MEDIUM COMPOSITION FOR CULTURING ANIMAL CELLS FOR PRODUCING RECOMBINANT EXTRACELLULAR MATRIX PROTEIN AND METHOD OF USING THE SAME

Abstract

Provided are a medium composition for culturing animal cells for producing a recombinant extracellular matrix protein, a method of producing the recombinant extracellular matrix protein with high purity, and a method of assaying a monomer of the recombinant extracellular matrix protein.

Claims

1. A method of assaying a monomer of a recombinant extracellular matrix protein, the method comprising: performing size exclusion chromatography on a sample comprising the recombinant extracellular matrix protein using a mobile phase comprising hydrochloride; and analyzing the monomer of the recombinant extracellular matrix protein in the sample, based on a result of the size exclusion chromatography.

2. The method of claim 1, wherein the recombinant extracellular matrix protein is collagen, elastin, fibronectin, laminin, vitronectin, tenascin, or hyaluronan and proteoglycan link (HAPLN) protein.

3. The method of claim 2, wherein the recombinant HAPLN protein is any one protein selected from the group consisting of HAPLN1, HAPLN2, HAPLN3, and HAPLN4.

4. The method of claim 1, wherein the hydrochloride is arginine hydrochloride, aniline hydrochloride, adenine hydrochloride, guanine hydrochloride, guanidine hydrochloride, histidine hydrochloride, or lysine hydrochloride.

5. The method of claim 1, wherein the mobile phase comprises hydrochloride at a concentration of more than 0.5 M.

6. The method of claim 1, wherein, in the analyzing, ratios of the monomer of the recombinant extracellular matrix protein and other impurities are analyzed.

Description

DESCRIPTION OF DRAWINGS

[0112] FIG. 1 shows a chromatogram of a linear gradient elution of anion exchange chromatography (AEX);

[0113] FIG. 2 shows a chromatogram of a step-wise elution of cation exchange chromatography (CEX);

[0114] FIG. 3 shows a result of SDS_PAGE_NR of the step-wise elution of CEX;

[0115] FIG. 4 shows a chromatogram of a step-wise elution of mixed-mode chromatography (MMC);

[0116] FIG. 5 shows a result of SDS_PAGE_NR of the step-wise elution of MMC;

[0117] FIGS. 6A and 6B show a chromatogram of hydrophobic interaction chromatography (HIC) comparative elution;

[0118] FIG. 7 shows a result of SDS_PAGE_NR of HIC comparative elution;

[0119] FIG. 8 shows a chromatogram showing a result of performing size exclusion chromatography (SEC) analysis for a sample including rhHAPLN1 using phosphate buffer(PB)+NaCl, 5 mM EDTA, or 5 mM EDTA+4 M Gdn-HCl as a mobile phase;

[0120] FIG. 9 shows a chromatogram showing a result of performing SEC analysis for a sample including rhHAPLN1 using 50 mM PB+150 mM NaCl+1M Arg-HCl (pH 6.3) as a mobile phase;

[0121] FIG. 10 shows a chromatogram showing a result of performing SEC analysis for Sample 3 using 50 mM PB+300 mM NaCl, 0.1 M Arg-HCl, 0.5 M Arg-HCl, 1.0 M Arg-HCl, or 1.0 M Gdn-HCl as a mobile phase;

[0122] FIG. 11 shows a chromatogram showing a result of performing SEC analysis for Sample 3 using 50 mM PB+300 mM NaCl, 0.1 M urea, 0.5 M urea, 1.0 M urea, 2.0 M urea, 4.0 M urea, or 6.0 M urea as a mobile phase; and

[0123] FIG. 12 shows a chromatogram showing a result of performing SEC analysis for Sample 3 using 1.0 M Gdn-HCl, 4.0 M urea, or 1.0 M Arg-HCl as a mobile phase.

BEST MODE

[0124] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1: Culture of Cells Producing Recombinant ECM Protein

[0125] A vector including a polynucleotide encoding a human HAPLN1 protein among ECM proteins was inserted to CHO-K1 cells to prepare a CHO-K1 cell line producing the recombinant human HAPLN1 protein. A cell line showing excellent protein production amount and quality was selected as a master cell bank (MCB).

[0126] The MCB was sub-cultured and inoculated at a concentration of 0.40±0.05×10.sup.6 cells/mL in a 250 L Hyperforma SUB bioreactor (Thermo), followed by fed-batch culture.

[0127] 22.36 g of ActiPro™ medium+0.5846 g of glutamine+10.00 g of HT Supplement (Thermo Fisher Scientific)+4.29 g of 10 N NaOH+1.80 g of NaHCO.sub.3 were used as a basic medium. A culture temperature, dissolved oxygen (DO), and pH were set to 36.5° C., 40.0%, and 7.00±0.20, respectively. 1 M sodium carbonate monohydrate was used as a pH control solution.

[0128] 181.04 g of HyClone™ Cell Boost 7a+12.28 g of FM020a of 10 N NaOH, and 94.60 g of HyClone™ Cell Boost 7b+105.93 g of FM020b of 10 N NaOH were used as a feeding medium (FM). A feeding strategy is shown as in Table 1 below.

TABLE-US-00001 TABLE 1 Fed-batch culture Feeding medium Day 3 Day 5 Day 6 Day 8 Day 10 FM020a (%(w/w)) 3 5 3 3 3 FM020b (%(w/w)) 0.3 0.5 0.3 0.3 0.3

[0129] As a feed additive, 50 μM CuSO.sub.4 was used, which was added to the bioreactor on day 0 of fed-batch culture.

[0130] A Glucose feed stock was prepared at 400 g glucose/kg. From day 3 to day 13 of fed-batch culture, when the glucose concentration dropped to less than 5.0 g/L, glucose was fed in such a way that it was raised to 6.0 g/L.

[0131] When a viable cell density (VCD) reached 20.00×10.sup.6 cells/mL, the temperature was changed to 31.0° C. Cells were harvested on 12 day of fed-batch culture or when viability dropped to less than 60%.

Experimental Example 1: Effect of Reducing Formation of Multimer of Recombinant ECM Protein According to Type of Additive

[0132] An experiment was performed to examine an effect of reducing formation of protein multimers according to the type of additive when cells producing the recombinant human HAPLN1 protein were cultured.

[0133] In detail, cells were cultured in the same manner as in Example 1, except for varying the type, concentration, and feeding strategy of the feeding additive. Results of cell culture and protein production according to the type and concentration of the feeding additive, and feeding strategy thereof in each experimental group are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Experimental Type of Concentration Feeding Titer Qp SEC_LC group additive* of additive strategy (g/L) (pg/cell/day) (Main)(%) Comparative — — — 2.66 21.08 51.1 Example 1 Comparative DMSO 0.5%(w/v) Feeding on 2.22 18.01 44.8 Example 2 days 4, 6, 8 Comparative DMSO 0.2%(w/v) Feeding on 2.42 19.15 44.4 Example 3 days 0, 3, 5, 7, 9 Comparative Glycerol 1.0%(w/v) Feeding on 2.46 20.46 43.5 Example 4 day 5 Comparative Poloxamer 0.2%(w/v) Feeding on 2.71 22.49 47.0 Example 5 188 day 5 Comparative EDTA 1 mM Feeding on 2.03 20.96 57.5 Example 6 day 5 Comparative PS80 0.04%(w/v)  Feeding on 2.75 23.77 43.8 Example 7 day 5 Comparative Cysteine 1 mM Feeding on 1.91 15.83 31.4 Example 8 days 5, 7, 9 Comparative GSH 0.2 mM Feeding on 2.36 19.48 43.5 Example 9 days 6, 8 Comparative GSH and 0.2 mM and Feeding on 2.52 20.31 47.0 Example 10 GSSG 0.2 mM days 6, 8 Comparative EDTA and 1 mM and Feeding on 2.16 22.34 — Example 11 MgCl.sub.2 50 μM day 5 Example 1 CuSO.sub.4 50 μM Feeding on 3.87 28.77 53.2 day 0 *DMSO: dimethyl sulfoxide, PS80: polysorbate 80, GSH: glutathione, GSSG: glutathione disulfide.

[0134] As shown in Table 2, when CuSO.sub.4 additive was used, a titer of the recombinant human HAPLN1 protein production was 3.87 g/L, which was the highest, and the production amount of the recombinant human HAPLN1 protein per cell was 28.77 pg/cell/day, which was the highest.

[0135] Therefore, it was confirmed that when a copper compound such as CuSO.sub.4 is used as an additive during culturing cells producing the recombinant human HAPLN1 protein, the effect of reducing formation of the protein multimer is excellent and the protein production amount is increased. Specifically, when CuSO.sub.4 at a concentration of 50 μM was fed on day 0 of cell fed-batch culture, the highest effect of reducing formation of the protein multimer and the highest protein production amount were observed. Accordingly, it is suggested that CuSO.sub.4 may be used in mass-production of the recombinant human HAPLN1 protein.

Experimental Example 2: Effect of Reducing Formation of Recombinant ECM Protein Multimer According to Concentration of Copper Compound

[0136] An experiment was performed to examine an effect of reducing formation of protein multimers according to concentrations of the copper compound when cells producing the recombinant human HAPLN1 protein were cultured.

[0137] In detail, cells were cultured in the same manner as in Example 1, except for varying the concentration of the copper compound. The concentration of the copper compound in each experimental group and results of protein production according to the concentration are shown in Table 3 below.

TABLE-US-00003 TABLE 3 SEC Titer Main HMW LMW Caliper_NR Experimental CuSO.sub.4 (D10) Peak Peak Peak Purity Peak Peak Peak group concentration (g/L) % % % % 1% 2% 3% Example 1 50 μM 2.57 50.3 38.5 11.2 85.7 20.6 65.1 0.68 Comparative 20 μM 2.42 51.8 38.8 9.4 86.7 20.9 65.8 0.67 Example 12 Comparative 5 μM 2.42 51.3 38.8 9.9 86.0 20.6 65.4 0.71 Example 13

[0138] As shown in Table 3, when CuSO.sub.4 was used at a concentration of more than 20 μM, a titer of the human HAPLN1 protein production was increased. In particular, Example 1, in which CuSO.sub.4 was used at a concentration of 50 μM or more, showed the excellent titer of the human HAPLN1 protein production. Therefore, when a copper compound such as CuSO.sub.4 is used at a concentration of more than 20 μM, particularly, 50 μM or more during culturing the cells producing the recombinant human HAPLN1 protein, the excellent effect of reducing formation of the protein multimer and the increased protein production amount were observed.

Example 2: Isolation and Purification of Recombinant ECM Protein

[0139] The recombinant human HAPLN1 protein was isolated and purified from the cells cultured according to Example 1. In detail, isolation and purification of the recombinant human HAPLN1 protein were performed according to the following procedures.

[0140] (1) Harvest and Clarification

[0141] For harvest and clarification, DOHC and A1HC depth filter of Millipore were used. Recommended loading volumes of DOHC and A1HC depth filters are 45 L/m.sup.2 and 90 L/m.sup.2, respectively.

[0142] (2) Ultrafiltration/Diafiltration 1 (UF/DF1)

[0143] Pellicon 3 (Ultracel, Type C Screen, 30 kDa) of Millipore was selected as a UF/DF1 membrane. A concentration of the loading sample was 5 g/L or less in the UF, and then diafiltration was performed with 6 times or more volume of a buffer containing 50 mM Tris-HCl and 5 mM EDTA at pH 9.0. A feed flow rate was 300 LMH or less and a transmembrane pressure (TMP) was 10 psi to 20 psi. A recommended loading amount is 70 L/m.sup.2 or less.

[0144] (3) Anion Exchange Chromatography (AEX)

[0145] Poros 50HQ resin of Life Tech was used as a capture resin. This step was performed in a bind-and-elute mode. A recommended protein loading amount is 10 g/L resin to 50 g/L resin. A selected elution buffer is a buffer containing 100 mM His-HCl and 5 mM EDTA at pH 5.0. A recommended UV peak collection range is 25 mAU/mm to 75 mAU/mm

[0146] (4) Solvent/Detergent (S/D) Virus Inactivation

[0147] S/D virus inactivation was performed according to a common method.

[0148] (5) Cation Exchange Chromatography (CEX)

[0149] This step was performed in a bind-and-elute mode. Capto S ImpAct resin of Cytiva (formerly GE Healthcare) was used as a CEX resin. A recommended CEX loading amount is 10 g/L resin to 15 g/L resin. By performing CEX, protein aggregates, host cell proteins (HCPs), and other impurities were removed. A buffer containing 50 mM Tris-HCl, 100 mM NaCl, and 5 mM EDTA at pH 8.0 is recommended as a washing buffer II, and a buffer containing 50 mM NaAc, 350 mM NaCl, and 5 mM EDTA at pH 5.5 is recommended as a washing buffer III. A recommended elution buffer is a buffer containing 50 mM Tris-HCl, 370 mM NaCl, and 5 mM EDTA at pH 8.0. A recommended UV peak collection range is 25 mAU/mm to 50 mAU/mm

[0150] (6) Mixed-Mode Chromatography (MMC)

[0151] This step was performed in a bind-and-elute mode. Capto adhere resin of Cytiva (formerly GE Healthcare) was used as an MMC resin. A recommended loading amount is 10 g/L resin to 15 g/L resin. To further remove protein aggregates and HCPs, a buffer containing 50 mM Tris-HCl, 0.5 M arginine(Arg), and 5 mM EDTA at pH 8.0 was used as an elution buffer.

[0152] (7) Hydrophobic Interaction Chromatography (HIC)

[0153] This step was performed in a bind-and-elute mode. Butyl Sepharose 4 Fast Flow resin of Cytiva (formerly GE Healthcare) was used as an HIC resin. A recommended loading amount is 3 g/L resin to 6 g/L resin. A buffer containing 50 mM Tris-HCl and 1.5 M NaCl at pH 8.0 is recommended as a washing buffer III. The target protein was eluted with high purity using a buffer containing 50 mM Tris-HCl and 0.5 M NaCl at pH 8.0.

[0154] (8) Ultrafiltration/Diafiltration 2 (UF/DF2)

[0155] Pellicon 3 (Ultracel, Type C Screen, 10 kDa) of Millipore was selected for UF/DF2. The loading sample was concentrated to 1 g/L to 3 g/L in UF, and subjected to diafiltration with 6 times or more volume of a buffer containing 20 mM NaAc at pH 5.0. A UF/DF2 pool concentration was 4.5 mg/mL to 5.5 mg/mL. A feed flow rate was 300 LMH or less, and a transmembrane pressure (TMP) was 10 psi to 20 psi. A load capacity is 70 g/m.sup.2 or less.

[0156] (9) Intermediate Depth Filtration (Int. DF)

[0157] For intermediate depth filtration, X0SP depth filter of Millipore was selected to remove HCPs. A recommended loading amount of the X0SP filter is 400 g/m.sup.2 to 800 g/m.sup.2.

[0158] (10) Formulation and Bulk Fill

[0159] A concentration of a drug substance (DS) is 2.0±0.2 g/L. A composition of a formulation buffer was determined by drug product development (DPD) and transferred to downstream process development (DSPD). PS80 and sucrose were added to VF pool samples at final concentrations of 0.04% (w/v) and 8% (w/v), respectively. DS was obtained after 0.2 μm final filtration.

Experimental Example 3: AEX Conditions for Specifically Isolating Specific Recombinant ECM Protein

[0160] Among ECM proteins, HAPLN1 protein has a molecular weight of 40 kDa to 50 kDa. In the anion exchange chromatography (AEX) of Example 2-(3), an experiment was performed to optimize AEX conditions for specifically isolating a recombinant human HAPLN1 protein.

[0161] (1) Elution conditions

[0162] (1.1) AEX linear gradient comparative elution

[0163] Materials: [0164] Column: Poros 50HQ, 3.024 mL (0.5 cm×15.4 cm) [0165] Loading material: concentration 2.64 g/L, pH 9.06, conductivity 1.44 mS/cm [0166] Loading amount: 30 g/L resin [0167] Column washing (sanitization) solution: 1.0 M NaOH [0168] Pre-equilibration buffer: 50 mM Tris-HCl, 1 M (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.0 [0169] Equilibration/washing buffer I: 50 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 9.0 [0170] Washing buffer II: 50 mM Tris-HCl, 10 mM NaCl, 5 mM EDTA, pH 9.0 [0171] Elution buffer: control: 50 mM Tris-HCl, 80 mM (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.5; condition 1-A: 20 mM His-HCl, pH 7.5, B: 50 mM His-HCl, 30 mM NaCl, 5 mM EDTA, pH 5.8; condition 2-A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.5; condition 3-A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.0; linear gradient elution from A to B for 20 column volumes (CV) [0172] Stripping buffer: 50 mM Tris-HCl, 1 M (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.0 [0173] Column storage solution: 20% ethanol

[0174] Experimental Procedure:

[0175] Eluates were collected at 25 mAU/mm to 25 mAU/mm. The elution conditions were verified using a chromatogram.

Results:

[0176] A yield of the recombinant human HAPLN1 protein, SEC analysis results, and HCP concentrations according to each elution condition are shown in Table 4 below. As shown in Table 4, when 100 mM His-HCl was added without salts to the elution buffer, better effect of capturing the recombinant human HAPLN1 protein was observed, as compared with those with salts. In addition, when 100 mM His-HCl was added, HCPs were decreased by 50%, as compared with a control.

TABLE-US-00004 TABLE 4 Elution conditions (linear Washing gradient elution from A to B) Yield* SEC(%) HCP Run buffer II A B (%) M/H/L (ppm) 1 (Control) — — 50 mM Tris-HCl, 79 53.1/46.7/0.2 164608 80 mM (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.5 2 (Elution 50 mM 20 mM 50 mM His-HCl, 74 56.7/43.3/ND 73112 condition 1) Tris-HCl, His-HCl, 30 mM NaCl, 10 mM NaCl, pH 7.5 5 mM EDTA, pH 5.8 3 (Elution 5 mM EDTA, 100 mM 100 mM His-HCl, 78 55.8/44.2/ND 79856 condition 2) pH 9.0 His-HCl, 5 mM EDTA, pH 5.5 4 (Elution pH 7.0 100 mM His-HCl, 85 54.4/45.6/ND 83654 condition 3) 5 mM EDTA, pH 5.0

[0177] (1.2) AEX linear gradient elution

[0178] Materials: [0179] Column: Poros 50HQ, 3.024 mL (0.5 cm×15.4 cm) [0180] Loading material: concentration 2.64 mg/mL, pH 9.06, conductivity 1.44 mS/cm [0181] Loading amount: 30 g/L resin [0182] Sanitization solution: 1.0 M NaOH [0183] Pre-equilibration buffer: 50 mM Tris-HCl, 1 M (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.0 [0184] Equilibration/washing buffer I: 50 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, pH 9.0 [0185] Washing buffer II: 50 mM Tris-HCl, 5 mM EDTA, pH 9.0 [0186] Elution buffer: A: 100 mM His-HCl, pH 7.0, B: 100 mM His-HCl, 5 mM EDTA, pH 5.0; linear gradient elution from A to B for 20 column volumes (CV). [0187] Stripping buffer: 50 mM Tris-HCl, 450 mM (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.0 [0188] Storage solution: 20% ethanol

[0189] Experimental Procedure:

[0190] Based on the results of linear gradient comparative elution, the material after UF/DF1 was loaded into a Poros 50HQ column, and an optimal elution buffer was determined using a step-wise elution method. Optimal elution conditions were confirmed using SEC purity analysis.

[0191] Results:

[0192] FIG. 1 shows a chromatogram of the linear gradient elution of AEX.

[0193] As shown in Table 4 and FIG. 1, 100 mM His-HCl efficiently captured the recombinant human HAPLN1 protein without additional salt addition. For a balance between purity and yield, pH 5.0 was chosen as pH for elution.

[0194] Therefore, it was found that about 100 mM His-HCl may be selected as the AEX elution buffer in order to specifically isolate the recombinant human HAPLN1 protein. In particular, when 100 mM His-HCl is used, the recombinant human HAPLN1 protein may be isolated with excellent purity and yield. For example, a buffer containing 100 mM His-HCl and 5 mM EDTA at pH 5.0 may be used as the AEX elution buffer.

Experimental Example 4: CEX Conditions for Removing Recombinant ECM Protein Aggregates, HCPs, and Other Impurities

[0195] The bind-and-elute mode CEX of Example 2-(5) was introduced to remove recombinant human HAPLN1 protein aggregates, HCPs, and other impurities. Therefore, an experiment was performed to optimize the CEX conditions for removing recombinant human HAPLN1 protein aggregates, HCPs, and other impurities.

[0196] (1) Elution Conditions

[0197] Materials: [0198] Column: Capto S ImpAct, 2.631 mL (0.5 cm×13.4 cm) [0199] Loading material: AEX eluate, concentration 11.844 mg/mL, pH 5.52, conductivity 11.70 mS/cm [0200] Loading amount: 10 mg/mL resin [0201] Equilibration/Washing I buffer: 50 mM NaAc-HAc, 5 mM EDTA, pH 5.5 [0202] Washing II buffer: 50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 8.0 [0203] Washing III buffer: 50 mM NaAC, 350 mM NaCl, 5 mM EDTA, pH 5.5 [0204] Elution buffer: (A) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, pH 8.0; step-wise elution: 20% B (100 mM NaCl), 5 CV; 40% B (200 mM NaCl), 5 CV; 60% B (300 mM NaCl), 5 CV; 75% B (375 mM NaCl), 5 CV; 85% B (425 mM NaCl) [0205] Stripping buffer: 50 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, pH 8.0

[0206] Experimental Procedure:

[0207] Optimal elution conditions were confirmed by performing a step-wise elution method. A loading amount was 10 g/L resin, and eluates were collected at 25 mAU/mm to 25 mAU/mm A protein concentration of each fraction was measured, and step-wise recovery amounts were calculated. In addition, sample purity was analyzed by SDS_PAGE_NR.

[0208] Results:

[0209] The elution conditions are important for product quality. Criteria for the optimal conditions are based on the removal of impurities.

[0210] FIG. 2 shows a chromatogram of the step-wise elution of CEX.

[0211] FIG. 3 shows a result of SDS_PAGE_NR of the step-wise elution of CEX.

[0212] The HCP test results in the CEX step-wise elution are shown in Table 5 below.

TABLE-US-00005 TABLE 5 NaCl concentration yield HCP Fraction (mM) (%) (ng/mg) Load — — 121669 F01 0 2.0 — E01 100 0.7 799110 E02 200 20.0 115447 E03 300 32.0 E04 375 9.8  35248 E05 425 1.6 — S01 500 9.2  11179

[0213] As shown in FIG. 3 and Table 5, when 100 mM NaCl (E01) was used, the largest amount of HCPs was removed, and loss of the target protein was only 0.7%. Yields of fraction E02-E04 and fraction E05 were 61.8% and 1.6%, respectively. The HMW content was increased with increasing NaCl. For a balance between purity and yield of the product, a buffer containing 50 mM Tris-HCl, 370 mM NaCl, 5 mM EDTA at pH 8.0 was recommended as the elution buffer.

Experimental Example 5: MMC Conditions for Removing Recombinant ECM Protein Aggregates and HCPs

[0214] The bind-and-elute mode MMC of Example 2-(6) was used to additionally remove recombinant human HAPLN1 protein aggregates and HCPs. Therefore, an experiment was performed to optimize the MMC conditions for removing recombinant human HAPLN1 protein aggregates and HCPs.

[0215] (1) Elution Conditions

[0216] The recombinant human HAPLN1 protein aggregates and HCPs were removed using a capto adhere. CEX eluates were loaded onto the Capto adhere column Based on the result of linear gradient elution for 50 L material production, optimal elution conditions were confirmed by a step-wise elution method.

[0217] Materials: [0218] Column: Capto adhere, 2.985 mL (0.5 cm×15.2 cm) [0219] Loading material: CEX eluate, concentration 3.105 mg/mL, HCP 155217 ng/mg, SEC purity 52.9% [0220] Loading amount: 7.5 g/L resin [0221] Pre-equilibration buffer: 50 mM NaAc-HAc, 1 M NaCl, 5 mM EDTA, pH 5.5 [0222] Equilibration/washing buffer I: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 [0223] Elution buffer: (A) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 1 M Arg, 5 mM EDTA, pH 8.0; step-wise elution: 20% B (200 mM Arg), 5 CV; 40% B (400 mM Arg), 10 CV; 50% B (500 mM Arg), 10 CV; 60% B (600 mM Arg), 10 CV; 70% B (700 mM Arg), 10 CV [0224] Stripping buffer: 50 mM HAc

[0225] Experimental Procedure:

[0226] The optimal elution conditions were determined by step-wise elution. CEX eluates were loaded into the Capto adhere column with 7.5 g/L resin, and the eluates were collected at 25 mAU/mm to 25 mAU/mm A protein concentration of each fraction was measured, and step-wise recovery amounts were calculated. HCP and SDS_PAGE_NR purity were also tested.

[0227] Results:

[0228] FIG. 4 shows a chromatogram of the step-wise elution of MMC.

[0229] FIG. 5 shows a result of SDS_PAGE_NR of the step-wise elution of MMC.

[0230] The results of testing HCPs in the MMC step-wise elution are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Arginine concentration Yield HCP Fraction (mM) (%) (ng/mg) Load — — 155217 Peak 1 (E01) 200 0.2 2711333 Peak 2 (E02-E03) 400 39.5 39995 Peak 3 (E04-E05) 500 28.1 4295 Peak 4 (E06) 600 6.6 5865 Peak 5 (E07) 700 0.7 — Peak 6 (S01) 1000 1.9 27198

[0231] As shown in FIG. 5 and Table 6, when 200 mM arginine (peak 1) was used, the largest amount of HCPs was removed, and loss of the target protein was only 0.2%. Yields of peak 2 and peak 3 were 39.5% and 28.1%, respectively. When the arginine concentration was high, the largest amount of HMW was eluted. For a balance between purity and yield of the product, a buffer containing 50 mM Tris-HCl, 500 mM arginine, and 5 mM EDTA at pH 8.0 was recommended as the elution buffer. 200 mM arginine may be used in washing.

Experimental Example 6: HIC Conditions for Improving Purity of Recombinant ECM Protein

[0232] The HIC of Example 2-(7) was used to remove recombinant human HAPLN1 protein multimers and HCPs. Therefore, an experiment was performed to optimize the HIC conditions for removing the recombinant human HAPLN1 protein multimers and HCPs.

[0233] (1) Elution Conditions

[0234] Materials: [0235] Column: Butyl Sepharose 4 Fast Flow, 2.631 mL (0.5 cm×13.4 cm) [0236] Loading materials: 1) MMC eluate, concentration 0.590 mg/mL, pH 8.09, 146.35 mS/cm, SEC purity 68.9%; 2) MMC eluate, concentration 0.543 mg/mL, pH 8.10, 145.82 mS/cm, SEC purity 68.9% [0237] Loading amount: 5 g/L resin [0238] Equilibration/washing buffer I: 50 mM Tris-HCl, 1 M (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.0 [0239] Washing buffer II: 50 mM Tris-HCl, 0.4 M (NH.sub.4).sub.2SO.sub.4, 5 mM EDTA, pH 8.0 [0240] Washing buffer III: 1) 50 mM Tris-HCl, 2 M NaCl, 5 mM EDTA, pH 8.0; 2) 50 mM Tris-HCl, 1.5 M NaCl, 5 mM EDTA, pH 8.0 [0241] Elution buffer:

[0242] 1) (A) 50 mM Tris-HCl, 2 M NaCl, 5 mM EDTA, pH 8.0; (B) 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; step-wise elution: 25% B (1.5 M NaCl), 10 CV; 50% B (1 M NaCl), 10 CV; 75% B (0.5 M NaCl), 10 CV; 90% B (0.2 M NaCl), 10 CV; 100% B (0 M NaCl), 10 CV;

[0243] 2) 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0 [0244] Stripping buffer: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0

[0245] Experimental Procedure:

[0246] Washing III and elution conditions were determined by performing a step-wise elution method. MMC eluates were adjusted with ˜1 M (NH.sub.4).sub.2SO.sub.4 before loading on the HIC column. A loading amount was 5 g/L resin, and eluates were collected at 25 mAU/mm to 25 mAU/mm A protein concentration of each fraction was measured, and step-wise recovery amounts were calculated. Purity was tested using SDS_PAGE_NR.

[0247] Results:

[0248] FIGS. 6A and 6B show a chromatogram of HIC comparative elution.

[0249] FIG. 7 shows a result of SDS_PAGE_NR of HIC comparative elution.

[0250] The results of the HIC step-wise elution are shown in Table 7 below.

TABLE-US-00007 TABLE 7 SEC Run Washing III Elution Yield purity No. conditions conditions Fraction (%) (%) 1) 2M NaCl Step-wise (1.5 .fwdarw. Washing 45.8 — 1 .fwdarw. 0.5 .fwdarw. Elution 15.3 — 0.2 .fwdarw. 0M NaCl) 2) 1.5M NaCl One-step (0.5M Washing 50.1 — NaCl) Elution 34.8 96.7/3.3/ND

[0251] As shown in FIG. 7 and Table 7, the largest amount of HMW was removed by the washing buffer II, and the target protein was not eluted in the presence of NaCl exceeding 1.5 M at pH 8.0. Therefore, in Run2, a buffer containing 50 mM Tris-HCl, 1.5 M NaCl, and 5 mM EDTA at pH 8.0 was used in Wash III, and a buffer containing 50 mM Tris-HCl, 0.5 M NaCl, and 5 mM EDTA at pH 8.0 was used in elution. Finally, a yield of the second experiment was 34.8%. Therefore, a buffer containing 50 mM Tris-HCl, 1.5 M NaCl, and 5 mM EDTA at pH 8.0 was used as the washing buffer III, and a buffer containing 50 mM Tris-HCl and 0.5 M NaCl at pH 8.0 was used as the elution buffer.

Example 3: Method of Assaying Recombinant ECM Proteins

[0252] The monomers of the recombinant ECM protein and other impurities in samples were analyzed by performing size exclusion chromatography (SEC) using a mobile phase containing hydrochloride. Specific conditions for SEC are as follows: [0253] Column: TSKgel G3000SWXL, 7.8×300 mm, 5 μm Steel (Manuf. TOSOH) [0254] Mobile phase: 50 mM phosphate buffer (PB), 300 mM NaCl, 1 M Gdn-HCl or Arg-HCl pH7.5 (±0.5) [0255] Detection wavelength: 280 nm [0256] Flow rate: 1.0 mL/min [0257] Column temperature: 25±3° C. [0258] Sample temperature: 5±3° C. [0259] Sample feeding amount: 100 μg

Experimental Example 7: Screening for Additives for Accurate Analysis of Recombinant ECM Proteins

[0260] An experiment was performed to screen for additives of a mobile phase, which is able to improve accuracy by reducing appearance of inaccurate peaks, when the monomers of the recombinant ECM protein and other impurities are analyzed using SEC. In detail, to analyze the monomers of the recombinant human HAPLN1 protein and other impurities using SEC, accuracy of SEC analysis according to the type and concentration of the additive used as the mobile phase was examined. As a sample, an intermediate product during the isolation and purification of the recombinant human HAPLN1 protein according to Example 2 was used.

[0261] FIG. 8 shows a chromatogram showing a result of performing SEC analysis for a sample including the recombinant human HAPLN1 protein using phosphate buffer(PB)+NaCl, 5 mM EDTA, or 5 mM EDTA+4 M Gdn-HCl as the mobile phase (MP).

[0262] FIG. 9 shows a chromatogram showing a result of performing SEC analysis for a sample including the recombinant human HAPLN1 protein using 50 mM PB+150 mM NaCl+1M Arg-HCl (pH 6.3) as the mobile phase.

[0263] As shown in FIGS. 8 to 9, when hydrochloride was added to the mobile phase, the monomer peak appeared most clearly.

[0264] FIG. 10 shows a chromatogram showing a result of performing SEC analysis using 50 mM PB+300 mM NaCl, 0.1 M Arg-HCl, 0.5 M Arg-HCl, 1.0 M Arg-HCl, or 1.0 M Gdn-HCl as the mobile phase. The analysis results are shown in Table 8 below.

TABLE-US-00008 TABLE 8 Mobile phase Monomer (%) HMW (%) LMW (%) 50 mM PB + 22.4 76.9 0.8 300 mM NaCl 0.1M Arg-HCl 26.1 73.9 ND 0.5M Arg-HCl 62.0 38.0 ND 1.0M Arg-HCl 71.8 28.2 ND 1.0M Gdn-HCl 77.2 22.8 ND

[0265] As shown in FIG. 10 and Table 8, the recombinant human HAPLN1 protein was well isolated with increasing concentration of Arg-HCl. In particular, when Arg-HCl and Gdn-HCl were used at a concentration of 1.0 M, the ability to isolate the recombinant human HAPLN1 protein was excellent.

[0266] FIG. 11 shows a chromatogram showing a result of performing SEC analysis using urea, which is known as a monomer assay additive, as a mobile phase, such as 50 mM PB+300 mM NaCl, 0.1 M urea, 0.5 M urea, 1.0 M urea, 2.0 M urea, 4.0 M urea, or 6.0 M urea. The analysis results are shown in Table 9 below.

TABLE-US-00009 TABLE 9 Mobile phase Monomer (%) HMW (%) LMW (%) 50 mM PB + 26.4 73.6 ND 300 mM NaCl 0.1M urea 26.4 73.6 ND 0.5M urea 26.7 73.3 ND 1.0M urea 27.7 72.3 ND 2.0M urea 32.1 67.9 ND 4.0M urea 75.6 24.4 ND 6.0M urea 75.6 24.4 ND

[0267] As shown in FIG. 11 and Table 9, the recombinant human HAPLN1 protein was well isolated with increasing concentration of urea. However, unlike hydrochloride, when urea was used at a high concentration of 4.0 M or more, it was possible to isolate the recombinant human HAPLN1 protein.

[0268] FIG. 12 shows a chromatogram showing a result of performing SEC analysis using 1.0 M Gdn-HCl, 4.0 M urea, or 1.0 M Arg-HCl as a mobile phase. The analysis results are shown in Table 10 below.

TABLE-US-00010 TABLE 10 Mobile phase Monomer (%) HMW (%) LMW (%) 1.0M Gdn-HCl 77.2 22.8 ND 4.0M urea 75.6 24.4 ND 1.0M Arg-HCl 71.8 28.2 ND

[0269] As shown in FIG. 12 and Table 10, when hydrochloride such as Gdn-HCl and Arg-HCl is used even at a low concentration of 1.0 M, the ability to isolate the recombinant human HAPLN1 protein was equivalent to or higher than that of using urea at a high concentration of 4.0 M.

[0270] Therefore, when SEC analysis was performed using 1.0 M hydrochloride as the additive of the mobile phase, the excellent ability to isolate the recombinant human HAPLN1 protein was observed, and inaccurate peaks in the chromatogram were significantly reduced, allowing accurate analysis of the monomer of the recombinant human HAPLN1 protein.

[0271] Taken together, it was confirmed that a ratio of the recombinant ECM protein monomer in the sample may be analyzed by performing size exclusion chromatography using the mobile phase containing hydrochloride.