Method for producing recombinant human nerve growth factor

Abstract

A method for producing recombinant human nerve growth factor, which can be used as a therapeutic drug, is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the production of highly pure, highly active, and non-deformed recombinant human nerve growth factor using genetic engineering methods.

Background Art

[0002] Oxervate (cenegermin, recombinant human nerve growth factor, rhNGF) is an eye drop initially developed by Dompe, an Italian pharmaceutical company. It is a rare pharmaceutical for treating adult patients with moderate to severe neurotrophic keratitis (NK) and is currently already on the market in the European Union and the United States. In addition, indications for treating Alzheimer's disease are also under research and development.

[0003] To produce recombinant human nerve growth factor, the original research company first expressed proNGF inclusion bodies, and then obtained recombinant human nerve growth factor through denaturation and regeneration, purification of the proNGF regeneration solution, enzymatic cleavage, and purification. However, since the above method requires enzymatic cleavage and further purification after enzymatic cleavage, the steps are relatively numerous and the process is relatively cumbersome.

[0004] Therefore, researchers have been searching for a simpler and more direct method that can denature and regenerate. Collins, et al. (US5,986,070) invented a direct denaturation and regeneration method for rhNGF using a method of denaturing with 8M urea using Tris as a buffer and regenerating under the condition of 8M urea, and obtained highly active rhNGF in such a method. However, as a result of measurement by mass spectrometry, rhNGF obtained by such a method has many impurities with a molecular weight close to that of rhNGF, and such impurities may be deformation products of rhNGF. Since both the molecular weight and properties of deformed rhNGF and non-deformed rhNGF are very similar, this causes difficulties in subsequent separation and purification, and it is very difficult to separate by general methods. Even if separation is performed, the yield is low.

Summary of the Invention

Problems to be Solved by the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a method for producing recombinant human nerve growth factor with high purity, high activity and no deformation. Through long-term research on rhNGF, the inventor of the present application found that when ammonium bicarbonate (NH4HCO3) is used as a buffer and denaturation and regeneration are carried out in the presence of urea (urea), the recombinant human nerve growth factor produced using the method of the present invention has significantly reduced impurities of non-target proteins, high purity, excellent biological activity, can be used as a therapeutic drug, and has good application prospects.

Means for Solving the Problems

[0006] To achieve the above object, the present invention adopts the following technical methods: The present invention provides a method for producing recombinant human nerve growth factor comprising the following steps: (a) Expressing and separating inclusion bodies of recombinant human nerve growth factor in Escherichia coli; (b) Washing the inclusion bodies using an inclusion body washing solution; (c) Adding a denaturing solution to dissolve the inclusion bodies; (d) Adding a regeneration solution to regenerate the inclusion bodies; (e) Purifying to obtain recombinant human nerve growth factor; Here, in step (d), ammonium bicarbonate is used as a buffer.

[0007] The inclusion bodies of recombinant human nerve growth factor can be expressed and separated using a commercialized or known Escherichia coli protein expression system. According to a preferred embodiment of the present invention, in the step (a), the amino acid sequence of human nerve growth factor is represented by SEQ ID NO: 1, and the full-length gene synthesis of the human nerve growth factor gene, vector insertion, transformation into Escherichia coli, expression, cell disruption, centrifugation, and precipitation collection steps are included. According to a preferred embodiment of the present invention, the vector is pET28a, the Escherichia coli is Escherichia coli expression strain BL21(DE3)plyss, the expression is IPTG-induced expression, and the cell disruption is by ultrasonic disruption or homogenizer disruption, etc.

[0008] After separating the inclusion bodies of recombinant human nerve growth factor, the inclusion bodies can be washed with an inclusion body washing solution to remove impurities. The inventors of the present application selected an inclusion body washing method particularly applicable to the recombinant human nerve growth factor of the present invention by conducting various experiments on the combination of the inclusion body washing solutions. According to a preferred embodiment of the present invention, the step (b) includes the following steps: resuspending the precipitate using inclusion body washing solution A, collecting the precipitate through ultrasonic treatment and centrifugation, where the inclusion body washing solution A contains 20-100 mM Tris, 50-150 mM NaCl, 1-10 mM EDTA, 0.5-3% Triton X-100, and pH 8.0-8.5; resuspending the precipitate using inclusion body washing solution B, collecting the precipitate through ultrasonic treatment and centrifugation, where the inclusion body washing solution B contains 20-100 mM Tris, 50-150 mM NaCl, 1-10 mM EDTA, 1-4 M urea, and pH 8.0-8.5; and further resuspending the precipitate using inclusion body washing solution C, collecting the precipitate through ultrasonic treatment and centrifugation, where the inclusion body washing solution C contains 20-100 mM Tris, 50-150 mM NaCl, 1-10 mM EDTA, and pH 8.0-8.5. More preferably, the step (b) includes the following steps: resuspending the precipitate using inclusion body washing solution A, collecting the precipitate through ultrasonic treatment and centrifugation, and repeating the washing step once, where the inclusion body washing solution A contains 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, and pH 8.5; resuspending the precipitate using inclusion body washing solution B, collecting the precipitate through ultrasonic treatment and centrifugation, and repeating the washing step once, where the inclusion body washing solution B contains 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 2 M urea, and pH 8.5; and further resuspending the precipitate using inclusion body washing solution C, collecting the precipitate through ultrasonic treatment and centrifugation, and repeating the washing step once, where the inclusion body washing solution C contains 50 mM Tris, 100 mM NaCl, 5 mM EDTA, and pH 8.5.

[0009] After purifying the inclusion bodies of recombinant human nerve growth factor, in order to obtain the target protein with the correct folding form and biological activity, the inclusion bodies must be subjected to denaturation and regeneration steps.

[0010] According to a preferred embodiment of the present invention, the step (c) includes: adding a denaturing solution containing 8 M urea, 15 - 25 mM citric acid, and pH 2.8 - 3.2 to the inclusion bodies at a ratio of wet weight (g) of the inclusion bodies: volume (mL) of the denaturing solution being 1:20 - 30, centrifuging after dissolution to remove the precipitate. According to a preferred embodiment of the present invention, the denaturing solution contains 8 M urea, 20 mM citric acid, and pH 3.0, and the denaturing solution is added to the inclusion bodies at a ratio of wet weight (g): volume (mL) being 1:25.

[0011] According to a preferred embodiment of the present invention, the step (d) includes the following steps: adding 1 / 4 volume of 0.8 - 1.0 M ammonium bicarbonate pH 8.0 - 9.0 and an 8 M urea solution to the denaturing solution, adding DTT to reach a final concentration of 5 - 10 mM, maintaining at 25 - 37 °C for 30 - 60 minutes; adding oxidized glutathione to reach a final concentration of 20 - 40 mM, maintaining at 25 - 37 °C for 10 - 15 minutes; further adding 19 volumes of a dilution buffer containing 50 - 150 mM Na2HPO4, 5 - 15 mM ethanolamine, 4.2 - 4.6 M urea, 14 - 18% PEG300, pH 8.3 - 8.5, then adding cysteine to reach a final concentration of 1 - 5 mM; degassing with argon gas, regenerating at 4 °C for 1 - 7 days; ultrafiltrating and concentrating the regeneration solution with a 3K membrane so that the final concentration of rhNGF reaches 1 - 2 mg / mL. More preferably, the concentration of ammonium bicarbonate is 1 M, the pH is 8.5, the final concentration of DTT is 5 mM, the final concentration of oxidized glutathione is 20 mM, the dilution buffer contains 100 mM Na2HPO4, 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, pH 8.3, and the final concentration of cysteine is 3 mM.

[0012] After denaturation and regeneration of the encapsulated body, the target protein can be purified using a known chromatography system. According to one embodiment of the present invention, the step (e) includes an SP purification and a C4 reverse phase chromatography step.

Advantages of the Invention

[0013] Beneficial effects of the present invention: 1. The operation is simple and enzyme cleavage is not required, so there are fewer steps, and the obtained product has fewer impurities and high purity. 2. The method of the present invention does not destroy the biological activity of the target protein, and the obtained product can be used as a candidate drug.

[0014] The present invention provides a method for producing recombinant human nerve growth factor. Because of fewer impurities, it has high purity, excellent biological activity, can be used as a therapeutic drug, and has good application prospects.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0016] The following examples and experimental examples are for more specifically explaining the present invention, but in no form do they limit the present invention.

[0017] Unless otherwise specified, all raw material components used in the following examples are commercially available.

[0018] Example 1: Production of rhNGF inclusion bodies 1.1 Construction of rhNGF expression strain Human nerve growth factor mature peptide (NGF) has two amino acids removed from the C-terminus, and its amino acid sequence is represented by SEQ ID NO:1. According to the codon preference of Escherichia coli, the codons were optimized to synthesize the full-length gene of the human nerve growth factor gene. Its gene sequence was inserted into the pET28a multiple cloning site (MCS) Xba I and Xho I as shown in SEQ ID NO:2 to construct the plasmid hNGFpET28a. A strong promoter T7 was used to express the protein. The plasmid hNGFpET28a was transformed into the Escherichia coli expression strain BL21(DE3)plyss (purchased from Promega) using a heat shock at 42°C. IPTG-induced expression and cell disruption by ultrasonic waves were performed on two colonies, followed by centrifugation at 12000 rpm × 5 min. The SDS-PAGE patterns of the supernatant and precipitate of the expression product are shown in Figure 1. The seed bacterial solution was put into 15% glycerol to prepare a bacterial glycerol stock, which was stored at -80°C.

[0019] 1.2 IPTG-induced expression The bacterial glycerol stock was inoculated into the antibiotic-containing medium at a volume ratio of 1:1000, and cultured overnight at 37 °C and 180 rpm. After that, the inoculum was inoculated into the antibiotic-containing medium at a volume ratio of 1:100, and cultured at 37 °C and 180 rpm until the OD600 reached 0.3 - 0.8. Then, IPTG was added to a final concentration of 1 mM, and the culture was continued at 25 °C and 170 rpm for 18 hours. After centrifugation at 8500×g for 10 minutes to remove the supernatant and collect the cells, the samples were extracted and sonicated. The precipitate was subjected to SDS-PAGE detection. The SDS-PAGE pattern of the induced expression product is shown in Figure 2.

[0020] 1.3 Isolation of inclusion bodies The cells and the disrupted cell lysate (2 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8.5) were added to the disrupted cell lysate at a ratio of wet weight (g): volume (mL) of 1:5 - 1:10, sonicated at 80% power for 2 seconds every 3 seconds for 5 minutes, and the sonication step was repeated 4 times. After centrifugation at 12000×g for 15 minutes at 4 °C, the precipitate was collected.

[0021] 1.4 Purification of inclusion bodies The inclusion bodies were resuspended using inclusion body washing solution A (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, pH 8.5), sonicated at 80% power for 2 seconds every 3 seconds for 5 minutes, and the sonication step was repeated 1 time. After centrifugation at 12000×g for 15 minutes at 4 °C, the precipitate was collected. The above washing step was repeated once.

[0022] The precipitate was resuspended using inclusion body washing solution B (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 2 M urea, pH 8.5), sonicated at 80% power for 2 seconds every 3 seconds for 5 minutes, and the sonication step was repeated 1 time. After centrifugation at 12000×g for 15 minutes at 4 °C, the precipitate was collected, and the above washing step was repeated once.

[0023] Furthermore, the precipitate was resuspended using inclusion body washing solution C (50 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8.5), sonicated at 80% power for 2 seconds at 3-second intervals for 5 minutes, with the ultrasonic wave stage repeated once, and centrifuged at 12,000 rpm for 15 minutes at 4°C to collect the precipitate.

[0024] Example 2: Denaturation, Renaturation and Purification of rhNGF Inclusion Bodies in Ammonium Bicarbonate 2.1 Denaturation of Inclusion Bodies The inclusion body precipitate in Example 1 and the denaturing solution (8 M urea, 20 mM citric acid, pH 3.0) were mixed at a ratio of wet weight (g): volume (mL) of 1:25, and after dissolution, centrifuged to remove the precipitate.

[0025] 2.2 Renaturation of Inclusion Bodies To the denaturing solution, 1 / 4 volume of 1 M ammonium bicarbonate pH 8.5 and 8 M urea solution were added, dithiothreitol (DTT) was added to reach a final concentration of 5 mM, and the mixture was maintained at 25°C for 30 - 60 minutes; then oxidized glutathione was added to reach a final concentration of 20 mM and maintained at 25°C for 10 - 15 minutes; 19 volumes of dilution buffer (100 mM Na2HPO4, 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, pH 8.3) were added; cysteine was added to reach a final concentration of 3 mM; degassed with argon gas and renatured at 4°C for 1 - 7 days; the renaturation solution was ultrafiltered and concentrated through a 3K membrane so that the final concentration of rhNGF reached 1 - 2 mg / mL.

[0026] At the same time, a control sample was constructed, and the difference was that 1 / 4 volume of 1 M Tris pH 8.5 and 8 M urea were added to the denaturing solution, and all other conditions were the same.

[0027] 2.3 Purification 2.3.1 SP Purification SP column: SP XL 1 mL from GE Sample loading: Buffer components: 20 mM NaAc pH 5.0, conductivity 4.5 Elution conditions: gradient elution A: 20 mM NaAc pH 5.0 B: 20 mM NaAc, 2 M NaCl pH 5.0 From A to B in 20 min.

[0028] 2.3.2 C4 Reverse Phase Chromatography Before sample loading, the protein was ultrafiltered and concentrated through a 3K membrane so that the final concentration of rhNGF reached 0.8 - 1.2 / mL. C4 column: Vydac 214TP 10μm C4 250×10 mM A: 0.1% trifluoroacetic acid H2O solution B: 0.1% trifluoroacetic acid acetonitrile solution Chromatography conditions: flow rate 5 mL / min Time (min) %B 0 5% 5 - 10 5 - 20% 10 - 40 20 - 50% 40 - 50 50 - 80%.

[0029] 2.4 Mass Spectrometry Detection The mass spectrometry conditions are as follows: Waters UPLC-XEVO G2 Q-TOF LC / MS system. The composition of the liquid phase part of the system is as follows: BSM binary high-pressure mixing pump, SM sample manager, TUV ultraviolet detector; the composition of the mass spectrum part is as follows: ESI source, Q-TOF detector. Data processing and analysis used Masslynx V4.1 and BiopharmaLynx analysis software (Version: 1.2).

[0030] Liquid Phase Conditions Chromatography column: Mass PREPTM Micro Desalting Column 2.1×5 mM (intact protein molecular weight analysis), column temperature: 80℃ Mobile phase A: 0.1% FA-H2O Mobile phase B: 0.1% FA-CAN Seal Wash Solution: 10% IPA Mass Spectrometry Cleaning Solution: 50% ACN Mass Spectrometry IntelliStart Valve Cleaning Solution: 50% MeOH Sample Injection Volume: 10 μL Sample Chamber Temperature: 10 °C

[0031]

Table 1

[0032] Mass Spectrometry Conditions MS data was collected in Resolution mode using the full continuum mode for all. LockSpray Collection Mode: Real-time collection, no calibration applied. Calibration Solution: Real-time calibration (LockSpray) solution: 2 ng / μL LE solution; Mass Axis Calibration Solution: 2 μg / μL sodium iodide solution

[0033]

Table 2

[0034] The mass spectrum of rhNGF produced with the control sample using Tris buffer is shown in Figure 3, and it can be seen that there are many impurity peaks with similar molecular weights to rhNGF. In contrast, as shown in Figure 4, the mass spectrum of rhNGF produced with the NH4HCO3 buffer of the present invention has almost no impurity peaks, and the purity of the rhNGF sample has been greatly improved.

[0035] Example 3: Measurement of rhNGF Biological Activity The biological activities of rhNGF produced in Example 2 and commercially available rhNGF (purchased from Sino Biological) were measured by the TF1 cell proliferation method.

[0036] The experimental steps are as follows: 1. Wash TF1 cells (ATCC@CRL-2003 TM ) in 1640 medium preheated to 37°C twice, and centrifuge at 300 - 500 g for 5 minutes; 2. Count the TF1 cells, suspend them in 1640 medium containing 10% FBS to reach an appropriate density, and then inoculate them into a 96-well plate at 10,000 cells / 150 μL / well; 3. Use 1640 medium in a 96-well plate to serially dilute the rhNGF sample in 9 gradients with a 3-fold dilution; after adding the diluted sample into a 96-well cell culture plate at 50 μL / well, fill the periphery of the 96-well plate with distilled water at 200 μL / well; 4. Incubate in an incubator at 37°C and 5% CO2 for 3 days (it can be extended to 4 days depending on the situation); 5. After 3 days, add 20 μL of CCK-8 solution to each well of the 96-well cell culture plate and continue to culture in an incubator at 37°C for 8 hours. 6. After mixing evenly, read the OD450 value with a microplate reader, perform data analysis with GraphPad Prism6, draw a graph, and calculate the EC50.

[0037] As shown in Figure 5, the results indicate that rhNGF produced using Tris buffer and rhNGF produced using the ammonium bicarbonate buffer of the present invention can both promote the growth of TF1 cells, similar to commercialized rhNGF. This suggests that rhNGF produced by the method of the present invention has excellent biological activity and can be used as a candidate drug.

Claims

1. In a method for producing recombinant human nerve growth factor, (a) expressing and isolating inclusion bodies of recombinant human nerve growth factor in Escherichia coli; (b) washing the inclusion bodies using an inclusion body washing solution; (c) adding a denaturing solution to dissolve the inclusion bodies; (d) adding a refolding solution to refold the inclusion bodies; and (e) purifying to obtain recombinant human nerve growth factor, wherein step (b) includes resuspending the precipitate using inclusion body washing solution A containing 20-100 mM Tris, 50-150 mM NaCl, 1-10 mM EDTA, 0.5-3% Triton (registered trademark) X-100, pH 8.0-8.5, collecting the precipitate through ultrasonic treatment and centrifugation; resuspending the precipitate using inclusion body washing solution B containing 20-100 mM Tris, 50-150 mM NaCl, 1-10 mM EDTA, 1-4 M urea, pH 8.0-8.5, collecting the precipitate through ultrasonic treatment and centrifugation; and further resuspending the precipitate using inclusion body washing solution C containing 20-100 mM Tris, 50-150 mM NaCl, 1-10 mM EDTA, pH 8.0-8.5, collecting the precipitate through ultrasonic treatment and centrifugation; step (c) includes adding a denaturing solution containing 8 M urea, 15-25 mM citric acid, pH 2.8-3.2 to the inclusion bodies at a ratio of wet weight (g) of the inclusion bodies to volume (mL) of the denaturing solution of 1:20-30, centrifuging after dissolution to remove the precipitate; step (d) uses ammonium bicarbonate as a buffer, and In the step (d), 1 / 4 volume of 0.8 - 1.0 M ammonium bicarbonate pH 8.0 - 9.0 and 8 M urea solution are added to the denaturing solution, DTT is added to reach a final concentration of 5 - 10 mM, and the mixture is maintained at 25 - 37 °C for 30 - 60 minutes; oxidized glutathione is added to reach a final concentration of 20 - 40 mM, and the mixture is maintained at 25 - 37 °C for 10 - 15 minutes; further, 19 volumes of a dilution buffer containing 50 - 150 mM Na ２ HPO ４ , 5 - 15 mM ethanolamine, 4.2 - 4.6 M urea, 14 - 18% PEG300, pH 8.3 - 8.5 is added, then cysteine is added to reach a final concentration of 1 - 5 mM; degassed with argon gas and regenerated at 4 °C for 1 - 7 days; the regenerated solution is ultrafiltered and concentrated with a 3K membrane so that the final concentration of rhNGF reaches 1 - 2 mg / mL A method for producing recombinant human nerve growth factor, characterized by the above.

2. In step (a), the amino acid sequence of human nerve growth factor is represented by SEQ ID NO: 1, and the method for production according to claim 1, characterized by including the steps of synthesizing the full-length gene of the human nerve growth factor gene, inserting it into a vector, transforming Escherichia coli, expressing, disrupting the bacteria, centrifuging, and collecting the precipitate.

3. The method for production according to claim 2, characterized in that the vector is pET28a, the Escherichia coli is Escherichia coli expression strain BL21(DE3)pLysS, the expression is IPTG-induced expression, and the disruption of the bacteria is ultrasonic disruption or homogenizer disruption.

4. The step (b) is to resuspend the precipitate using the inclusion body washing solution A containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton (registered trademark) X-100, pH 8.5, collect the precipitate through ultrasonic treatment and centrifugation, and repeat the washing step once; resuspend the precipitate using the inclusion body washing solution B containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 2 M urea, pH 8.5, collect the precipitate through ultrasonic treatment and centrifugation, and repeat the washing step once; and further resuspend the precipitate using the inclusion body washing solution C containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8.5, collect the precipitate through ultrasonic treatment and centrifugation, and repeat the washing step once. The manufacturing method according to claim 1 is characterized by including these steps.

5. The denaturing solution contains 8 M urea, 20 mM citric acid, and pH 3.

0. The manufacturing method according to claim 1 is characterized in that the inclusion body and the denaturing solution are introduced in a ratio of wet weight (g): volume (mL) of 1:

25.

6. The concentration of the ammonium hydrogen carbonate is 1 M, the pH is 8.5, the final concentration of the DTT is 5 mM, the final concentration of the oxidized glutathione is 20 mM, and the dilution buffer is 100 mM of Na ２ HPO ４ , 10 mM of ethanolamine, 4.6 M of urea, 15.8% of PEG 300, with a pH of 8.3, and the final concentration of the cysteine is 3 mM. The production method according to claim 1, characterized in that.

7. The step (e) includes an SP purification and a C4 reverse phase chromatography step. The manufacturing method according to claim 1 is characterized by this.

8. 1) A step of expressing and separating inclusion bodies of recombinant human nerve growth factor in Escherichia coli. The amino acid sequence of the human nerve growth factor is represented by SEQ ID NO: 1, the expression vector is pET28a, the Escherichia coli is Escherichia coli expression strain BL21(DE3)pLyss, and the expression is IPTG-induced expression; 2) A step of washing the inclusion bodies in step 1) using an inclusion body washing solution; here: 2a) Wash the inclusion bodies using the inclusion body washing solution A containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton (registered trademark) X-100, pH 8.5; 2b) Wash the inclusion bodies obtained in step 2a) using the inclusion body washing solution B containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, 2 M urea, pH 8.5; 2c) Wash the inclusion bodies obtained in step 2b) using the inclusion body washing solution C containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8.5; Optionally repeat steps 2a) to 2c); 3) A denaturing solution containing 8 M urea, 20 mM citric acid, and pH 3.0 is introduced at a ratio such that the wet weight (g) of the encapsulated body to the volume (mL) of the denaturing solution is 1:25, and the encapsulated body obtained by the washing in step 2) is dissolved; 4) A step of performing encapsulation body regeneration on the encapsulation body obtained in step 3), wherein 1 / 4 volume of 1.0 M ammonium bicarbonate pH 8.5 and 8 M urea solution are added to the denaturing solution of step 3), DTT is added to reach a final concentration of 5 mM, and the mixture is maintained at 25 - 37 °C for 30 - 60 minutes; oxidized glutathione is added to reach a final concentration of 20 mM, and the mixture is maintained at 25 - 37 °C for 10 - 15 minutes; 19 volumes of 100 mM Na ２ HPO ４ , 10 mM ethanolamine, 4.6 M urea, 15.8% PEG300, a dilution buffer containing pH 8.3 are added; cysteine is added to reach a final concentration of 3 mM; degassed with argon gas and regenerated at 4 °C for 1 - 7 days; the regenerated solution is ultrafiltered and concentrated with a 3K membrane so that the final concentration of rhNGF reaches 1 - 2 mg / mL. The production method according to claim 1, characterized by including this step.

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