A purification method suitable for collagen, hyaluronidase and coenzymes.

Abstract

This invention discloses a purification method applicable to collagen, hyaluronidase, and coenzymes, belonging to the field of bioengineering technology. This invention provides a universal purification system of "directional pretreatment - gradient membrane separation - multi-mode chromatography - precise purification - low-temperature molding," integrating innovative technologies such as hypotonic-mild fragmentation coupling, composite membrane-chromatographic synergistic enhancement, in-situ online cleaning enhancement, and real-time monitoring and control of product stability, achieving efficient separation and purification of various bioactive substances. It solves the problems of traditional purification processes, such as strong targeting, poor versatility, significant product activity loss, unstable purity, and high industrialization costs. Product purity is ≥98%, activity retention rate is ≥97%, and media lifespan is extended by more than 3 times. It is suitable for high-end fields such as pharmaceuticals, cosmetics, and food, and has significant industrialization value.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a purification method suitable for collagen, hyaluronidase and coenzyme substances. Background Technology

[0002] Collagen, hyaluronidase, coenzyme A, and coenzyme I are high-value bioactive substances that play an important role in the pharmaceutical, cosmetic, and food industries. Collagen is used for skin repair and tissue engineering scaffolds; hyaluronidase is used to enhance drug penetration and relieve edema; coenzyme A is used to treat metabolic diseases and protect liver function; and coenzyme I is used for anti-aging, neuroprotection, and metabolic regulation. The purity and activity of these substances directly determine the quality and safety of the final product, and separation and purification are the core steps in determining product quality.

[0003] Currently, existing separation and purification technologies face several bottlenecks: First, they have extremely poor versatility. Traditional processes are customized for single products, with purification equipment, reagents, and parameters completely independent for different products. This leads to redundant construction of production lines, high R&D costs, and difficulty in adapting to flexible production of multiple products. Second, they suffer severe activity loss. High temperatures, strong acids and alkalis, and high concentrations of organic reagents during purification easily cause denaturation and degradation of bioactive substances, resulting in an activity retention rate generally below 85%. Third, purity is unstable. When the composition of the fermentation broth fluctuates, traditional processes have weak anti-interference capabilities, easily resulting in residual proteins and small molecule impurities, leading to a large fluctuation range in product purity (85%~90%). 2%); Fourth, industrialization efficiency is low, the process steps are cumbersome (up to 8-10 steps), the operation is complicated, and some key steps rely on imported reagents and equipment, resulting in high production costs and difficulty in large-scale production; Fifth, the chromatography media has a short lifespan and is difficult to clean thoroughly after contamination, increasing consumable costs and production risks; Sixth, the intracellular product fragmentation process is prone to product denaturation, while releasing a large amount of impurities and nucleic acids, increasing the burden on subsequent purification; Seventh, the purification process lacks real-time activity monitoring and dynamic control, making it impossible to respond to the problem of decreased product activity in a timely manner; Eighth, tag affinity purification is prone to tag residue, affecting product safety and applicability.

[0004] Therefore, developing a universal, highly active, high-purity, low-cost, pollution-resistant, and low-loss separation and purification process is of great practical significance for breaking through the industrialization bottlenecks of collagen, hyaluronidase, coenzyme A, and coenzyme I, and enhancing the market competitiveness of these products. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a method for purifying bioactive substances, constructing a universal purification system of "directional pretreatment - gradient membrane separation - multi-mode chromatography - precise purification - low-temperature molding," integrating innovative technologies such as hypotonic-mild fragmentation coupling, composite membrane-chromatographic synergistic enhancement, in-situ online cleaning enhancement, and real-time monitoring and control of product stability, to achieve efficient separation and purification of various bioactive substances. This solves the problems of traditional purification processes, such as strong targeting, poor versatility, significant loss of product activity, unstable purity, high industrialization costs, short media lifetime, intracellular product fragmentation and denaturation, and delayed activity regulation.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a method for purifying bioactive substances, comprising: S1: Targeted pretreatment: Take the fermentation broth containing bioactive substances, centrifuge to remove impurities and collect the crude extract, or centrifuge to collect the cells, perform hypotonic-mild disruption coupled treatment and centrifuge to remove impurities and collect the crude extract; S2: Gradient membrane separation: The pretreated solution is sequentially passed through a microfiltration membrane, a composite membrane-chromatographic synergistic modified ultrafiltration membrane, and a nanofiltration membrane for gradient separation. The molecular weight cutoff of the ultrafiltration membrane and the operating parameters of the nanofiltration are selected according to the molecular weight of the target product. The target product is enriched and impurities are removed to obtain a membrane separation enrichment solution. S3: Multimode chromatography: The membrane separation enrichment solution is sequentially purified by multimode ligand complex ion chromatography, hydrophobic interaction chromatography and gel filtration chromatography. The chromatography process integrates in-situ online cleaning enhancement technology. The chromatography medium and elution conditions are selected according to the differences in charge characteristics, hydrophobicity and molecular weight of the target product. The target component is collected to obtain the chromatographic purification solution. S4: Precision purification: The chromatographic purification solution is desalted and decolorized to remove residual salt ions and pigment impurities, and then sterilely filtered to obtain a purified solution; the purification process is integrated with a real-time product stability monitoring and control system to dynamically maintain product activity; S5: Low-temperature molding: The refined liquid is molded using a low-temperature drying process to obtain a high-purity bioactive substance product.

[0007] In one embodiment, the centrifugation in S1 above uses a disc centrifuge or a tube centrifuge. If it is an extracellular product, the supernatant is collected and centrifuged using a disc centrifuge at 10,000-14,000 rpm or continuous flow centrifugation. If it is an intracellular product, cells are obtained using a tube centrifuge at 10,000-14,000 rpm for 20-30 min. Cell disruption employs a hypotonic-mild disruption coupling treatment: the cells are suspended in 0.05 mol / L Tris-HCl buffer (pH 7.0, containing 5 mmol / L EDTA) and incubated at 4°C for 30 min for hypotonic pretreatment; subsequently, ultrasonic gradient disruption is performed with parameters set to "low power (100W) 30s + stop 30s" for 20 cycles, then increased to medium power (200W) for 10 cycles, controlling the cell disruption rate to ≥95% and the target product activity loss to ≤2%.

[0008] To address the different characteristics of fermentation broths, targeted treatment methods such as "centrifugation-crushing-centrifugation" or "direct centrifugation" are employed, combined with low-osmotic-mild crushing technology, to reduce product denaturation and impurity release, thus adapting to the raw material characteristics of different products.

[0009] In one embodiment, the microfiltration membrane in S2 has a pore size of 0.1~0.45μm, used to remove fine suspended impurities; for macromolecular products (collagen, hyaluronidase): a 100-200kDa composite membrane-chromatographic synergistic modification ultrafiltration membrane is used: a polyethersulfone ultrafiltration membrane is selected, coupled with sodium carboxymethyl cellulose (dynamic binding capacity 90-100mg / ml) to form an ion exchange modified membrane, achieving targeted adsorption. Simultaneously, utilizing the "molecular sieve effect," the polyethersulfone ultrafiltration membrane retains macromolecular impurities (such as fibrin, polysaccharides), achieving… Dual purification of "sieving + adsorption"; small molecule products (coenzyme A / I): matched with 2~10kDa composite membrane-chromatographic synergistic modification ultrafiltration membrane + 0.3~0.5kDa nanofiltration membrane, composite membrane-chromatographic synergistic modification ultrafiltration membrane: selected polyethersulfone ultrafiltration membrane, coupled with sodium quaternary amino cellulose (dynamic binding capacity 100-115mg / ml) to form ion exchange modified membrane to achieve targeted adsorption. At the same time, utilizing the "molecular sieve effect", the polyethersulfone ultrafiltration membrane retains large molecular protein impurities, and the nanofiltration membrane retains the target product and removes small molecule impurities such as amino acids and inorganic salts. Synergistic optimization of membrane pore size and operating pressure: For 0.1~0.45μm microfiltration membranes, it is adapted to low-pressure operation of 0.05MPa (to avoid adsorption of target products on the membrane surface), and ultrafiltration membranes are adapted to medium-pressure operation of (0.05~0.1)MPa (to improve impurity filtration efficiency); in the nanofiltration stage, the pressure is adjusted to (0.4~0.6)MPa according to the concentration requirements, and the concentration factor is precisely controlled at 8~15 times to avoid over-concentration leading to product aggregation.

[0010] The three-stage gradient separation of "microfiltration-composite modified ultrafiltration-nanofiltration" is adopted, combined with targeted impurity adsorption, to achieve integrated synergy of "impurity removal-target enrichment-precision concentration", which greatly improves separation efficiency and reduces the load of subsequent chromatography.

[0011] In one embodiment, the multimode ligand complex ion chromatography in S3 is a highly efficient separation technique that combines strong anion exchange with hydrophobic interactions. Multimode ligand complex anion chromatography is a highly efficient separation technique that combines weak cation exchange with hydrophobic interactions. Its core lies in a single column with multiple mechanisms. It is tolerant to high salt and high pH in the loading solution and removes a wider range of impurities. The eluent for multimode ligand complex ion chromatography is a Tris-HCl buffer (50 mM) with a pH gradient (4.0–9.0) containing a 0–1.0 mol / L NaCl gradient. The elution flow rate is a linear flow rate of 100–200 cm / h. The target elution peak is collected to obtain the purification intermediate. In one embodiment, the medium for hydrophobic interaction chromatography is phenyl agarose gel or butyl agarose gel, or a special packing material with a polymer backbone and linear styrene as a functional group. The equilibration buffer is a phosphate buffer (pH 6.0-7.5) containing 1.0-2.0 mol / L ammonium sulfate, and the elution buffer is a phosphate buffer (pH 6.0-7.5) containing 0-1.2 mol / L ammonium sulfate, with an elution flow rate of 100-200 cm / h (linear flow rate). Gel filtration chromatography: Sephadex G-75 gel was used, the eluent was phosphate buffer at pH 5.0-6.5, the elution flow rate was linear at 30-100 cm / h, the main peak was collected, and the chromatographic purification solution was obtained. In-situ online cleaning enhancement: After each chromatography cycle, the cleaning program is started. The first stage uses 0.5 mol / L NaOH + 0.1% sodium dodecyl sulfate (SDS) and is circulated at 40℃ for 30 min. The second stage uses 0.1 mol / L Tris-HCl buffer (pH 8.0) + 0.2 mg / mL proteinase K and is circulated at 37℃ for 20 min. The third stage uses 0.05 mol / L citric acid + 0.01% sodium sulfite and is circulated for 15 min. After cleaning, the media regeneration rate is ≥98%, and the service life is increased from 50 cycles to more than 150 cycles.

[0012] The innovative process combines "multi-mode ligand composite ion chromatography, hydrophobic interaction chromatography, and gel filtration chromatography" with in-situ online cleaning technology to achieve high-purity separation step by step, while extending the service life of the media and reducing production costs.

[0013] In one embodiment, the desalination in S4 is performed using ultrafiltration or nanofiltration membranes, with ultrafiltration membranes retaining a molecular weight cutoff of 5-10 kDa and nanofiltration membranes retaining a molecular weight cutoff of 0.3-0.5 kDa; the decolorization is performed using activated carbon adsorption or decolorizing resin adsorption, with a decolorization temperature of 4-25°C and a decolorization time of 0.5-2 h; and the aseptic filtration uses a aseptic filter element with a pore size of 0.22 μm.

[0014] Real-time monitoring and control of product stability: The characteristic absorption peaks of the product are detected in real time by an online circular dichroism chromatograph. When the peak shape change rate is ≥5%, stabilizers are added to the feed solution (0.05% trehalose + 0.01% vitamin E for collagen; 0.03% mannitol + 0.01% dextran for hyaluronidase; 0.1 mol / L trehalose + 0.02% DTT for coenzymes). At the same time, the process temperature is reduced by 5~10℃ until the peak shape returns to stability.

[0015] By desalting and decolorizing, residual salt ions and pigment impurities from the chromatography process are removed, preventing salt ions from affecting product stability and pigments from affecting product appearance. Then, sterile filtration ensures that the product meets pharmaceutical-grade sterility requirements and is suitable for high-end applications.

[0016] In one embodiment, the low-temperature drying process in S5 is freeze drying or vacuum low-temperature drying; the freeze drying conditions are: pre-freezing temperature -30 to -40°C, pre-freezing time 2 to 6 hours, primary drying sublimation temperature -5 to -10°C, drying time 10 to 20 hours, and vacuum degree 0.25 mbar; the desorption drying temperature is 20 to 35°C, vacuum degree 0 mbar, and drying time 8 to 24 hours.

[0017] Freeze-drying or vacuum low-temperature drying is used to avoid the degradation of bioactive substances in a low-temperature environment throughout the process. At the same time, drying parameters are controlled to obtain a finished product with low moisture content and good stability, ensuring that the product is not easily deteriorated during long-term storage.

[0018] In one embodiment, the purification process of the target bioactive substance is controlled at a temperature of 4~35℃ throughout, and adjusted according to the thermal stability of the product; the temperature for coenzyme A and coenzyme I is controlled at 4~20℃, and the temperature for collagen and hyaluronidase is controlled at 10~35℃.

[0019] In another aspect, the present invention provides the application of the above-described purification method in the preparation of collagen, hyaluronidase, coenzyme A and / or coenzyme I.

[0020] In another aspect, the present invention also provides collagen, hyaluronidase, coenzyme A and / or coenzyme I, obtained by the above purification method.

[0021] (III) Beneficial Effects This invention provides a purification method suitable for hyaluronidase, collagen, and coenzymes. Compared with existing technologies, it has the following advantages: 1. High degree of versatility: By adapting five major process modules and key parameters (such as the molecular weight cutoff of ultrafiltration membrane and the selection of chromatography media), the purification of collagen, hyaluronidase, coenzyme A and coenzyme I can be achieved without changing the process framework, reducing the duplication of production line construction, reducing equipment investment and R&D costs, and adapting to the production of multiple products.

[0022] 2. High product activity and purity: Low temperature control throughout the process, combined with low osmosis-mild crushing, real-time activity regulation, low temperature drying and other technologies, avoids product denaturation and degradation, and the activity retention rate is ≥97%; through a combination of gradient membrane separation and multi-mode chromatography and other processes, impurities such as proteins, small molecules, pigments and specific impurities are completely removed, the product purity is ≥98% and the purity fluctuation range is ≤1%, and the stability is extremely strong.

[0023] 3. High industrial efficiency: The process is simplified to a 5-step core process, which is easy to operate, has strong anti-interference ability, and is adaptable to fluctuations in fermentation broth composition; technologies such as in-situ online cleaning enhancement and composite membrane-chromatography synergistic enhancement significantly improve production efficiency and reduce industrial production costs.

[0024] 4. Wide product applicability: The purified product meets pharmaceutical-grade standards in terms of sterility, purity, and activity, and can be directly applied to pharmaceutical preparations, high-end cosmetic raw materials, and functional food additives, covering high-value application areas with broad market prospects. Long media lifespan and low cost: In-situ online cleaning and enhancement technology significantly extends the lifespan of chromatography media, reduces the frequency of consumable replacement, and lowers production costs by more than 30%.

[0025] 5. Significant advantages in intracellular product purification: The low-osmotic-mild fragmentation coupling technology reduces product denaturation and the release of impurities such as proteins and nucleic acids, thereby reducing the subsequent purification load and improving separation efficiency. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Terms and Definitions As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0029] Example 1: Purification of collagen: 1. Targeted pretreatment: Take collagen fermentation broth (Pichia pastoris extracellular fermentation), centrifuge continuously at 11500 rpm using a disc centrifuge to remove cell precipitate, collect crude extract, sample turbidity 158 NTU; 2: Gradient membrane separation: The crude extract is filtered through a 0.22μm precision filter cartridge; then filtered through a composite modified ultrafiltration membrane (polyethersulfone material, coupled with sodium carboxymethyl cellulose ligand) with a molecular weight cutoff of 200kDa, at an operating pressure of 0.05MPa, and the permeate is collected. 3: Multimodal chromatography purification: 3.1: Multimode ligand complex ion chromatography: The membrane permeate was loaded onto a multimode ligand complex cation chromatography system. The equilibration buffer was 50 mM Tris-HCl buffer with pH 4.0. The elution flow rate was 150 cm / h linear flow rate. A gradient elution of 0–1.0 mol / L NaCl was used. The target peak was collected to obtain the purified intermediate solution. 3.2: Hydrophobic interaction chromatography: The purified intermediate solution was loaded onto a phenyl agarose gel chromatography column. The equilibration buffer was pH 6.0 phosphate buffer containing 1.5 mol / L ammonium sulfate, and the flow rate was 150 cm / h linearly. Gradient elution was performed using 1.2~0 mol / L ammonium sulfate, and the target peak was collected to obtain the hydrophobic purified solution. 3.3: Gel filtration chromatography: The hydrophobic purification solution was loaded onto a Sephadex G-75 gel resin column, the equilibration buffer was pH 6.5 phosphate buffer, and the linear flow rate was 50 cm / h; the main peak was collected to obtain the chromatographic purification solution.

[0030] 3.4: In-situ online cleaning: After chromatography, start the cleaning program and operate according to the steps of "alkali washing + enzymatic hydrolysis + neutralization". After completion, it is ready for use. The media regeneration rate is ≥98% and the media service life is up to 160 times.

[0031] 4. Precise purification: The chromatographically purified solution is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa at an operating pressure of 0.1 MPa, and the concentrate is collected. 0.1% (w / v) activated carbon is added, and the solution is decolorized at 10°C for 1 hour. The activated carbon is then removed by filtration. The characteristic absorption peaks are monitored using an online circular dichroism chromatograph. 0.05% trehalose and 0.01% vitamin E are added, and the solution is cooled to 15°C. After the peak shape stabilizes, the solution is filtered through a 0.22 μm sterile filter to obtain the purified solution. 5. Low-temperature molding: The refined liquid is freeze-dried at a pre-freezing temperature of -40℃ for 4 hours. The first drying sublimation temperature is -10℃, the drying time is 18 hours, and the vacuum degree is 0.25mbar. The desorption drying temperature is 30℃, the vacuum degree is 0mbar, and the drying time is 20 hours to obtain the finished collagen product.

[0032] The product was tested and found to have a purity of 98.8%, an activity retention rate of 97.5%, a miscellaneous protein content of 1.0%, a moisture content of 3.2%, and a bacterial count of ≤50 CFU / g.

[0033] Example 2 Purification of hyaluronidase: 1. Targeted pretreatment: Take the hyaluronidase fermentation broth (Pichia pastoris expression), centrifuge continuously at 11500 rpm using a disc centrifuge to remove cell precipitate, collect the crude extract, and the sample turbidity is 139 NTU. 2: Gradient membrane separation: The pretreated solution is filtered through a 0.22μm microfiltration membrane; then filtered through a composite modified ultrafiltration membrane (polyethersulfone material, coupled with sodium carboxymethyl cellulose ligand) with a molecular weight cutoff of 150kDa, at an operating pressure of 0.06MPa, and the permeate is collected. 3: Multimodal chromatography purification: 3.1: Multimode ligand composite ion chromatography: The membrane permeate was loaded onto a multimode ligand composite cation chromatography column. The equilibration buffer was 50 mM Tris-HCl buffer at pH 7.0, and the elution flow rate was 200 cm / h linearly. Elution was performed using a pH gradient (5.0~7.0) containing 0~1.0 mol / L NaCl. The target peak was collected to obtain the purified intermediate solution. 3.2: Hydrophobic interaction chromatography: The purified intermediate solution was loaded onto a phenyl agarose gel chromatography column. The equilibration buffer was pH 7.0 phosphate buffer containing 1.2 mol / L ammonium sulfate, and the flow rate was 150 cm / h linearly. The column was eluted with a gradient of 1.0-0 mol / L ammonium sulfate, and the target peak was collected to obtain the hydrophobic purified solution. 3.3: Gel filtration chromatography: The hydrophobic purification solution was loaded onto a Sephadex G-75 gel resin column, the equilibration buffer was pH 6.5 phosphate buffer, and the linear flow rate was 30 cm / h; the main peak was collected to obtain the chromatographic purification solution.

[0034] 3.4: In-situ online cleaning: The chromatography column is cleaned according to the preset program, with a media regeneration rate of ≥98% and a media service life of up to 155 cycles.

[0035] 4. Precise Purification: The chromatographically purified solution is passed through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa at an operating pressure of 0.1 MPa, and the concentrate is collected. 0.08% (w / v) decolorizing resin is added, and the solution is decolorized at 10°C for 0.5 h. The resin is then removed by filtration. The solution is monitored by an online circular dichroism chromatograph. When the peak shape change rate reaches 5%, 0.03% mannitol + 0.01% dextran is added, and the temperature is lowered to 15°C. After the peak shape stabilizes, the solution is filtered through a 0.22 μm sterile filter to obtain the purified solution. 5. Low-temperature molding: The freeze-drying conditions are: pre-freezing temperature -30℃, pre-freezing time 3h, first drying sublimation temperature -5℃, drying time 15h, vacuum degree 0.25mbar; desorption drying temperature 30℃, vacuum degree 0mbar, drying time 18h, to obtain the hyaluronidase product.

[0036] The finished product has a purity of 98.2%, an activity retention rate of 97.8%, a moisture content of 3.5%, and a bacterial count of ≤80 CFU / g.

[0037] Example 3 Purification of Coenzyme A: 1. Targeted pretreatment: Take the coenzyme A fermentation broth (intracellular fermentation of Escherichia coli), centrifuge at 14000 rpm for 30 min using a tube centrifuge to collect the bacterial cells, suspend the bacterial cells in 0.05 mol / L Tris-HCl buffer (pH 7.0, containing 5 mmol / L EDTA), and incubate at 4℃ for 30 min for hypotonic pretreatment. Then, use ultrasonic gradient disruption (100W 30s / 30s × 20 times, 200W × 10 times), and continuously feed centrifuge at 11500 rpm using a disc centrifuge to obtain the pretreated solution. The sample turbidity is 175 NTU. 2. Gradient membrane separation: The pretreated solution was filtered through a 0.22 μm microfiltration membrane; then filtered through a composite modified ultrafiltration membrane (polyethersulfone material, coupled with sodium quaternary amino cellulose ligand) with a molecular weight cutoff of 3 kDa, at an operating pressure of 0.08 MPa, and the permeate was collected; the permeate was concentrated through a nanofiltration membrane (molecular weight cutoff of 0.5 kDa) at an operating pressure of 0.4 MPa, with a concentration factor of 10 times, to obtain the membrane separation enrichment solution; 3: Multimodal chromatography purification: 3.1: Multimode ligand complex ion chromatography: The membrane separation enrichment solution was loaded onto a multimode ligand complex anion chromatography system. The equilibration buffer was 50 mM Tris-HCl buffer with pH 4.0, and the elution flow rate was a linear flow rate of 200 cm / h. A gradient elution of 0–1.0 mol / L NaCl was used, and the target peak was collected to obtain the purification intermediate solution. 3.2: Hydrophobic Interaction Chromatography: The purified intermediate solution was loaded onto a special packing material with a polymer backbone and linear styrene as functional groups. The equilibration buffer was a pH 6.0 phosphate buffer containing 0.8 mol / L ammonium sulfate, and the flow rate was 100 cm / h linearly. A gradient elution of 0.8–0 mol / L ammonium sulfate was used, and the target peak was collected to obtain the hydrophobic purified solution. 3.3: In-situ online cleaning: Complete the chromatography column cleaning process with a media regeneration rate of ≥98%; media lifespan reaches 158 cycles.

[0038] 4. Precision purification: The chromatographic purified solution is passed through a nanofiltration membrane with a molecular weight cutoff of 0.5 kDa at an operating pressure of 0.4 MPa, and the concentrate is collected. When the peak shape change rate reaches 4% by online circular dichroism chromatograph, 0.1 mol / L trehalose + 0.02% DTT is added, the temperature is lowered to 10℃, and after the peak shape stabilizes, it is filtered through a 0.22 μm sterile filter to obtain the purified solution. 5. Low-temperature molding: The purified liquid is freeze-dried at a pre-freezing temperature of -30℃ for 3 hours, a first-stage drying sublimation temperature of -10℃ for 16 hours, and a vacuum degree of 0.25 mbar; the desorption drying temperature is 15℃, the vacuum degree is 0 mbar, and the drying time is 6 hours to obtain the finished coenzyme A product.

[0039] The product was tested and found to have a purity of 99.1%, a potency of 410 U / mg, an activity retention rate of 97.8%, a moisture content of 2.8%, and a bacterial count of ≤30 CFU / g.

[0040] Example 4 Purification of Coenzyme I: 1. Targeted pretreatment: Take the coenzyme I fermentation broth (intracellular fermentation of Escherichia coli), centrifuge at 14000 rpm for 30 min using a tube centrifuge, collect the cells, suspend the cells in 0.05 mol / L Tris-HCl buffer (pH 7.0, containing 5 mmol / L EDTA), incubate at 4℃ for 30 min for hypotonic pretreatment, then use ultrasonic gradient disruption (100W 30s / 30s × 20 times, 200W × 10 times), and continuously feed centrifuge at 11500 rpm using a disc centrifuge to obtain the pretreated solution. The sample turbidity is 167 NTU. 2. Gradient membrane separation: The pretreated solution was filtered through a 0.22 μm microfiltration membrane; then filtered through a composite modified ultrafiltration membrane (polyethersulfone material, coupled with sodium quaternary amino cellulose ligand) with a molecular weight cutoff of 2 kDa, at an operating pressure of 0.08 MPa, and the permeate was collected; the permeate was concentrated through a nanofiltration membrane (molecular weight cutoff of 0.5 kDa) at an operating pressure of 0.4 MPa, with a concentration factor of 12 times, to obtain the membrane separation enrichment solution; 3: Multimodal chromatography purification: 3.1: Multimode ligand complex ion chromatography: The membrane separation enrichment solution was loaded onto a multimode ligand complex anion chromatography system. The equilibration buffer was 50 mM Tris-HCl buffer with pH 4.0, and the elution flow rate was a linear flow rate of 150 cm / h. A gradient elution with 0~1.0 mol / L NaCl was used, and the target peak was collected to obtain the purification intermediate solution. 3.2: Hydrophobic interaction chromatography: The purified intermediate solution was loaded onto a phenyl agarose gel chromatography column. The equilibration buffer was pH 6.0 phosphate buffer containing 1.0 mol / L ammonium sulfate, and the flow rate was 100 cm / h linearly. Gradient elution was performed using 1.0~0 mol / L ammonium sulfate, and the target peak was collected to obtain the hydrophobic purified solution. 3.3: In-situ online cleaning: Complete the chromatography column cleaning procedure, with a media regeneration rate of ≥98% and a media lifespan of up to 152 cycles.

[0041] 4. Precision purification: The chromatographic purified solution is passed through a nanofiltration membrane with a molecular weight cutoff of 0.3 kDa at an operating pressure of 0.4 MPa, and the concentrate is collected. When the peak shape change rate reaches 5% by online circular dichroism chromatograph, 0.1 mol / L trehalose + 0.02% DTT is added, the temperature is lowered to 8°C, and after the peak shape stabilizes, it is filtered through a 0.22 μm sterile filter to obtain the purified solution. 5. Low-temperature molding: The refined liquid is freeze-dried at a pre-freezing temperature of -40℃ for 3 hours, followed by a first-stage drying sublimation temperature of -10℃ for 18 hours and a vacuum degree of 0.25 mbar. The desorption drying temperature is 20℃, the vacuum degree is 0 mbar, and the drying time is 10 hours to obtain the NAD product.

[0042] The finished product has a purity of 99.5%, an activity retention rate of 97.5%, a moisture content of 3.0%, and a bacterial count of ≤40 CFU / g.

[0043] Comparative Example 1: Collagen Purification: 1. Targeted pretreatment: Take collagen fermentation broth (Pichia pastoris extracellular fermentation), centrifuge continuously at 11500 rpm using a disc centrifuge to remove cell precipitate, collect crude extract, sample turbidity 153 NTU; 2: Gradient membrane separation: The crude extract is filtered through a 0.22μm precision filter cartridge; then filtered through a composite modified ultrafiltration membrane (polyethersulfone material, coupled with sodium quaternary amino cellulose ligand) with a molecular weight cutoff of 200kDa, at an operating pressure of 0.05MPa, and the permeate is collected. 3: Multimodal chromatography purification: 3.1: Multimode ligand complex ion chromatography: The membrane permeate was loaded onto a multimode ligand complex cation chromatography system. The equilibration buffer was 50 mM Tris-HCl buffer with pH 4.0. The elution flow rate was 150 cm / h linear flow rate. A gradient elution of 0–1.0 mol / L NaCl was used. The target peak was collected to obtain the purified intermediate solution. 3.2: Hydrophobic interaction chromatography: The purified intermediate solution was loaded onto a phenyl agarose gel chromatography column. The equilibration buffer was pH 6.0 phosphate buffer containing 1.5 mol / L ammonium sulfate, and the flow rate was 150 cm / h linearly. Gradient elution was performed using 1.2~0 mol / L ammonium sulfate, and the target peak was collected to obtain the hydrophobic purified solution. 3.3: Gel filtration chromatography: The hydrophobic purification solution was loaded onto a Sephadex G-75 gel resin column, the equilibration buffer was pH 6.5 phosphate buffer, and the linear flow rate was 50 cm / h; the main peak was collected to obtain the chromatographic purification solution.

[0044] 3.4: After chromatography, wash with 0.5 mol / L NaOH solution at room temperature for 3 CV. The media can be used directly after washing and has a service life of up to 50 cycles.

[0045] 4. Precise purification: Pass the chromatographic purified solution through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa at an operating pressure of 0.1 MPa, collect the concentrate; add 0.1% (w / v) activated carbon, decolorize at 10°C for 1 h, filter to remove the activated carbon, and then filter through a 0.22 μm sterile filter cartridge to obtain the purified solution; 5. Low-temperature molding: The refined liquid is freeze-dried at a pre-freezing temperature of -40℃ for 4 hours. The first drying sublimation temperature is -10℃, the drying time is 18 hours, and the vacuum degree is 0.25mbar. The desorption drying temperature is 30℃, the vacuum degree is 0mbar, and the drying time is 20 hours to obtain the finished collagen product.

[0046] The finished product was tested and found to have a purity of 95.4%, an activity retention rate of 87%, a contaminant protein content of 7.0%, a moisture content of 3.8%, and a bacterial count ≤50 CFU / g. A comparison of the processes in Comparative Example 1 and Example 1 is shown in Table 1.

[0047] Table 1. Comparison of processes between Comparative Example 1 and Example 1 Comparative Example 2: Coenzyme A Purification: 1: Fermentation broth pretreatment: Take the same coenzyme A fermentation broth (intracellular fermentation of Escherichia coli) as in Example 3, centrifuge at 14000 rpm for 30 min to collect the cells, homogenize and crush under high pressure (100 MPa, 3 times), and centrifuge to remove impurities to obtain crude extract; 2. Preliminary purification: The crude extract was adjusted to pH 3.5 using 6 mol / L hydrochloric acid. The sample was then continuously centrifuged at 11500 rpm using a disc centrifuge to obtain a pretreated solution with a turbidity of 167 NTU. The crude extract was then filtered through a 0.22 μm precision filter to obtain the chromatographic loading solution. 3. Purification: Traditional strong anion exchange chromatography (without multi-mode ligand complex chromatography, without in-situ online washing) is used. The eluent is Tris-HCl buffer (pH 8.0) containing 0~1.0 mol / L NaCl, and the flow rate is 100 cm / h. After chromatography, the media is washed with 0.5 mol / L NaOH solution at room temperature for 3CV. The media can be used directly after washing and has a service life of up to 61 cycles.

[0048] 4. Concentration and Desalting: Pass the chromatographic purified solution through a nanofiltration membrane with a molecular weight cutoff of 0.5 kDa at an operating pressure of 0.4 MPa, collect the concentrate, and then filter it through a 0.22 μm sterile filter cartridge to obtain the purified solution. 5. Drying: The purified solution was freeze-dried at a pre-freezing temperature of -30℃ for 3 hours, a first-stage drying sublimation temperature of -10℃ for 16 hours, and a vacuum degree of 0.25 mbar; the desorption drying temperature was 15℃, the vacuum degree was 0 mbar, and the drying time was 6 hours to obtain the coenzyme A product.

[0049] The finished product was tested and found to have a purity of 56.1%, a potency of 198 U / mg, an activity retention rate of 73.8%, a moisture content of 4.8%, and a bacterial count ≤30 CFU / g. A comparison of the processes in Comparative Example 2 and Example 3 is shown in Table 2.

[0050] Table 2 Comparison of processes between Comparative Example 2 and Example 3 It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying a bioactive substance, characterized in that, include: S1: Targeted pretreatment: Collect the fermentation broth containing bioactive substances, centrifuge to remove impurities and obtain crude extract; or collect the fermentation broth containing bioactive substances, centrifuge to collect the cells, perform hypotonic-mild disruption coupled treatment, centrifuge to remove impurities and collect crude extract; S2: Gradient membrane separation: The crude extract is passed through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane to obtain a membrane separation enrichment; S3: Multimode chromatography: The membrane separation enrichment solution is sequentially purified by multimode ligand complex ion chromatography, hydrophobic interaction chromatography and gel filtration chromatography. The chromatography process integrates in-situ online cleaning to obtain the chromatographic purified solution. S4: Precision purification: The chromatographic purification solution is desalted and decolorized, and the product stability is adjusted by circular dichroism chromatograph to obtain a purified solution; S5: Low-temperature molding: The refined liquid is dried at low temperature to obtain a high-purity bioactive substance product.

2. The purification method according to claim 1, characterized in that, In step S1, if the bioactive substance is an extracellular product, the centrifugation conditions are disc centrifuge at 10,000-14,000 rpm or continuous flow centrifugation; if the bioactive substance is an intracellular product, the centrifugation conditions are tubular centrifuge at 10,000-14,000 rpm for 20-30 min.

3. The purification method according to claim 1, characterized in that, The hypotonic treatment method is as follows: place in Tris-HCl buffer containing 4~6 mmol / L EDTA at a concentration of 0.04~0.06 mol / L, and incubate at 2~8℃ for 15~45 min.

4. The purification method according to claim 1, characterized in that, The pore size of the microfiltration membrane is 0.1~0.45μm; And / or the ultrafiltration membrane has a molecular weight cutoff of 2~200kDa, and the ultrafiltration membrane is a polyethersulfone coupled with sodium carboxymethyl cellulose ligand and / or sodium quaternary amino cellulose ligand, with a dynamic binding loading of 90~115mg / ml; And / or the nanofiltration membrane has a molecular weight cutoff of 0.2~1 kDa.

5. The purification method according to claim 1, characterized in that, The chromatography conditions for the multimode ligand complex ion chromatography are as follows: Elution buffer: Tris-HCl buffer containing 0~1.0 mol / L NaCl, 50 mM, pH 4.0~9.0; Elution flow rate: 100~200 cm / h, linear flow rate; And / or the chromatographic conditions for the hydrophobic interaction chromatography are: Equilibrium buffer: phosphate buffer containing 1.0~2.0 mol / L ammonium sulfate, pH 6.0~7.5; Eluent: Phosphate buffer containing 0-1.0 mol / L ammonium sulfate, pH 6.0-7.5; Elution flow rate: 100~200 cm / h, linear flow rate; And / or the chromatography conditions for the gel filtration chromatography are: Gel: Sephadex G-75 gel; Elution buffer: phosphate buffer, pH 5.0~6.5; Elution flow rate: 30~100cm / h, linear flow rate.

6. The purification method according to claim 1, characterized in that, The in-situ online cleaning includes: Alkaline washing: 0.1~1.0 mol / L NaOH + 0.05~0.15% sodium dodecyl sulfate (SDS), circulate at 35~50℃ for 15~45 min; And / or enzymatic digestion: 0.05~0.15mol / L Tris-HCl buffer + 0.1~0.3mg / mL proteinase K, cycle at 25~45℃ for 10~30min; Neutralization: 0.01~0.1mol / L citric acid + 0.01~0.05% sodium sulfite, circulate for 10~30min.

7. The purification method according to claim 1, characterized in that, The adjustment of the circular dichroism chromatograph includes: when the peak shape change rate is ≥5%, adding a stabilizer and lowering the temperature by 5~10℃.

8. The purification method according to any one of claims 1-7, characterized in that, The purification temperature is 4~35℃.

9. The use of the purification method according to any one of claims 1-8 in the preparation of collagen, hyaluronidase, coenzyme A and / or coenzyme I.

10. A collagen, hyaluronidase, coenzyme A and / or coenzyme I, characterized in that, Obtained by the purification method described in any one of claims 1 to 8.

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