (c) is passed through a virus filter to remove the virus.

[0111] Unlike conventional methods in the prior art, the antibody preparation method of the present invention performs viral filtration before filtration using ultrafiltration, percolation, and / or multilayer filters. In conventional methods, viral filtration is performed after filtration using ultrafiltration, percolation, and multilayer filters, making it difficult to prepare high concentrations of the drug. Conversely, in the method of the present invention, viral filtration is performed first, thereby reducing the loading amount in the ultrafiltration, percolation, and / or multilayer filtration steps to ensure protein quality while minimizing yield loss, and offering the advantage of easily preparing high concentrations of the drug.

[0112] Specifically, in this invention, virus filtering can use Modus 1.3 (Merck product). More specifically, PES material is preferably used.

[0113] The method for preparing the antibody population of the present invention includes step (e), which involves concentrating and buffer exchanging the antibody eluent eluted in step (d) above to prepare antibody populations containing residual DNA and host cell proteins at concentrations of less than 10 ppb and 10 ppm, respectively.

[0114] In this invention, the concentration and buffer exchange of the prepared antibody eluent can refer to the conventional concentration and buffer exchange process used for storing antibodies in commercial products.

[0115] Next, the solution containing the filtered antibodies (eluted antibody eluent) from the virus filtration process described above can be subjected to ultrafiltration, diafiltration, etc. Ultrafiltration and diafiltration are as described above.

[0116] The virus removal and filtration process described above removes viral impurities and can further remove HCP and residual DNA. Furthermore, this subsequent ultrafiltration and diafiltration can be used for concentration and / or buffer exchange.

[0117] The buffer solution may contain conventional antibodies or components required for the preparation of the desired dosage form. For example, as an example of pharmaceutically acceptable excipients, the buffer solution may contain conventional buffer components for preparing excipients at known concentrations, including any number of nonionic or ionic excipients, as described in the reference cited herein [Ref: Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)]. The aforementioned buffer exchange may, for example, be an exchange with nonionic excipients, such as sugars including polysorbate and poloxamer, or nonionic surfactants, without altering the antibody concentration.

[0118] The host cell protein content of the antibody group prepared according to the present invention, after concentration and buffer exchange, can be less than 10 ppm, for example, from 0.0001 ppm to 10 ppm, more specifically, from 0.001 ppm to 5 ppm. Furthermore, the residual DNA can be less than 10 ppb, for example, from 0.0001 ppb to 10 ppb, more specifically, from 0.001 ppb to 1 ppb.

[0119] In embodiments of the present invention, the antibody purification method according to the present invention confirms that antibodies can be purified to a high purity of 99.9%. In particular, the prepared antibody population contains antibodies comprising residual DNA and host cell proteins at concentrations of 0.1 ppb and 5 ppm or less, respectively.

[0120] According to another embodiment, the present invention provides an antibody population prepared by the above method, which contains more than 60% major active antibodies.

[0121] The methods, antibody groups, and antibody groups containing more than 60% of the main active antibodies are described above.

[0122] Invention Effects

[0123] By utilizing the antibody preparation method of the present invention, expensive protein A columns are not used to remove impurities, thereby enabling the preparation of target antibody groups with high purity and high quality. In particular, the method of the present invention provides a method with the following excellent advantages: it significantly reduces production costs while achieving process automation. Attached Figure Description

[0124] Figure 1 A flowchart illustrating the preparation process of the adalimumab antibody of the present invention is provided.

[0125] Figure 2The results of cation exchange chromatography performed by a method similar to that described in Korean Patent No. 10-1498771 are shown.

[0126] Figure 3 The results of cation exchange chromatography in Example 2-1 of the present invention are shown.

[0127] Figure 4 The results of cation exchange chromatography in Example 2-2 of the present invention are shown.

[0128] Figure 5 The results of hydrophobic interaction chromatography performed according to the present invention are shown.

[0129] Figure 6 The results of anion exchange resin chromatography performed according to the present invention are shown. Detailed Implementation

[0130] The present invention will now be described in detail through examples. However, the following examples are merely illustrative and the present invention is not limited to these examples.

[0131] Below, in Figure 1 A flowchart detailing the antibody preparation process of the specific embodiments is provided. Specifically, the antibody-containing culture medium sample of the present invention is recovered, filtered, and then treated with a cation exchange resin. Afterwards, virus inactivation is performed, followed by hydrophobic reaction with a resin. Then, ultrafiltration is performed once, followed by anion exchange resin treatment. Finally, virus filtration is performed, followed by a second ultrafiltration, and then dosage formization is carried out. The specific processes briefly mentioned above are described in detail in the following embodiments.

[0132] Example 1: Culture medium pretreatment method for antibody purification

[0133] After culturing recombinant CHO cells expressing adalimumab antibody and expressing adalimumab antibody, the pH was lowered to below 6 to adsorb the antibody onto a cation exchange column.

[0134] In this embodiment, the degree of impurity removal is confirmed according to the culture medium pretreatment method.

[0135] In this embodiment, the following method was used: the pH was lowered to 5 in a culture medium containing cells and then filtered to prepare a sample for injection into a cation exchange column. The specific conditions are shown in Table 1.

[0136] Table 1

[0137]

[0138] The culture medium was pretreated according to the above method, and the turbidity of the culture medium in sequence (1), (2) supernatant, (4) acid-treated culture medium, and (6) pretreated culture medium after filtration and sterilization was analyzed. As a result, the turbidity of the culture medium in (1) was confirmed to be 6810 NTU, and the turbidity of the supernatant in (2) was confirmed to be 2.98 NTU. Furthermore, the turbidity of the acid-treated culture medium in (4) was confirmed to be 5475 NTU, and the turbidity of the pretreated culture medium after filtration and sterilization in (6) was confirmed to be 5.13 NTU.

[0139] Furthermore, during the filtration step, the average Flux showed a large filtration capacity of approximately 150 LMH, thus confirming minimal loss during the culture medium pretreatment step.

[0140] The filtration process in each step showed a turbidity removal rate of over 99%, thus confirming that the pretreated culture medium was suitable for use in subsequent steps.

[0141] The above results demonstrate that the pretreatment method of the present invention, followed by a single removal of cells using an initial filter, is suitable for the process. Furthermore, it is shown that if the precipitate is removed by lowering the pH to below 6 (preferably pH 5), a culture supernatant with higher purity than the initial culture medium can be obtained.

[0142] Example 2: Cation Exchange Chromatography

[0143] Select Fractogel COO - A column with functional groups that are advantageous in terms of purity and yield, serving as an alternative to column processes utilizing protein A in cation exchange columns.

[0144] An experiment to regulate isomeric antibodies was carried out using the aforementioned cation exchange column, as follows.

[0145] To achieve equilibration, the column was equilibrated by passing 14 column volumes of a buffer solution containing pH 5.0, 20 mM acetate, and 40 mM sodium chloride (6.0 mS / cm). The pretreated supernatant was then loaded at a capacity of CM (25 mg / mL column).

[0146] After loading, the first washing step is performed as follows: 5 column volumes of the first washing buffer (containing pH 5.0, 20 mM acetate and 40 mM sodium chloride, 6.0 mS / cm) are processed to attach any antibodies not attached to the column, and the remaining portion is washed with the supernatant.

[0147] Then, a second washing step was performed, which involved using a total of 15 column volumes of first buffer (pH 6.0, 30 mM acetate, 2.4 mS / cm) and second buffer (a buffer containing pH 6.0, 30 mM acetate and 100 mM sodium chloride, 12.5 mS / cm), with a ratio of 43% by weight of the first buffer and 57% by weight of the second buffer.

[0148] The second washing step was performed using a mixture of 43% by weight of the first buffer and 57% by weight of the second buffer.

[0149] Subsequently, in order to perform the desorption step, the first buffer solution was reduced and increased in three stages at 40%, 30%, and 20% by weight, and the second buffer solution was reduced and increased in three stages at 60%, 70%, and 80% by weight.

[0150] As described in the second washing step above, after each mixture is preferentially executed, three elution (desorption) steps are performed using the mixed buffer solution.

[0151] Unlike conventional methods of cation exchange chromatography, the types of buffer solutions used in this invention are simplified to three, and the washing steps are greatly reduced to two compared to existing technologies.

[0152] Furthermore, the desorption step only requires adjusting the ratio of the first and second buffer solutions to perform the reaction, thereby automating and simplifying the entire process.

[0153] Furthermore, in the cation exchange chromatography of this embodiment, a flow rate of 180 cm / hr is used, which is more than 1.5 times faster than conventional methods. At this high flow rate, higher productivity relative to time is achieved compared to existing methods.

[0154] The following is a detailed description of the use of the cation exchange resin Fractogel COO in the present invention, as described in Table 2. - The purification process of heterogeneous antibodies.

[0155] Table 2

[0156]

[0157] In addition, for comparison, an equilibration step without NaCl was added in a manner similar to that of prior Korean Patent 10-1498771 to perform elution. Specifically, in the first washing step, 20 mM acetate at pH 5.0 and 40 mM sodium chloride were treated with a total of 5 column volumes; in the second washing step, 30 mM acetate at pH 6.0 was treated with a total of 10 column volumes; in the first desorption step, 30 mM acetate at pH 6.0 and 50 mM sodium chloride were treated with a total of 10 column volumes; in the second desorption step, 30 mM acetate at pH 6.0 was treated with a total of 1.5 column volumes; and in the third desorption step, 30 mM acetate at pH 6.0 and 80 mM sodium chloride were treated with a total of 8 column volumes (comparative example).

[0158] exist Figures 2 to 4 The experimental results above are shown.

[0159] Figure 2 The cation exchange chromatography results of the above comparative examples are shown. Figure 3 The results of Embodiment 2-1 of the present invention are shown. Figure 4 The results of Embodiment 2-2 of the present invention are shown.

[0160] Table 3 below shows the proportions and yields of the acidic isomeric antibodies, major active antibodies, and basic isomeric antibodies confirmed by the above cation exchange chromatography.

[0161] Table 3

[0162] Example 2-1 Example 2-2 Comparative example Acidic isomeric antibody peak (%) 16～19 14～19 15～16 Major active antibody peak (%) 66～69 65～69 68 Basic isomeric antibody peak (%) 13～15 13～19 16 Yield (%) 60～67 67～89 52～56

[0163] The purification process utilizing the cation exchange column conditions described above significantly increases the yield while simultaneously increasing the content of the main active antibody. Furthermore, the process is automated by using only three buffer solutions to collect the eluted protein. This advantage demonstrates the additional benefit of reducing buffer preparation and footprint costs when producing antibodies.

[0164] The greatest advantage is that by injecting the above three buffers in sequence, proteins are collected based on column volume during elution. Therefore, it is easier to obtain antibodies in high yield under established conditions, and it can be appropriately applied to process automation.

[0165] Example 3: Virus inactivation

[0166] The elution buffer obtained according to Examples 2-1 and 2-2 was added to 1M citric acid buffer to inactivate the virus at pH 3.8 for 1 hour. After inactivation, 2M Trizma base buffer was added to adjust the pH of the sample to 6.0. The inactivated sample was then filtered through a 0.2μm filter.

[0167] Example 4: Hydrophobic reactive resin

[0168] A process to improve the purity of antibodies is performed using phenyl sepharose fast flow, a type of hydrophobic interaction chromatography (HIC).

[0169] Specifically, in order to perform HIC, the various eluents prepared by the method described in Example 3 above were used.

[0170] Based on the pH 6.0 acetate, adsorption is performed in a 0.6 M citrate concentration. When eluting, a concentration gradient is provided until the elution buffer reaches 5 column volumes.

[0171] The basic buffer solution in cation exchange resins utilizes acetate at a pH below 6.0. Therefore, the use of a pH below 6.0 has the advantage of simplifying the preparation process in buffer solution preparation.

[0172] The 5-column volume concentration gradient method in the elution process showed excellent results in terms of yield, and in the case of buffer, the acetate buffer at pH 6.0 also showed excellent results in terms of yield and pH stability of the antibody.

[0173] The specific conditions for the above hydrophobic interaction chromatography (HIC) are shown in Table 4 below.

[0174] Table 4

[0175]

[0176] Perform hydrophobic interaction chromatography under the above conditions. Figure 5 The results are shown.

[0177] Example 5: Single-stage ultrafiltration

[0178] Under a feed pressure ≤ 1 bar, the Pellicon 3 Cassette (membrane) was equilibrated using 25 mM Tris-HCl buffer (pH 7.5, 2.0 mS / cm) and the process solution was concentrated to 10 mg / mL. Subsequently, the process solution was exchanged using buffer to achieve a pH of 7.5 and a conductivity ≤ 3.0 mS / cm, followed by one ultrafiltration.

[0179] Example 6: Anion Exchange Resin Chromatography

[0180] In the preparation method of the present invention, a process for preparing antibody populations with higher purity using anion exchange resin chromatography was determined.

[0181] Specifically, the anion exchange column adsorbs anion-carrying proteins above their isoelectric point. Therefore, in the case of antibodies with an isoelectric point of 7 or higher (e.g., 7 to 10 for adalimumab, and approximately 8.4 for hmirtrazine), when using a neutral pH buffer, the antibody does not adhere to the anion exchange resin and overflows into the flow-through. Thus, to confirm the anion exchange resin and buffer conditions suitable for the preparation process of this invention, the following experiments were performed.

[0182] Specifically, in this embodiment, Q Fastflow (QFF, GE), a quaternary amine series widely used in production-scale processes, was used as the anion exchange resin for purification. First, as sample preparation for loading onto the anion exchange resin, a cation exchange column, a hydrophobic interaction column, and a single ultrafiltration were performed in the culture supernatant to replace the buffer solution, thereby preparing the sample with appropriate conductivity and pH. Purity, host cell protein content, residual DNA content, and yield were confirmed under the conditions of 25 mM Tris HCl in buffer at pH 7.5.

[0183] exist Figure 6 The results of chromatography using anion exchange resin are shown.

[0184] The prepared antibody population showed a purity of 100%, an HCP content of 6.8 ng / mg, a residual DNA content of 0.04 pg / mg, and a yield of 94% to 97%. As a result, in anion exchange resin chromatography, a buffer containing TrisHCl and a pH of 7 to 8 were shown to be beneficial for preparing the antibody population of the present invention.

[0185] Example 7: Virus filtration, secondary ultrafiltration and percolation

[0186] The samples subjected to anion exchange resin chromatography in Example 6 above were filtered using a virus filter Modus 1.3 (Merck).

[0187] Subsequently, a second ultrafiltration was performed using a Pellicon 3 Cassette (membrane) ultrafiltration filter. Specifically, under a feed pressure ≤1 bar, the Pellicon 3 Cassette (membrane) was equilibrated with a 14 mM phosphate buffer (pH 5.2, ≥12.0 mS / cm), and the process solution was concentrated to 55.5 mg / mL. Afterward, an exchange was performed using a buffer solution to adjust the pH and conductivity of the process solution to 5.2 and ≥11.0 mS / cm, respectively.

[0188] Unlike the method in the prior art Korean patent 10-1498771, the virus filtration described above is performed before the secondary ultrafiltration. In the aforementioned prior art, virus filtration is performed after both primary and secondary ultrafiltration, making it difficult to prepare high concentrations of the drug. In contrast, in the method of the present invention, virus filtration is performed before the secondary ultrafiltration, thereby providing the advantages of a reduced loading to ensure protein quality, reduced yield loss, and easier preparation of high concentrations of the drug.

[0189] Example 8: Confirmation based on changes in the content of host cell-derived proteins and DNA throughout the entire process in large batches.

[0190] Based on the steps of Examples 1 to 7 above, the content of host cell-derived proteins and DNA in the entire process was confirmed, and the results are shown in Table 5 below.

[0191] In the case of the samples described in Table 5 below, the results are for samples obtained according to the preparation process of Examples 2-2.

[0192] Table 5

[0193]

[0194]

[0195] As confirmed in Table 5 above, the HCP and DNA contents were verified according to the steps outlined. As a result, the cation exchange chromatography described in Examples 2-2 significantly reduced the amount of DNA derived from host cells. Subsequently, virus inactivation was prioritized, and protein quality was ensured through ultrafiltration / percolation in the previous dosage form step, while simultaneously reducing yield loss, thus facilitating the preparation of high-concentration drugs. Furthermore, an antibody population with minimized host cell-derived protein and DNA contents can be prepared through the aforementioned series of steps.

[0196] Example 9: Confirmation of virus removal capability based on the entire process in large-scale production.

[0197] In the entire process according to the steps of Examples 1 to 7 above, the virus removal rates in the low pH treatment, anion exchange chromatography, and virus reduction filtration processes were confirmed, and the results are shown in Table 6 below.

[0198] Table 6

[0199]

[0200]

[0201] As can be confirmed in Table 6 above, the inactivation and removal capabilities of murine leukemia virus, pseudorabies virus, reovirus type 3, and mouse parvovirus were verified according to the steps outlined. Under the above results, it is shown that almost no virus is expressed in the antibody population produced by the process of the present invention. In particular, when considering the virus inactivation and removal capabilities in the above processes, even if unexpected virus expression or contamination occurs in the purification process of the present invention, it can be removed.

[0202] As can be understood from the above description, those skilled in the art can implement this invention in other specific embodiments without changing its technical concept or essential features. Relatedly, it should be understood that the embodiments described above are merely illustrative in all respects and are not limiting. The scope of this invention should be interpreted as including within the scope of the appended claims and all modifications or variations derived therefrom, compared to the detailed description above.

Claims

1. A method for preparing an antibody population, characterized in that, Step (a): Load the sample containing the antibody mixture into an equilibrated cation exchange column, wash the cation exchange column, and then use elution buffer to elute the antibody bound to the column, thereby removing host cell protein and heterologous antibody from the sample containing the antibody mixture. Step (b) involves loading the sample containing the mixed salts of the antibody eluent eluted in step (a) above onto a hydrophobic interaction column and eluting the antibody bound to the column using an elution buffer to remove host cell proteins and residual DNA from the antibody eluent. Step (c) involves passing the antibody elution buffer from step (b) that has removed host cell proteins and residual DNA through an anion exchange column to collect the permeate, thereby removing host cell proteins and residual DNA. Step (d) involves passing the liquid from step (c) above through a virus filter to remove the virus; as well as Step (e) involves concentrating and buffer-exchanging the permeate from which the virus was removed in step (d) above to prepare antibody populations containing residual DNA and host cell proteins at concentrations of less than 10 ppb and 10 ppm, respectively. The above-mentioned cation exchange column has COO - Pillars of the functional group The step of washing the cation exchange column in step (a) includes: Step 1), a first wash was performed using a buffer solution containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride; and Step 2), a second wash is performed using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 55 mM to 59 mM sodium chloride. The step of eluting the antibody bound to the column using elution buffer in step (a) includes: Step 1), the antibody is first eluted using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 57 mM to 63 mM sodium chloride. Step 2), the antibody is eluted a second time using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 67 mM to 73 mM sodium chloride; and Step 3) The antibody is eluted a third time using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 77 mM to 83 mM sodium chloride. The buffer solutions for the first, second, and third elution steps are prepared by adding a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and sodium chloride at a predetermined molar concentration to a 25 mM to 35 mM acetate buffer at pH 5.5 to 6.5, thereby achieving the aforementioned predetermined molar concentration of sodium chloride for the first, second, and third elution steps. The antibody mentioned above is adalimumab.

2. The method for preparing the antibody population according to claim 1, characterized in that, The antibody-containing mixture sample from step (a) above is prepared by a method including the following steps: adjusting the pH of the culture supernatant to 4 to 6 to remove precipitates.

3. The method for preparing antibody populations according to claim 1, characterized in that, In the antibody-containing mixture of the sample from step (a) above, the conductivity of the solution is 5 mS / cm to 7 mS / cm.

4. The method for preparing antibody clusters according to claim 1, characterized in that, The antibody eluent eluted in step (a) above contains more than 60% of the major active antibody, less than 20% of acidic isomeric antibodies and less than 20% of basic isomeric antibodies.

5. The method for preparing antibody populations according to claim 1, characterized in that, The step of loading the antibody-containing mixture onto an equilibrated cation exchange column includes the following steps: equilibrating the sample with a Fractogel COO2 column using an equilibration buffer containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride. - The column is loaded with a sample containing a mixture of antibodies.

6. The method for preparing the antibody population according to claim 1, characterized in that, In the second washing step, the buffer is prepared by mixing a 25mM to 35mM acetate buffer at pH 5.5 to 6.5 with a buffer containing 25mM to 35mM acetate at pH 5.5 to 6.5 and sodium chloride at a set molar concentration to achieve a sodium chloride molar concentration of 55mM to 59mM.

7. The method for preparing the antibody population according to claim 1, characterized in that, Step (a) above includes: Step 1), Fractogel COO2 equilibrated with an equilibration buffer containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride. - The column is loaded with a sample containing a mixture of antibodies; Step 2), perform the first wash with a buffer solution containing 15 mM to 30 mM acetate at pH 4.5 to 5.5 and 35 mM to 45 mM sodium chloride; Step 3), a second wash is performed using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 55 mM to 59 mM sodium chloride. Step 4) The antibody is first eluted using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 57 mM to 63 mM sodium chloride. Step 5), the antibody is eluted a second time using a buffer solution containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 67 mM to 73 mM sodium chloride; and Step 6) The antibody is eluted a third time using a buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 and 77 mM to 83 mM sodium chloride.

8. The method for preparing the antibody population according to claim 1, characterized in that, Step (b) above involves eluting the antibody using a concentration gradient.

9. The method for preparing the antibody population according to claim 8, characterized in that, The concentration gradient method described above includes the following steps: loading a hydrophobic interaction column equilibrated with an equilibration buffer containing 25 mM to 35 mM acetate and 0.3 M to 1.0 M citrate at pH 5.5 to 6.5, loading a sample containing the antibody elution buffer eluted in step (a) with the same citrate concentration as the equilibration buffer, and applying the elution buffer containing 25 mM to 35 mM acetate at pH 5.5 to 6.5 in a concentration gradient manner to elute the antibody.

10. The method for preparing the antibody population according to claim 1, characterized in that, The hydrophobic interaction column in step (b) above is a phenyl agarose gel column.

11. The method for preparing the antibody population according to claim 1, characterized in that, The anion exchange column in step (c) above is equilibrated with an equilibration buffer at pH 7.0 to 8.0 before the sample is injected.

12. The method for preparing the antibody population according to claim 11, characterized in that, The above equilibration buffer contains Tris-HCl with a pH of 7.0 to 8.

0.

13. The method for preparing the antibody population according to claim 1, characterized in that, The anion exchange column used in step (c) above is a Q Fast Flow column.

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