WO2019184368A1 - 一种高纯度rhNGF的制备方法 - Google Patents

一种高纯度rhNGF的制备方法 Download PDF

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WO2019184368A1
WO2019184368A1 PCT/CN2018/114538 CN2018114538W WO2019184368A1 WO 2019184368 A1 WO2019184368 A1 WO 2019184368A1 CN 2018114538 W CN2018114538 W CN 2018114538W WO 2019184368 A1 WO2019184368 A1 WO 2019184368A1
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chromatography
elution
cation exchange
nacl
conductivity
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French (fr)
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刘文超
孙洪亮
张怡
王跃生
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江苏中新医药有限公司
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Priority to US17/030,297 priority Critical patent/US20210070821A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • the invention relates to a method for preparing high-purity rhNGF, in particular to a method for obtaining high-purity rhNGF from CHO cell culture.
  • rhNGF Genetically engineered and produced in host cells, rhNGF usually contains a variety of impurities, including host cell proteins, nucleic acids, as well as variants of the expression product rhNGF, such as precursors, N-terminal truncations and abnormalities; There are impurities of various organic and inorganic components such as endotoxin, viral contaminants, and cell culture medium components. Among them, N-terminal truncation and abnormal variants and precursors are the most important impurities affecting the quality of recombinant human nerve growth factor and must be removed.
  • Patent CN102702341A uses a two-step cation exchange and molecular sieve (Superdex 75) method to prepare rhNGF with a purity greater than 98%.
  • the molecular sieve (Superdex 75) is used to remove precursor variants, the patent does not mention, and the molecular sieve requires samples. It is highly concentrated, and the sample loading can only be between 1% and 4% of the column volume. The resin itself is also expensive, which is not suitable for large-scale industrial production.
  • Patent CN1268639C uses high-efficiency cation exchange to separate rhNGF oxidation, isomerization, deamidation and other variants by linear gradient elution. Hydrophobic interaction chromatography (preferably phenyl) is used to remove the precursors, but the method uses a linear gradient. Elution method.
  • the patent CN106478801A adopts a two-step method of cation exchange and hydrophobic chromatography (preferably phenyl) to prepare rhNGF with a purity greater than 99%.
  • the hydrophobic interaction chromatography of the method also adopts a linear gradient elution method. Linear gradient elution methods usually require a two-pump chromatography system, which requires high equipment and is not conducive to large-scale industrial production.
  • each of the chromatographic methods of the prior art is capable of removing a portion of the relevant impurities in rhNGF, all of the major impurities cannot be satisfactorily removed using a single method.
  • cation exchange chromatography is mainly used to remove N-terminal truncations and abnormal variants
  • hydrophobic interaction chromatography is mainly used to remove precursor variants.
  • the present invention adopts two chromatographic methods of cation exchange chromatography and hydrophobic interaction chromatography in sequence to obtain high purity rhNGF.
  • cation exchange chromatography is mainly used to remove N-terminal truncation and abnormal variants
  • hydrophobic interaction chromatography is mainly used to remove precursor variants.
  • the present invention analyzes the physicochemical properties of rhNGF and its precursors. Since the NGF precursor contains a glycosylation modification, the mature rhNGF does not contain a glycosylation modification. The precursor is less hydrophobic than mature rhNGF due to the presence of sugar chains.
  • the present invention utilizes this property to separate precursors from mature rhNGF using hydrophobic interaction chromatography and stage cleaning using hydrophobic interaction chromatography to remove precursor variants. Moreover, the present invention adopts a dynamic cleaning method to improve the efficiency of purifying rhNGF.
  • cation exchange chromatography The purpose of cation exchange chromatography is to eliminate N-terminal truncations and abnormal variants in recombinant human nerve growth factor.
  • N-terminal truncation and abnormal variants are the most critical impurities affecting the quality of recombinant human nerve growth factor and must be removed.
  • the invention analyzes the physicochemical properties of rhNGF and its variants, and finds that in the weak cation exchange high performance liquid chromatography (WCX-HPLC) analysis, the N-terminal truncation and abnormal variant peaks are in front of the main peak, indicating that it has a lower The isoelectric point. Therefore, in the purification process using the cation exchange chromatography of the present invention, the N-terminal truncation and abnormal variants are removed by the method of increasing the conductivity at a stage, and the effect is good.
  • WCX-HPLC weak cation exchange high performance liquid chromatography
  • the operation method is as follows:
  • a method for preparing high-purity rhNGF comprises sequentially performing hydrophobic interaction chromatography and cation exchange chromatography on CHO cell culture;
  • N-terminal truncation and abnormal variants were removed prior to cation exchange chromatography.
  • CHO cell culture is purified by column chromatography, one or more times;
  • the CHO cell culture is a recombinantly produced human nerve growth factor expressed by a Chinese hamster ovary (CHO) cell recombinant host cell culture, containing a large amount of impurities;
  • the resulting material still contains various contaminants such as recombinant human nerve growth factor variants (such as N-terminal truncations, precursors, and abnormal variants) that are difficult to remove by conventional means.
  • recombinant human nerve growth factor variants such as N-terminal truncations, precursors, and abnormal variants
  • the hydrophobic chromatography cleaning solution is an aqueous solution of an alcohol and NaCl, and simultaneously meets the following conditions:
  • the content of alcohol is lower than the content of alcohol in the eluent used in step 2.2;
  • the alcohol preferably ethanol; in the example of the present invention, the washing buffer used is 4% - 6% ethanol (volume ratio);
  • 2NaCl content is 200 ⁇ 400mM
  • 3pH is the same as the range of the substance obtained in step 1;
  • the cation chromatography cleaning solution is a NaCl-containing buffer having a conductivity higher than that of the cation chromatography sample material.
  • step 2
  • the cleaning solution is higher than the chromatography eluent
  • the concentration of alcohol in the solution is lower than the chromatography eluent.
  • Step 2.1 adopts the "dynamic cleaning" method, that is, according to the linear relationship of the peak area of the elution product of the column chromatography in step 1, the cleaning volume is obtained, and is calculated according to the following formula:
  • Washing volume (CV) 8.5 - peak area / ml resin / 1000.
  • the eluent is an aqueous solution of an alcohol, or an aqueous solution of an alcohol and NaCl, containing 7%-20% alcohol, 0-100 mM NaCl.
  • the conductivity of the eluent used in step 3.2 is higher than the conductivity of the cleaning solution described in step 3.1.
  • the eluent used in step 3.2 is a buffer containing NaCl.
  • the NaCl content is 350 to 600 mM, and the conductivity is 35 to 60 ms/cm.
  • step 2 and step 3 can be reversed, that is, the order of operation of the purification method can be: 1-2-3, or 1-3-2.
  • Hydrophobic interaction chromatography material It has been found that hydrophobic interaction chromatography materials with large particle size of solid phase particles, such as Octyl FF, Capto Butyl, Capto Phenyl HS, Butyl FF and Phenyl FF from GE, remove precursors. no significant effect.
  • the hydrophobic interaction chromatography mediator used in the present invention is a phenyl or butyl group, preferably using Butyl sepharose High Performance.
  • Cation exchange material Contains a highly crosslinked agarose solid phase, such as SP HP or styrene-divinylbenzene solid phase from GE, such as the POROS 50HS column from Applied Biosystems, for other cations with larger solid particle size Exchange materials, such as Capto S from GE, have no significant effect on the removal of variants. It has been found through experiments that the cation exchange ligand of the chromatographic medium is preferably a propyl sulfonate group.
  • the hydrophobic interaction chromatography purification scheme comprises the following sequential steps: (1) balancing the hydrophobic interaction chromatography material; (2) loading the crude product into the hydrophobic interaction chromatography material; (3) using an equilibration buffer Top wash; (4) intermediate wash using wash buffer; (5) elution of the desired recombinant human nerve growth factor using elution buffer.
  • the present invention employs hydrophobic interaction chromatography, and the recombinant human nerve growth factor precursor (mainly precursor) can be washed by various mobile phase conditions, and the mobile phase conditions include lowering the salt concentration, and also increasing the concentration of the polar solvent, and the pH is also the same. It has an effect on the binding of recombinant human nerve growth factor to the resin, preferably neutral pH.
  • the buffer salt used in the buffer comprises sodium acetate, phosphate, MES, MOPSO, preferably phosphate is selected as the buffer salt.
  • the elution salts used in the buffer include, but are not limited to, sodium chloride, sodium acetate, potassium chloride, and ammonium sulfate.
  • the organic solvent used in the buffer includes, but is not limited to, ethanol, propylene glycol, ethylene glycol, and hexanediol.
  • ethanol is selected as the organic solvent to be used.
  • an equilibration buffer is passed through the material prior to loading the crude product comprising recombinant human nerve growth factor and one or more molecular variants onto the hydrophobic interaction chromatography material.
  • the equilibration buffer has a pH of from about 5.5 to about 7.0, such as about pH 6.0, and too low a pH (less than 5.0) results in enhanced hydrophobic action.
  • the equilibration buffer salt concentration is controlled in the range of about 0.8 M to 1.2 M NaCl, such as about 1.1 M NaCl.
  • An exemplary equilibration buffer comprises 20 mM MES, 1.1 M NaCl, pH 6.0 or 20 mM PB, 1 M NaCl, pH 7.0.
  • the crude product comprising recombinant human nerve growth factor and one or more molecular variants is loaded onto a hydrophobic interaction chromatography material, the pH of which is in the range of pH 5.5 to pH 7.0, such as pH 6.0. Or at pH 7.0, the salt concentration is controlled in the range of about 0.8 M to 1.2 M NaCl, such as about 1.1 M NaCl.
  • the crude product eluted from the hydrophobic chromatography is loaded into a hydrophobic interaction chromatography with a loading density of about 5-10 g/L resin, and the recombinant human nerve growth factor is bound to the hydrophobic interaction chromatography packing.
  • the top wash is performed using an equilibration buffer.
  • the top wash conditions are the same as the equilibration step, and the top wash is generally carried out in 2-3 column volumes.
  • the wash buffer flows through the hydrophobic interaction chromatography material during the cleaning process.
  • the wash buffer composition is typically selected as a molecular variant (precursor) that elutes as much of the impurities as possible from the resin without eluting the desired recombinant human nerve growth factor.
  • the pH of the wash buffer is controlled in the range of 5.5-7.0, such as about pH 6.0 or pH 7.0, the salt concentration is controlled in the range of about 0.2 to about 0.4 M NaCl, for example about 0.25 M, and the organic solvent is controlled at about 4% to about 6% ethanol, for example about 5%.
  • the cleaning volume is determined by dynamic control according to the elution peak area of the previous step of hydrophobic interaction chromatography, generally 5-7 CV.
  • Preferred wash buffers include 20 mM PB, 0.4 M NaCl, 6% ethanol, pH 6.0 or 20 mM PB, 0.25 M NaCl, 5% ethanol, pH 7.
  • the desired recombinant human nerve growth factor is eluted from the hydrophobic interaction chromatography material. Elution of recombinant human nerve growth factor can be achieved by reducing the salt concentration or increasing the concentration of the organic solvent.
  • the elution buffer comprises from about 0 to about 100 mM NaCl, from about 7% to about 20% ethanol.
  • the elution buffer typically has approximately the same pH as the wash buffer.
  • a preferred elution buffer comprises 20 mM PB, 0.1 M NaCl, 7% ethanol, pH 7.0.
  • Another preferred elution buffer comprises 20 mM PB, 20% ethanol, pH 6.0.
  • the hydrophobic interaction chromatography purification method herein consists of only the following steps: equilibration, loading of a crude product comprising recombinant human nerve growth factor and molecular variants for elution of molecular variants The washing step, and the elution step of eluting recombinant human nerve growth factor.
  • the recombinant human nerve growth factor preparation obtained according to the hydrophobic interaction chromatography method herein can be subjected to other purification. Exemplary further purification steps have been discussed above.
  • the "conductance" described above regulates conductance by means of salt addition, including but not limited to sodium chloride, potassium chloride, sodium sulfate, sodium acetate, preferably sodium chloride.
  • the buffer salts used in the washing buffer and the elution buffer include sodium acetate, phosphate, MES, MOPSO, and preferably, MES is used as a buffer salt.
  • cleaning it is meant that the cleaning buffer flows through the cation exchange material and the effluent liquid (which can carry away some of the impurities) is discarded.
  • the "elution” means that the elution buffer flows through the cation exchange material to collect the effluent liquid (containing the purified target product).
  • Contaminant refers to process-related impurities that are different from the desired recombinant human nerve growth factor. Contaminants include, but are not limited to, host cell materials such as Chinese hamster ovary cell proteins, nucleic acids; endotoxins; viral contaminants; cell culture components.
  • Cation exchange material refers to a solid phase that is negatively charged and has free cations for exchange of cations in an aqueous solution flowing through the solid phase.
  • Commercially available cation exchange materials include immobilized propyl sulfonate (SP), sulfonyl (S) or crosslinked polystyrene-divinylbenzene coated with a sulfopropyl functionalized polyhydroxylated polymer on agarose Solid phase particles, etc.
  • Load refers to a composition that is loaded onto a cation exchange material.
  • “Equilibrium buffer” refers to a buffer used to balance the cation exchange material prior to loading the composition onto the cation exchange material.
  • Regeneration Buffer can be used to regenerate the cation exchange packing to enable it to be reused.
  • the regeneration buffer has the ability to remove substantially all of the contaminants and the electrical conductivity and pH of the recombinant human nerve growth factor from the cation exchange packing.
  • Conductivity refers to the ability of an aqueous solution to conduct electrical current between two electrodes.
  • the conductivity of the solution can be varied by changing the ion concentration of the solution.
  • Top wash refers to the process of washing the composition from a cation exchange column using an equilibration buffer after loading of the composition.
  • the cation exchange purification scheme generally comprises the following sequential steps: (1) balancing the cation exchange material; (2) loading the composition to the cation exchange material; (3) performing an overhead wash using an equilibration buffer; (4) Intermediate washing using washing buffer; (5) Elution using elution buffer to obtain a desired recombinant human nerve growth factor purified product.
  • an equilibration buffer is passed through the material prior to loading the crude product comprising recombinant human nerve growth factor and one or more molecular variants onto the cation exchange material.
  • the equilibration buffer has a pH of from about 5.5 to 6.5, such as about pH 6.2.
  • An exemplary equilibration buffer comprises 20 mM MES, 110 mM NaCl, pH 6.2.
  • a composition comprising recombinant human nerve growth factor and one or more molecular variants is loaded onto a cation exchange material, the pH of the composition being in the range of pH 5.5 to pH 6.5, such as pH 5.8 Or pH 6.2, the conductance is in the range of 10-14 ms/cm, for example 13 ms/cm.
  • the composition from the hydrophobic chromatography elution is loaded onto a cation exchange chromatography with a loading density of about 1-5 g/L resin, and the recombinant human nerve growth factor and variant are bound to a cation exchange packing, most of the hosts.
  • the top wash is performed using an equilibration buffer.
  • the top wash conditions are the same as the equilibration step, and the top wash is generally carried out in 2-3 column volumes.
  • the wash buffer flows through the cation exchange material during the cleaning process.
  • the wash buffer composition is typically selected to elute as many molecular variants as possible from the resin (N-terminal truncation and abnormality) without eluting the desired recombinant human nerve growth factor.
  • the pH of the wash buffer is controlled in the range of 5.5-6.5, such as about pH 5.8 or pH 6.2, and the conductivity is controlled in the range of 20-30 ms/cm, for example about 29 ms/cm.
  • buffer salts buffered in this pH range include, but are not limited to, MES, MOPOS, sodium acetate, phosphate, and the like.
  • Preferred wash buffers include 20 mM MES, 290 mM NaCl, pH 5.8 or 20 mM PB, 220 mM NaCl, pH 6.2.
  • the desired recombinant human nerve growth factor is eluted from the cation exchange material. Elution of recombinant human nerve growth factor can be achieved by increasing conductivity or ionic strength.
  • the conductivity of the elution buffer needs to be greater than about 35 ms/cm, and the increased conductivity can be achieved by including a relatively high salt concentration in the elution buffer. Examples of salts useful for this purpose include, but are not limited to, sodium chloride, potassium chloride, sodium acetate.
  • the elution buffer comprises from about 350 to about 600 mM NaCl.
  • the elution buffer typically has approximately the same pH as the wash buffer.
  • a preferred elution buffer comprises 20 mM MES, 0.4 M NaCl, pH 6.2.
  • Another preferred elution buffer comprises 20 mM PB, 0.5 M NaCl, pH 6.2.
  • the cation exchange purification method herein consists of only the following steps: equilibration, loading a composition comprising recombinant human nerve growth factor and molecular variants for eluting molecular variants A washing step, and an elution step of eluting recombinant human nerve growth factor.
  • the recombinant human nerve growth factor preparation obtained according to the cation exchange chromatography method herein can be subjected to further purification. Exemplary further purification steps have been discussed above.
  • Stage cleaning + elution is used, which is different from the linear gradient elution of the prior art
  • phase conductance ie, in the wash phase, the conductivity of the wash buffer is higher than the crude product to be purified; in the elution phase, the conductivity of the elution buffer is higher than the wash buffer
  • cation exchange chromatography is mainly used to remove N-terminal truncations and abnormal variants
  • hydrophobic interaction chromatography is mainly used to remove precursor variants.
  • cation exchange chromatography can also remove a small number of precursor variants with a scavenging rate of 30% ⁇ 10%. Although it has a scavenging effect, it has less contribution to precursor scavenging than hydrophobic chromatography.
  • Hydrophobic interaction chromatography can also N-terminal truncation and abnormal variants are removed. This is because abnormal variants usually have strong hydrophobicity.
  • the principle of controlling hydrophobic chromatography can remove some N-terminal truncations and abnormal variants, and the clearance rate is 46% ⁇ 4.
  • the combination of ion exchange chromatography and hydrophobic chromatography can remove about 74% of N-terminal truncations and abnormal variants, which is improved compared to the use alone.
  • Fig. 1 and Table 3 of Example 1 are comparisons of the scavenging effects of rhNGF variants in the case of two methods, single and combined use, respectively. This includes the removal of N-terminal truncations and abnormalities, precursor variants. Among them, method A: cation exchange chromatography; B method: hydrophobic interaction chromatography; A+B: two methods combined. It can be seen from the data that the combination of the two methods can achieve the cleaning effect that cannot be achieved by a single method.
  • Figure 1 shows the N-terminal truncation and abnormal variant removal effect. among them:
  • the combination of the two methods can achieve the N-terminal truncation and variant removal effects that cannot be achieved by using A or B alone.
  • Figure 2 Precursor variant removal effect.
  • a method cation exchange chromatography
  • B method hydrophobic interaction chromatography
  • the figure illustrates that the removal of precursor variants is primarily dependent on hydrophobic interaction chromatography, which is not helpful for scavenging.
  • Figure 3 is a process of hydrophobic chromatography purification of recombinant human nerve growth factor, including equilibration, loading, washing and elution.
  • SP HP provides superior variant clearance compared to Capto S;
  • Figure 9 Process diagram of purification of recombinant human nerve growth factor by cation exchange material
  • a purification process for cation exchange chromatography is provided, which is generally divided into equilibration, loading, washing, and elution.
  • Multi-batch cation exchange chromatography data statistics are provided, including variant clearance and product recovery.
  • MES 2-(N-morpholine)ethanesulfonic acid
  • MOPOS is 3-(N-morpholine)-2-hydroxypropanesulfonic acid
  • SEC-HPLC is a molecular exclusion high performance liquid chromatography
  • PB is a phosphate buffer
  • WCX-HPLC is a weak cation exchange high performance liquid chromatography
  • TFA is trifluoroacetic acid.
  • Example 1 Two methods of cation exchange chromatography and hydrophobic interaction chromatography were used to purify rhNGF.
  • the crude rhNGF which has been subjected to at least one column purification, is loaded to the ion exchange packing.
  • the packing is SPHP, the column height is 100mm, and the residence time is 6min.
  • the loading buffer 20 mM MES, 0.11 M NaCl, pH 6.2 was used to equilibrate 4 CV, and after the end of the loading, the 2 CV was equilibrated, and the mixture was washed with 20 mM MES, 0.28 M NaCl, pH 6.2 buffer, and washed 8 CV. Elution was carried out using 20 mM MES, 0.4 M NaCl, pH 6.2 buffer, and 5 CV was eluted.
  • the collection principle is that the starting point is based on the UV slope greater than 30, and the end point is the second peak at 40 mAu. This step can eliminate N-terminal truncation, abnormal structure and other variants, and can remove most of the HCP.
  • the ion exchange chromatography elution product was added with 0.7 M NaCl for the next hydrophobic interaction chromatography.
  • the packing is Butyl HP with a column height of 100 mm and a residence time of 6 min.
  • the column was pre-balanced with 4 mM MES, 1.1 M NaCl, pH 7 at 4 CV, and 2 CV was equilibrated after loading, and intermediate washing was performed using 20 mM PB, 0.25 M NaCl, 5% ethanol, and pH 7 buffer.
  • the intermediate cleaning volume is used for dynamic decision making.
  • Elution was carried out using 20 mM PB, 0.1 M NaCl, 7% ethanol, pH 7 buffer.
  • the principle of collection was that the starting point was less than -2.9999 according to the conductance slope and the end point was 100-150 mAu. This step is mainly used to clear the precursor.
  • A cation exchange chromatography method
  • B hydrophobic interaction chromatography method
  • the value in the table is the percent clearance for the purge.
  • the column was operated in a binding-elution mode and was carried out at ambient temperature.
  • the column was subjected to a hydrophobic interaction chromatography resin (Butyl Sepharose High Performance).
  • the resin consists of a highly crosslinked agarose matrix coupled to a butyl functional group.
  • the hydrophobic interaction chromatography resin was loaded into the column to a bed height of 9-11 cm. Prior to loading the hydrophobic chromatography elution product, the stock solution in the hydrophobic interaction column was washed out using equilibration buffer and column equilibration was performed. The ion exchange chromatography eluted product was loaded onto a balanced chromatography column and the product was bound to the resin.
  • top wash was performed using equilibration buffer and the unbound load was washed out. After the top wash is finished, wash with a washing buffer to remove molecular variants. The elution buffer was then used for elution up to 3 CV and the eluted product was collected. After elution, the column was cleaned using regeneration buffer (20% ethanol) and cleaning solution (0.5 N NaOH) and then stored in stock solution until the next use (see Figure 3).
  • Table 2 shows the specific process conditions of the recombinant human nerve growth factor hydrophobic interaction chromatography of the present invention.
  • the cleaning volume of the isocratic cleaning process is variable depending on the loading amount of the loading, and the loading amount has a certain relationship with the intermediate cleaning volume.
  • the intermediate wash volume decision can be made online in real time by taking the peak area of the sample eluted one step ahead of the hydrophobic chromatography instead of the sample load.
  • the cleaning volume and the peak area of the eluted sample in the first step of hydrophobic chromatography are calculated according to the data of the multi-batch hydrophobic chromatography purification data.
  • the relationship is shown in Figure 4. As can be seen from Figure 4, the maximum cleaning volume can reach 8.5 CV, and as the loading amount increases, the cleaning volume decreases. Typically, the normal cleaning volume needs to be larger than the first peak.
  • Recombinant human nerve growth factor recovery and precursor variant clearance were analyzed by SEC-HPLC.
  • the column was TSK gel G2000SWXL column, 7.8 x 300 mm.
  • the sample volume was 20 ⁇ L
  • the flow rate was 0.5 mL/min
  • the column temperature was 25 degrees
  • the detection wavelength was 280/214 nm
  • the analysis time was 40 min.
  • the scale calculation uses the area normalization method. Since the solution system is mild, it does not cause dissociation of the two subunits of recombinant human nerve growth factor, so the peak corresponds to the dimer.
  • SEC-HPLC can better distinguish between mature and precursor variants.
  • SEC-HPLC analysis was performed on the loading and elution samples during the purification process, and the results are shown in Fig. 5. As can be seen, the precursor variants in the product are removed by purification of the process of the invention.
  • Precursor clearance and product recovery were calculated according to SEC-HPLC analysis of crude and eluted products before loading.
  • Data analysis was performed on a multi-batch hydrophobic interaction chromatography process, as shown in FIG. The removal rate of the precursor variant by this process was 98.0% ⁇ 0.9%, and the recovery was 58% ⁇ 7%.
  • This example describes a cation exchange chromatography process for purifying recombinant human nerve growth factor.
  • the column was operated in a binding-elution mode and was carried out at ambient temperature.
  • the column used a cation exchange resin (SP Sepharose High Performance).
  • the resin consists of a highly crosslinked agarose matrix coupled with a negatively charged functional group.
  • the cation exchange resin was loaded into the column to a bed height of 9-11 cm.
  • the stock solution in the cation exchange column was washed out using equilibration buffer and column equilibration was performed.
  • the hydrophobic chromatography elution product is loaded onto a balanced chromatography column and the product is bound to the resin. After loading, top wash was performed using equilibration buffer and the unbound load was washed out.
  • Recombinant human nerve growth factor recovery and molecular variant clearance were analyzed by RP-HPLC.
  • the specific method was: analysis using a Thermo UltiMate 3000 binary HPLC system.
  • the column is an Agilent C3RRHD with a specification of 2.1*100mm.
  • the mobile phase A is an aqueous solution containing 0.1% TFA
  • the mobile phase B is a 0.1% TFA acetonitrile solution.
  • the gradient is 0 min 95%, 2 min 95%, 4 min 73%, 16 min 63%, 18 min 5%, 20 min. 5%, 22 min 95%, 24 min 95%.
  • the flow rate was 0.5 mL/min
  • the detection wavelength was 280/214 nm.
  • the scale calculation uses the area normalization method.
  • a recombinant human nerve growth factor molecule is composed of two subunits (peptide chains) joined by non-covalent bonds. In the reverse phase analysis, the two subunits will dissociate due to the organic solvent, so the peak corresponds to the subunit type.
  • Variant clearance (1 - ratio of eluted product variant / proportion of composition variant before loading) * 100%;
  • Product recovery (eluting product unit injection amount main peak area * elution volume) / (comparative unit unit injection amount main peak area * loading volume) *100%. Data analysis was performed on a multi-batch cation exchange chromatography process.

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Abstract

提供了一种从CHO细胞培养物中得到高纯度rhNGF的方法。结合了阳离子交换层析和疏水作用层析方法，其特征是，在两种方法层析前，分别增加了清洗步骤。

Description

一种高纯度rhNGF的制备方法 技术领域：
本发明涉及一种高纯度rhNGF的制备方法，特别涉及一种从CHO细胞培养物中得到高纯度rhNGF的方法。
背景技术：
基因工程表达并在宿主细胞中产生的rhNGF中通常含有多种杂质，包括宿主细胞的蛋白质、核酸，还有表达产物rhNGF的变异体，诸如前体、N端截短及异常等；另外，还会存在内毒素、病毒污染物和细胞培养基成分等各种有机和无机成分的杂质。其中，N端截短及异常变异体与前体是影响重组人神经生长因子质量最关键的杂质，必须加以清除。
以上这些杂质的理化性质各不相同，最有效的清除方法也各不相同。
目前，对rhNGF进行纯化的报道有：
专利CN102702341A采用阳离子交换及分子筛(Superdex 75)两步方法制备了纯度大于98％的rhNGF，虽然猜测分子筛(Superdex 75)用以清除前体变异体，但该专利并未提及，且分子筛要求样品高度浓缩，上样量只能在柱体积1％-4％之间，树脂本身也较贵，规模化工业生产中不太适用。
专利CN1268639C采用高效阳离子交换以线性梯度洗脱方式分离rhNGF氧化、异构、脱酰胺等变异体，采用疏水作用层析(优选苯基)用以去除前体，但该方法均采用了线性梯度的洗脱方式。
专利CN106478801A采用阳离子交换及疏水层析(优选苯基)两步方法制备的纯度大于99％的rhNGF，该方法疏水作用层析同样采用了线性梯度的洗脱方式。线性梯度洗脱方式通常需要双泵层析系统，对设备要求较高，不利于大规模工业生产。
以上这些方法均未涉及清除N端截短及异常变异体，及采用阶段动态清洗方式清除前体变异体，且层析步骤采用线性梯度的洗脱方式，不利于大规模工业放大。
现有技术的每种层析方法虽然都能够除去rhNGF中一部分相关杂质，但使用单一方法都不能满意地去除所有主要杂质。比如，阳离子交换层析主要用于清除N端截短及异常变 异体，疏水作用层析主要用于清除前体变异体。
因此，有必要研究不同方法的联合使用，才能得到高纯度的rhNGF。
发明内容
本发明的目的是结合不同手段，提供一种从CHO细胞培养物中得到高纯度rhNGF的方法。
本发明采用依次进行阳离子交换层析和疏水作用层析两种层析方法，可以得到高纯度rhNGF。其中，阳离子交换层析主要用于清除N端截短及异常变异体，疏水作用层析主要用于清除前体变异体。
关于疏水作用层析
本发明分析了rhNGF及其前体的理化性质。由于NGF前体中含有糖基化修饰，而成熟rhNGF不含糖基化修饰。前体由于存在糖链，比成熟rhNGF疏水性更弱。本发明利用该性质，使用疏水作用层析分离前体与成熟rhNGF，并使用疏水作用层析进行阶段清洗以清除前体变异体。而且本发明采用动态清洗的方式，提高了纯化rhNGF的效率。
关于阳离子交换层析
阳离子交换层析的目的是清除重组人神经生长因子中N端截短及异常变异体。
N端截短及异常变异体是影响重组人神经生长因子质量最关键的杂质，必须加以清除。
本发明分析了rhNGF及其变异体的理化性质，发现在弱阳离子交换高效液相色谱(WCX-HPLC)分析中，N端截短及异常变异体出峰在主峰前，显示了其具有较低的等电点。因此，在本发明在用阳离子交换层析的纯化工艺中，采用阶段升高电导率的方法清除N端截短及异常变异体，效果良好。
操作方法如下：
一种高纯度rhNGF的制备方法，包括对CHO细胞培养物依次进行疏水作用层析和阳离子交换层析；
其特征是，在两种方法层析前，分别具有清洗步骤：
疏水作用层析前，清除前体；
阳离子交换层析前，清除N端截短及异常变异体。
具体地，按以下3个步骤进行：
1前处理：将CHO细胞培养物经过柱层析纯化，进行一次或多次；
所述CHO细胞培养物是中国仓鼠卵巢(CHO)细胞重组宿主细胞培养物表达的重组生产的人神经生长因子，含有大量杂质；
所述柱层析的方式不限，按本领域技术人员熟知的所有柱层析方法均可进行，目的是除去一般性的杂质。
虽然经过常规方法前处理，所得物质中仍含有常规手段难以去除的重组人神经生长因子变异体(如N端截短、前体及异常变异体等)等多种污染物。
2在疏水层析柱中清洗和洗脱
2.1清除前体：用“疏水层析清洗液”清洗步骤1的产物，弃去流出的液体；这个步骤可以除去前体类变异体；
2.2层析：从流出的洗脱液中得到疏水层析产物；
3在阳离子层析柱中清洗和洗脱
3.1清除N端截短及异常变异体：用“阳离子层析清洗液”清洗步骤2的产物，弃去流出的液体；
3.2层析：从流出的洗脱液中得到rhNGF纯品；
步骤2.1中，所述疏水层析清洗液是醇和NaCl的水溶液，且同时满足以下条件：
①醇的含量低于步骤2.2所用的洗脱液中醇的含量；所述醇，优选乙醇；在本发明的实例中，所用的清洗缓冲液是4％-6％乙醇(体积比)；
②NaCl的含量是200～400mM；
③pH与步骤1所得物质范围相同；
步骤3.1中，所述阳离子层析清洗液是含NaCl的缓冲液，其电导率高于阳离子层析样品原料的电导率。
步骤2中：
电导率：清洗液高于层析洗脱液；
醇在溶液中的浓度：清洗液低于层析洗脱液。
步骤2.1采用“动态清洗”方式，即根据步骤1柱层析洗脱产物峰面积线性关系式得出清洗体积，按下式计算：
清洗体积(CV)＝8.5-峰面积/ml树脂/1000。
步骤2.2中，洗脱液是醇的水溶液，或醇和NaCl的水溶液，含7％-20％醇，0-100mMNaCl。
步骤3.2所用的洗脱液的电导率高于步骤3.1所述清洗液的电导率
步骤3.2中所用的洗脱液，是含NaCl的缓冲液。所述NaCl的含量是350～600mM，电导率为35-60ms/cm。
步骤2和步骤3的顺序可以调换，即纯化方法的操作顺序可以是：1—2—3，也可以 是1—3—2。
本发明对层析材料进行了研究：
疏水作用层析材料：经实验发现，固相颗粒粒径较大的疏水作用层析材料，例如来自GE的Octyl FF、Capto Butyl、Capto Phenyl HS、Butyl FF及Phenyl FF等，对前体的清除效果不明显。本发明所用的疏水作用层析介质配基为苯基或丁基，优选使用Butyl sepharose High Performance。
阳离子交换材料:包含高交联琼脂糖固相，例如来自GE的SP HP或苯乙烯-二乙烯基苯固相，例如来自Applied Biosystems的POROS 50HS柱，对于固相颗粒粒径较大的其它阳离子交换材料，例如来自GE的Capto S，对变异体的清除效果不明显。经实验发现，层析介质阳离子交换配基为丙磺基较好。
在本发明的一个实例中，疏水作用层析纯化方案包括以下按序步骤：(1)平衡疏水作用层析材料；(2)将粗品加载到疏水作用层析材料；(3)使用平衡缓冲液进行顶洗；(4)使用清洗缓冲液进行中间清洗；(5)使用洗脱缓冲液洗脱期望的重组人神经生长因子。
本发明采用疏水作用层析，可用各种流动相条件进行清洗重组人神经生长因子前体(主要为前体)，该流动相条件包括降低盐浓度，也包括增加极性溶剂的浓度，pH同样对重组人神经生长因子与树脂的结合有影响，优选中性pH。本发明的实施方案中，缓冲液所用缓冲盐包括乙酸钠、磷酸盐、MES、MOPSO，优选地，选用磷酸盐作为缓冲盐。缓冲液所用洗脱盐包括但不限于氯化钠、乙酸钠、氯化钾及硫酸铵，优选地，选用氯化钠作为洗脱盐。缓冲液中所用有机溶剂包括但不限于乙醇、丙二醇、乙二醇及己二醇，优选地，选用乙醇作为所用有机溶剂。
通常，在将包含重组人神经生长因子和一种或多种分子变异体的粗品加载到疏水作用层析材料上之前，使平衡缓冲液流过所述材料。在本发明的优选实施方案中，平衡缓冲液具有约5.5至约7.0的pH，例如约pH6.0，过低的pH(小于5.0)会导致疏水作用增强。平衡缓冲液盐浓度控制在约0.8M至1.2M NaCl范围内，例如约1.1M NaCl。一种示例性的平衡缓冲液包含20mM MES，1.1M NaCl，pH6.0或20mM PB，1M NaCl，pH7.0。
平衡后，将包含重组人神经生长因子和一种或多种分子变异体的粗品加载到疏水作用层析材料上，所述粗品的pH在pH5.5至pH7.0范围中，例如pH6.0或pH7.0，盐浓度控制在约0.8M至1.2M NaCl范围内，例如约1.1M NaCl。在一个实施方案中，将来自疏水层析洗脱的粗品加载到疏水作用层析，加载密度约5-10g/L树脂，重组人神经生长因子与前体结合至疏水作用层析填料。
加载后，使用平衡缓冲液进行顶洗，顶洗条件与平衡步骤相同，一般进行顶洗2-3个柱体积。
顶洗结束后，使用清洗缓冲液清洗疏水作用层析材料。在清洗过程中清洗缓冲液流过疏水作用层析材料。清洗缓冲液组成一般选择成自树脂洗脱尽可能多的杂质的分子变异体(前体)，而不洗脱期望的得到的重组人神经生长因子。清洗缓冲液pH控制在5.5-7.0范围内，例如约pH6.0或pH7.0，盐浓度控制在约0.2至约0.4M NaCl范围内，例如约0.25M，有机溶剂控制在约4％至约6％乙醇，例如约5％。清洗体积采用动态控制的方式，根据疏水作用层析上一步层析的洗脱峰面积决定，一般为5-7CV。优选的清洗缓冲液包括20mM PB，0.4M NaCl，6％乙醇，pH6.0或20mM PB，0.25M NaCl，5％乙醇，pH7。
在所述清洗步骤后，自疏水作用层析材料洗脱期望的重组人神经生长因子。重组人神经生长因子的洗脱可以通过降低盐浓度或提高有机溶剂浓度来实现。在一个实施方案中，所述洗脱缓冲液包含约0至约100mM NaCl，约7％至约20％的乙醇。洗脱缓冲液一般与清洗缓冲液具有大致相同的pH。一种优选的洗脱缓冲液包含20mM PB，0.1M NaCl，7％乙醇，pH7.0。另一种优选的洗脱缓冲液包含20mM PB，20％乙醇，pH6.0。
虽然涵盖别的其它步骤，但优选的是，本文中的疏水作用层析纯化方法只由下列步骤组成：平衡，加载包含重组人神经生长因子及分子变异体的粗品，用于洗脱分子变异体的清洗步骤，和洗脱重组人神经生长因子的洗脱步骤。
如果必要，依照本文中的疏水作用层析方法获得的重组人神经生长因子制备物可以进行别的纯化。上文已经讨论了示例性的进一步纯化步骤。
以上所述”电导”,通过加盐方式调节电导，所述盐包括但不限于氯化钠、氯化钾、硫酸钠、乙酸钠，优选地选用氯化钠。
清洗缓冲液和洗脱缓冲液所用的缓冲盐包括乙酸钠、磷酸盐、MES、MOPSO，优选地，选用MES作为缓冲盐。
本文中提到的术语解释如下：
所述“清洗”，指清洗缓冲液流过阳离子交换材料，流出的液体(可带走部分杂质)弃去。
所述“洗脱”，指洗脱缓冲液流过阳离子交换材料，收集流出的液体(含纯化目标产品。
“污染物”指与期望的重组人神经生长因子不同的过程相关杂质。污染物包括但不限于：宿主细胞物质，诸如中国仓鼠卵巢细胞蛋白质，核酸；内毒素；病毒污染物；细胞培 养基成分。
“阳离子交换材料”指带有负电荷且有游离阳离子供与流过该固相的水溶液中阳离子交换的固相。商品化的阳离子交换材料包括在琼脂糖上固定化丙磺基(SP)、磺酰基(S)或经磺丙基官能化多羟基化聚合物包被的交联聚苯乙烯-二乙烯基苯固相颗粒等。
“载荷”指加载到阳离子交换材料上的组合物。
“平衡缓冲液”指用在将所述组合物加载到阳离子交换材料上之前平衡阳离子交换材料的缓冲液。
“再生缓冲液”可用于再生阳离子交换填料，使它能够再次使用。再生缓冲液具有自阳离子交换填料清除基本上所有污染物及重组人神经生长因子的电导率和pH。
“电导率”指水溶液在两个电极之间传导电流的能力。溶液电导率可以通过改变溶液的离子浓度来改变。
“顶洗”指在所述组合物加载后，使用平衡缓冲液将所述组合物从阳离子交换柱洗出的过程。
在本发明的一个实例中，阳离子交换纯化方案通常包括以下按序步骤：(1)平衡阳离子交换材料；(2)将组合物加载到阳离子交换材料；(3)使用平衡缓冲液进行顶洗；(4)使用清洗缓冲液进行中间清洗；(5)使用洗脱缓冲液洗脱，得到期望的重组人神经生长因子纯化产物。
通常，在将包含重组人神经生长因子和一种或多种分子变异体的粗品加载到阳离子交换材料上之前，使平衡缓冲液流过所述材料。在本发明的优选实施方案中，平衡缓冲液具有约5.5至6.5的pH，例如约pH6.2。一种示例性的平衡缓冲液包含20mM MES，110mM NaCl，pH6.2。
平衡后，将包含重组人神经生长因子和一种或多种分子变异体的组合物加载到阳离子交换材料上，所述组合物的pH在pH5.5至pH6.5范围中，例如pH5.8或pH6.2，电导在10-14ms/cm范围中，例如13ms/cm。在一个实施方案中，将来自疏水层析洗脱的组合物加载到阳离子交换层析，加载密度约1-5g/L树脂，重组人神经生长因子与变异体结合至阳离子交换填料，大部分宿主细胞蛋白(HCP)流穿。
加载后，使用平衡缓冲液进行顶洗，顶洗条件与平衡步骤相同，一般进行顶洗2-3个柱体积。
顶洗结束后，使用清洗缓冲液清洗阳离子交换材料。在清洗过程中清洗缓冲液流过阳离子交换材料。清洗缓冲液组成一般选择成自树脂洗脱尽可能多的分子变异体(N端截短 及异常)，而不洗脱期望的得到的重组人神经生长因子。清洗缓冲液pH控制在5.5-6.5范围内，例如约pH5.8或pH6.2，电导率控制在20-30ms/cm范围内，例如约29ms/cm。在此pH范围中缓冲的缓冲盐例子包括但不限于MES、MOPOS、乙酸钠、磷酸盐等。优选的清洗缓冲液包括20mM MES，290mM NaCl，pH5.8或20mM PB，220mM NaCl，pH6.2。
在所述清洗步骤后，自阳离子交换材料洗脱期望的重组人神经生长因子。重组人神经生长因子的洗脱可以通过提高电导率或离子强度来实现。洗脱缓冲液电导率需大于约35ms/cm，升高的电导率可以通过在洗脱缓冲液中包含相对较高的盐浓度来实现。用于此目的的盐的例子包括但不限于氯化钠、氯化钾、乙酸钠。在一个实施方案中，所述洗脱缓冲液包含约350至约600mM NaCl。洗脱缓冲液一般与清洗缓冲液具有大致相同的pH。一种优选的洗脱缓冲液包含20mM MES，0.4M NaCl，pH6.2。另一种优选的洗脱缓冲液包含20mM PB，0.5M NaCl，pH6.2。
虽然涵盖别的其它步骤，但优选的是，本文中的阳离子交换纯化方法只由下列步骤组成：平衡，加载包含重组人神经生长因子及分子变异体的组合物，用于洗脱分子变异体的清洗步骤，和洗脱重组人神经生长因子的洗脱步骤。
如果必要，依照本文中的阳离子交换层析方法获得的重组人神经生长因子制备物可以进行别的纯化。上文已经讨论了示例性的进一步纯化步骤。
本发明阳离子交换层析方法的优点：
采用阶段清洗+洗脱的方式，区别于现有技术的线性梯度洗脱；
通过阶段电导增加的方式(即，在清洗阶段，清洗缓冲液的电导率高于待纯化的粗品；在洗脱阶段，洗脱缓冲液的电导率高于清洗缓冲液)清除分子变异体。
经实验证实，用本发明方法对N端截短(6-117)及异常分子变异体的清除效果好(见实施例)。
本发明人通过研究发现，虽然阳离子交换层析主要用于清除N端截短及异常变异体，疏水作用层析主要用于清除前体变异体。但阳离子交换层析同样能够清除少部分前体变异体，清除率为30％±10％，虽然具有清除效果，但相较疏水层析对前体清除贡献较小；而疏水作用层析同样能够清除N端截短及异常变异体，这是由于异常变异体通常具有较强的疏水性，因此控制疏水层析收集原则可清除部分N端截短及异常变异体，清除率为46％±4％，离子交换层析及疏水层析联合使用可清除约74％的N端截短及异常变异体，较单独使用有所提高。
实施例1的图1和表3是两种方法分别单用和联合使用情况下，rhNGF变异体清除 效果的对比。包括清除N端截短及异常、前体变异体。其中，A方法：阳离子交换层析；B方法：疏水作用层析；A+B：两种方法联合使用。从数据上可见两种方法联合使用可以达到单独方法不能达到的清除效果。
附图说明
图1N端截短及异常变异体清除效果。其中：
A方法：阳离子交换层析；
B方法：疏水作用层析。
该图可以看出，两种方法联用能够达到单独使用A或B不能达到的N端截短及变异体清除效果。
图2前体变异体清除效果。其中：A方法：阳离子交换层析；B方法：疏水作用层析；
该图说明前体变异体的清除主要取决于疏水作用层析，阳离子交换层析对清除帮助不大。
图3是疏水层析纯化重组人神经生长因子过程，包括平衡、加载、清洗及洗脱。
图4疏水层析前样品峰面积与清洗柱体积关系
揭示了疏水作用层析之前一步洗脱样品峰面积与疏水作用层析清洗体积之间的关系，峰面积越大，清洗体积越小。
图5装载上样前样品与洗脱样品SEC-HPLC分析
提供了疏水作用层析过程样品的SEC-HPLC分析结果，结果显示清洗过程清除了大部分前体变异体。
图6前体清除率及产品回收率数据总结
提供了多批次疏水作用层析数据统计结果，包括前体变异体清除率及产品回收率；
图7和图8：Capto S及SP HP填料清除变异体能力对比；
两种离子交换层析材料清除变异体(N端截短及异常)能力的对比，相较于Capto S，SP HP提供了卓越的变异体清除能力；
图9：阳离子交换材料纯化重组人神经生长因子过程图；
提供了阳离子交换层析的纯化过程，该过程一般分为平衡、加载、清洗及洗脱。
图10：阳离子交换纯化过程清洗样品及洗脱样品RP-HPLC对比分析
提供了阳离子交换层析过程样品的RP-HPLC分析结果，结果显示清洗过程清除了N端截短及异常变异体。
图11：变异体清除率及样品回收率数据总结
提供了多批次阳离子交换层析数据统计结果，包括变异体清除率及产品回收率。
具体实施方式
以下实施例仅用于举例说明本发明的方法和装置，并不限定本发明的范围。其中：
MES为2-(N-吗啉)乙磺酸；
MOPOS是3-(N-吗啉)-2-羟基丙磺酸；
SEC-HPLC是分子排阻高效液相色谱；
PB是磷酸盐缓冲液，
RP-HPLC是反相高效液相色谱，
WCX-HPLC为弱阳离子交换高效液相色谱，
TFA是三氟乙酸。
实施例1 阳离子交换层析和疏水作用层析两种方法联用纯化rhNGF
以下是操作方法的简述，阳离子交换层析和疏水作用层析两种方法的详细说明和操作见后附的对比实验。
1、方法
将经过至少一步柱纯化的rhNGF粗品加载到离子交换填料。填料为SPHP，柱高100mm，停留时间为6min。上样前预先使用上样缓冲液20mM MES，0.11M NaCl，pH6.2平衡4CV，上样结束后平衡2CV，使用20mM MES，0.28M NaCl，pH6.2缓冲液中间清洗，清洗8CV。使用20mM MES，0.4M NaCl，pH6.2缓冲液进行洗脱，洗脱5CV。收集原则为起始点依照UV斜率大于30，结束点为第二个峰40mAu处，该步可以清除N端截短、异常结构等变异体，并可清除大部分HCP。
离子交换层析洗脱产品添加0.7M NaCl，进行下一步疏水作用层析。填料为Butyl HP，柱高100mm，停留时间6min。柱子预先使用上样缓冲液20mM MES，1.1M NaCl，pH7平衡4CV，上样结束后平衡2CV，使用20mM PB，0.25M NaCl，5％乙醇，pH7缓冲液进行中间清洗。中间清洗体积进行动态决策方式。使用20mM PB，0.1M NaCl，7％乙醇，pH7缓冲液进行洗脱，收集原则为起始点依照电导斜率小于-2.9999，结束点为100-150mAu。该步主要用来清除前体。
2、结果
见图1、图2，及表1数据。
表1 两种方法单用和联用对rhNGF分子变异体清除的比较
Figure PCTCN2018114538-appb-000001
上表中，A：阳离子交换层析方法；B：疏水作用层析方法。
表中数值为清除的百分清除率。
3、结论
从图1、图2和表1数据都可清楚看出，阳离子交换层析和疏水作用层析两种方法联用，能够达到单独使用不能达到的N端截短及变异体清除效果，得到适于临床使用的高纯度产品。
对比实验1 重组人神经生长因子的疏水作用层析
1.1 总过程
以结合-洗脱模式操作层析柱，在环境温度中进行。所述层析柱使用疏水作用层析树脂(Butyl Sepharose High Performance)。该树脂由偶联丁基官能团的高交联琼脂糖基质组成。将疏水作用层析树脂装填入柱至9-11cm的床高度。在加载疏水层析洗脱产物前，使用平衡缓冲液将疏水作用层析柱中的贮存液清洗出，并且进行柱平衡。将离子交换层析洗脱产物加载到经过平衡的层析柱上，产物结合在树脂上。加载后，使用平衡缓冲液进行顶洗，将未结合载荷洗出。顶洗结束后，使用清洗缓冲液进行清洗，以清除分子变异体。然后使用洗脱缓冲液进行最多3CV的洗脱，收集洗脱产物。洗脱后，使用再生缓冲液(20％乙醇)及清洁液(0.5N NaOH)清洁柱子，之后在贮存液中贮存，直至下次使用(见图3)。
表2是本发明的重组人神经生长因子疏水作用层析的具体工艺条件。
表2 重组人神经生长因子工艺
Figure PCTCN2018114538-appb-000002
1.2 动态控制中间清洗体积
根据装载上样量的不同，等度清洗过程的清洗体积是可变的，上样量与中间清洗体积存在着一定的关系。以疏水层析之前一步洗脱样品峰面积代替上样量，可以实时在线进行中间清洗体积决策。以清洗过程中第一个峰谷处(即图3圆圈处)清洗体积为基准，根据多批次疏水层析纯化数据统计清洗体积与疏水层析之前一步洗脱样品峰面积，两者之间的关系如图4所示。由图4可见，最高清洗体积可达8.5CV，随着上样量的增加，清洗体积随之减少。通常，正常清洗体积需大于第一峰谷。
1.3 层析前后样品的分析对比
采用SEC-HPLC方法分析重组人神经生长因子回收率及前体变异体清除率。色谱柱为TSK gel G2000SWXL column,7.8x 300mm。流动相为0.15M磷酸氢二钠0.1M磷酸二氢 钠溶液/乙腈＝85:15(体积比)。分析时上样体积20μL，流速0.5mL/min，柱温25度，检测波长280/214nm，分析时间40min。比例计算采用面积归一化方法。由于溶液体系温和，不会引起重组人神经生长因子两个亚基的解离，所以峰对应二聚体。SEC-HPLC可以较好的分辨成熟体和前体变异体。对纯化过程中的加载上样前及洗脱样品进行SEC-HPLC分析，结果如图5所示。由图可见，经过本发明所示工艺的纯化，产品中的前体变异体得到了清除。
1.4 数据统计分析
根据SEC-HPLC对上样前粗品及洗脱产物的分析，统计前体的清除率及产品的回收率。前体变异体清除率＝(1-洗脱产物前体变异体比例/上样前粗品前体变异体比例)*100％；产品回收率＝(洗脱产物单位进样量主峰峰面积*洗脱体积)/(上样前粗品单位进样量主峰峰面积*装载上样体积)*100％。对多批次疏水作用层析工艺进行数据分析，如图6所示。该工艺条件对前体变异体的清除率为98.0％±0.9％，回收率为58％±7％。
对比实验2 重组人神经生长因子的阳离子交换层析工艺
1.1 此实施例描述用于纯化重组人神经生长因子的阳离子交换层析工艺。
此实例总结了对改良重组人神经生长因子阳离子交换步骤实施的发展研究。在这些研究中评估了两种阳离子交换填料：Capto S及SP Sepharose High Performance。对两种离子交换填料进行清除分子变异体(N端截短及异常)研究，发现SP Sepharose High Performance具有显著地清除分子变异体的工艺性能(见图7和图8)，用于改良的纯化重组人神经生长因子的阳离子交换树脂。
以结合-洗脱模式操作层析柱，在环境温度中进行。所述层析柱使用阳离子交换树脂(SP Sepharose High Performance)。该树脂由偶联有带负电荷的官能团的高交联琼脂糖基质组成。将阳离子交换树脂装填入柱至9-11cm的床高度。在加载疏水层析洗脱产物前，使用平衡缓冲液将阳离子交换柱中的贮存液清洗出，并且进行柱平衡。将疏水层析洗脱产物加载到经过平衡的层析柱上，产物结合在树脂上。加载后，使用平衡缓冲液进行顶洗，将未结合载荷洗出。顶洗结束后，使用清洗缓冲液进行清洗，以清除分子变异体。然后使用更高电导率的洗脱缓冲液进行最多5CV的洗脱，收集洗脱产物。洗脱后，使用再生缓冲液(1M NaCl)及清洁液(0.5N NaOH)清洁柱子，之后在贮存液中贮存，直至下次使用(见图9)。
下表提供了本文中发明的重组人神经生长因子工艺条件的描述。
表3 重组人神经生长因子工艺
Figure PCTCN2018114538-appb-000003
1.2 纯化产物分析
采用RP-HPLC方法分析重组人神经生长因子回收率及分子变异体清除率。具体方法为：使用Thermo UltiMate 3000二元HPLC系统分析。色谱柱为Agilent C3RRHD，规格为2.1*100mm。流动相A为含0.1％TFA的水溶液，流动相B为含0.1％TFA乙腈溶液，梯度以A相比例计为0min 95％，2min 95％，4min 73％，16min 63％，18min 5％，20min 5％，22min 95％，24min 95％。流速0.5mL/min，检测波长280/214nm。比例计算采用面积归一化方法。重组人神经生长因子分子由两个亚基(肽链)通过非共价键结合而成。在反相分析中，由于有机溶剂，两个亚基会解离，故峰对应的是亚基类型。对纯化过程中的清洗及洗脱样品进行RP-HPLC分析。
结果如图10所示。由图中可见清洗样品与洗脱样品在N端截短及异常变异体上的差异，经过本发明方法的纯化，产品中的N端截短及异常变异体含量大幅降低。
1.3 数据统计分析
根据RP-HPLC对上样前组合物及洗脱产物的分析，统计变异体的清除率及产品的回收率，按下式计算：
变异体清除率＝(1-洗脱产物变异体比例/上样前组合物变异体比例)*100％；
产品回收率＝(洗脱产物单位进样量主峰峰面积*洗脱体积)/(上样前组合物单位进 样量主峰峰面积*装载上样体积)*100％。对多批次阳离子交换层析工艺进行数据分析。
分析结果为：对变异体的清除率52％±9％，产品回收率76％±7％。如图11所示。

Claims (10)

  1. 一种高纯度rhNGF的制备方法，包括对CHO细胞培养物依次进行疏水作用层析和阳离子交换层析；其特征是：两种方法的层析洗脱操作前，均分别具有清洗步骤：
    疏水作用层析洗脱操作前，清除前体；
    阳离子交换层析洗脱操作前，清除N端截短及异常变异体。
  2. 权利要求1所述的方法，每种层析方法的清洗和层析两个阶段中，均设定不同的电导率，具体为：
    疏水作用层析：清洗液的电导率高于层析洗脱液的电导率；
    阳离子层析：清洗液的电导率高于样品原料的电导率；层析洗脱液的电导率高于清洗液的电导率。
  3. 权利要求1或2所述的方法，按以下3个步骤进行：
    1前处理：将CHO细胞培养物经过柱层析纯化，进行一次或多次；
    2在疏水层析柱中清洗和洗脱
    2.1清除前体：用“疏水层析清洗液”清洗步骤1的产物，弃去流出的液体；
    2.2层析：从流出的洗脱液中得到疏水层析产物；
    3在阳离子层析柱中清洗和洗脱
    3.1清除N端截短及异常变异体：用“阳离子层析清洗液”清洗步骤2的产物，弃去流出的液体；
    3.2层析：从流出的洗脱液中得到rhNGF纯品；
    步骤2.1中，所述疏水层析清洗液是醇和NaCl的水溶液，且同时满足以下条件：
    ①醇的含量低于步骤2.2所用的洗脱液中醇的含量；
    ②NaCl的含量是200～400mM；
    ③pH与步骤1所得物质范围相同；
    步骤3.1中，所述阳离子层析清洗液是含NaCl的缓冲液。
  4. 权利要求3所述的方法，步骤2.1采用“动态清洗”方式，即根据步骤1柱层析洗脱产物峰面积线性关系式得出清洗体积，按下式计算：
    清洗体积(CV)＝8.5-峰面积/ml树脂/1000。
  5. 权利要求3所述的方法，步骤2.2中，洗脱液是醇的水溶液，含7％-20％醇；或醇和NaCl的水溶液，含7％-20％醇，0-100mM NaCl。
  6. 权利要求3所述的方法，步骤3.2中所用的洗脱液，是含NaCl的缓冲液。
  7. 权利要求6所述的方法，所述NaCl的含量是350～600mM。
  8. 权利要求3所述的方法，步骤3.2中所用的洗脱液，电导率为35-60ms/cm。
  9. 权利要求1所述的方法，所用的疏水作用层析介质配基为苯基或丁基；阳离子层析介质的阳离子交换配基为丙磺基。
  10. 权利要求3所述的方法，操作顺序为：步骤1—步骤3—步骤2。
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