New method for purifying heparan-N-sulfatase

Abstract

The present invention relates to a method of purifying heparan-N-sulfatase from a solution comprising heparan-N-sulfatase comprising at least one impurity, the method comprising performing multi-mode chromatography (MMC) to obtain an eluate; and performing caprylate precipitation to obtain a supernatant. According to the method disclosed by the invention, HCP (host cell protein) can be very effectively removed, and the purity and the stability of the purified heparan-N-sulfatase are greatly improved.

Description

Technical Field

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

[0002] Mucopolysaccharidoses (MPS) are rare, inherited lysosomal storage diseases caused by the deficiency or absence of certain lysosomal enzymes.

[0003] Normal people who normally express lysosomal enzymes convert polysaccharide molecules into substances that can be used in the body through the metabolic process of lysosomal enzymes. However, patients who lack or are deficient in lysosomal enzymes suffer from various types of diseases due to the accumulation of polysaccharide molecules in cells, tissues, and organelles called lysosomes.

[0004] Sanfilippo syndrome, named after the American doctor "Sanfilippo" who first discovered the disease in 1963, is a mucopolysaccharidosis. Sanfilippo syndrome is an autosomal recessive disease, called MPS III, and is characterized by clinically absent corneal opacities, and mild physical changes such as hepatosplenomegaly or changes in the skeletal system, but very severe and progressive central nervous system symptoms.

[0005] Sanfilippo syndrome is caused by defects in four different enzymes required for the degradation of polysaccharides, particularly glycosaminoglycans (GAGs), and is divided into MPS IIIA (Sanfilippo A), MPS IIIB (Sanfilippo B), MPS IIIC (Sanfilippo C), and MPS IIID (Sanfilippo D) based on the defective enzyme. The defective enzymes and genetic map loci for each Sanfilippo syndrome are listed below.

[0006] Type A (MPS IIIA): Heparan N-sulfatase - chromosome 17 (17q25.3)

[0007] Type B (MPS IIIB): N-acetyl-α-D-glucosaminidase - chromosome 17 (17q21)

[0008] Type C (MPS IIIC): Acetyl-CoA:α-glucosyl-N-acetyltransferase - chromosome 14

[0009] Type D (MPS IIID): n-acetyl-α-D-glucosamine-6-sulfatase - chromosome 12 (12q14)

[0010] As can be seen above, MPS IIIA is caused by a deficiency of heparan-N-sulfatase, an enzyme involved in the degradation of heparan sulfate, and hydrolyzes the sulfate moiety attached to the amino group of glucosamine residues. Symptoms of MPS IIIA (Sanfilippo A) usually 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 as well as difficulty in long-term survival.

[0011] Currently, there is no approved treatment for MPS IIIA, and only symptomatic treatment is being performed to relieve symptoms. Enzyme replacement therapy (ERT), a therapy in which prepared heparan-N-sulfatase is administered externally to MPS IIIA patients, is expected to be very useful in treating MPS IIIA.

[0012] In order to treat MPS IIIA patients using heparan-N-sulfatase via enzyme replacement therapy, large-scale production of heparan-N-sulfatase is necessary, which requires a process for culturing and isolating / purifying recombinant cells.

[0013] Separation and purification processes including immobilized metal affinity chromatography (IMAC) (also known as metal chelate affinity chromatography (MCAC)), cation exchange chromatography (CEX) and anion exchange chromatography (AEX) have been developed for the separation and purification of recombinantly produced heparan-N-sulfatase (see Korean Patent No. 2,286,260).

[0014] In addition, separation and purification processes including anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), hydroxyapatite chromatography (HA), and cation exchange chromatography (CEX) have also been developed (see U.S. Patent Publication No. 2012 / 0329133).

[0015] However, conventional technology still has the problem of low stability of heparan-N-sulfatase during the purification process, and due to not fully removing HCP (host cell protein) and other impurities during the production of heparan-N-sulfatase using recombinant cells, the quality and safety of the heparan-N-sulfatase finally produced deteriorate. Therefore, there is still an urgent need for new heparan-N-sulfatase separation / purification processes that can solve these problems. Summary of the invention

[0016] In order to solve the problems of conventional methods for separating and purifying heparan-N-sulfatase, an object of the present invention is to provide a novel method and process for purifying heparan-N-sulfatase, which can minimize the stability deterioration of heparan-N-sulfatase during the separation and purification process and greatly remove HCP and other impurities; a pharmaceutical composition comprising heparan-N-sulfatase prepared by the method; and a method for treating Sanfilippo syndrome using the same.

[0017] To achieve the above object, the present invention provides a method for purifying heparan-N-sulfatase from a solution containing heparan-N-sulfatase including at least one impurity, the method comprising performing multi-mode chromatography (MMC) to obtain an eluate; and performing caprylate precipitation to obtain a supernatant.

[0018] Heparan-N-sulfatase is a lysosomal enzyme known in the art as N-sulphoglucosamine sulphohydrolase (SGSH; EC 3.10.1.1); N-sulfoglucosamine sulfohydrolase; 2-desoxy-D-glucoside-2-sulfamate sulphohydrolase (sulphamate sulphohydrolase); heparin sulfamidase; sulfoglucosamine sulfamidase; sulfamidase; as well as HNS, rhHNS, sulfamidase, rhNS and rhSGSH, and is particularly preferably derived from human.

[0019] In the present invention, human heparan-N-sulfatase has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or more amino acid residues are deleted at the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO: 1, and has the activity of heparan-N-sulfatase, and in particular, is interpreted as a sequence having a sequence homology of 90% or more, preferably 95% or more, more preferably 99% or more with the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or more amino acid residues are deleted at the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO: 1.

[0020]

[0021] The solution comprising heparan-N-sulfatase comprising one or more impurities is preferably, but not limited to, a cell culture fluid, wherein the cell culture fluid is a culture fluid of a host cell capable of recombinantly producing heparan-N-sulfatase.

[0022] The host cell can be any host cell known in the art, and examples of host cells include, but are not limited to, Bacillus strains such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, prokaryotic host cells such as Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus), fungi such as Aspergillus species, yeasts such as Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces, and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as cells derived from insects, and cells derived from plants or mammals.

[0023] Preferably, the host cell can be monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, Syrian baby hamster kidney (BHK) cells, MDCK, myeloma cell line cells, HuT 78 cells or 293 cells.

[0024] Additionally, cell culture fluids can be clarified by removing cells and cell debris, for example, by depth filtration.

[0025] In the present invention, the multimodal chromatography may be achieved by simultaneously performing cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC), but is not limited thereto.

[0026] Various impurities can be removed by multimodal chromatography. In particular, enzymes with different contents of M6P (mannose-6-phosphate) can be selectively eluted, and by removing impurities with properties similar to those of heparan-N-sulfatase, the purity of the final purified heparan-N-sulfatase can be improved, and its stability can be significantly increased.

[0027] In the present invention, the resin for multimodal chromatography can be used without limitation as long as it is applicable to both cation exchange chromatography and hydrophobic interaction chromatography. For example, Capto MMC and Captoadhere resins having the structures of Formula 1 and Formula 2 can be used, but are not limited thereto.

[0028]

[0029] When multimodal chromatography is performed according to the present invention, the pH of the buffer (solution) used for loading and equilibration is 4.5±1.0, preferably 4.5±0.5, more preferably 4.5±0.1, but not limited thereto.

[0030] The buffer contains 200±100 mM, preferably 200±50 mM, more preferably 200±20 mM NaCl, and 5 to 100 mM, preferably 10 to 50 mM, more preferably 15 to 30 mM conventional acetate, phosphate, citrate, histidine, glycine or Tris buffer, etc., but is not limited thereto.

[0031] In the step of performing the multimodal chromatography according to the present invention, the buffer used for washing and elution after the resin is bound to heparan-N-sulfatase is 5 to 100 mM, preferably 10 mM to 50 mM, more preferably 15 to 30 mM conventional histidine, glycine, acetate, phosphate, citrate or Tris buffer etc., but not limited thereto.

[0032] The pH of the buffer used in the washing step may be 5.0 to 8.0, preferably 6.0 to 7.5, more preferably 6.3 to 7.0.

[0033] In particular, washing according to the invention may comprise two or more washing steps. In this case, the pH of the buffer used in the subsequent washing step may be higher than the pH of the buffer used in the preliminary washing step.

[0034] Specifically, when washing includes two washing steps, the pH of the first washing step is 6.0 to 6.6, preferably 6.2 to 6.6, more preferably 6.4 to 6.5, and the pH of the second washing step is 6.6 to 7.2, preferably 6.7 to 7.0, more preferably 6.75 to 6.9, but is not limited thereto.

[0035] Furthermore, the pH of the buffer used in the elution step may be 6.6 to 9.0, preferably 6.9 to 8.8, more preferably 8.0 to 8.4, but is not limited thereto.

[0036] The step of performing caprylate precipitation to obtain a supernatant according to the present invention has the effect of improving impurity removal efficiency by effectively removing HCP having a relatively low pH.

[0037] Caprylate precipitation is performed by adding caprylic acid or a salt thereof, preferably sodium caprylate, to the target solution, in a specific example, to the multimodal chromatography eluent or the affinity chromatography eluent, to obtain a concentration of 1 to 50 mM, preferably 5 to 30 mM, more preferably 8 to 12 mM at a pH of 3.5 to 6.0, preferably 4.0 to 5.5, more preferably 4.3 to 5.0.

[0038] The method for purifying heparan-N-sulfatase according to the present invention further comprises performing affinity chromatography to obtain an eluate between performing multimodal chromatography to obtain an eluate and performing caprylate precipitation to obtain a supernatant.

[0039] Thus, the method can include performing multimodal chromatography to obtain an eluate; performing affinity chromatography to obtain an eluate; and performing caprylate precipitation to obtain a supernatant.

[0040] According to the present invention, the step of using affinity chromatography includes removing impurities, in particular, HCP such as cathepsin X, which has similar physical and chemical properties such as pI (isoelectric point) or hydrophobicity to heparan-N-sulfatase, to enhance the effect of removing impurities.

[0041] The resin used for affinity chromatography according to the present invention is preferably heparin sepharose or blue sepharose, but is not limited thereto.

[0042] The pH of the buffer (solution) used for loading, equilibration, washing and elution can be 4.5±1.0, preferably 4.5±0.5, more preferably 4.5±0.1, but not limited thereto, and the buffer is 5 to 100 mM, preferably 10 to 50 mM, more preferably 15 to 30 mM conventional acetate, phosphate, citrate, histidine, glycine or Tris buffer, etc., but not limited thereto.

[0043] In particular, the buffer for washing may contain 150±100 mM, preferably 150±50 mM, more preferably 150±20 mM NaCl, and the buffer for elution may contain 300±100 mM, preferably 300±50 mM, more preferably 300±20 mM NaCl.

[0044] According to the method for purifying heparan-N-sulfatase of the present invention, before performing multimodal chromatography to obtain an eluate, it can also include performing primary anion exchange chromatography (AEX) to obtain an eluate, performing solvent / detergent treatment, and performing cation exchange chromatography (CEX) to obtain an eluate.

[0045] Thus, the method may include performing primary anion exchange chromatography (AEX) to obtain an eluent; solvent / detergent treatment; performing cation exchange chromatography (CEX) to obtain an eluent; performing multimodal chromatography to obtain an eluent; performing affinity chromatography to obtain an eluent; and performing caprylate precipitation to obtain a supernatant.

[0046] The primary anion exchange chromatography is a step for maximally recovering heparan-N-sulfatase from the cell culture fluid, and the resin used for the primary anion exchange chromatography according to the present invention is a weak anion exchange resin or a strong anion exchange resin, and examples of the resin 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, Fractogel TMAE and PorosXQ, and the like.

[0047] During the primary anion exchange chromatography according to the present invention, the pH of the buffer (solution) used for loading, balancing, washing and elution is 7.5±1.0, preferably 7.5±0.7, more preferably 7.5±0.5, but not limited thereto, and without limitation, the resin is 5 to 100 mM, preferably 10 to 70 mM, more preferably 40 to 60 mM conventional Tris, acetate, phosphate, citrate, histidine or glycine buffer, etc., but not limited thereto. In addition, the elution buffer may contain 200±100 mM, preferably 200±70 mM, and more preferably 200±50 mM NaCl.

[0048] Solvent / detergent treatment is a step for inactivating viruses, preferably using a combination of polysorbate (particularly polysorbate 20 and / or 80) and tri-n-butyl phosphate (TnBP), but not limited thereto.

[0049] The solvent / detergent treatment is carried out at a pH of 7.5±1.0, preferably 7.5±0.5, more preferably 7.5±0.2, at 18 to 28°C, preferably 20 to 25°C, for 1 hour or more or 6 hours or less.

[0050] Cation exchange chromatography is a process that maximizes the recovery of heparan-N-sulfatase and removes impurities such as process-related impurities and HCPs formed during solvent / detergent processing.

[0051] The resin used in the cation exchange chromatography according to the present invention may be a weak cation exchange resin or a strong cation exchange resin, examples of which include carboxymethyl (CM), sulfopropyl (SP) and methanesulfonic acid (S) resins, and the resin is preferably SP agarose gel, etc., but is not limited thereto.

[0052] During the cation exchange chromatography according to the present invention, the pH of the buffer (solution) used for loading, equilibration, washing and elution is 4.5±0.7, preferably 4.5±0.5, more preferably 4.5±0.1, but not limited thereto, and without limitation, the resin is 5 to 100 mM, preferably 10 to 50 mM, more preferably 15 to 30 mM conventional acetate, phosphate, citrate, histidine, glycine or Tris buffer etc., but not limited thereto.

[0053] Furthermore, the buffer used for loading or equilibration 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 NaCl, more preferably 200±20 mM NaCl.

[0054] According to the method for purifying heparan-N-sulfatase of the present invention, after performing caprylate precipitation to obtain a supernatant, the method may further include performing secondary anion exchange chromatography (AEX) to obtain an eluate.

[0055] Thus, the method may include performing a primary anion exchange chromatography (AEX) to obtain an eluent; solvent / detergent treatment; performing a cation exchange chromatography (CEX) to obtain an eluent; performing a multimodal chromatography to obtain an eluent; performing an affinity chromatography to obtain an eluent; performing an octanoate precipitation to obtain a supernatant; and performing a second anion exchange chromatography (AEX) to obtain an eluent.

[0056] Secondary anion exchange chromatography is a process for maximally recovering heparan-N-sulfatase and removing process-related impurities such as heparin, caprylate or solvent / detergent. The resin used in the anion exchange chromatography according to the present invention is preferably a strong anion exchange resin such as Q Sepharose, Fractogel TMAE (M), Fractogel TMAE (S) or Poros XQ, but is not limited thereto.

[0057] During the secondary anion exchange chromatography according to the present invention, the pH of the buffer (solution) used for loading, equilibration, washing and elution is 7.5±1.0, preferably 7.5±0.7, more preferably 7.5±0.5, but not limited thereto, and without limitation, the resin is 5 to 100 mM, preferably 10 to 50 mM, more preferably 15 to 30 mM conventional histidine, glycine, acetate, phosphate, citrate or Tris buffer etc., but not limited thereto.

[0058] Furthermore, the equilibration buffer may comprise 50±20 mM, preferably 50±10 mM, more preferably 50±5 mM NaCl, and the elution buffer may comprise 150±50 mM, preferably 150±30 mM, more preferably 150±10 mM NaCl.

[0059] The method for purifying heparan-N-sulfatase according to the present invention may further include nanofiltration after performing secondary anion exchange chromatography (AEX) to obtain an eluate.

[0060] Thus, the method may include performing a primary anion exchange chromatography (AEX) to obtain an eluent; solvent / detergent treatment; performing a cation exchange chromatography (CEX) to obtain an eluent; performing a multimodal chromatography to obtain an eluent; performing an affinity chromatography to obtain an eluent; performing an octanoate precipitation to obtain a supernatant; performing a second anion exchange chromatography (AEX) to obtain an eluent; and nanofiltration.

[0061] Nanofiltration aims at removing viruses and can be performed using nanofilters commonly used for removing viruses.

[0062] The method for purifying heparan-N-sulfatase according to the present invention may further comprise ultrafiltration / diafiltration (UF / DF); performed at least once in a step selected from the following constituent steps: before performing primary anion exchange chromatography (AEX) to obtain an eluate; between performing caprylate precipitation to obtain a supernatant and performing a second anion exchange chromatography (AEX) to obtain an eluate; and after nanofiltration.

[0063] Thus, when ultrafiltration / diafiltration (UF / DF) is included in all steps before performing primary anion exchange chromatography (AEX) to obtain an eluate, between performing caprylate precipitation to obtain a supernatant and performing secondary anion exchange chromatography (AEX) to obtain an eluate, and after nanofiltration, the overall method may include:

[0064] Primary ultrafiltration / diafiltration (UF / DF);

[0065] performing primary anion exchange chromatography (AEX) to obtain an eluent;

[0066] Solvent / detergent handling;

[0067] performing cation exchange chromatography (CEX) to obtain an eluent;

[0068] performing multimodal chromatography to obtain an eluent;

[0069] performing affinity chromatography to obtain an eluate;

[0070] Caprylate precipitation was performed to obtain a supernatant;

[0071] Two-stage ultrafiltration / diafiltration (UF / DF);

[0072] performing secondary anion exchange chromatography (AEX) to obtain an eluent;

[0073] Nanofiltration; and

[0074] Three-stage ultrafiltration / diafiltration (UF / DF).

[0075] Primary ultrafiltration / diafiltration is intended to concentrate cell culture fluid, preferably cell culture fluid that has undergone a purification process, and to exchange buffers to reduce the volume of the target culture fluid, shorten loading time, and improve the convenience of subsequent processes.

[0076] The cut-off value of the membrane used for primary ultrafiltration / diafiltration may be 10 to 100 kDa, preferably 20 to 70 kDa, more preferably 30 to 50 kDa, but is not limited thereto.

[0077] The pH of the buffer used for primary ultrafiltration / diafiltration can be 7.5±1.0, preferably 7.5±0.7, more preferably 7.5±0.5, but not limited thereto, and 5 to 100 mM, preferably 10 to 80 mM, more preferably 30 to 70 mM conventional Tris, histidine, glycine, acetate, phosphate or citrate buffer or the like is used, but not limited thereto.

[0078] A secondary ultrafiltration / diafiltration was performed between the caprylate precipitation and the secondary anion exchange chromatography in order to shift the pH and conductivity.

[0079] The cutoff value of the membrane used for secondary ultrafiltration / diafiltration may be 10 to 100 kDa, preferably 20 to 70 kDa, more preferably 30 to 50 kDa, but is not limited thereto.

[0080] The pH of the buffer used for secondary ultrafiltration / diafiltration can be 7.5±1.0, preferably 7.5±0.7, more preferably 7.5±0.5, but not limited thereto, and 5 to 50 mM, preferably 10 to 40 mM, more preferably 15 to 30 mM conventional histidine, glycine, acetate, phosphate, citrate or Tris buffer is used, but not limited thereto.

[0081] When the conductivity of the secondary ultrafiltration / diafiltration is 10 mS / cm or less, preferably 8 mS / cm or less, and more preferably 6 mS / cm or less, the process solution is recovered and the pH is adjusted to 7.5±1.0.

[0082] A tertiary ultrafiltration / diafiltration is performed after the nanofiltration and aims to finally concentrate the result to a high concentration and exchange the buffer.

[0083] The cutoff value of the membrane used for tertiary ultrafiltration / diafiltration may be 10 to 100 kDa, preferably 20 to 70 kDa, more preferably 30 to 50 kDa, but is not limited thereto.

[0084] The pH of the buffer used for tertiary ultrafiltration / diafiltration can be 8.0±1.0, preferably 8.0±0.7, more preferably 8.0±0.5, but not limited thereto, and 1 to 20 mM, preferably 2 to 10 mM, more preferably 3 to 8 mM conventional histidine, glycine, acetate, phosphate, citrate or Tris buffer is used, but not limited thereto.

[0085] Unless otherwise stated, all purification steps according to the present invention are carried out at room temperature, specifically 15 to 30°C, preferably 18 to 25°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is a chromatogram illustrating the purification of heparan-N-sulfatase using primary anion exchange chromatography.

[0087] Figure 2 The results of SDS-PAGE of the solutions of each step of the primary anion exchange chromatography are shown.

[0088] Figure 3 is a chromatogram illustrating the purification of heparan-N-sulfatase using cation exchange chromatography.

[0089] Figure 4 The results of SDS-PAGE of the solution at each step of cation exchange chromatography are shown.

[0090] Figure 5 In multimodal chromatography, the chromatogram is based on the pH of the eluent.

[0091] Figure 6 It is a figure which shows the SDS-PAGE result based on the pH of the eluent in multimodal chromatography.

[0092] Figure 7 The elution results in affinity chromatography are shown.

[0093] Figure 8 The entire process of purifying heparan-N-sulfatase according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0094] Hereinafter, the present invention will be described in more detail with reference to Examples. However, it is obvious to those skilled in the art that these Examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0095] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0096] [Example]

[0097] Example 1: Clarification of cell culture fluid

[0098] A cell culture fluid comprising recombinantly produced heparan-N-sulfatase and one or more impurities is depth filtered using a depth filter.

[0099] Specifically, distilled water was allowed to completely flow into depth filters Cat. No. MD0HC054H1 or Cat. No. MX0HC027H1 from Merck KGaA and depth filters Cat. No. NP6PDK516 or Cat. No. NP5LPDD16 from Pall Corporation to remove the solution contained in the filter, pH 7.5, 50 mM Tris buffer was allowed to flow to reach equilibrium, and then the cell culture fluid was filtered at ≤ 2.0 bar.

[0100] The recovery rate was 95% or more, and the HCP removal capacity was about 0.4 LRV (log reduction value).

[0101] Example 2: Primary ultrafiltration / diafiltration (UF / DF)

[0102] The clarified cell culture fluid is subjected to primary ultrafiltration / diafiltration for concentration and buffer exchange.

[0103] Specifically, a pH 7.5, 50 mM Tris buffer is flowed into a Pellicon 3 Ultracel C screen (Cat. No. P3C030C01) having a cutoff value of 30 to 50 kDa from Merck KGaA or an Omega T-series 30 kDa (OS030T12) membrane from Poll Corporation to reach an equilibrium state, and the culture filtrate is concentrated 10 times compared with the initial volume, and the culture filtrate is concentrated up to 13 times compared with the initial volume by buffer exchange with a pH 7.5, 50 mM Tris buffer of 3DV (diafiltration volume) or higher, and then recovered.

[0104] The concentration factor is 7 to 13 times, and the buffer exchange volume is 3DV or more. Finally, the recovery rate is 95% or more, and the HCP removal capacity is about 0.1 LRV (log reduction value) or more.

[0105] Example 3: Primary Anion Exchange Chromatography

[0106] 3.1 Validation of basic anion exchange chromatography (AEX)

[0107] The preliminary ultrafiltered / diafiltered solution was subjected to anion exchange chromatography using Q Sepharose 6 Fast Flow, Fractogel EMD TMAE(M) or Poros XQ as strong anion exchange resin and DEAE Sepharose Fast Flow as weak anion exchange resin.

[0108] In particular, the resin was CIP (cleaning in place) with 5CV (column volume) of 0.5N NaOH, 15CV of pH 7.5 ± 0.5, 50mM Tris equilibration buffer (EQ buffer) was allowed to flow to reach equilibrium, and the primary UF / DF solution was loaded. Then, 5CV of pH 7.5 ± 0.5, 50mM Tris equilibration buffer was injected to reach equilibrium again, 5CV of 50mMTris elution buffer (pH 7.5 ± 0.5, 200 ± 50mM NaCl) was injected, and when the UV signal during chromatography was 50mAu, the eluate was collected up to 2.5CV (132.5mL).

[0109] Then, 5 CV of pH 7.5±0.5, 2000±200 mM NaCl and 50 mM Tris column wash buffer (CW buffer) was injected and CIP was performed using 5 CV of 0.5 N NaOH, and 15 CV of equilibration buffer was allowed to flow to reach equilibrium.

[0110] The results of anion exchange chromatography under the above conditions showed that when unbound, in addition to elution in the chromatogram, CW and NaOH were injected and UV signals were detected, which meant that the impurities were removed (see Figure 1 ), and the results of SDS-PAGE showed that in the eluate (lane 3), at least as much impurity as in lanes 2 and 4 was removed compared to the loaded sample (lane 1) (see Figure 2 ).

[0111] In general, the yield was about 90% or higher, the purity was about 70% or higher, and the HCP removal capacity was about 0.2LRV or higher. All the resins used had similar recoveries after process optimization. Therefore, considering process stability and economic feasibility, Q Sepharose is preferred.

[0112] 3.2 Optimization of anion exchange chromatography (AEX)

[0113] As shown in Table 1, the performance of the AEX process was tested while varying the process pH and NaCl concentration in the elution buffer.

[0114] In particular, the test was performed with a difference of ±0.5 based on the initial experimental pH (pH 7.5), and stepwise elution was performed with NaCl concentrations of 100, 150 and 200 mM to determine the elution performance for the target protein (i.e., heparan-N-sulfatase) according to the NaCl concentration in the elution buffer at each pH.

[0115] [Table 1]

[0116] Performance of AEX according to process pH and NaCl concentration in elution buffer

[0117]

[0118] The results show that heparan-N-sulfatase binds to the resin at pH 7.0 to 8.0, and most of the heparan-N-sulfatase is eluted from 100mM NaCl. In addition, most of the heparan-N-sulfatase is recovered from the 150mM NaCl fraction. Therefore, it is found that when the NaCl concentration in the elution buffer is 100mM, preferably 150mM or higher, satisfactory results can be obtained.

[0119] Example 4: Solvent / detergent treatment

[0120] Viruses in the eluate obtained by primary AEX are inactivated by solvent / detergent treatment (S / D treatment).

[0121] Specifically, the S / D stock solution was added to the eluate obtained by primary AEX to obtain 1% polysorbate 80 and 0.3% TnBP, and stirred at 20 to 25°C for 1 hour.

[0122] At this time, S / D treatment was carried out for up to 6 hours under three conditions of pH 7.3, 7.5 and 7.7, and it was confirmed that sufficient S / D treatment could be carried out within the pH range defined above. However, when the treatment time exceeds 6 hours, the turbidity of the eluent may cause process risks. Therefore, it is preferred that the treatment time is adjusted to less than 6 hours.

[0123] Example 5: Cation Exchange Chromatography

[0124] 5.1 Validation of Basic Cation Exchange Chromatography (CEX)

[0125] Cation exchange chromatography was performed using SP Sepharose Fast Flow resin to maximize the recovery of heparan-N-sulfatase from the S / D treated solution and to remove impurities, particularly solvents and detergents, which are process impurities.

[0126] Specifically, the resin was CIP (cleaning in place) with 5 CV (column volume) of 0.5 N NaOH, 10 CV of 20 mM sodium acetate (SA) equilibration buffer (EQ buffer, pH 4.5±0.1, 100±20 mM NaCl) was allowed to flow to reach equilibrium, and the S / D-treated solution was loaded.

[0127] Then, 5CV of 20mM sodium acetate equilibration buffer (pH 4.5±0.1, 100±20mM NaCl) was injected to reach equilibrium again, 5CV of 20mM sodium acetate elution buffer (pH 4.5±0.1, 200±20mM NaCl) was injected, and the eluate was initially collected in an amount of 3CV and then the remaining amount of 2CV was discarded as waste.

[0128] Then, 5 CV of 20 mM sodium acetate column wash buffer (CW buffer, pH 4.5±0.1, 2000±200 mM NaCl) was injected, CIP was performed with 5 CV of 0.5 N NaOH, and 10 CV of equilibration buffer was flowed to reach an equilibrium state.

[0129] The results of cation exchange chromatography under the above conditions showed that when unbound, CW and NaOH were injected in addition to the elution in the chromatography, UV signals were detected, which meant that the impurities were removed (see Figure 3 ), which can be seen from the results of SDS-PAGE. In the eluate (lane 3), at least as much impurity as in lanes 2 and 4 was removed compared to the loaded sample (lane 1) (see Figure 4 ), and the purity was increased by about 20% compared to the primary anion exchange chromatography eluate.

[0130] In general, the yield is about 90% or higher, the purity is about 93% or higher, and the HCP removal capacity is about 1.2 LRV or higher.

[0131] 5.2 Optimization of Cation Exchange Chromatography (CEX)

[0132] As shown in Table 2, the performance of the CEX process was tested while varying the process pH and NaCl concentration in the elution buffer.

[0133] In particular, based on the initial experimental pH (pH 4.5), testing was performed in the range of 4.0 to 5.4, and testing was performed according to concentrations of 2 to 300 mM to determine the elution performance for the target protein (i.e., heparan-N-sulfatase) according to the NaCl concentration in the elution buffer at each pH.

[0134] [Table 2]

[0135] Performance of CEX according to process pH and NaCl concentration in elution buffer

[0136]

[0137] As can be seen from Table 3, the results show that the optimal NaCl concentration in the elution buffer at this time is 50 (at pH 5.4) to 210 mM NaCl (at pH 4.6 or lower).

[0138] [Table 3]

[0139] Elution results according to process pH and NaCl concentration in elution buffer

[0140]

[0141] Example 6: Multimodal Chromatography

[0142] Multimodal chromatography was performed using Capto MMC resin capable of simultaneous cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) to more efficiently remove impurities from the CEX eluate and selectively purify heparan-N-sulfatase containing M6P (mannose 5-phosphate).

[0143] Specifically, the resin was CIPed with 5 CV of 0.5 N NaOH, 10 CV of 20 mM sodium acetate (SA) equilibration buffer (EQ buffer, pH 4.5, 200 mM NaCl) was allowed to flow to reach equilibrium, and the CEX eluate was loaded.

[0144] Then, 5CV of 20mM histidine equilibration buffer (pH 5.5±0.1) was injected to reach a re-equilibrium state, the column was initially washed with 5CV of 20mM histidine primary wash buffer (pH 6.5±0.1), and then a second wash was performed with 10CV of 20mM histidine secondary wash buffer (pH 6.85±0.1).

[0145] When the pH of the washing buffer was 6.5 to 6.9, the washed solution contained a large amount of HCP, but the content of heparan-N-sulfatase was quite low. The washing was performed in two steps, with the pH of the first washing buffer being 6.45 and the pH of the second washing buffer being increased to 6.85 to more effectively remove HCP and increase the recovery rate of heparan-N-sulfatase.

[0146] Then, 10 CV of histidine elution buffer (pH 6.8±0.1 to 8.3±0.1, 20 mM) was injected and all of the eluate was recovered.

[0147] The results showed that the target protein (i.e., heparan-N-sulfatase) was primarily recovered at an elution buffer pH of 6.7, while heparan-N-sulfatase could be properly recovered at elution buffer pHs up to 8.4 (see Figure 5 ).

[0148] In addition, as the pH of the elution buffer increased, the pI range of heparan-N-sulfatase gradually increased, and there was no significant difference in FGly content between the fractions, but the M6P content decreased as the elution pH increased. That is, heparan-N-sulfatase with a lower pI value contained a larger amount of M6P (see Figure 6 and Table 4).

[0149] [Table 4]

[0150] Purification efficiency of M6P according to the pH of the elution buffer

[0151]

[0152] Then, 5 CV of 20 mM histidine column wash buffer (CW buffer, pH 7.5, 2000 mM NaCl) was injected, CIP was performed with 5 CV of 0.5 N NaOH, and 10 CV of equilibration buffer was flowed to reach an equilibrium state.

[0153] In general, the yield is about 70-90% or higher, the purity is about 97% or higher, and the HCP removal capacity is about 1.8 LRV or higher.

[0154] Example 7: Affinity chromatography

[0155] 7.1 Validation of basic affinity chromatography

[0156] Affinity chromatography using heparin sepharose or blue sepharose resin was performed to remove impurities such as cathepsin X, which was mostly contained in the MMC eluate.

[0157] Specifically, the resin was CIPed with 5 CV of 0.1 N NaOH, 10 CV of 20 mM sodium acetate (SA) equilibration buffer (EQ buffer, pH 4.5, 200 mM NaCl) was allowed to flow to reach equilibrium, and the MMC eluate was loaded.

[0158] Subsequently, 5 CV of 20 mM sodium acetate (SA) equilibration buffer (pH 4.5±0.1) was injected to achieve re-equilibrium, and the column was washed twice with 10 CV of 20 mM sodium acetate wash buffer (pH 4.5±0.1, 150±20 mM NaCl).

[0159] Then, inject 10CV of 20mM sodium acetate elution buffer (pH 4.5±0.1, 300±20mM NaCl), recover all the eluate, inject 5CV of 20mM sodium acetate wash buffer (CW buffer, pH 4.5±0.1, 2000±200mMNaCl), perform CIP with 5CV of 0.1N NaOH, and allow 10CV of equilibrium buffer to flow to reach equilibrium.

[0160] The results of affinity chromatography according to the above process showed that heparan-N-sulfatase was purified to a very high purity, such as Figure 7 as shown in .

[0161] In general, the yield is about 90% or higher, the purity is about 99% or higher, and the HCP removal capacity is about 1.0 LRV or higher.

[0162] 7.2 Optimization of affinity chromatography

[0163] As shown in Table 5, the performance of affinity chromatography was tested while varying the process pH and NaCl concentration in the elution buffer.

[0164] In particular, based on the initial condition test, the pH was set to pH 4.5±0.1, the central NaCl concentration of the wash buffer was set to 150 mM, and the central NaCl concentration of the elution buffer was set to 300 mM.

[0165] [Table 5]

[0166] Elution results according to process pH and NaCl concentration in elution buffer

[0167]

[0168] As can be seen in Table 5, the results show that when the NaCl concentration of the wash buffer was in the range of 130 to 170 mM and the NaCl concentration in the elution buffer was in the range of 280 to 320 mM, HCP was effectively removed and high-purity heparan-N-sulfatase was obtained.

[0169] Example 8: Caprylate Precipitation

[0170] Caprylate precipitation was performed to precipitate and remove HCPs with lower pis.

[0171] Specifically, a 500 mM stock solution of sodium octanoate was prepared and added to the affinity chromatography eluate to adjust the concentration to 10 mM.

[0172] Then, precipitation is performed at a pH of 4.5±0.1 and at a temperature of 20 to 25° C. for 1 to 3 hours while stirring at a speed of 200±20 rpm, and the precipitated impurities are removed by filtration.

[0173] As can be seen from Table 6, the results show that when caprylate precipitation was performed, the content of HCP, which is the main impurity, was greatly reduced compared to the case where caprylate precipitation was not performed. Cathepsin X, the main HCP, was hardly detected, and lysosomal Pro-X carboxypeptidase was greatly reduced.

[0174] [Table 6]

[0175] Impurity removal effect by octanoate precipitation

[0176]

[0177] ND: Not Determined

[0178] In general, the yield is about 90% or higher, the purity is about 99% or higher, and the HCP removal capacity is about 1.0 LRV or higher.

[0179] Example 9: Two-stage ultrafiltration / diafiltration (UF / DF)

[0180] The supernatant of the caprylate precipitation is adjusted to a pH and conductivity suitable for secondary anion exchange chromatography by secondary ultrafiltration / diafiltration. After secondary ultrafiltration / diafiltration, the concentration factor is increased by about 2 times, the buffer exchange volume is 3DV or more, the pH is increased from 4.5 to about 7.5, and the conductivity is reduced from 25 mS / cm to 6 mS / cm or less.

[0181] Specifically, using a Pellicon 3 Ultracel Cscreen (Cat No. P3C030C01) membrane with a cutoff value of 30 to 50 kDa from Merck, a pH 7.5, 20 mM histidine buffer was flowed to reach an equilibrium state, the supernatant of the caprylate precipitation was concentrated 2 times compared to the initial volume, the buffer was exchanged with a pH 7.5, 20 mM histidine buffer of 2DV or more, the treatment solution was recovered when the conductivity reached 6 mS / cm or less, and then the pH value of the recovered treatment solution was adjusted to 7.5.

[0182] Example 10: Secondary Anion Exchange Chromatography

[0183] In order to remove process-related impurities such as heparin, caprylate, solvents and detergents from the secondary ultrafiltration / diafiltration solution, secondary anion exchange chromatography was performed using Fractogel EMD TMAE (M) and Fractogel EMD TMAE (S) as strong anion exchange resins.

[0184] Specifically, the resin was CIPed with 5CV of 0.5N NaOH, 15CV of 20mM histidine equilibration buffer (pH 7.5±0.5, 50mM NaCl) was allowed to flow to reach equilibrium, and the secondary UF / DF solution was loaded. Then, 10CV of 20mM histidine equilibration buffer (pH 7.0±0.5, 50mM NaCl) was injected to reach equilibrium, and 7CV of 20mM histidine elution buffer (pH 7.0±0.5, 150±10mM NaCl) was injected.

[0185] Then, 5 CV of 20 mM histidine column wash buffer (CW buffer, pH 7.0±0.5, 2000±200 mM NaCl) was injected, CIP was performed with 5 CV of 0.5 N NaOH, and equilibration buffer was allowed to flow for 10 CV to reach equilibrium.

[0186] In general, the yield was about 90% or higher, the purity was about 99% or higher, and there was little difference between the resins.

[0187] Furthermore, the results of the tests performed when the NaCl concentration in the eluent was fixed at 150 mM and the pH was changed from 6.8 to 7.7 showed that heparan-N-sulfatase could be effectively purified in all cases, as shown in Table 7.

[0188] [Table 7]

[0189] Purification effect of AEX according to pH value

[0190]

[0191] Furthermore, the results of the tests performed while fixing the pH at 7.5 and changing the NaCl concentration in the eluent to 130 to 150 mM showed that heparan-N-sulfatase could be efficiently purified in all cases, as shown in Table 8.

[0192] [Table 8]

[0193] The purification effect of AEX depends on the concentration of NaCl in the eluent

[0194]

[0195] Example 11: Nanofiltration

[0196] Finally, for virus removal by filtration, nanofiltration was performed using nanofilters from Merck KGaA, Asahi, and Sartorius. Although there were no differences between the manufacturers’ products, the recovery rates of Sartorius’ nanofilters were quite high, with an overall yield of more than 90%.

[0197] Example 12: Three-stage ultrafiltration / diafiltration (UF / DF)

[0198] The final formulation was subjected to three stages of ultrafiltration / diafiltration to achieve high concentration and buffer exchange.

[0199] Specifically, using a Pellicon3 Ultracel C screen (Cat. No. P3C030C01) membrane with a cutoff value of 30 to 50 kDa from Merck KGaA, 4.2 mM histidine buffer (pH 8.2, 93.75 mM NaCl) was allowed to flow to reach equilibrium, the secondary AEX eluate was concentrated to about 5 mg / mL, and then recovered.

[0200] Figure 8 The overall process flow chart according to a specific embodiment of the present invention is shown.

[0201] Although the specific configuration of the present invention has been described in detail, it will be appreciated by those skilled in the art that the present invention can be implemented in a modified form without departing from the basic features of the present invention. Therefore, the above embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined in the claims rather than in the foregoing description, and all differences equivalent thereto will be interpreted as falling within the scope of the present invention.

[0202] Industrial Applicability

[0203] The method for purifying heparan-N-sulfatase according to the present invention can greatly improve the purity, safety and stability of the produced heparan-N-sulfatase by effectively removing HCP and other impurities, and is therefore very suitable for the effective production of heparan-N-sulfatase for enzyme replacement therapy.

[0204] Sequence Listing Free Text

[0205] Attached as an electronic document.

Claims

1. A method for purifying heparan-N-sulfatase from a solution comprising heparan-N-sulfatase comprising at least one impurity, the method comprising: performing multimodal chromatography (MMC) to obtain an eluent; as well as Caprylate precipitation was performed to obtain the supernatant.

2. The method according to claim 1, wherein: The solution containing heparan-N-sulfatase is a cell culture solution.

3. The method according to claim 1, wherein: The multimodal chromatography is achieved by simultaneously performing cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC).

4. The method according to claim 3, wherein: The resin used for the multimodal chromatography (MMC) was Capto MMC resin or Capto adhere resin.

5. The method according to claim 3, wherein: After the heparan-N-sulfatase is bound to the resin, the multimodal chromatography (MMC) includes two or more washing steps.

6. The method according to claim 5, wherein: In both washing steps, the pH of the buffer used in the subsequent washing step is higher than the pH of the buffer used in the initial washing step.

7. The method according to claim 1, wherein: The caprylate precipitation is performed by adding caprylate at a concentration of 1 to 20 mM.

8. The method according to claim 1, further comprising: Between performing multimodal chromatography (MMC) to obtain an eluent and performing caprylate precipitation to obtain a supernatant, Affinity chromatography was performed to obtain an eluate.

9. The method according to claim 8, further comprising: performing primary anion exchange chromatography (AEX) to obtain an eluent; Solvent / detergent handling; as well as Cation exchange chromatography (CEX) is performed to obtain an eluent, This was followed by multimodal chromatography to obtain the eluent.

10. The method according to claim 9, wherein: The resin used for the primary anion exchange chromatography (AEX) is a weak anion exchange resin or a strong anion exchange resin.

11. The method according to claim 9, further comprising: After caprylate precipitation to obtain the supernatant, Secondary anion exchange chromatography (AEX) was performed to obtain an eluent.

12. The method according to claim 11, further comprising performing nanofiltration after performing secondary anion exchange chromatography (AEX) to obtain an eluate.

13. The method of claim 12, further comprising ultrafiltration / diafiltration (UF / DF), at least once in a step selected from the group consisting of: before performing a primary anion exchange chromatography to obtain an eluate; between performing caprylate precipitation to obtain a supernatant and performing a second anion exchange chromatography (AEX) to obtain an eluate; and after nanofiltration.

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