Art
[1]
The present invention relates to a method for controlling an object, the method for producing a TNFR-Fc fusion protein mixture and a hydrophobic chromatogram peak 3 content comprising a peak 3 of the hydrophobic chromatogram in an amount of, specifically, (a) in mammalian cells a sample containing the produced TNFR-Fc fusion protein mixture with equilibration buffer containing sodium chloride or ammonium sulfate before-production method and a predetermined TNFR-Fc fusion protein mixture using a hydrophobic chromatography medium containing equilibrated aromatic functionality of the equilibration buffer containing sodium chloride or ammonium sulfate at a concentration related to a method for controlling the hydrophobic chromatogram peak 3 content by hydrophobic chromatography.
[2]
BACKGROUND
[3]
Etanercept (Etanercept) has biological inflammatory modulators which serves to the competitive inhibition in that the coupling TNF-α receptor (TNF-α receptor) and TNF-α on the cell surface in vivo, inhibiting an immune response involved with TNF-α ( a biological inflammation modulator). These etanercept is a soluble human p75 TNF (tumor nectosis factor) receptor (TNFR) with human immunoglobulin G subclass (subclass) as a TNFR-Fc fusion protein, the Fc region binding the first, the fusion protein the two are disulfide bond (disulfide bond ) with the same type connected to the dimer (having the form of a homodimer), a molecular weight of a macromolecule kDa up to 150 (Goldenberg, Clinical Therapeutics , 21 (1): 75-87, 1999; Moreland . et al , .. Ann Intern Med . , 130 (6): 478-486, 1999).
[4]
A typical form of such a fusion protein in the early 1990s, the University of Texas, Southwestern Medical Center was first synthesized by Bruce A Boy teulreo (Bruce A. Beutler) of the (University of Texas Southwestern Medical Center) , Amgen (Amgen) in 2002 Enbrel (Enbrel by four ® was launched under the name of). Etanercept has clinical studies are underway for application to the rheumatoid arthritis, psoriasis, ankylosing spondylitis etc. has been used, vasculitis, Crohn's disease and Alzheimer's disease as TNF-α inhibitors.
[5]
TNFR-Fc fusion proteins can be produced by fusing a 232 amino acid Fc portion, including the 235 amino acid TNFR with the hinge of which shows the biological activity in in the form dimers (dimer) when produced using recombinant DNA technology. The TNFR is 4 domain and the membrane (transmembrane) total 235 as the number of cysteine ​​22 of these amino acids that make up this cysteine ​​divided into regions and all form a three-dimensional structure made of a disulfide bond. However, there occurs a case the cysteine ​​is combined randomly case of producing TNFR-Fc fusion protein by using a mammalian cells do not form a disulfide bond with the same native protein. In addition, some TNFR part is cut off there occurs a case that does not produce the correct form of the TNFR-Fc dimer. For the correct disulfide bonds that fulfill the TNFR-Fc binding ability with the TNF-α dramatically reduced by not exhibit the proper biological activity. In addition, some or all of TNFR may not have biological activity, even if, like the cut.
[6]
Thus, when produced using recombinant DNA technology and animal cell culture techniques to TNFR-Fc dimer because include active-form protein, disulfide inactive-type protein binding is not correct, the aggregates and the truncated form of the protein production at the same time, these protein techniques for separation and purification is necessary.
[7]
[8]
Moreover, according to the patent (US patent 7,294,481) relates to a production method of the disclosed recombinant proteins by the developer, Immunex Corporation Inc. of etanercept, hydrophobic chromatography was via (hydrophobic interaction chromatography HIC) analysis confirmed the presence of three peaks confirmed, the peaks are in turn adapted to S186 and from D235 location Clipped forms of etanercept, active form of TNFR-Fc fusion protein and aggregate, disulfide scrambled TNFR-Fc and various forms of etanercept with very low biological activity, including It was. This discloses the same peak pattern in Korea Patent Registration No. 10-1454316 discloses a method for producing a call-activatable TNFR-Fc fusion proteins using a recombinant protein production method, used for the isolation of existing TNFR-Fc Fusion Protein hydrophobic chromatography is impurities, for example, agglomerates, disulfide scrambled TNFR-Fc fusion proteins, type cutting TNFR-Fc fusion protein and remove the components other than the active form of TNFR-Fc fusion protein with a minimum of impurities and active form TNFR- It was the sole purpose of obtaining the Fc fusion protein in high purity.
[9]
[10]
Meanwhile, the bio-similar may be used to mean the equivalent material in terms of quality, safety, efficacy, side compared to the existing licensed product, more detail is a composition similar also in the soil and the content of the Originator product have a biological product. For example, well-known as forming the hydrophobic chromatography peak 3 through the low, the above prior art biologically active for etanercept (Pfizer), the component (hereinafter referred to as peak 3) and contains from about 9 to 18% these components may also affect, as well as efficacy (potency) Pharmacology and Therapeutics. Therefore, in order to develop it as a biosimilar product, it is necessary to adjust the amount of peak 3 to roughly Originator product. However, conventional recombinant protein but Although the method for purifying a sample by using a HIC containing the TNFR-Fc produced by the production method attempts (Korea Patent Registration No. 10-1454316 call), which remove impurities and active form of a TNFR- Fc that is, there was no attempt to be only for obtaining a component of peak 2 in high purity, to adjust the content of a low biological activity of a type of impurity peak 3 to roughly Originator product.
[11]
Detailed Description of the Invention
SUMMARY
[12]
The present inventors have intensively studied efforts result, an aromatic functional group to develop the production of TNFR-Fc fusion protein method by adjusting the amount of Peak 3 contains it at a constant level according as producing TNFR-Fc fusion protein using hydrophobic chromatography hydrophobic chromatography including a; used (hydrophobic interaction chromatography HIC) medium (medium), and this prior to the equilibration buffer containing sodium chloride having a predetermined concentration of the - by the load and then equilibrated sample dissolution of existing Originator product and of It confirmed that the content of the peak to roughly 3 controlled to produce a TNFR-Fc fusion protein, thereby completing the present invention.
[13]
Problem solving means
[14]
An object of the present invention to provide a method for controlling the content object in a manner that, to prepare a TNFR-Fc fusion protein mixture containing the three peaks of the chromatogram and a hydrophobic content of hydrophobic chromatography peak 3 grams to.
[15]
Effects of the Invention
[16]
The present invention relates to as producing TNFR-Fc fusion protein using hydrophobic chromatography, adjusting the content of the hydrophobic chromatographic peak 3 by using equilibrated with the equilibration buffer containing sodium chloride or ammonium sulfate with a predetermined concentration of performing a hydrophobic chromatography and it is possible to provide a method for producing a TNFR-Fc fusion protein to include an amount which purpose it. Thus, the proposed method can be effectively used for the production of biological products, including recombinant proteins such as etanercept produced by recombinant DNA technology from an animal cell culture.
[17]
Brief Description of the Drawings
[18]
1 is a diagram showing the Phenyl Sepharose HIC Flow-Through the process chromatogram using a high-performance resin (Phenyl Sepharose High Performance resin) according to an embodiment of the present invention.
[19]
Best Mode for Carrying Out the Invention
[20]
Provides a method of an aspect of the present invention for solving the foregoing problems, the manufacture, TNFR-Fc fusion protein mixture containing the three peaks of the chromatogram for the purpose of hydrophobic content to.
[21]
[22]
The method preferably includes: (a) equilibration buffer containing sodium chloride or ammonium sulfate, a sample containing the TNFR-Fc fusion protein mixture produced in mammalian cells (equilibration buffer; EQ buffer) to pre-include equilibrated aromatic functionality step a; (HIC hydrophobic interaction chromatography) medium injected into a packed column hydrophobic chromatography; And (b) it can be carried out, including the step of collecting the eluate to elute the protein eluted with buffer containing sodium chloride or ammonium sulfate at the same concentration as the equilibration buffer.
[23]
[24]
Wherein the TNFR-Fc transfected with fusion protein with an expression vector comprising a polynucleotide encoding the case of producing TNFR-Fc fusion protein as a switch in which the host cell, the dimer (dimer) type that indicates the biological activity of binding to TNF-α for TNFR-Fc fusion protein in addition to being formed TNFR cysteine ​​is combined randomly native TNFR form a disulfide bond that is not identical to the protein, or, TNFR TNFR-Fc dimer formed body of the part is cut the correct form of the protein of the protein this was not a problem. These components appear as each discrete peak at a hydrophobic chromatogram, the old study, is defined as this, each peak 1, peak 2 and peak 3, each peak of the cutting-type protein, active proteins and other aggregates or disulfide scrambled the biological activity of such TNFR-Fc was found to comprise a lower component. Such a TNFR-Fc fusion proteins, including etanercept, sold by blockbuster drugs. However, licensed by conventional clinical and commercial product contains the peak 3 to 9 to 18%, which is at a level that could affect the efficacy and / or pharmacological, in particular to permit the bio-similar drugs It requires the equivalence with an existing licensed product or originator product, in addition, when the bio-similar according to separation and purification processes of the manufacturing process used for the production becomes contains about such impurities vary, which may affect the efficacy of the drug It is equally adjust the peak 3 content of hydrophobic chromatogram is very important. However, when using the above recombinant protein production techniques produce a TNFR-Fc fusion protein, the content of each component is provided is not constant, it is impossible to control. Thus, as well as to eliminate the component is simply a low biological activity over the course of purifying a mixture containing the TNFR-Fc fusion proteins of the various types needs a way to adjust it to contain a certain level. The method of the present invention and the like in as using hydrophobic chromatography from the mixture of the TNFR-Fc fusion protein, separating the active form of TNFR-Fc fusion protein, an object content in the aggregates or disulfide scrambled TNFR-Fc fusion protein It can be usefully used to purification so as to contain a peak 3 of the hydrophobic chromatogram that.
[25]
The method of the invention the peak 3 of the hydrophobic chromatography, the production in mammalian cells TNFR-Fc fusion protein mixture variety of salts having a concentration of the sample containing the release of only the removal of impurities existing, especially those to be equilibrated with sodium chloride hydrophobic chromatogram the level can be adjusted to the desired content was first identified, these results are not reported.
[26]
[27]
The term, "TNFR (tumor necrosis factor receptor) protein" in the present invention means the receptor protein which binds to TNF-α. The TNFR proteins TNFRI (p55), and include all of the protein or TNFRII (p75) protein, preferably not proteins or TNFRII, limited to this. In addition, the TNFRII may be mixed with TNFRSF1B (Tumor necrosis factor receptor superfamily member 1B). TNFRII the protein is divided into four domains, and a transmembrane (transmembrane) domain. Examples TNFRII may be a protein comprising the four domain and transmembrane region, which consists of 235 amino acids, but is not limited thereto. The TNFRI protein and can be obtained from known databases such as GenBank National Institutes of Health has information on TNFRII proteins. Examples Accession number that may be a protein NP_001056, or P20333, but not limited thereto.
[28]
The TNFR proteins can be used in the treatment of diseases which are so Genie an activity of binding to TNF-α is known to cause various diseases when overexpressed in the human body, by using this, mediated by TNF-α, such as autoimmune diseases. By fusing the Fc region of an immunoglobulin to the TNFR protein it can be used to do this by making the form of a fusion protein which increases the half-life.
[29]
In the present invention, the term, "TNFR (tumor necrosis factor receptor) -Fc fusion protein" is one of the TNFR protein or all or a portion of which is connected by the Fc region and the enzyme action of the immunoglobulin, or the two polypeptides by genetic manipulation It means the result expressed as a polypeptide. Wherein the TNFR-Fc fusion protein, but may be connected via a direct connection the Fc region of the TNFR protein and an immunoglobulin or a peptide linker (peptide linker), but is not limited thereto. Examples of the TNFR-Fc fusion protein may include etanercept.
[30]
Wherein the TNFR-Fc fusion proteins can be produced by fusing all or a portion of the TNFR protein and the immunoglobulin Fc region, and examples thereof include an immunoglobulin Fc domain that one to 235 11 of TNFRⅡ protein comprising the amino acid region and the hinge (hinge) region the fusion to the 232 amino acids, but is not limited thereto. In addition, the TNFR-Fc fusion protein may be codon optimized according to the host cell (codon optimization) to be expressed, in the example the TNFR-Fc fusion protein is specific for CHO cells that is defined by the amino acid sequence of SEQ ID NO: 1 enemy It is a codon optimized TNFR-Fc fusion protein, but not limited to this. Wherein the TNFR-Fc fusion protein as well as the amino acid sequence of SEQ ID NO: 1, the sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, and most preferably includes both if the protein having substantially the activity of binding to the TNF-α amino acid sequence shown as more than 98% similarity. Furthermore, if a sequence having such a similarity amino acid sequence having a biological activity the same as or correspond to the TNFR-Fc fusion protein, within the scope of the present invention, protein variants, some sequence having a deletion, modified, substituted or added in the amino acid sequence included is obvious. Further, the polynucleotide encoding a TNFR-Fc fusion protein is that as well as the nucleotide sequence of SEQ ID NO: 2, the sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably more than 95%, and most preferably all, if substantially a polynucleotide encoding a protein having an activity of binding to TNF-α as a nucleotide sequence representing at least 98% similarity. Furthermore, if a sequence having such similarities nucleotide sequence encoding an amino acid sequence having a biological activity the same as or correspond to the TNFR-Fc fusion protein, the part sequence deletion, modification, the protein variants with a substituted or added in the amino acid sequence included is apparent within the scope of the present invention the nucleotide sequences encoding. In one embodiment of the present invention, to optimize codon typically specific for CHO cells.
[31]
The term, in the present invention, "immunoglobulin; Fc region (immunoglobulin Ig)" is other than the portion other than the variable regions of the heavy and light chains of immunoglobulin, the heavy chain constant region 1 (CH1) and light chain constant domain (CL1), a heavy chain constant means a region 2 (CH2), heavy chain constant region 3 (CH3) and a hinge (hinge) portion of an immunoglobulin comprising part. In addition, the immunoglobulin Fc regions of the present invention includes derivatives thereof as well as the sequence of natural-type amino acid sequence. It means that the amino acid sequence derivative is one or more amino acid residues in the native amino acid sequence that has a different sequence by a deletion, insertion, or entirely vivo conservative substitutions, or a combination thereof. In addition, the immunoglobulin Fc region may be an Fc region of the IgG, IgM, IgE, IgA or IgD-derived or a combination of both (combination) or a mixed (hybrid). Preferably an IgG known to be derived from improving the half-life of the binding protein, but are not more preferably IgG1-derived or, limited to this.
[32]
On the other hand, the term "combination (combination)" in the present invention is meant to form a bond with the dimer or the time of forming the oligomer, the same origin short chain immunoglobulin single-chain polypeptide of a polypeptide coding for the Fc regions of different origin do. That is, it is possible to produce dimers or multimers Fc from IgG, IgA Fc, IgM Fc, IgD Fc and two or more fragments selected from the group consisting of the Fc fragment of IgE.
[33]
The term "mixed (hybrid)" in the present invention is a term that means that the existing sequence corresponding to the immunoglobulin Fc fragment of two or more different origins within the immunoglobulin Fc regions of the single chain. For the present invention, it is possible that different forms of the hybrid. In other words, possible to IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc of CH1, CH2, CH3 and the domain hybrid consisting of one to four domains from the group consisting of CH4, and it may comprise a hinge. On the other hand, IgG is also divided into subclasses of IgG1, IgG2, IgG3 and IgG4 can also be a combination of both, or their hybridization to the invention.
[34]
Wherein the TNFR-Fc fusion proteins can be obtained by expression by introducing the expression vector into mammalian cells comprising the polynucleotide encoding the fusion protein.
[35]
According to the present invention was used pCUCBin-mSig-TNFcept vector as an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein was transduced to this, CHO cells expressing TNFR-Fc fusion protein. TNFR-Fc fusion protein obtained by the above method, the active form of TNFR-Fc fusion proteins, type cutting TNFR-Fc fusion proteins, inactive form TNFR-Fc fusion protein, such as various types of TNFR- and / or TNFR-Fc fusion protein aggregates there is Fc fusion protein is mixed, as well as the active form of TNFR-Fc fusion protein therefrom, there is a need to be purified to contain the inactive form TNFR-Fc fusion protein at a predetermined ratio. When using the production method of the present invention can adjust the amount of peak 3, or the like agglomerates to be purified with the fragment, the active form of the fusion protein, can be adjusted so that it contains a content of interest them.
[36]
In order to generally remove them, because the culture of the mammalian cells are additionally include various proteins in addition to the desired protein, which, preferably, the sample containing the TNFR-Fc fusion protein mixture produced in the mammalian cell is a chemical conversion chromatography striking of the cell culture section using both chromatography, anion chromatography or they can be purified by preparing.
[37]
In the specific embodiment of the present invention it was used for transfection was performed the first affinity chromatography for the culture of CHO cells was infected obtained by performing anion chromatography for its eluent as the eluent sample.
[38]
[39]
For example, a medium filled in the column to be used for the hydrophobic chromatography can be used a medium containing a phenyl group as an aromatic functional group. In a particular embodiment of the invention but using a column filled with a phenyl-Sepharose High Performance resin (Phenyl Sepgarose High Performance resin) produced from GE Healthcare captured, but is not limited thereto.
[40]
[41]
For example, an object that the content of hydrophobic chromatography of peak 3 g of the object may be 9 to 18%. For example, the (a) peak 3 content of hydrophobic chromatogram of TNFR-Fc fusion protein mixture of step a, if it exceeds 20% peak 3 content purified by applying the method of the present invention can lower the 9 to 18% is.
[42]
[43]
For example, equilibration buffer and elution buffer in which the use may be a buffer containing 7 to 15 mM sodium phosphate. Although the embodiments of the present invention using a buffer containing 10 mM sodium phosphate, but is not limited thereto.
[44]
[45]
For example, pH of the equilibration buffer and elution buffer may be used in which the 6 to 8.5 range. Buffer having a pH outside the above range, it is possible to cause the denaturation of the target protein may be difficult to use in hydrophobic chromatography of the recombinant protein.
[46]
[47]
For example, a method which comprises controlling the concentration of sodium chloride in the equilibration buffer in accordance with the content of the hydrophobic chromatographic peak 3 of the manufacturing method according to the present invention is the purpose, the purpose hydrophobic chromatography content of grams Peak 3 to 2 to 17 If the% can be achieved sikimeuroseo 1 to 1.4 equilibrated with a buffer containing sodium chloride in M ​​concentrations. Specifically, when the case of using the equilibration buffer containing 1.1 M sodium chloride for loading sample peak of 3 content of 21% and to lower the peak 3 content of 5.3 ~ 17.0%, using the equilibration buffer containing 1.2 M sodium chloride, 2.9 ~ to 15.7%, 1.3 M the case of using the equilibration buffer containing sodium chloride to 2.5 ~ 14.8%, 1.4 M the case of using the equilibration buffer containing sodium chloride was reduced to 2.2 ~ 13.7%.
[48]
[49]
Further, there is provided a method further comprising a step of adding ammonium sulfate in equilibration buffer according to the content of the hydrophobic chromatographic peak 3 of the manufacturing method according to the present invention object, where the hydrophobic chromatography amount of grams the peak 3 of the sample exceeds 45% If, it is characterized by solidifying the equilibrium buffer containing ammonium sulfate of from 0.45 to 0.55 M concentration.
[50]
For example, recombinant protein is obtained culture solution by a method for producing an excess of a hydrophobic chromatogram peak 3 for example, when contained is greater than 45% and equilibrated with equilibration buffer containing ammonium sulfate to perform the hydrophobic chromatography, 20-30% of after the lower the amount of peak 3, by carrying out the hydrophobic chromatography with equilibration buffer containing the above-described sodium chloride it is possible to lower the amount of peak 3, as 10%.
[51]
[52]
The hydrophobic chromatography can be carried out through a series of steps consisting of the sequence of loading, equilibrating, strips and CIP (cleaning-in-place) process.
[53]
By the strip process is performed to remove all of the protein remaining on the column so as to elute, except that the strip process, which does not contain sodium chloride or ammonium sulfate may be carried out using a buffer solution of the same composition as the equilibration buffer is.
[54]
CIP is in production as a biological product for safe and efficient removal of impurities from the chromatography medium in the process for the cost-effective management device makes it possible to reuse them. The CIP, but it can be carried out using a sodium hydroxide solution of about 0.5 N concentration, but is not limited thereto. As needed, CIP, but it can be carried out several times, but is not limited thereto.
[55]
[56]
In yet one embodiment, before the hydrophobic chromatography medium with equilibration buffer containing sodium chloride or ammonium sulfate in 0.45 to 0.55 M concentration of 1.0 to 1.5 M concentration comprising the steps of equilibration; It provides a method of adjusting the content of a step of loading a sample containing the TNFR-Fc fusion protein prepared to have the same salt concentration in the equilibration chromatography medium, and the peak of the hydrophobic chromatogram contained in the etanercept 3 -, and the former do.
[57]
[58]
For example,, the pH of the equilibration buffer as described above may be from 6 to 8.5 days.
[59]
Method using the equilibration buffer containing sodium chloride or ammonium sulfate of the present invention is characterized by the adjustment in a direction to decrease as compared with the samples of the three peak load content in the final product.
[60]
[61]
For example, it is possible to reduce the amount of peak 3 from 2 to 17% in using the equilibration buffer containing sodium chloride in the sample to a peak of 3 content exceeds 20%.
[62]
As another example, it is possible to reduce the amount of peak 3 to a level of less than 18% in using the equilibrium buffer containing ammonium sulfate in the sample to a peak of 3 content exceeds 45%.
[63]
Mode for the Invention
[64]
The present invention will be described in further detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it is not the scope of the present invention is limited by these examples.
[65]
[66]
Example 1: etanercept of manufacture and HIC Flow-Through
[67]
[68]
To prepare a etanercept, CHO cells, the TNFR-Fc transfected with vector (pCUCBin-mSig-TNFcept) containing the gene encoding the fusion protein and was incubated infection and purification by affinity for a CHO cell culture screening using MTX Photography the (affinity chromatography) with an anion chromatography (anion chromatography) were applied sequentially.
[69]
First, affinity chromatography was performed as described below in detail. Affinity resin is MabSelect SuRe ™ (GE Healthcare) a XK26 column (GE Healthcare) and then filled in a column equilibrated given sufficiently flushed with 20 mM Tris -HCl pH 8.0 buffer solution containing 100 to 200 mM sodium chloride, prepared by the culture given the flow was combined with the affinity resin and eluted with eluting buffer of pH 3.4 to pH 3.0. 2 by using the M Tris adjust the pH of the eluate to 7.0 to 8.0.
[70]
Through the affinity chromatography it was performed as follows for anion chromatography for the obtained leaching solution. And then to the specific anion resin is Fractogel EMD TMAE (Merck) filling the LRC15 columns (Pall Life Sciences) given enough flow to a buffer solution containing tris of pH 7.5 to 8.5 to equilibrate the column, the parent eluate from Mars chromatography a given flow combines the protein with an anion resin and eluted with 100 to 200 mM sodium chloride, tris elution buffer.
[71]
[72]
As it follows on the obtained leaching solution by following the affinity chromatography, and anion chromatography, and then was applied to HIC Flow-Through process. At this time, the amount of Peak 3 is recovered is butyl -NPR (TSK, 14947) linearly by 0.1 M sodium phosphate pH using an analytical column 6.0, 1.8 M ammonium (A buffer) and 0.1 sodium phosphate pH6.0 (buffer B) It was measured with a gradient.
[73]
[74]
Experimental Example Peak 3 content adjusted using sodium chloride: 1
[75]
[76]
HIC Flow-Through was the sodium chloride in 10 mM sodium phosphate for the process of preparing each of 1.1 M to 1.4 M containing equilibration buffer (buffer EQ) that, all equilibrium buffer was adjusted to a pH of 6.3. Sample loading the HIC were also prepared to contain a sodium phosphate and sodium chloride having the same concentration and equilibration buffer.
[77]
Phenyl Sepharose High Performance resin (GE Healthcare) for using HIC Flow-Through the process as shown in Figure 1, loading and equilibration (EQ), a strip and made of a CIP process, the strip and is 10 mM respectively to the buffer the CIP step a sodium phosphate pH 6.3 with 0.5 N sodium hydroxide were used.
[78]
The eluate (elution pool) was in the loading phase, based on the absorbance at 280 nm to the equilibration buffer phase were collected from more than 50 mAU evaluated by collecting 5CV (column volume) fractions, to depending on sodium chloride concentration used and the results are shown in Table 1 It is shown.
[79]
TABLE 1
Equilibration buffer conditions Peak 3 content (%)
Loading sample 21
10 mM sodium phosphate pH 6.3, 1.1 M sodium chloride 5.3 ~ 17.0
10 mM sodium phosphate pH 6.3, 1.2 M sodium chloride 2.9 ~ 15.7
10 mM sodium phosphate pH 6.3, 1.3 M sodium chloride 2.5 ~ 14.8
10 mM sodium phosphate pH 6.3, 1.4 M sodium chloride 2.2 ~ 13.7

[80]
As, as shown in Table 1, the sodium chloride concentration is increased with the content of Peak 3 was confirmed that showed a tendency to decrease, In conclusion, the amount of Peak 3 depending on the concentration of sodium chloride can be adjusted from 2.2 ~ 17.0% of It was.
[81]
[82]
Experimental Example 2: Ammonium sulfate Peak 3 content of control with
[83]
[84]
The equilibration buffer (buffer EQ) to contain 0.5 M sodium sulfate in 20 mM Tris -HCl pH 8.0 buffer solution for HIC Flow-Through process using ammonium sulfate was prepared. HIC load sample also was prepared by loading the sample to contain ammonium 20 mM Tris -HCl pH 8.0 and 0.5 M sulfuric acid.
[85]
A phenyl-Sepharose High Performance resin (GE Healthcare) with 5 cm packed into a XK16 (GE Healthcare) was subjected to HIC Flow-Through process. As in Experimental Example 1 The process was used as the loading, equilibrating, and the strip is respectively 20 mM Tris -HCl pH 7.0 with 0.5 N sodium hydroxide were made by CIP process, the step of the strip and CIP buffer.
[86]
The eluate (elution pool) was evaluated in the loading phase, based on the absorbance at 280 nm was collected from more than 50 mAU to equilibration buffer stage collecting 5CV fraction, and as a result, HIC Flow for loading a sample containing a Peak 3 46.1% when performing -Through process, after the process was confirmed that the amount of Peak 3 was adjusted to 12%.

Claims
[Claim 1]
Method for producing, TNFR-Fc fusion protein mixture containing the peak 3 of the hydrophobic chromatogram in an amount that an object comprising the steps of: (a) a sample containing the TNFR-Fc fusion protein mixture produced in mammalian cells equilibration buffer containing sodium chloride or ammonium sulfate, (equilibration buffer; EQ buffer) around - hydrophobic chromatography including the equilibrium aromatic functionality; comprising: (hydrophobic interaction chromatography HIC) medium is injected into a packed column; And (b) a step of collecting the eluate to elute the protein eluted with buffer containing sodium chloride or ammonium sulfate at the same concentration as the equilibration buffer.
[Claim 2]
The method of claim 1, wherein the hydrophobic chromatography method object of the aimed content of the peak of 3 grams is of 9 to 18% of.
[Claim 3]
The method of claim 1, wherein said aromatic functional group like a phenyl group.
[Claim 4]
2. The method of claim 1, wherein (a) TNFR-Fc fusion protein mixture of step is a method that the content of the peak 3 of the hydrophobic chromatographic than 20%.
[Claim 5]
The method of claim 1, wherein the equilibration buffer and elution buffer is a buffer containing 7 to 15 mM sodium phosphate.
[Claim 6]
The method of claim 1, wherein the equilibration buffer and elution buffer will be the method with a pH of 6 to 8.5.
[Claim 7]
According to claim 1, a method which comprises controlling the concentration of sodium chloride in the equilibration buffer in accordance with the object content of the hydrophobic chromatographic peak 3 which, hydrophobic chromatography amount of grams the peak 3 of the sample 20% excess to 45% or less and, when the objective hydrophobic chromatography amount of peak 3 grams of 2 to 17% also characterized in that the solidifying equilibrated with 1 to buffer containing 1.4 M of sodium chloride concentration.
[Claim 8]
According to claim 1, for the purpose characterized in that the addition of ammonium sulfate in equilibration buffer according to the content of the hydrophobic chromatographic peak 3 which, hydrophobic chromatography amount of grams the peak 3 of the sample exceeds 45%, and the desired when the content of the hydrophobic chromatographic peak 3 from 10 to 20% also characterized in that the solidifying equilibrated with buffer containing ammonium sulfate of from 0.45 to 0.55 M concentration.
[Claim 9]
The method of claim 1, wherein the mammalian cell is pCUCBin-mSig TNFcept-way vector to the transfection of CHO cells were.
[Claim 10]
2. The method of claim 1, wherein the sample containing the TNFR-Fc fusion protein produced in the mammalian cell mixture is not refined portion using both the cell culture medium affinity chromatography, anion chromatography or a combination thereof.
[Claim 11]
The method of claim 1, wherein said hydrophobic chromatography is composed of a sequence of loading, equilibrating, strips and CIP process.
[Claim 12]
Before the hydrophobic chromatography medium with equilibration buffer containing sodium chloride or ammonium sulfate in 0.45 to 0.55 M concentration of 1.0 to 1.5 M concentration comprising the steps of equilibration; Method for controlling the amount of a step of loading a sample containing the TNFR-Fc fusion protein prepared to have the same salt concentration in the equilibration chromatography medium, a hydrophobic peak in the chromatogram contained in the etanercept 3 - and the former.
[Claim 13]
The method of claim 12, wherein the equilibration buffer containing sodium chloride, the method would with a pH of 6 to 8.5.
[Claim 14]
The method is compared to the sample loading and in the direction in which the amount of peak 3 decreased adjustment to the claim 12.
[Claim 15]
14. The method of claim 13, the method of the content of peak 3 decreased the amount of Peak 3 from 2 to 17% in the sample greater than 20%.
[Claim 16]
14. The method of claim 13, the method of the content of peak 3 decreased the amount of Peak 3 from 10 to 20% in the sample exceeds 45%.