CN106279397A - A kind of extracting method of nerve growth factor - Google Patents

Abstract

The invention provides a method for extracting nerve growth factor, the method comprising: after homogenizing mouse submandibular gland tissue, centrifuging to collect the homogenate supernatant; performing CM reverse adsorption cation exchange chromatography on the homogenate supernatant Finally, collect the flow-through liquid; acidify and centrifuge the flow-through liquid and collect the acid hydrolysis supernatant; perform adsorption cation exchange chromatography on the acid hydrolysis supernatant to collect the target protein liquid corresponding to the target protein peak; Hydrophobic chromatography to obtain the target protein solution after hydrophobic chromatography. The process of the present invention simplifies the process steps, the process is simple, not only greatly shortens the time used for preparation, but also improves the uniformity and activity of the product, and the purity of the sample is further improved, providing a new method for the separation and purification of NGF way.

Description

A kind of extraction method of nerve growth factor

technical field

The invention relates to the field of biotechnology, in particular to a method for extracting nerve growth factor.

Background technique

Mouse nerve growth factor (mNGF) is the first discovered NGF and also the most thoroughly studied NGF. The complete mNGF consists of α, β, and γ subunits in the form of α ₂ β ₂ γ ₂ . Among them, the two β subunits are the biologically active center of mNGF, and have all the biological functions of mNGF; Binding of receptors. The two β subunits form a dimer, β-NGF, in a non-covalent bond.

The establishment of separation and purification process of β-NGF is the basis of its application and development. In 1960, Cohen first proposed and established a method for the separation and purification of β-NGF in the mouse submandibular gland. Subsequently, many literatures reported improved methods for isolating and purifying NGF from rat submandibular gland. According to the differences in their isolation strategies, these methods can be divided into two categories: one is to isolate the intact 7S NGF first, then depolymerize the 7S NGF to release the biologically active β-subunit dimer (β-NGF), and then β-NGF is obtained by further separation; the other is the direct separation of β-subunit dimers of NGF with biological activity, namely 2.5S NGF.

At present, there are four domestic injection mouse NGF on the market. The purification methods of each company are mostly based on two-step cation (CM sepharose FF) exchange chromatography. The main process of the method includes: tissue fragmentation --- full dialysis --- CM reverse adsorption chromatography---full dialysis or ultrafiltration---acidification dissociation---CM adsorption chromatography-size exclusion chromatography. For the detailed process, please refer to the method introduced in the article "Preparation of Freeze-dried Mouse Nerve Growth Factor Pilot Scale and Discussion on Some Issues" published by Ren Wanqiong et al. Specifically, the mouse submandibular gland tissue was homogenized, and the homogenate supernatant was obtained after centrifugation. After fully dialyzing the homogenate supernatant, reverse adsorption ion exchange chromatography was performed to collect the flow-through liquid; the flow-through liquid was dialyzed or ultrafiltered again and then subjected to acidification dissociation and centrifugation to obtain the acid hydrolysis supernatant; the acid hydrolysis supernatant was Adsorption ion exchange chromatography is carried out to collect the target protein peak, and the target protein solution is subjected to size exclusion chromatography to obtain NGF extract.

However, the process involves two steps of dialysis, which is time-consuming and cumbersome. Each step of dialysis takes more than 20 hours, and leakage and rupture are prone to occur when the dialysis volume is large. In addition, because the mouse submandibular gland is rich in β-NGF endopeptidase and carboxypeptidase B protease, these two enzymes can cut amino acids at the amino or carboxyl terminals of β-NGF, so the longer the dialysis step, the more NGF is cut. The greater the probability of cleavage, the greater the proportion of missing amino acids at both ends of NGF will increase, which will affect the uniformity and activity of the product. At the same time, the existence of multiple cleavage forms is not conducive to the quality control of the product.

Therefore, it is necessary to provide a fast and simple method for extracting nerve growth factor in order to obtain nerve growth factor with high uniformity, strong activity and good quality.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned defects in the prior art, and provide a preparation method of mouse NGF.

The inventors of the present invention discovered unexpectedly during the research process that adding a hydrophobic chromatography process in the NGF extraction step can achieve an extraction effect similar to or even better than the conventional extraction method without using the two-step dialysis in the conventional method , can greatly save the time required for extraction, and at the same time, the uniformity and activity of the product can also be improved.

Based on the above findings, the present invention provides a method for extracting nerve growth factor, the method comprising: after homogenizing mouse submandibular gland tissue, centrifuging to collect the homogenate supernatant; performing CM back-adsorption of cations on the homogenate supernatant Collect the flow-through after exchange chromatography; acidify and centrifuge the flow-through and collect the acid hydrolysis supernatant; perform adsorption cation exchange chromatography on the acid hydrolysis supernatant to collect the target protein solution corresponding to the target protein peak; The protein solution is subjected to hydrophobic chromatography to obtain the target protein solution after hydrophobic chromatography.

Optionally, the equilibrium liquid of the hydrophobic column used in the hydrophobic chromatography is a buffer solution with a salt concentration of 0.5-3.0 mol/L and a pH value of 6.0-9.0.

Optionally, the equilibrium solution of the hydrophobic column used in the hydrophobic chromatography is obtained by dissolving a salt selected from sodium chloride, ammonium sulfate and sodium sulfate in phosphate buffer or Tris-HCl buffer, The final concentration of the salt dissolved in the equilibrium solution is 0.5-3.0 mol/L, and the pH value is 6.0-9.0.

Optionally, when the salt is sodium chloride, the final concentration of sodium chloride is 0.5-3.0mol/L; when the salt is ammonium sulfate, the final concentration of ammonium sulfate is 0.5-2.0mol/L; When the salt is sodium sulfate, the final concentration of sodium sulfate is 0.5-2.0mol/L.

Optionally, the pH value of the phosphate buffer is 6.0-8.0, and the pH value of the Tris-HCl buffer is 7.1-9.0.

Optionally, the hydrophobic column medium in the hydrophobic chromatography is Butyl-S-FF, Phenyl FF, Phenyl HP, Butyl FF, OctylFFButyl HP, Phenyl HS FF or FractogelEMD Phenyl(S).

Optionally, the sample loading flow rate during the hydrophobic chromatography is 5-300 cm/hr, preferably 80-200 cm/hr.

Optionally, the column temperature in the hydrophobic chromatography process is 2-8°C.

Optionally, the method also includes the step of performing size exclusion chromatography.

Optionally, the pH value of the homogenate supernatant is 6.0-7.0.

Optionally, the method for obtaining the homogenate supernatant comprises the following steps:

Mix the submandibular gland of the mouse with purified water and homogenate fully, collect the supernatant by centrifugation, adjust the pH of the supernatant with a 0.5mol/L phosphate buffer solution of pH 6.0-7.0, and obtain the homogenate supernatant .

Optionally, the equilibrium liquid used in the CM back-adsorption cation exchange chromatography is a phosphate buffer solution with a concentration of 0.01-0.1 mol/L and a pH of 6.0-7.0; the sample loading flow rate is 5-700 cm/h.

Optionally, the acidifying centrifugation includes the following steps: adding acid buffer solution to the flow-through solution to adjust the pH value to 3.5-4.5, and then adding NaCl to make the final concentration of NaCl in the system 0.1-0.5mol/L, and standing Centrifuge to obtain the acid hydrolyzed supernatant.

Optionally, the acidic buffer added to the flow-through is acetate buffer or citrate buffer with a pH of 3.5-4.5.

Optionally, the step of adsorption cation exchange chromatography comprises:

Equilibrate the filler with the adsorption cation exchange chromatography equilibrium solution, after loading the acid hydrolysis supernatant, first elute with the cation exchange chromatography equilibrium solution, the adsorbed protein is eluted with pH8.5～9.5, concentration 0.01～0.10mol/L Washing with Tris-HC1 washing buffer, and then performing gradient elution with 0.01-0.10mol/L Tris-HC1-0.2-0.8mol/L NaCl elution buffer at pH 8.5-9.5; wherein, the adsorbed cation The exchange chromatography equilibrium solution contains acetate with a concentration of 0.01-0.1 mol/L and NaCl with a concentration of 0.1-0.5 mol/L, and the pH value is 3.5-4.5. The method provided by the present invention has the following advantages:

1. From the perspective of the process flow, the present invention directly loads the mandibular gland tissue after homogenization, and directly acidifies it after CM anti-adsorption salt ion exchange chromatography, does not involve dialysis solution replacement or ultrafiltration steps, and greatly reduces the production and operation time. Compared with the conventional method, it saves 14-48 hours.

2. The proportion of intact β-NGF obtained by extraction is significantly increased, and the uniformity of the sample is greatly improved: judging from the isoelectric point, it is reduced from the previous three bands to two bands; from the results of mass spectrometry, it is reduced from the previous four peaks to Two peaks; the results of isoelectric point and mass spectrometry molecular weight analysis show that the process of the present invention can effectively inhibit the degradation of the C-terminal arginine of NGF by carboxypeptidase, and can also effectively reduce the ratio of the cut form of N-terminal AA.

3. The purity of the target protein solution obtained after hydrophobic chromatography can reach more than 99% or even 100%.

In summary, the process of the present invention simplifies the process steps, and the process is simple, which not only greatly shortens the time used for preparation, but also improves the uniformity and activity of the product, and the purity of the sample is further improved, providing for the separation and purification of NGF. a new way.

Description of drawings

Fig. 1 is HPLC purity detection result figure among the embodiment 1;

Fig. 2 is HPLC purity detection result figure in comparative example 1;

Fig. 3 is SDS-PAGE purity detection result figure in embodiment 1;

Fig. 4 is SDS-PAGE molecular weight detection result figure among the embodiment 1;

Fig. 5 is embodiment 1 and comparative example medium electric focusing electrophoresis comparative figure;

Fig. 6 is mass spectrometry molecular weight detection result figure in embodiment 1;

Fig. 7 is mass spectrometry molecular weight detection result figure in comparative example 1;

Figure 8 is the result of isoelectric focusing electrophoresis.

detailed description

The present invention will be described in detail below through specific embodiments. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

The invention provides a method for extracting nerve growth factor, which comprises the steps of centrifuging and collecting the homogenate supernatant after homogenizing mouse submandibular gland tissue; performing CM reverse adsorption cation exchange chromatography on the homogenate supernatant and collecting the flow-through ; carry out acidification and centrifugation to the flow-through liquid and collect the acid hydrolysis supernatant; carry out adsorption cation exchange chromatography on the acid hydrolysis supernatant to collect the target protein solution corresponding to the target protein peak; carry out hydrophobic chromatography on the target protein solution to obtain Target protein solution after hydrophobic chromatography.

In one embodiment of the present invention, the method further includes adjusting the pH value of the homogenate supernatant to 6.0-7.0 before performing CM reverse adsorption cation exchange chromatography. The method for obtaining the homogenized supernatant comprises the following steps: mixing the mouse submandibular gland with purified water pre-cooled to 4°C according to the mass volume ratio of 1:1-20, and then performing pressure homogenization, and the homogenized group The supernatant was collected by centrifugation at 8000-12000 g for 20-60 min, and the pH of the homogenate supernatant was adjusted with a 0.5 mol/L phosphate solution of pH 6.0-7.0. Wherein, after mixing the submandibular gland of the mouse with purified water, it can be first crushed with a tissue grinder, and then pressure homogenized with a high-pressure homogenizer.

In the embodiment of the present invention, there is no particular limitation on the method of CM back-adsorption cation exchange chromatography, and conventional methods in the art can be used, for example, CM32, CM Sepharose FF or other equivalent media can be used. The equilibrium liquid used in the CM back-adsorption cation exchange chromatography process is 0.01-0.1mol/L, pH6.0-7.0 phosphate buffer solution; the sample loading flow rate is 5-700cm/h. In a preferred embodiment of the present invention, the homogenate supernatant can be put on the CM-32 column (4.5 × 15cm) after equilibrating with the balance liquid, and then use a concentration of 0.02mol/L, pH6. 8 phosphate buffer was eluted at a flow rate of 300ml/h, and the collected non-adsorbed protein peak was the flow-through solution.

In the present invention, the acidifying centrifugation may include the following steps: adding an acidic buffer solution to the flow-through solution to adjust the pH value to 3.5-4.5, and then adding NaCl so that the final concentration of NaCl in the system is 0.1-0.5 mol/L, After standing still, centrifuge at 8000-16000g for 20-60min to obtain acid hydrolysis supernatant. Preferably, the acidic buffer added to the flow-through solution is acetate buffer or citrate buffer with a pH of 3.5-4.5. Specifically, in order to obtain a better acidifying centrifugation effect, acetate buffer solution with a pH of 4.0 and a concentration of 0.5 mol/L can be directly added to the flow-through solution to lower the pH of the system to below 4.5, and then NaCl is added to make the system The final concentration of NaCl in the solution was 0.4mol/L. After standing still for 10 minutes, centrifuge at 16000g at 4°C for 40 minutes, and collect the supernatant to obtain the acid hydrolysis supernatant.

In one embodiment of the present invention, the adsorption cation exchange chromatography can be performed by conventional methods. That is to say, the acid hydrolysis supernatant is loaded to a fully balanced cation exchange chromatography column, after the sample is loaded, it is washed to the baseline with the balance solution, and the impurity peak is eluted to the baseline with the washing buffer, and then the gradient is carried out with the elution buffer. For elution, collect when the number displayed on the UV detector rises from the baseline, and stop collecting the target protein peak when it falls to the baseline. For example, the method described in the article "Preparation of Freeze-dried Mouse Nerve Growth Factor in Pilot Scale and Discussion of Some Issues" published by Ren Wanqiong et al. can be used to carry out the adsorption cation exchange chromatography step.

Wherein, the adsorption cation exchange chromatography filler includes but not limited to CM Sepharose FF, SP Sepharose HP, Capto MMC and Fractogel EMD SO3-650.

In one embodiment of the present invention, the adsorption cation exchange chromatography can be carried out through the following steps: equilibrate the filler with adsorption cation exchange chromatography equilibrium solution, and load the acid hydrolysis supernatant at a flow rate of 70-400cm/hr After that, firstly, it was eluted with cation exchange chromatography equilibrium solution, and the adsorbed protein was washed with Tris-HCl washing buffer with pH 8.5-9.5 and concentration 0.01-0.10mol/L, and then with 0.01 ～0.10mol/LTris-HC1-0.2～0.8mol/LNaCl elution buffer for gradient elution; wherein, the adsorption cation exchange chromatography equilibrium solution contains acetate with a concentration of 0.01-0.1mol/L and 0.1 -0.5mol/L of NaCl, the pH value is 3.5-4.5.

In one embodiment of the present invention, the equilibrium solution of the hydrophobic column used in the hydrophobic chromatography is to dissolve a salt selected from sodium chloride, ammonium sulfate and sodium sulfate in phosphate buffer or Tris- The final concentration of salt dissolved in the equilibrium solution of the hydrophobic column is 0.5-3.0 mol/L obtained by the HCl buffer solution, and the pH value of the equilibrium solution of the hydrophobic column is 6.0-9.0.

In one embodiment of the present invention, sodium chloride is dissolved in phosphate buffer or Tris-HCl buffer to obtain the equilibrium solution of the hydrophobic column, and the final concentration of sodium chloride is 0.5-3.0mol/L, preferably 1- 2mol/L.

In another embodiment of the present invention, ammonium sulfate is dissolved in phosphate buffer or Tris-HCl buffer to obtain the equilibrium liquid of the hydrophobic column, and the final concentration of ammonium sulfate is 0.5-2.0mol/L, preferably 0.8-1.6 mol/L

In yet another embodiment of the present invention, sodium sulfate is dissolved in phosphate buffer or Tris-HCl buffer to obtain the equilibrium solution of the hydrophobic column, and the final concentration of sodium sulfate is 0.5-2.0mol/L, preferably 0.8-1.6 mol/L.

Wherein, the pH value of the phosphate buffer solution is 6.0-8.0, and the concentration is 0.01-0.1 mol/L; the pH value of the Tris-HCl buffer solution is 7.1-9.0, and the concentration is 0.01-0.1 mol/L.

Before hydrophobic chromatography, first adjust the concentration of salt ions in the target protein liquid, the adjustment method can be to make the type and concentration of ions in the target protein liquid and the hydrophobic column by adding the same solid salt. The concentration and type of ions in the equilibrium solution are consistent.

After sample loading, rinse with the balance solution of the hydrophobic column to the baseline, and then elute with the corresponding buffer solution without salt. When the number displayed on the UV detector starts to rise, start collecting, and stop collecting the target protein peak when it drops to the baseline.

Wherein, the hydrophobic column medium in the hydrophobic chromatography is Butyl-S-FF, Phenyl FF, Phenyl HP, Butyl FF, OctylFF, Butyl HP, Phenyl HS FF or FractogelEMD Phenyl(S). Preferably, the hydrophobic column medium in the hydrophobic chromatography is ButylFF.

In the present invention, the sample loading flow rate in hydrophobic chromatography is 5-300 cm/h, preferably 80-200 cm/h, and the column temperature is 2-8°C.

Preferably, the method provided by the present invention further includes performing exclusion chromatography after hydrophobic chromatography to obtain NGF extract.

Conventional methods can be used for size exclusion chromatography, and the equilibrium solution selected during size exclusion chromatography can be a common size exclusion chromatography buffer, preferably 0.01 to 0.1 mol/L phosphate (containing 0.1 to 0.2 mol/L chlorinated Sodium, pH6.5～7.0) buffer solution, collect the target protein peak when the number displayed on the ultraviolet detector rises from the baseline, and stop collecting the target protein peak when it drops to the baseline.

As the size exclusion chromatography medium, Superdex 75 prep grade chromatography, G25 chromatography and other equivalent media can be used. For example, using the method introduced in the article "Pilot-scale preparation of freeze-dried mouse nerve growth factor and discussion of some problems" published by Ren Wanqiong et al., Sephadex G-75 column chromatography column 2.6 × 110cm, with 0.05mol/L, pH4 .0 acetate elution, flow rate 78ml/h, collect the main peak of the elution peak, which is the obtained 2.5SNGF of purification route B.

Preferably, after the obtained NGF extract is pre-filtered with a 0.1 μm membrane, it is also necessary to filter out viruses with a virus-removing membrane, and the filtrate is collected. After passing the inspection, it is the product NGF extract.

The chromatography described in this article is all carried out in a conventional chromatography device. The most common chromatography column is a cylindrical or nearly cylindrical hollow tube, which is generally made upright when used. The height and diameter of the chromatographic column can be selected according to the specific production scale. The medium filled in the chromatography column is selected according to needs.

The method provided by the present invention may also include some conventional steps for extracting and preparing medicines, such as steps of virus inactivation, virus removal and filtration.

The virus inactivation method can be carried out using conventional virus inactivation methods in the art, for example, low pH value virus inactivation method, S/D virus inactivation method and sodium octanoate virus inactivation method can be used. The present invention has no particular limitation on the timing of virus inactivation, which can be carried out at any stage of the method of the present invention.

The virus-removing filtration step can use conventional filtration methods in the field, and membrane filtration is often used. Generally, the final filtration is performed after the extraction step is completed.

The method provided by the present invention obtains the beneficial effect of saving time and improving the uniformity and activity of the product by adding a hydrophobic chromatography step in the NGF preparation step. In order to make the whole extraction method form an efficient whole and better realize this Invention purpose of the invention, the inventor has carried out a large amount of adjustments as mentioned above to the collection method of the homogenate supernatant, the CM reverse adsorption cation exchange chromatography method, the acidification centrifugation method, and the adsorption cation exchange chromatography steps, so that the entire preparation The system and the hydrophobic chromatographic step form an integrated whole, which is better applicable to the technical scheme of extracting nerve growth factor through the hydrophobic chromatographic step. However, it should be understood that the above-mentioned specific implementation is only the preferred situation of the present invention rather than the only situation, and those skilled in the art can reasonably predict that other equivalent alternatives or obvious deformations other than the specific implementation given in the specification are all achievable.

The following examples will further illustrate the present invention.

Example 1

1) Homogenate: Take the submandibular gland of the mouse, add pre-cooled purified water to fully homogenate, and centrifuge the homogenate at 11000×g, 1hr, 4°C, collect the centrifuged supernatant, add an appropriate amount of 0.5mol/L phosphoric acid Salt solution (pH 6.5) was used to adjust the pH of the centrifuged supernatant to 6.8±0.2 for later use.

2) CM reverse adsorption: The centrifuged supernatant obtained in step 1) is subjected to CM Sepharose Fast Flow cation exchange chromatography, and the CM Sepharose Fast Flow chromatography column is fully balanced with 0.02mol/L, pH6.8 phosphate buffer before loading the sample , After loading the sample, rinse with the balance solution, collect the protein outflow peak, and the flow rate is 150cm/h.

3) Acidification: Add 1mol/L acetate buffer solution (pH4.0) to the flow-through solution to quickly reduce the pH to 4.0, then add NaCl to make the final concentration of NaCl in the system reach 0.4mol/L, and let it stand for about 5 minutes After centrifugation at 10000g at 4°C for 30min, the acid hydrolysis supernatant was taken.

4) CM2 column: CM Sepharose FF chromatography column is fully equilibrated with 0.05mol/L acetate buffer solution (pH4.0) before loading the sample. -HC1 (pH9.0) buffer to elute the impurity peaks to the baseline, and then use 0.05mol/L Tris-HC1 and 0.05mol/L Tris-HC1-0.4mol/L NaCl (pH9.0) gradient elution, according to the UV absorption Collect the target peak when the number displayed on the UV detector starts to rise, and stop collecting the target protein peak when it drops to the baseline.

5) Hydrophobic chromatography: Butyl Sepharose 4 FF chromatography column is fully equilibrated with 0.02mol/L phosphate (pH6.8)-1.5mol/L sodium chloride buffer. Add solid sodium chloride to the feed liquid obtained in step 4) so that the final concentration of sodium chloride in the feed liquid is 1.5mol/L, after the sodium chloride is fully dissolved, load the sample at a speed of 120cm/h, after the sample loading is completed Rinse to the baseline with equilibrium solution, then elute with 0.02mol/L phosphate (pH6.8) to collect the target peak.

6) Virus removal: filter the hydrophobic chromatography elution peak feed solution in 5) with a 0.1 μm membrane, then filter the virus with a 20 nm pore size virus removal membrane, and collect the filtrate, which is the NGF extract.

Comparative example 1

1) Homogenate: take the submandibular gland, add pre-cooled purified water to fully homogenate, and centrifuge the homogenate at 11000×g, 1 hr, 4°C, and collect the centrifuged supernatant.

2) Dialysis: The centrifuged supernatant was fully dialyzed in 20 mol/L, pH 6.8 phosphate buffer for 24 hours.

3) CM reverse adsorption chromatography: CM Sepharose FF chromatographic column was fully equilibrated with 20mmol/L, pH6.8 phosphate buffer solution before loading the sample. After sample loading, rinse with equilibrium solution and collect protein elution peaks.

4) Dialysis: The protein flow-through was dialyzed in 0.25mmol/L, pH6.8 phosphate buffer for 24 hours, during which the dialysate was replaced twice

5) Acidification: Add 1 mol/L acetate buffer (pH 4.0) to the solution after dialysis to quickly reduce the pH to 4.0, then add NaCl to make it 0.4 mol/L, let stand for about 5 minutes, then centrifuge at 10,000 g for 30 minutes, and take the supernatant.

6) CM2 column: CM Sepharose FF chromatography column is fully equilibrated with 50mmol/L acetate buffer (pH4.0) before loading the sample. (pH9.0) buffer to elute the miscellaneous peaks to the baseline, then use 50mmol/L Tris-HC1 and 50mmol/L Tris-HC1-0.4mol/L NaCl (pH9.0) gradient elution, collect the target peak according to the UV absorption , start collecting when the number displayed on the UV detector starts to rise, and stop collecting the target protein peak when it drops to the baseline.

7) Superdex 75 prep grade chromatographic column is fully equilibrated with pH6.8, 0.05mol/L phosphate-0.15mol/L sodium chloride buffer solution, and then the sample is loaded, and the number displayed on the UV detector starts to collect when it starts to rise from the baseline , stop collecting the target protein peak when it drops to the baseline.

8) The target protein solution is pre-filtered with a 0.1 μm membrane, and then filtered with a 20 nm pore size virus-removing membrane to remove viruses, and the filtrate is collected, which is the NGF extract.

Test example 1 The various index detections of the NGF extract of embodiment 1 and comparative example 1

1. HPLC detection of the purity of the NGF extract

The NGF extracts prepared in Example 1 and Comparative Example 1 were tested by HPLC. The detection method refers to the patent ZL200510130348.3. The test results of the NGF extracts in Example 1 and Comparative Example 1 are shown in Figure 1 and Figure 2 respectively.

It can be seen from the HPLC detection result chart that the sample purity of Example 1 is 100%, and the sample purity of Comparative Example 1 is 93.29%.

2. SDS-PAGE electrophoresis to detect the molecular weight and purity of NGF

Using the SDS-PAGE electrophoresis loading buffer, with or without adding mercaptoethanol, load the low molecular weight Marker and 10 μg of the mNGF obtained in Example 1, respectively, and perform electrophoresis. The electrophoresis condition is 200V constant voltage, 45 minutes . Stained with Coomassie Brilliant Blue G-25 to detect the molecular weight and purity of NGF, the results are shown in Figures 3 and 4.

It can be seen from Figures 3 and 4 that the molecular weight of mNGF is about 13kDa, indicating that the obtained target protein is correct, there is only one band without other impurities, and the purity can reach 100%.

3. Isoelectric focusing electrophoresis

According to the method introduced in the article "Pilot-scale preparation of freeze-dried mouse nerve growth factor and discussion of several issues" published by Ren Wanqiong et al., the isoelectric focusing electrophoresis is performed. The results are shown in Figure 5. The analysis of the isoelectric focusing electrophoresis results is shown in the table 1.

Table 1

From the results in Figure 5 and Table 1, it can be seen that lane 1 is the NGF obtained by using the method of Example 1, with isoelectric points of 9.1 and 8.9, mainly two bands, and the content of full-length NGF (the isoelectric point is 9.1 bands) Reached 77.8%; 4 swimming lanes are the NGF that comparative example 1 method obtains, and isoelectric point is 9.1,8.9,8.6, and isoelectric point is 3 bands, and homogeneity can only reach 51.5%, and wherein full-length NGF (isoelectric point The band with point 9.1) can only reach 13.5%, indicating that the mNGF prepared by the present invention has higher uniformity.

4. Molecular weight determination and homogeneity verification of mNGF by LC-MS mass spectrometry

The liquid chromatograph uses Agilent 1290 Infinity LC reverse phase chromatography system, the chromatographic column uses Poroshell 300 SB-C8, 2.1mm×75mm, 5μm, and the column temperature is room temperature. Mobile phase composition: A: 0.1% formic acid/water, B: 0.1% formic acid/acetonitrile. Injection volume: 0.1 mg/ml, 20 μl. The column was equilibrated with 5% B for 5 min and eluted linearly from 5% B to 95% B in 30 min. The mass spectrometer used Agilent 6530 ESI-Q-TOF MS. Scanning range: m/z 100~3200. The wavelength of ultraviolet-visible light is A280nm.

The total ion spectrum and ultraviolet spectrum of the target protein mNGF obtained in Example 1 and Comparative Example 1 were measured, and the results are shown in Figure 6 and Figure 7 respectively. It can be seen from Figure 6 that it contains two peaks, among which: the measured molecular weight of peak 1 is 12357.18, the theoretical molecular weight is 12358.04, and the corresponding monomer form is NGF with 8 AA cut at the N-terminal. The measured molecular weight of peak 2 is 13251.78, and the theoretical molecular weight is 13251.01, which corresponds to NGF with a full length of 118AA in the monomeric form. It can be seen from Figure 7 that it contains 4 peaks, of which: in addition to NGF (peak 2) with 8 AA cuts at the N-terminus corresponding to Figure 6 and NGF with 118AA in full length (peak 4), there are two other form of monomer.

As can be seen from the above, the content of the intact NGF in the target protein NGF obtained in Example 1 is much higher than that of the intact NGF in Comparative Example 1, and the NGF extracted by the method provided by the present invention is superior in product uniformity and integrity. Better than NGF in Comparative Example 1.

5. Measurement of mNGF activity by chicken embryo dorsal root ganglion method and TF-1 cell method

1) Measurement of mNGF activity by chicken embryo dorsal root ganglion method

Dilute the NGF sample: solution A: add 6 ng of extracted mNGF sample to 1ml of serum-free DMEM culture solution; solution B: take 50 μl of solution A and add 4.95 ml of serum-free DMEM solution; solution C: take 60 μl of solution B and add serum-free DMEM 2.94ml of culture solution (3ml in total) to make the final concentration (3AU/ml). Solution A and B are diluted in centrifuge tubes, solution C is in the cell bottle, and solution C is used as bottle No. 1, and then diluted 3 times into: No. 2, No. 3, No. 4, No. 5, and No. 6 test solution. Add 1 culture bottle for each solution to be tested, 2ml/bottle. At the same time, the serum-free DMEM culture solution was used as the blank control, and the standard product purchased from the National Institute of Inspection and Quarantine was used as the positive control (reference product). After adding 8-day-old chick embryo dorsal root ganglia, place it in a 5% CO ₂ , 37°C saturated humidity incubator, and observe the results after 24 hours.

The content of NGF per milliliter of the test sample when the growth is the best is taken as 1 activity unit (AU). From the dilution where the negative control result appeared, count back to the 3rd and 4th two dilutions and take the best growth as the determination end point to calculate the titer. The reference product is the standard product purchased from the China Inspection Institute, and the volume of each tube is 1000AU.

The calculation formula of NGF specific activity is:

Specific activity of the sample to be tested (AU/mg) = reference product activity (AU/ml) × [sample pre-dilution factor × activity at the corresponding reference product dilution point (AU/ml) / reference product measured activity (AU/ml) ]

The activity of the target protein extracted in Example 1 and Comparative Example 1 was simultaneously measured using the above method. As a result, the NGF activities of Example 1 and Comparative Example 1 both reached 500,000 AU/mg, and the results are shown in Table 2.

2) TF-1 cell method to measure mNGF activity

The detailed operation method is operated according to the method in Example 1 in the patent publication number CN103376248A, and the patent name is "Quantitative Assay Method for Nerve Growth Factor Activity". The specific activity test results are shown in Table 2.

Table 2

batch number chicken embryo method TF-1 cell specific activity Example 1 5×10 ⁵ AU/mg 11.7×10 ⁵ U/mg Comparative example 1 5×10 ⁵ AU/mg 5.0×10 ⁵ U/mg

As can be seen from Table 2, due to the low sensitivity of the dorsal root ganglion method, there is no difference in the specific activity of mNGF between the two examples, but the specific activity of the TF-1 cell method with high sensitivity can be used to detect the specific activity of Example 1. greatly improved.

6. Comparison of the purity of the target protein peak before and after hydrophobic chromatography

The aforementioned HPLC was used to detect the peak purity of the target protein before and after the hydrophobic chromatography step. The data statistics are shown in Table 3 below. The purity increased from about 70% to 100%, and the effect of the hydrophobic chromatography step was obvious.

table 3

Liquid purity Target protein peak collected before hydrophobic step 72.7% Target protein peaks collected after hydrophobic chromatography 100%

7. The comparison table of the overall detection results of Example 1 and Comparative Example 1 is shown in Table 4 below

Table 4

Example 2

Prepare mNGF according to the same method as in Example 1, the difference is that except for the hydrophobic chromatography step, the steps of CM reverse adsorption cation exchange chromatography, acidification centrifugation, adsorption cation exchange chromatography and size exclusion chromatography are all referred to Ren Wanqiong et al. The route B in the method introduced in the article "Pilot-scale preparation of freeze-dried mouse nerve growth factor and discussion of several issues" prepares mNGF.

test case 2

Test Example 2 is used to illustrate the various indicators of the mNGF extract of Example 2. The detection method is the same as Test Example 1, and the results of each detection item are listed in Table 5 and Table 6 (Table 6 is the uniformity test result).

table 5

Table 6

From the above results, it can be seen that the isoelectric point of NGF obtained by using the method of Example 2 is 9.1, 8.9, mainly two bands, and the uniformity reaches 69.4%, which shows that the mNGF prepared by the present invention has high uniformity and is mainly integrity NGF.

Example 3

Prepare mNGF according to the same method as in Example 1, the difference is that the following steps are inserted after the hydrophobic chromatography step: Superdex 75 prep grade chromatography column is buffered with pH6.8, 0.05mol/L phosphate-0.15mol/L sodium chloride After the liquid was fully balanced, the sample was loaded, and the collection was started when the number displayed on the ultraviolet detector began to rise, and the collection of the target protein peak was stopped when it dropped to the baseline, and the gel filtration chromatography sample was obtained.

Test case 3

Test Example 3 is used to illustrate the detection of various indicators of the NGF extract in Example 3. The gel filtration chromatography samples were detected, and compared with the results of Comparative Example 1, the results are summarized in Table 7 below.

Table 7

Test items Example 3 Example 1 Comparative example 1 HPLC purity 100.0% 100.0% 93.00% Isoelectric point 8.9, 9.1 8.9, 9.1 8.6, 8.9, 9.1 Molecular weight (kD) 13.0 13.0 12.1 Residual host protein (ng/mg) 80 250 300 DNA residue (ng/mg) 8 100 310

As can be seen from the data in the table, compared with Example 1, the process of Example 3 has significantly reduced residual host protein and DNA residues in the product; It is also significantly reduced, and the advantages of the patented process are obvious, especially because gel filtration chromatography can significantly reduce the amount of endotoxin and DNA residues in the product.

Example 4-9

Prepare mNGF according to the same method as in Example 1, except that the parameters given in Table 8 are used to carry out the hydrophobic chromatography step:

Table 8

(2) Experimental results

1. The purity, isoelectric point and cell specific activity of the samples obtained in each hydrophobic experiment were tested, and the results are shown in Table 9 below.

Table 9

Example HPLC purity Isoelectric point TF-1 cell activity (10,000 U/mg) 4 99.5% 9.1, 8.9 9.9×10 ⁵ 5 100.0% 9.1, 8.9 10.8×10 ⁵ 6 99.6% 9.1, 8.9 9.7×10 ⁵ 7 100.0% 9.1, 8.9 10.0×10 ⁵ 8 100.0% 9.1, 8.9 10.2×10 ⁵ 9 99.7% 9.1, 8.9 9.9×10 ⁵

It can be seen from the data in the table that the purity of the samples in each hydrophobic chromatography experiment has reached above 99.0%, and the activity is above 9.5×10 ⁵ U/mg. There are only two bands in the isoelectric focusing analysis, and the product uniformity is lower than that of the old technology There is a significant improvement. The results of isoelectric focusing electrophoresis are shown in Figure 8, in which the lanes with three bands on the right side of Figures A and B are the samples in Comparative Example 1.

2. Uniformity test results

It can be seen from Fig. 8 (Fig. 8A and Fig. 8B) that Experiments 1-6 are NGF obtained by using the method in Examples 4-9 respectively, with isoelectric points of 9.1 and 8.9, mainly two bands, and the total length of NGF is The content (the isoelectric point is 9.1 bands) has reached more than 65%; the NGF obtained by the method of comparative example 1 has isoelectric points of 9.1, 8.9, and 8.6, and the isoelectric point is 3 bands, with poor uniformity, and the full-length NGF (The strip with an isoelectric point of 9.1) content can only reach 13.5%, indicating that the mNGF prepared by the present invention has higher uniformity.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (15)

1. the extracting method of a nerve growth factor, it is characterised in that described method includes: Homogenate supernatant is collected by centrifugal after mouse submandibular gland tissue homogenate；Described homogenate supernatant is entered Collect after the attached cation-exchange chromatography of row CM back suction and flow through liquid；The described liquid that flows through is acidified Collected after centrifugation acidolysis supernatant；Described acidolysis supernatant is carried out Liquidity limit displacement chromatography Collect the destination protein liquid that target protein peak is corresponding；Described destination protein liquid is carried out hydrophobic layer Analysis, it is thus achieved that the destination protein liquid after hydrophobic chromatography.

Extracting method the most according to claim 1, it is characterised in that described hydrophobic chromatography The balance liquid of the drainage column of middle use be salt concentration be the pH value of 0.5-3.0mol/L be 6.0-9.0 Buffer.

Extracting method the most according to claim 2, it is characterised in that described hydrophobic chromatography The balance liquid of the drainage column of middle use is by the one in sodium chloride, ammonium sulfate and sodium sulfate Salt is dissolved in what phosphate buffer or Tris-HCl buffer obtained, dissolves in balance liquid The final concentration of 0.5-3.0mol/L of salt, pH value are 6.0-9.0.

Extracting method the most according to claim 3, it is characterised in that when described salt is chlorine When changing sodium, the final concentration of 0.5-3.0mol/L of sodium chloride；When described salt is ammonium sulfate, sulfur The final concentration of 0.5-2.0mol/L of acid ammonium；When described salt is sodium sulfate, the end of sodium sulfate is dense Degree is 0.5-2.0mol/L.

5. according to the extracting method described in claim 3 or 4, it is characterised in that described phosphoric acid The pH value of salt buffer is 6.0-8.0, and the pH value of described Tris-HCl buffer is 7.1-9.0.

6. according to the extracting method described in any one in claim 1-5, it is characterised in that Drainage column medium in described hydrophobic chromatography be Butyl-S-FF, Phenyl FF, Phenyl HP, Butyl FF, OctylFFButyl HP, Phenyl HS FF or Fractogel EMD Phenyl (S).

7. according to the extracting method described in any one in claim 1-6, it is characterised in that During hydrophobic chromatography, loading flow velocity is 5～300cm/hr, preferably 80～200cm/hr.

Extracting method the most according to claim 7, it is characterised in that hydrophobic chromatography process Column temperature be 2～8 DEG C.

9. according to the extracting method described in any one in claim 1-8, it is characterised in that Described method also includes exclusion chromatography step.

10. according to the extracting method described in any one in claim 1～9, it is characterised in that The pH value of described homogenate supernatant is 6.0-7.0.

11. extracting method according to claim 10, it is characterised in that homogenate supernatant Preparation method comprise the following steps:

Fully it is homogenized after mouse submandibular gland is mixed with purified water, centrifugal collection supernatant, uses pH The concentration of 6.0～7.0 be 0.5mol/L phosphate buffer regulation supernatant pH after obtain even Slurry supernatant.

12. according to the extracting method described in any one in claim 1-11, it is characterised in that The balance liquid used during the attached cation-exchange chromatography of CM back suction is concentration The phosphate buffer of 0.01-0.1mol/L, pH6.0-7.0.

13. according to the extracting method described in any one in claim 1-12, it is characterised in that Described acidifying is centrifugal to be comprised the following steps: add acidic buffer regulation pH value in liquid to flowing through To 3.5-4.5, add NaCl, make the final concentration of 0.1-0.5mol/L of NaCl in system, quiet Put centrifugal acquisition acidolysis supernatant.

14. extracting method according to claim 13, it is characterised in that flow through in liquid and add The acidic buffer entered be pH be acetate buffer or the citrate buffer of 3.5-4.5.

15. according to the extracting method described in any one in claim 1-14, it is characterised in that The step of described Liquidity limit displacement chromatography includes:

With Liquidity limit displacement chromatography balance liquid, filler is balanced, by acidolysis supernatant Sample, first with cation-exchange chromatography balance liquid eluting, the protein pH8.5～9.5 of absorption, The Tris-HC1 of concentration 0.01～0.10mol/L washes miscellaneous buffer and washes miscellaneous, then with pH8.5's～9.5 0.01～0.10mol/L Tris-HC1-0.2～0.8mol/L NaCl elution buffer carry out gradient and wash De-；Wherein, described Liquidity limit displacement chromatography balance liquid containing concentration is The acetate of 0.01-0.1mol/L and the NaCl of 0.1-0.5mol/L, pH value is 3.5-4.5.