Purification method for novel heparan-N-sulfatase

Abstract

The present invention relates to a method for purifying heparan-N-sulfatase from a heparan-N-sulfatase-containing solution containing one or more impurities, the method comprising the steps of: obtaining an eluate by multi-mode chromatography (MMC); and obtaining a supernatant by caprylic acid precipitation. The heparan-N-sulfatase purification method according to the present invention not only enables highly efficient removal of HCPs (host cell proteins), but also significantly improves the purity and stability of the purified heparan-N-sulfatase.

Description

[Technical Field]

[0001] The present invention relates to a novel method for purifying heparan-N-sulfatase (HNS), which can significantly reduce the content of impurities and significantly improve the stability of HNS, a pharmaceutical composition containing the heparan-N-sulfatase produced by this method, and a method for treating Sanfilippo syndrome using the same.

[0002] [Background technology]

[0003] Mucopolysaccharidoses (MPS), or mucopolysaccharidosis, are a group of rare inherited lysosomal storage disorders caused by a deficiency or insufficiency of specific lysosomal enzymes.

[0004] In normal individuals, where lysosomal enzymes are normally expressed, polysaccharide molecules can be converted into bioavailable substances through metabolic processes by lysosomal enzymes. However, in patients with a deficiency or lack of these lysosomal enzymes, polysaccharide molecules accumulate in cells, tissues, and organelles called lysosomes, which can lead to various forms of disease.

[0005]

[0006] Sanfilippo syndrome is a type of mucopolysaccharidoses, named after the American physician Sanfilippo, who first discovered the disease in 1963. Sanfilippo syndrome, also known as MPS III, is an autosomal recessive genetic disorder characterized by the absence of corneal opacity and mild physical changes such as hepatosplenomegaly and skeletal changes, but by severe and progressive central nervous system symptoms.

[0007] Sanfilippo syndrome is caused by a deficiency of four other enzymes necessary for breaking down polysaccharides, particularly glycosaminoglycans (GAGs), and is classified into MPS IIIA (Sanfilippo A), MPS IIIB (Sanfilippo B), MPS IIIC (Sanfilippo C), and MPS IIID (Sanfilippo D) depending on the enzyme that is deficient. The deficient enzymes and gene locations (genetic map locus) for each Sanfilippo syndrome are as follows:

[0008]

[0009] Type A (MPS IIIA): Heparan N-sulfatase - chromosome 17 (17q25.3)

[0010] Type B (MPS IIIB): N-acetyl-α-D-glucosaminidase - chromosome 17 (17q21)

[0011] Type C (MPS IIIC): Acetyl-CoA:α-glucosaminide-N-acetyltransferase - chromosome 14

[0012] Type D (MPS IIID): N-acetyl-α-D-glucosaminide-6-sulfatase (N-acetyl-α-D-glucosaminide-6-sulfatase) - chromosome 12 (12q14)

[0013]

[0014] As mentioned above, MPS IIIA is caused by a deficiency of heparan-N-sulfatase, an enzyme involved in the degradation of heparan sulfate. Heparan-N-sulfatase hydrolyzes the sulfate moiety attached to the amino group of the glucosamine residue of heparan sulfate. Symptoms of MPS IIIA (Sanfilippo A) typically appear between the ages of 2 and 6, but may be diagnosed after the age of 13. In general, patients with MPS IIIA are known to have significant developmental delays and poor long-term survival.

[0015]

[0016] Currently, there is no approved treatment for MPS IIIA, and only symptomatic treatment is available to alleviate symptoms. Enzyme replacement therapy (ERT), which involves administering exogenously produced heparan-N-sulfatase to MPS IIIA patients, is expected to be very useful in treating MPS IIIA.

[0017] Thus, in order to treat MPS IIIA patients with enzyme replacement therapy using heparan-N-sulfatase, mass production of heparan-N-sulfatase is essential, and the development of recombinant cell culture processes and separation and purification processes for mass production is essential.

[0018]

[0019] For the separation and purification of recombinantly produced heparan-N-sulfatase, a separation and purification process has been developed, including immobilized metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC), cation exchange chromatography (CEX), and anion exchange chromatography (AEX) (see Korean Patent No. 2,286,260).

[0020] In addition, separation and purification processes including anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), hydroxyapatite chromatography (HA), and cation exchange chromatography (CEX) have been developed (see U.S. Patent Publication No. 2012 / 0329133).

[0021]

[0022] However, the existing processes still have problems such as a decrease in the stability of heparan-N-sulfatase during the purification process, and the quality and safety of the final heparan-N-sulfatase are reduced because HCPs (host cell proteins) and other impurities generated when heparan-N-sulfatase is produced using recombinant cells are not completely removed. Therefore, there remains a strong demand for a new heparan-N-sulfatase isolation and purification process that solves these problems.

[0023]

[0024] Summary of the Invention [Problem to be solved by the invention]

[0025] In order to solve the above-mentioned problems of the existing heparan-N-sulfatase isolation and purification process, the present invention provides a new heparan-N-sulfatase purification process and method that can minimize the decrease in stability of heparan-N-sulfatase during the isolation and purification process and can significantly remove HCP and other impurities, a pharmaceutical composition containing heparan-N-sulfatase prepared by such a method, and a method for treating Sanfilippo syndrome using the same. [Means for solving the problem]

[0026]

[0027] To achieve the above object, the present invention provides a method for purifying heparan-N-sulfatase from a heparan-N-sulfatase-containing solution containing one or more impurities, the method comprising the steps of obtaining an eluate by multi-mode chromatography (MMC) and obtaining a supernatant by caprylate precipitation.

[0028]

[0029] The heparan-N-sulfatase is a lysosomal enzyme known in the art as N-sulfoglucosamine sulfohydrolase (SGSH; EC 3.10.1.1; N-sulfoglucosamine sulfohydrolase; 2-desoxy-D-glucoside-2-sulfamate sulfohydrolase; heparin sulfamidase; sulfoglucosamine sulfamidase; sulfamidase; HNS, rhHNS, sulfamidase, rhNS, or rhSGSH), and is particularly preferably derived from a human.

[0030] In the present invention, human heparan-N-sulfatase is understood to include the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 having heparan-N-sulfatase activity but with some amino acid residues deleted at the N-terminus and / or C-terminus, and in particular, the human heparan-N-sulfatase is understood to include the amino acid sequence of SEQ ID NO: 1 having 90% or more, preferably 95% or more, and more preferably 99% or more sequence identity with SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 having some amino acid residues deleted at the N-terminus and / or C-terminus.

[0031] JPEG2025531586000002.jpg105170

[0032] The heparan-N-sulfatase-containing solution containing one or more impurities is preferably, but not limited to, a cell culture medium, and the cell culture medium is preferably a culture medium of host cells capable of recombinantly producing heparan-N-sulfatase.

[0033] The host cell may be any host cell known in the art, and examples thereof include prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus), fungi, such as Aspergillus species, Pichia pastoris, and Saccharomyces cerevisiae. Cells that can be used include, but are not limited to, yeast such as Bacillus cerevisiae, Schizosaccharomyces, and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.

[0034] Preferably, the host cells may be monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, a myeloma cell line, HuT 78 cells or 293 cells.

[0035]

[0036] In addition, the cell culture medium may be clarified by removing cells and cell debris through depth filtration or the like.

[0037]

[0038] In the present invention, the multimode chromatography may be characterized by simultaneously performing cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC), but is not limited thereto.

[0039] The multimode chromatography allows for the removal of various impurities, particularly selective elution of enzymes with different M6P (Mannose-6-phosphate) contents. By removing impurities with properties similar to those of heparan-N-sulfatase, not only can the purity of the final purified heparan-N-sulfatase be improved, but its stability can also be significantly increased.

[0040]

[0041] In the present invention, the resin for multimode chromatography may be any resin capable of simultaneously performing cation exchange chromatography and hydrophobic interaction chromatography. For example, Capto MMC and Capto adhere resins having the structures of Chemical Formula 1 and Chemical Formula 2, respectively, may be used, but are not limited thereto.

[0042] JPEG2025531586000003.jpg68170

[0043] When performing multimode chromatography according to the present invention, the pH of the buffer (solution) used for loading and equilibration may be, but is not limited to, 4.5±1.0, preferably 4.5±0.5, and more preferably 4.5±0.1.

[0044] The buffer contains 200±100 mM, preferably 200±50 mM, more preferably 200±20 mM NaCl, and 5 mM to 100 mM, preferably 10 mM to 50 mM, more preferably 15 mM to 30 mM of common acetate, phosphate, citrate, histidine, glycine, Tris buffer, etc., can be used without limitation, but is not limited thereto.

[0045]

[0046] In the step of performing the multimode chromatography according to the present invention, the buffer used in the washing and elution steps after heparan-N-sulfatase is bound to the resin may be, but is not limited to, 5 mM to 100 mM, preferably 10 mM to 50 mM, and more preferably 15 mM to 30 mM of common histidine, glycine, acetate, phosphate, citrate, Tris buffer, etc.

[0047] The pH of the buffer used in the washing step may be between 5.0 and 8.0, preferably between 6.0 and 7.5, more preferably between 6.3 and 7.0.

[0048] In particular, the washing step according to the present invention may be characterized by including two or more washing steps, and in this case, the pH of the buffer used in the subsequent washing steps may be higher than that of the first washing step.

[0049] Specifically, when two washing steps are included, the pH of the first washing step may be, but is not limited to, 6.0 to 6.6, preferably 6.2 to 6.6, and more preferably 6.4 to 6.5, and the pH of the second washing step may be, but is not limited to, 6.6 to 7.2, preferably 6.7 to 7.0, and more preferably 6.75 to 6.9.

[0050] The pH of the buffer used in the elution step may be, but is not limited to, 6.6 to 9.0, preferably 6.9 to 8.8, and more preferably 8.0 to 8.4.

[0051]

[0052] The step of obtaining a supernatant by caprylic acid precipitation according to the present invention has the effect of efficiently removing HCPs having a relatively low pH, thereby increasing the efficiency of impurity removal.

[0053] The caprylic acid precipitation is characterized in that it is carried out by adding caprylic acid or a salt thereof, preferably sodium caprylate, to a solution to be treated, specifically, an eluate from multimode chromatography or an eluate from affinity chromatography, to a concentration of 1 mM to 50 mM, preferably 5 mM to 30 mM, more preferably 8 mM to 12 mM, under conditions of pH 3.5 to 6.0, preferably 4.0 to 5.5, more preferably 4.3 to 5.0.

[0054]

[0055] The method for purifying heparan-N-sulfatase according to the present invention may further include a step of performing affinity chromatography to obtain an eluate, between the step of performing multimode chromatography to obtain an eluate and the step of performing caprylic acid precipitation to obtain a supernatant.

[0056] Thus, the method can include the steps of obtaining an eluate by performing multimodal chromatography; obtaining an eluate by performing affinity chromatography; and obtaining a supernatant by performing caprylic acid precipitation.

[0057]

[0058] The affinity chromatography step of the present invention uses a resin that specifically binds only to heparan-N-sulfatase to remove impurities that have similar physicochemical properties to heparan-N-sulfatase, such as pI and hydrophobicity, particularly HCPs such as cathepsin X, thereby enhancing the impurity removal effect.

[0059] The resin used in the affinity chromatography according to the present invention is preferably, but not limited to, heparin sepharose or blue sepharose.

[0060] The pH of the buffer (solution) used for loading, equilibration, washing, and elution may be, but is not limited to, 4.5±1.0, preferably 4.5±0.5, and more preferably 4.5±0.1. Normal acetate, phosphate, citrate, histidine, glycine, Tris buffer, etc., at 5 mM to 100 mM, preferably 10 mM to 50 mM, and more preferably 15 mM to 30 mM, may be used without limitation, but is not limited to these.

[0061] In particular, the buffer used for washing may contain 150±100 mM, preferably 150±50 mM, more preferably 150±20 mM NaCl, and the buffer used for elution may contain 300±100 mM, preferably 300±50 mM, more preferably 300±20 mM NaCl.

[0062]

[0063] The method for purifying heparan-N-sulfatase according to the present invention may further include, before the step of obtaining an eluate by performing multimode chromatography, a step of performing a primary anion exchange chromatography (AEX) to obtain an eluate; a solvent / detergent treatment step; and a step of performing a cation exchange chromatography (CEX) to obtain an eluate.

[0064] Thus, the method may include the steps of: performing a primary anion exchange chromatography to obtain an eluate; a solvent / detergent treatment step; performing a cation exchange chromatography to obtain an eluate; performing multimode chromatography to obtain an eluate; performing affinity chromatography to obtain an eluate; and performing caprylic acid precipitation to obtain a supernatant.

[0065]

[0066] The primary anion exchange chromatography step is a step for maximizing recovery of heparan-N-sulfatase from the cell culture medium. The resin used in the primary anion exchange chromatography according to the present invention may be either a weak anion exchange resin or a strong anion exchange resin. Examples of resins that may be used include, but are not limited to, weak anion exchange resins such as DEAE Sepharose, and strong anion exchange resins such as Q Sepharose, Fractogel TMAE(M), Fractogel TMAE(S), and Poros XQ.

[0067]

[0068] When performing primary anion exchange chromatography according to the present invention, the pH of the buffer (solution) used for loading, equilibration, washing, and elution may be, but is not limited to, 7.5±1.0, preferably 7.5±0.7, and more preferably 7.5±0.5. Examples of suitable buffers include, but are not limited to, 5 mM to 100 mM, preferably 10 mM to 70 mM, and more preferably 40 mM to 60 mM Tris, acetate, phosphate, citrate, histidine, and glycine buffers. The elution buffer may contain 200±100 mM, preferably 200±70 mM, and more preferably 200±50 mM NaCl.

[0069]

[0070] The solvent / detergent treatment step is a process for virus inactivation, and preferably uses a polysorbate, specifically, polysorbate 20 and / or 80 in combination with TnBP (Tri-n-butyl-phosphate), but is not limited thereto.

[0071] The solvent / detergent treatment step is preferably carried out at a pH of 7.5±1.0, preferably 7.5±0.5, more preferably 7.5±0.2, and at 18°C ​​to 28°C, preferably 20°C to 25°C, for at least 1 hour and up to 6 hours.

[0072]

[0073] The cation exchange chromatography step is a step for maximizing recovery of heparan-N-sulfatase while simultaneously removing process-related impurities generated in the solvent / detergent treatment step and impurities such as HCP.

[0074] For the cation exchange chromatography according to the present invention, either a weak cation exchange resin or a strong cation exchange resin can be used. Examples of resins that can be used include, but are not limited to, carboxymethyl (CM), sulfopropyl (SP), or methyl sulfonate (S) resins, preferably SP Sepharose.

[0075] When performing cation exchange chromatography according to the present invention, the pH of the solution (buffer) used for loading, equilibration, washing, and elution may be, but is not limited to, 4.5±0.7, preferably 4.5±0.5, and more preferably 4.5±0.1. Conventional acetate, phosphate, citrate, histidine, glycine, Tris buffers, etc., having concentrations of 5 mM to 100 mM, preferably 10 mM to 50 mM, and more preferably 15 mM to 30 mM, may be used without limitation, but are not limited to these.

[0076] Additionally, the loading and equilibration buffer may contain 100±70 mM, preferably 100±50 mM, more preferably 100±20 mM NaCl, and the elution buffer may contain 200±100 mM, preferably 200±50 mM, more preferably 200±20 mM NaCl.

[0077]

[0078] The method for purifying heparan-N-sulfatase according to the present invention may further include, after the step of obtaining a supernatant by caprylic acid precipitation, a step of obtaining an eluate by performing a secondary anion exchange chromatography (AEX).

[0079] Thus, the method may include the steps of: performing a first anion exchange chromatography to obtain an eluate; a solvent / detergent treatment step; performing a cation exchange chromatography to obtain an eluate; performing multimode chromatography to obtain an eluate; performing affinity chromatography to obtain an eluate; performing caprylic acid precipitation to obtain a supernatant; and performing a second anion exchange chromatography to obtain an eluate.

[0080]

[0081] The secondary anion exchange chromatography step is a step for increasing purity by removing process-related impurities such as heparin, caprylic acid, and solvents / surfactants. Preferably, a strong anionic resin such as Q Sepharose, Fractogel TMAE(M), Fractogel TMAE(S), or Poros XQ is used, but is not limited to these.

[0082]

[0083] When performing the secondary anion exchange chromatography according to the present invention, the pH of the solution (buffer) used for loading, equilibration, washing, and elution may be, but is not limited to, 7.5±1.0, preferably 7.5±0.7, and more preferably 7.5±0.5. Examples of common buffers that can be used without limitation include 5 mM to 100 mM, preferably 10 mM to 50 mM, and more preferably 15 mM to 30 mM histidine, glycine, acetate, phosphate, citrate, and Tris buffers.

[0084] The equilibration buffer may contain 50±20 mM, preferably 50±10 mM, and more preferably 50±5 mM NaCl, and the elution buffer may contain 150±50 mM, preferably 150±30 mM, and more preferably 150±10 mM NaCl.

[0085]

[0086] The method for purifying heparan-N-sulfatase according to the present invention may further include a nanofiltration step after the step of obtaining an eluate by performing the second anion exchange chromatography.

[0087] Thus, the method may include a step of obtaining an eluate by performing a first anion exchange chromatography; a solvent / detergent treatment step; a step of obtaining an eluate by performing a cation exchange chromatography; a step of obtaining an eluate by performing a multimode chromatography; a step of obtaining an eluate by performing affinity chromatography; a step of obtaining a supernatant by performing caprylic acid precipitation; and a step of obtaining an eluate by performing a second anion exchange chromatography; and a nanofiltration step.

[0088]

[0089] The nanofiltration step is aimed at removing viruses and can usually be carried out using a nanofilter for virus removal.

[0090]

[0091] The method for purifying heparan-N-sulfatase according to the present invention may further include an ultrafiltration / diafiltration (UF / DF) step at one or more positions selected from the following: before the step of obtaining an eluate by performing primary anion exchange chromatography; between the step of obtaining a supernatant by performing caprylic acid precipitation and the step of obtaining an eluate by performing secondary anion exchange chromatography; and after the nanofiltration step.

[0092]

[0093] Therefore, when an ultrafiltration / diafiltration step is included at every position between the step of obtaining a supernatant by caprylic acid precipitation and the step of obtaining an eluate by performing a second anion exchange chromatography before the step of obtaining an eluate by performing a first anion exchange chromatography, the overall process is as follows:

[0094] primary ultrafiltration / diafiltration (UF / DF) stage;

[0095] Obtaining an eluate by performing a primary anion exchange chromatography (AEX);

[0096] solvent / surfactant treatment stage;

[0097] Obtaining an eluate by performing cation exchange chromatography (CEX);

[0098] obtaining an eluate by performing multi-mode chromatography;

[0099] Obtaining an eluate by performing affinity chromatography;

[0100] Obtaining a supernatant by caprylic acid precipitation;

[0101] secondary ultrafiltration / diafiltration (UF / DF) stage;

[0102] Obtaining an eluate by performing a second anion exchange chromatography (AEX);

[0103] a nanofiltration stage; and

[0104] A third ultrafiltration / diafiltration (UF / DF) stage may be included.

[0105]

[0106] The primary ultrafiltration / diafiltration step is a step for concentrating the cell culture medium, preferably the culture medium that has undergone a clarification process, and exchanging the buffer, thereby reducing the volume of the culture medium to be treated, shortening the column loading time, and improving the convenience of subsequent processes.

[0107] The cut-off value of the membrane used in the first ultrafiltration / diafiltration step may be, but is not limited to, 10 kDa to 100 kDa, preferably 20 kDa to 70 kDa, and more preferably 30 kDa to 50 kDa.

[0108] The pH of the buffer used in the first ultrafiltration / diafiltration step may be, but is not limited to, 7.5±1.0, preferably 7.5±0.7, and more preferably 7.5±0.5. Examples of common buffers that may be used without limitation include tris, histidine, glycine, acetate, phosphate, and citrate buffers, each having a pH of 5 mM to 100 mM, preferably 10 mM to 80 mM, and more preferably 30 mM to 70 mM.

[0109]

[0110] The second ultrafiltration / diafiltration step is located between the caprylic acid precipitation step and the second anion exchange chromatography step, and is a step for pH and conductivity conversion.

[0111] The cut-off value of the membrane used in the second ultrafiltration / diafiltration step may be, but is not limited to, 10 kDa to 100 kDa, preferably 20 kDa to 70 kDa, and more preferably 30 kDa to 50 kDa.

[0112] The pH of the buffer used in the second ultrafiltration / diafiltration step may be, but is not limited to, 7.5±1.0, preferably 7.5±0.7, and more preferably 7.5±0.5. Examples of buffers that can be used without limitation include common histidine, glycine, acetate, phosphate, citrate, and Tris buffers, each having a concentration of 5 mM to 50 mM, preferably 10 mM to 40 mM, and more preferably 15 mM to 30 mM.

[0113] When the conductivity of the secondary ultrafiltration / diafiltration step reaches 10 mS / cm or less, preferably 8 mS / cm or less, and more preferably 6 mS / cm or less, the process solution can be recovered and the pH adjusted to 7.5±1.0.

[0114]

[0115] The third ultrafiltration / diafiltration step follows nanofiltration and is a final step for highly concentrated and buffer exchange.

[0116] The cut-off value of the membrane used in the third ultrafiltration / diafiltration step may be, but is not limited to, 10 kDa to 100 kDa, preferably 20 kDa to 70 kDa, and more preferably 30 kDa to 50 kDa.

[0117] The pH of the buffer used in the third ultrafiltration / diafiltration step may be, but is not limited to, 8.0±1.0, preferably 8.0±0.7, and more preferably 8.0±0.5. Examples of buffers that may be used without limitation include common histidine, glycine, acetate, phosphate, citrate, and Tris buffers, each having a concentration of 1 mM to 20 mM, preferably 2 mM to 10 mM, and more preferably 3 mM to 8 mM.

[0118]

[0119] Unless otherwise specified, all purification steps according to the present invention are carried out at room temperature, specifically between 15°C and 30°C, preferably between 18°C ​​and 25°C.

[0120] [Brief explanation of the drawings]

[0121] [Figure 1] FIG. 1 shows a chromatogram of the heparan-N-sulfatase purification process using primary anion exchange chromatography.

[0122] [Figure 2] FIG. 1 shows the results of SDS-PAGE of each stage solution of primary anion exchange chromatography.

[0123] [Figure 3] FIG. 1 shows a chromatogram of the heparan-N-sulfatase purification process using cation exchange chromatography.

[0124] [Figure 4] FIG. 1 shows the results of SDS-PAGE of each stage solution of cation exchange chromatography.

[0125] [Figure 5] FIG. 1 shows chromatograms of the eluate in multimode chromatography according to pH.

[0126] [Figure 6] FIG. 1 shows the results of SDS-PAGE depending on the pH of the eluate in multimode chromatography.

[0127] [Figure 7] FIG. 1 shows elution results in affinity chromatography.

[0128] [Figure 8] FIG. 1 is a diagram showing the overall steps of a method for purifying heparan-N-sulfatase according to one embodiment of the present invention.

[0129]

[0130] DETAILED DESCRIPTION OF THE INVENTION

[0131] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0132] Furthermore, unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs.

[0133]

[0134] [Example]

[0135] Example 1: Purification of cell culture medium

[0136] The cell culture medium containing recombinantly produced heparan-N-sulfatase and one or more impurities was subjected to depth filtration using a depth filter.

[0137] Specifically, distilled water was thoroughly passed through a Merck Cat. No. MD0HC054H1 or Cat. No. MX0HC027H1 deep layer filter and a Poll Cat. No. NP6PDK516 or Cat. No. NP5LPDD16 deep layer filter to remove the internal liquid of the filter, and then 50 mM Tris buffer (pH 7.5) was passed through the filter to equilibrate the filter. The cell culture solution was then filtered at a pressure of ≦2.0 bar.

[0138] The recovery rate was over 95%, and the HCP removal rate was approximately 0.4 LRV (Log reduction value).

[0139]

[0140] Example 2: Primary Ultrafiltration / Diafiltration (UF / DF)

[0141] The clarified cell culture medium was subjected to a first UF / DF process for concentration and buffer exchange.

[0142] Specifically, a Merck Pellicon 3 Ultracel C screen (Cat No. P3C030C01) with a cutoff value of 30 kDa to 50 kDa or a Poll Omega T-series 30 kDa (OS030T12) membrane was used, and equilibrated while flowing 50 mM Tris buffer, pH 7.5, to concentrate the culture filtrate 10-fold compared to the initial volume. After that, buffer exchange was performed with 50 mM Tris buffer, pH 7.5, at a rate of 3DV (diafiltration volume) or more, and the resulting concentrate was concentrated to 13-fold compared to the initial volume and recovered.

[0143] The concentration ratio was 7-13 times, and the buffer exchange volume was 3 DV or more. The final recovery rate was 95% or more, and the HCP removal rate was approximately 0.1 LRV (Log reduction value) or more.

[0144]

[0145] Example 3: Primary Anion Exchange Chromatography

[0146] 3.1 Basic Anion Exchange Chromatography (AEX) Process Validation

[0147] The solution obtained through the first ultrafiltration / diafiltration was subjected to anion exchange chromatography using strong anion exchange resins Q Sepharose 6 Fast Flow, Fractogel EMD TMAE (M), and Poros XQ, and weak anion exchange resin DEAE Sepharose Fast Flow.

[0148] Specifically, the resin was cleaned in place (CIP) with 0.5N NaOH for 5 CV (column volume), equilibrated with 15 CV of 50mM Tris equilibration buffer (EQ buffer), pH 7.5±0.5, and loaded with the primary UF / DF solution. Then, 5 CV of 50mM Tris equilibration buffer, pH 7.5±0.5, was injected for re-equilibration. 5 CV of 50mM Tris elution buffer, pH 7.5±0.5, 200±50mM NaCl, 50mM Tris elution buffer was injected. Collection of the eluate was initiated when the UV signal reached 50mAu, and only 2.5 CV (132.5mL) was collected.

[0149] Then, 5 CV of pH 7.5±0.5, 2000±200 mM NaCl, 50 mM Tris column washing buffer (CW buffer) was injected, followed by 5 CV of clean-up with 0.5 N NaOH, followed by equilibration by flowing 15 CV of equilibration buffer.

[0150] Anion exchange chromatography was performed under the above conditions. When unbound, CW, and NaOH were injected in addition to the elution, UV signals were detected in the chromatogram, confirming the removal of impurities (see Figure 1). SDS-PAGE confirmed that the eluate (lane 3) had at least as many impurities removed as lanes 2 and 4 compared to the load sample (lane 1) (see Figure 2).

[0151] Overall, the yield was approximately 90% or higher, the purity was approximately 70% or higher, and the HCP removal capacity was approximately 0.2 LRV or higher. Furthermore, all of the resins used had similar recovery rates during process optimization, so Q Sepharose was preferred when considering process robustness and economy.

[0152]

[0153] 3.2 Optimization of the Anion Exchange Chromatography (AEX) Step

[0154] The performance of the AEX step was tested at different step pHs and NaCl concentrations in the elution buffer, as described in Table 1.

[0155] Specifically, the test was performed with a difference of ±0.5 based on the initial experimental pH (pH 7.5), and stepwise elution was performed at NaCl concentrations of 100 mM, 150 mM, and 200 mM to confirm the elution performance of the target protein, heparan-N-sulfatase, at each pH depending on the NaCl concentration in the elution buffer.

[0156] [Table 1]

[0157] The results confirmed that heparan-N-sulfatase bound to the resin over the entire pH range from 7.0 to 8.0, and a large proportion of heparan-N-sulfatase was eluted with 100 mM NaCl. Furthermore, most of the heparan-N-sulfatase was recovered in the 150 mM NaCl fraction. Therefore, we confirmed that satisfactory results were obtained when the NaCl concentration in the elution buffer was 100 mM, preferably 150 mM NaCl or higher.

[0158]

[0159] Example 4: Solvent / Surfactant Treatment

[0160] The eluate obtained from the first AEX was subjected to solvent / detergent treatment (S / D treatment) to inactivate viruses.

[0161] Specifically, an S / D stock solution containing 1% Polysorbate 80 and 0.3% TnBP was added to the eluate obtained from the first AEX, and the mixture was stirred at 20 to 25°C for 1 hour.

[0162] The S / D treatment was carried out for up to 6 hours under three pH conditions: 7.3, 7.5, and 7.7, and it was confirmed that sufficient S / D treatment was possible within these pH ranges. However, if the treatment time exceeds 6 hours, the product becomes cloudy and there is a risk of process risk. Therefore, it is preferable to adjust the treatment time to within 6 hours.

[0163]

[0164] Example 5: Cation Exchange Chromatography

[0165] 5.1 Basic Cation Exchange Chromatography (CEX) Process Validation

[0166] To maximize recovery of heparan-N-sulfatase from the S / D-treated solution and to remove impurities, particularly process impurities such as solvents and detergents, cation exchange chromatography was performed using SP Sepharose Fast Flow resin.

[0167] Specifically, the resin was cleaned with 0.5 N NaOH for 5 CV, then equilibrated by running 10 CV of equilibration buffer (EQ buffer) containing 100 mM NaCl and 20 mM sodium acetate (SA) at pH 4.5 ± 0.1, and the S / D-treated solution was loaded.

[0168] This was followed by a 5 CV injection of 20 mM sodium acetate equilibration buffer, 100 ± 20 mM NaCl, pH 4.5 ± 0.1, to re-equilibrate the column, and a 5 CV injection of 200 ± 20 mM NaCl, pH 4.5 ± 0.1, to eluate the column. After collecting the initial 3 CV of eluate, the remaining 2 CV were discarded as waste.

[0169] Then, 5 CV of pH 4.5±0.1, 2000±200 mM NaCl, 20 mM sodium acetate column wash buffer (CW buffer) was injected, followed by 5 CV of clean-up with 0.5 N NaOH, followed by equilibration with 10 CV of equilibration buffer.

[0170] Cation exchange chromatography was performed under the above conditions. When unbound, CW, and NaOH were injected in addition to the elution, UV signals were detected in the chromatogram, confirming the removal of impurities (see Figure 3). This was confirmed by SDS-PAGE. Furthermore, the eluate (lane 3) was confirmed to have removed at least as many impurities as lanes 2 and 4 compared to the load sample (lane 1) (see Figure 4), demonstrating an approximately 20% increase in purity compared to the primary anion exchange chromatography eluate.

[0171] Overall, the yield was about 90% or more, the purity was about 93% or more, and the HCP removal capacity was about 1.2 LRV or more.

[0172]

[0173] 5.2 Optimization of the Cation Exchange Chromatography (CEX) Step

[0174] The performance of the CEX step was tested at different step pHs and NaCl concentrations in the elution buffer, as described in Table 2.

[0175] Specifically, the test was carried out in the range of 4.0 to 5.4, based on the initial experimental pH (pH 4.5). To confirm the elution performance of the target protein, heparan-N-sulfatase, at each pH, ​​depending on the NaCl concentration in the elution buffer, the test was also carried out at concentrations of 2 mM to 300 mM.

[0176] [Table 2]

[0177] As a result, as shown in Table 3, it was confirmed that the optimal NaCl concentration in the elution buffer was 50 (at pH 5.4) to 210 mM NaCl (at pH 4.6 or lower).

[0178] [Table 3]

[0179]

[0180] Example 6: Multimode Chromatography

[0181] To more efficiently remove impurities from the CEX eluate and selectively purify heparan-N-sulfatase containing M6P (mannose 5-phosphate), multimode chromatography was performed using Capto MMC resin, which allows simultaneous cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC).

[0182]

[0183] Specifically, the resin was cleaned with 0.5 N NaOH for 5 CV, then equilibrated by running 10 CV of pH 4.5, 200 mM NaCl, 20 mM sodium acetate (SA) equilibration buffer (EQ buffer), and the CEX eluate was loaded.

[0184] The column was then re-equilibrated by injecting 5 CV of 20 mM histidine equilibration buffer at pH 5.5±0.1, and then washed with 5 CV of 20 mM histidine primary wash buffer at pH 6.5±0.1. The column was then washed with 10 CV of 20 mM histidine secondary wash buffer at pH 6.85±0.1.

[0185] When the washing buffer had a pH of 6.5 to 6.9, the solution after washing contained a large amount of HCP, but the content of heparan-N-sulfatase was somewhat low. Therefore, by dividing the washing step into two stages, with the pH of the first washing buffer being 6.45 and the second washing buffer being 6.85, HCP could be removed more efficiently and the recovery rate of heparan-N-sulfatase could be increased.

[0186] Then, 10 CV of 20 mM histidine elution buffer at pH 6.8±0.1 to 8.3±0.1 was injected, and the entire eluate was collected.

[0187] As a result, it was confirmed that the target protein, heparan-N-sulfatase, began to be recovered when the elution buffer pH was 6.7, and that heparan-N-sulfatase could be properly recovered at all pH levels up to 8.4 (see Figure 5).

[0188] Furthermore, as the pH of the elution buffer increases, the pI range of heparan-N-sulfatase gradually increases. While there is little difference in the FGly content among the fractions, the M6P content decreases with increasing elution pH. That is, heparan-N-sulfatases with low pI values ​​contain a large amount of M6P (see Figure 6 and Table 4).

[0189] [Table 4]

[0190] Then, 5 CV of pH 7.5, 2,000 mM NaCl, 20 mM histidine column washing buffer (CW buffer) was injected, followed by 5 CV of CIP with 0.5 N NaOH, followed by 10 CV of equilibration buffer to achieve equilibration.

[0191] Overall, the yield was about 70-90% or more, the purity was about 97% or more, and the HCP removal capacity was about 1.8 LRV or more.

[0192]

[0193] Example 7: Affinity Chromatography

[0194] 7.1 Basic Affinity Chromatography Process Validation

[0195] To remove the most common impurities in the MMC eluate, such as cathepsin X, affinity chromatography was performed using heparin sepharose or blue sepharose resin.

[0196] Specifically, the resin was cleaned with 0.1 N NaOH for 5 CV, then equilibrated by running 10 CV of pH 4.5, 200 mM NaCl, 20 mM sodium acetate (SA) equilibration buffer (EQ buffer), and the MMC eluate was loaded.

[0197] The column was then re-equilibrated with a 5 CV injection of 20 mM sodium acetate (SA) equilibration buffer, pH 4.5 ± 0.1, and washed a second time with a 10 CV injection of 150 ± 20 mM NaCl, 20 mM sodium acetate wash buffer, pH 4.5 ± 0.1.

[0198] Next, 10 CV of elution buffer (pH 4.5 ± 0.1, 300 ± 20 mM NaCl, 20 mM sodium acetate) was injected, and all eluate was collected. Five CV of column wash buffer (CW buffer) (pH 4.5 ± 0.1, 2,000 ± 200 mM NaCl, 20 mM sodium acetate) was then injected. After 5 CV of clean-up with 0.1 N NaOH, the column was equilibrated by running 10 CV of equilibration buffer.

[0199] As a result of affinity chromatography performed according to the above steps, it was confirmed that heparan-N-sulfatase was purified with very high purity, as shown in FIG.

[0200] Overall, the yield was about 90% or more, the purity was about 99% or more, and the HCP removal capacity was about 1.0 LRV or more.

[0201]

[0202] 7.2 Affinity Chromatography Process Optimization

[0203] The performance of the affinity chromatography step was tested at different step pHs and NaCl concentrations in the elution buffer, as described in Table 5.

[0204] Specifically, based on the initial condition experiment, the pH was set to pH 4.5±0.1, the center value of the NaCl concentration of the washing buffer was set to 150 mM, and the center value of the NaCl concentration of the elution buffer was set to 300 mM.

[0205] [Table 5]

[0206] As a result, as shown in Table 5, it was confirmed that HCP was efficiently removed over the entire range of NaCl concentrations in the washing buffer from 130 mM to 170 mM and in the elution buffer from 280 mM to 320 mM, and that heparan-N-sulfatase could be obtained with high purity.

[0207]

[0208] Example 8: Caprylic Acid Precipitation

[0209] A caprylic acid precipitation step was performed to precipitate and remove the relatively low pI HCPs.

[0210] Specifically, a stock solution of sodium caprylate at a concentration of 500 mM was prepared and added to the affinity chromatography eluate to a concentration of 10 mM.

[0211] Thereafter, precipitation was allowed to occur for 1 to 3 hours under conditions of a pH of 4.5±0.1 and a temperature of 20° C. to 25° C. while stirring at a speed of 200±20 rpm, and the precipitated impurities were removed by filtration.

[0212]

[0213] As a result, as shown in Table 6, it was confirmed that the content of most impurities, HCPs, was significantly reduced when the caprylic acid precipitation step was included compared to when the caprylic acid precipitation step was not included. In particular, cathepsin X, a major HCP, was barely detected, and lysosomal Pro-X carboxypeptidase was also significantly reduced.

[0214] [Table 6]

[0215] Overall, the yield was about 90% or more, the purity was about 99% or more, and the HCP removal capacity was about 1.0 LRV or more.

[0216]

[0217] Example 9: Secondary Ultrafiltration / Diafiltration (UF / DF)

[0218] The supernatant of the caprylic acid precipitate was subjected to secondary ultrafiltration / diafiltration to adjust the pH and conductivity to those suitable for secondary anion exchange chromatography. Through secondary ultrafiltration / diafiltration, the concentration factor was adjusted to approximately 2x, the buffer exchange volume to 3DV or more, the pH to 4.5 to approximately 7.5, and the conductivity to 25mS / cm to 6mS / cm or less.

[0219] Specifically, a Merck Pellicon 3 Ultracel C screen (Cat No. P3C030C01) membrane with a cutoff value of 30 kDa to 50 kDa was used, and equilibrated while passing a pH 7.5, 20 mM histidine buffer. The supernatant of the caprylic acid precipitate was concentrated to twice its initial volume, and then buffer exchanged with a pH 7.5, 20 mM histidine buffer for at least 2 DV. When the conductivity reached 6 mS / cm or less, the process solution was recovered, and the pH of the recovered process solution was adjusted to 7.5.

[0220]

[0221] Example 10: Secondary Anion Exchange Chromatography

[0222] In order to remove process-related impurities such as heparin, caprylic acid, solvents, and detergents from the second ultrafiltration / diafiltration solution, a second anion exchange chromatography was performed using strong anion exchange resins, Fractogel EMD TMAE(M) and Fractogel EMD TMAE(S).

[0223] Specifically, the resin was cleaned with 0.5N NaOH for 5 CV, equilibrated with 50 mM NaCl, 20 mM histidine equilibration buffer (pH 7.5 ± 0.5) for 15 CV, and then loaded with the secondary UF / DF solution. This was followed by re-equilibration with 10 CV of 50 mM NaCl, 20 mM histidine equilibration buffer (pH 7.0 ± 0.5), and then 7 CV of 150 mM NaCl, 20 mM histidine elution buffer (pH 7.0 ± 0.5).

[0224] Then, 5 CV of pH 7.0±0.5, 2000±200 mM NaCl, 20 mM histidine column washing buffer (CW buffer) was injected, followed by 5 CV of CIP with 0.5 N NaOH, followed by equilibration by flowing 10 CV of equilibration buffer.

[0225] Overall, the yield was approximately 90% or more, and the purity was approximately 99% or more, with almost no difference depending on the resin.

[0226] Furthermore, the eluate NaCl concentration was fixed at 150 mM, and the pH was varied from 6.8 to 7.7. As a result, as shown in Table 7, it was confirmed that heparan-N-sulfatase could be efficiently purified in all cases.

[0227] [Table 7]

[0228] Furthermore, we fixed the pH at 7.5 and varied the NaCl concentration of the eluate from 130 mM to 150 mM. As a result, as shown in Table 8, we confirmed that heparan-N-sulfatase could be efficiently purified in all cases.

[0229] [Table 8]

[0230]

[0231] Example 11: Nanofiltration

[0232] Finally, to remove the virus by filtration, nanofiltration was performed using nanofilters from Merck, Asahi Co., Ltd., and Sartorius Co. There was no difference between manufacturers, but the recovery rate of the Sartorius nanofilter was slightly higher, and the overall yield was over 90%.

[0233]

[0234] Example 12: Tertiary Ultrafiltration / Diafiltration (UF / DF)

[0235] For final formulation, a third round of ultrafiltration / diafiltration was performed to achieve high concentration and buffer exchange.

[0236] Specifically, a Merck Pellicon 3 Ultracel C screen (Cat No. P3C030C01) membrane with a cutoff value of 30 kDa to 50 kDa was used, and equilibrated with a pH 8.2, 93.75 mM NaCl, 4.2 mM histidine buffer, and the secondary AEX eluate was concentrated to a concentration of approximately 5 mg / mL and collected.

[0237]

[0238] The overall process flow according to a specific embodiment of the present invention is as shown in FIG.

[0239]

[0240] The present invention has been described above with reference to preferred embodiments thereof. Those skilled in the art will recognize that the present invention can be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the above-described embodiments should be considered as illustrative rather than restrictive. The scope of the present invention is defined by the appended claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

[0241] [Industrial Applicability]

[0242] The method for purifying heparan-N-sulfatase according to the present invention can significantly improve the purity, safety, and stability of the produced heparan-N-sulfatase by efficiently removing HCP and other impurities, and is highly suitable for the efficient production of heparan-N-sulfatase for use in enzyme replacement therapy.

[0243] [Sequence List Free Text]

[0244] Electronic file attached.

Claims

1. 1. A method for purifying heparan-N-sulfatase from a heparan-N-sulfatase-containing solution containing one or more impurities, the method comprising: Obtaining an eluate by performing multi-mode chromatography (MMC); and Obtaining a supernatant by caprylic acid precipitation; A method for purifying heparan-N-sulfatase, comprising:

2. 2. The method for purifying heparan-N-sulfatase according to claim 1, wherein the sulfatase-containing solution containing one or more impurities is a cell culture medium.

3. 2. The method for purifying heparan-N-sulfatase according to claim 1, wherein the multimode chromatography (MMC) comprises simultaneously performing cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC).

4. 4. The method for purifying heparan-N-sulfatase according to claim 3, wherein the resin used in the multimode chromatography (MMC) is Capto MMC or Capto adhere.

5. 4. The method for purifying heparan-N-sulfatase according to claim 3, wherein the multimode chromatography (MMC) comprises two or more washing steps after heparan-N-sulfatase is bound to a resin.

6. 6. The method for purifying heparan-N-sulfatase according to claim 5, wherein the pH of the buffer used in the second washing step is higher than that of the first washing step.

7. 2. The method for purifying heparan-N-sulfatase according to claim 1, wherein the caprylic acid precipitation is carried out by adding caprylic acid so that the concentration of caprylic acid becomes 1 mM to 20 mM.

8. Between the step of obtaining an eluate by performing multimode chromatography (MMC) and the step of obtaining a supernatant by performing caprylic acid precipitation, Obtaining an eluate by performing affinity chromatography; The method for purifying heparan-N-sulfatase according to claim 1, further comprising:

9. Before obtaining an eluate by performing multimode chromatography (MMC), Obtaining an eluate by performing a primary anion exchange chromatography (AEX); a solvent / detergent treatment step; and Obtaining an eluate by performing cation exchange chromatography (CEX); The method for purifying heparan-N-sulfatase according to claim 8, further comprising:

10. The method for purifying heparan-N-sulfatase according to claim 9, wherein the resin used in the primary anion exchange chromatography (AEX) is a weak anion exchange resin or a strong anion exchange resin.

11. After the step of obtaining the supernatant by caprylic acid precipitation, Obtaining an eluate by performing a second anion exchange chromatography (AEX); The method for purifying heparan-N-sulfatase according to claim 9, further comprising:

12. After the step of obtaining an eluate by performing the second anion exchange chromatography (AEX), nanofiltration stage; The method for purifying heparan-N-sulfatase according to claim 11, further comprising:

13. before obtaining an eluate by performing a first anion exchange chromatography; between the steps of obtaining a supernatant by performing caprylic acid precipitation and obtaining an eluate by performing a second anion exchange chromatography; and After the nanofiltration stage: an ultrafiltration / diafiltration (UF / DF) step at one or more locations selected from: The method for purifying heparan-N-sulfatase according to claim 12, further comprising:

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