A method for industrial purification and freeze-drying of recombinant proteinase K

Abstract

The invention discloses a recombinant proteinase K industrial purification and freeze-drying method. After the recombinant proteinase K fermentation liquid expressed by Pichia pastoris is clarified and filtered, a diluent is added to dilute, and concentrated and dialyzed through an ultrafiltration step to obtain a proteinase K dialyzate containing trace metal ions and pigments, and the dialyzate is separated and purified by cation exchange chromatography to obtain an eluent containing recombinant proteinase K; the eluent containing recombinant proteinase K is placed in a room temperature crystallization precipitation for 4-6h, and poured into a positive pressure filtration device after stirring and mixing, and a proteinase K filter cake is obtained by filter pressing; after the filter cake is redissolved, a freeze-drying excipient is added, and freeze-dried according to an optimized freeze-drying process, and a proteinase K finished product is obtained after sub-packaging. The method has simple process steps, high yield, low cost, and is easy to industrially scale up production. The obtained proteinase K target has high yield and good activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for industrial purification and freeze-drying of recombinant proteinase K. Background Art

[0002] Proteinase K is a serine protease that was first purified from an extract of Tritirachiumalbum limber in 1974. The main cleavage site is the peptide bond at the carboxyl end of hydrophobic amino acids such as aliphatic or aromatic amino acids. Studies have shown that the enzyme is active at pH 4-12 and 20℃-60℃, and also has the ability to cleave proteins in SDS, urea, chelating agents (such as EDTA) and sulfhydryl reagents (such as trypsin inhibitors or chymotrypsin inhibitors). Therefore, it has a wide range of applications, including the preparation of chromosomal DNA for pulsed electrophoresis, protein blotting, and the removal of nucleases in DNA and RNA preparation.

[0003] The application of proteinase K is not limited to the fields of medical diagnosis and scientific research. It can also replace the traditional chemical methods that may cause pollution to process raw materials in the fields of industrial leather, papermaking, feed, etc. Therefore, using genetic engineering technology and protein engineering technology to obtain proteinase K with high activity and low cost has great social and economic value.

[0004] At present, the industrially produced gene recombinant proteinase K uses prokaryotic cells (such as Escherichia coli) and eukaryotic cells (yeast) as hosts. The advantage of using Escherichia coli as a host is that it is simple and economical. However, the proteinase K expressed by this system is not active and the yield is too low, which is difficult to meet market demand. The hexahistidine tag added to the C-terminus of the recombinant proteinase K using yeast as a host requires the fermentation broth to undergo steps such as centrifugation, metal chelate column chromatography and weak cation exchange column chromatography separation, and macroporous decolorizing gel decolorization treatment, and the process steps are relatively complicated. In view of the application value of proteinase K, it is necessary to develop a separation and purification technology that is fast and economically suitable for industrial scale and continuous production. Summary of the invention

[0005] The object of the present invention is to provide an industrial purification and freeze-drying method for recombinant proteinase K.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A chromatography method for separating and purifying recombinant proteinase K from Pichia pastoris comprises the following steps in sequence:

[0008] 1) The supernatant of the recombinant proteinase K fermentation broth expressed by Pichia pastoris is clarified, and then an equal volume of diluent is added to dilute and mix. The diluted fermentation supernatant is concentrated using an ultrafiltration membrane package, and after concentration to the end point, an equal volume of dialysate is added for multiple dialysis to obtain a proteinase K dialysate containing trace metal ions and pigments. The ultrafiltration membrane package can be selected from Millipore 5kD, 10kD or Sartorius 5kD, 10kD. The preferred membrane package has a retention pore size of 5kD.

[0009] 2) The dialyzate of the recombinant proteinase K containing a small amount of metal ions and pigments is subjected to cation exchange chromatography after adjusting the pH and conductivity to obtain an eluate containing a higher concentration of proteinase K. The filler of the cation exchange chromatography is selected from NanoGel 30 / 50SP, UniGel 30 / 80SP, SP Bestarose FF, SP Bestarose HP, BestaroseDiomond MMC, Uniphere S, MacroPrep S, POROS XS, SP-6FF, SP-6HP, SP Sepharose TM FastFlow. In one embodiment, the filler of the cation exchange chromatography is preferably SP Bestarose FF. The cation exchange chromatography can be eluted by pH or salinity gradient, preferably sodium chloride gradient elution.

[0010] 3) The obtained proteinase K eluate is subjected to crystallization precipitation at room temperature. Before the crystallization precipitation, the proteinase K eluate is preferably adjusted to a protein concentration in the range of 40 to 50 mg / ml. After the precipitation is completed, a positive pressure filtration device is used to obtain a proteinase K precipitation filter cake. The filter paperboard is Shenyang Great Wall filter paperboard, and the filter plate pore size is 0.2 to 5 μm, preferably 2.5 to 5 μm.

[0011] 4) Add the obtained proteinase K precipitate filter cake to a reconstitution buffer for reconstitution. In one embodiment, the reconstitution buffer is 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2, pH 4.5-5.5. After mixing with the preferred freeze-drying formula, freeze-drying is performed according to the preferred freeze-drying process. The obtained freeze-dried powder is packaged to finally obtain a finished product of proteinase K freeze-dried powder.

[0012] The proteinase K activity of the supernatant of the recombinant proteinase K fermentation broth is 30-40 U / mgP.

[0013] The present invention also discloses a specific implementation method, in which, after process amplification, the protein purification yield of the obtained proteinase K lyophilized powder is not less than 70%, and the activity is 30-40 U / mgP. The specific implementation steps are:

[0014] 1) The supernatant of the fermentation broth of the recombinant proteinase K expressed by Pichia pastoris is clarified, the pH and conductivity of the clarified supernatant are measured, and then an equal volume of diluent is added to dilute and mix; the diluent is: 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0;

[0015] The ultrafiltration membrane package is installed on the ultrafiltration system, the inlet pressure and the reflux pressure are controlled, the transmembrane pressure is adjusted and the water flux is measured, and ultrafiltration concentration is performed after the membrane package is installed; the ultrafiltration membrane package has a retention pore size of 5 to 10 kD, and the transmembrane pressure is 1 to 1.25 bar or 14.5 to 18 psi or 0.1 to 0.125 MPa.

[0016] The concentrated solution concentrated to the endpoint is dialyzed multiple times with an equal volume of dialysate until the dialysis endpoint is reached; the dialysate comprises: 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0; the effluent at the dialysis endpoint is colorless, has a conductivity of 0.5-2 mS / cm, and a pH of 7.5-9.0.

[0017] 2) The cation exchange chromatography medium used is Bogelon SP Bestarose FF cation exchange filler, and the chromatography steps are as follows:

[0018] Using 5 to 15 column volumes of an equilibrium buffer having a composition of 10 to 50 mM Tris, 0 to 5 mM CaCl2, and a pH of 7.5 to 9.0, the chromatography column is equilibrated at a flow rate of 50 to 200 cm / hour;

[0019] The proteinase K dialyzate of step 1) is used as a sample to be loaded at a flow rate of 50 to 200 cm / hour; wherein the sample conductivity is 0.5 to 2 mS / cm, the pH is 7.5 to 9.0, and the loading capacity is 56 to 90 mg protein / ml filler;

[0020] Re-equilibrate the column with an equilibration buffer of 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0 at a flow rate of 50-200 cm / hour;

[0021] Using an elution buffer having a composition of 10-50 mM Tris, 0-5 mM CaCl2, 0-100 mM NaCl, and a pH of 7.5-9.0, the chromatography column is eluted at a flow rate of 50-200 cm / hour to finally obtain a proteinase K eluate;

[0022] The protein recovery of the cationic chromatography step-by-step amplification chromatography is greater than 70%.

[0023] 3) Cut the filter paper of appropriate size and install it into the positive pressure filter, and rinse the positive pressure filter device and the filter paper with an elution buffer containing 10-50 mM Tris, 0-5 mM CaCl2, 0-100 mM NaCl, and pH 7.5-9.0;

[0024] The proteinase K eluate in step 2) is adjusted to a pH of 7.5 to 9.0, a Cond of 0 to 10 mS / cm, and a protein concentration of 40 to 50 mg / ml, and poured into a stainless steel barrel, and the ambient temperature is controlled at 20 to 25° C., and allowed to stand for more than 4 hours until the proteinase K crystals are completely precipitated;

[0025] Pour the completely precipitated proteinase K into the positive pressure filter tank in batches, connect the compressed air to the filter press tank, control the pressure gauge of the filter press tank to 0-0.15MPa to start filtration, and finally obtain the proteinase K filter cake;

[0026] The protein recovery in the proteinase K filter cake is greater than 90%.

[0027] 4) adding 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2, pH 4.5-5.5 to the filter cake containing recombinant proteinase K obtained in step 3) to redissolve, adding mannitol at a ratio of W / V=1:20 and mixing evenly to obtain a mixed solution;

[0028] Pour the mixture into a freeze-drying plate, set the pre-freezing temperature to -40 to -45°C, the cooling time to 30 minutes, and maintain for 3 to 6 hours;

[0029] Set the temperature of the first stage of primary drying to -35~-40℃, the heating time to 30min, maintained for 1~2h, and the vacuum degree to 10~20Pa; the temperature of the second stage is 0~10℃, the heating time is 4~6h, maintained for 40~45h.

[0030] The invention adopts industrial process steps such as ultrafiltration-cation exchange-filter pressing, etc., so that the batch production time is shortened to 1-2 days, and the total yield of proteinase K in the freeze-dried preparation obtained after purification is as high as more than 70%, thereby realizing large-scale and continuous production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Color change of proteinase K fermentation broth before and after ultrafiltration.

[0032] Figure 2 : GE DEAE anion exchange chromatography.

[0033] Figure 3 :SP Bestarose FF cation exchange chromatography 1ml column linear elution chromatogram.

[0034] Figure 4 :SP Bestarose FF cation exchange chromatography 84ml column gradient elution chromatogram.

[0035] Figure 5 : Comparison of proteinase K crystal precipitation and stratification.

[0036] Figure 6 : Morphological changes of proteinase K after freeze-drying.

[0037] Figure 7 : Dissolution of proteinase K in sodium acetate-acetic acid buffer at different pH values.

[0038] Figure 8 : L-tyrosine standard curve. DETAILED DESCRIPTION

[0039] The following will describe the technical solution of the present invention in detail through examples and drawings, so as to better illustrate the characteristics and advantages of the present invention. The provided examples should be interpreted as an illustration of the method of the present invention, and do not limit the technical solution disclosed by the present invention in any way.

[0040] Unless otherwise specified, the reagents and instruments used in the following examples are commercially available.

[0041] Example 1: Proteinase K purification and lyophilization process optimization

[0042] 1.1 Experimental reagents and instruments

[0043] Proteinase K fermentation broth was purchased from Jinan Baisijie Bioengineering Co., Ltd.;

[0044] The ultrafiltration system was purchased from Merck Millipore, model CUF 100;

[0045] Ultrafiltration membrane package, purchased from Sartorius;

[0046] Chromatographic filler, SP Bestarose FF, was purchased from Shanghai Bogeron; DEAE anion exchange filler, was purchased from General Electric Company;

[0047] Other chemical reagents were commercially available.

[0048] 1. Tangential flow ultrafiltration

[0049] 1.1 Ultrafiltration process exploration

[0050] Yeast fermentation broth is dark red-brown and contains a large amount of pigment. According to the molecular weight of proteinase K, the present invention uses a suitable ultrafiltration membrane package to remove pigment from yeast fermentation broth. The color of the solution after ultrafiltration becomes lighter than that before and after ultrafiltration, indicating that a large amount of pigment is removed. The color change of the fermentation broth before and after ultrafiltration is shown in the attached Figure 1 .

[0051] The present invention selects ultrafiltration membrane packages with different cut-off pore sizes and membrane areas for comparative experiments. The results are shown in Table 1. The ultrafiltration membrane packages with different cut-off pore sizes and membrane areas have protein recovery rates greater than 90%, and both membrane packages can meet the needs of scaled-up production. In addition, after the membrane area is enlarged to 33 times in the comparative experiment, the protein recovery after ultrafiltration is relatively stable, indicating that the process can be stably scaled up.

[0052] Table 1 Ultrafiltration results of ultrafiltration membrane packages with different pore sizes and membrane areas

[0053]

[0054] 1.2 Ultrafiltration process optimization

[0055] To adapt to the larger scale of fermentation broth processing, the ultrafiltration process parameters need to be optimized. Take 500 ml of fermentation broth, add an equal volume of ultrapure water to dilute it while stirring and set aside.

[0056] Using Sartorius 0.1m 2 5kD ultrafiltration membrane package for ultrafiltration concentration, the normal water flux of this membrane package product is 42L / h / m 2 , Pin(max)=4bar. After optimization, the final control is Pin(inlet pressure)=2bar, Pret(reflux pressure)=0.5bar, Pout(permeation pressure)=0bar. The TMP(transmembrane pressure) of the membrane package is calculated as (Pin+Pret) / 2-Pout=1.25bar=18psi.

[0057] The process parameters of the ultrafiltration concentration and dialysis steps were optimized according to the above transmembrane pressure. The results are shown in Table 2 and Table 3. The batch processing capacity and the actual membrane package usage area were calculated based on the average water flux of the concentration and ultrafiltration steps after optimization. The final result is: 250L / batch feed, 8h ultrafiltration required membrane area

[0058] ={(250L / 17LMH)+(250L*7 / 28LMH} / 8h=9.65m 2 , the number of membrane packages required = 9.65m 2 / 0.66m 2 ≈15 yuan.

[0059] Table 2 Concentration step process parameter control table

[0060]

[0061] Average flux of concentration step = 0.5L / (18 / 60)h / 0.1m 2 ≈17LMH

[0062] Table 3 Dialysis step process parameter control table

[0063]

[0064] Average dialysis step flux = 3.5 L / (74 / 60) h / 0.1 m 2 ≈28LMH

[0065] 2. Ion exchange chromatography

[0066] According to the isoelectric point of proteinase K, proteinase K can be separated and purified by two purification methods: anion penetration mode or cation binding mode.

[0067] 2.1 Anion exchange chromatography

[0068] The anion exchange chromatography conditions were explored using a GE DEAE column. The ultrafiltration treated proteinase K fermentation broth was adjusted to pH 7.6 and conductivity to 0.9 mS / cm before loading. 10 mM Tris-HCl, 1 mM CaCl2, pH 7.5 was used as the balance solution; 10 mM Tris-HCl, 1 M NaCl, 1 mM CaCl2, pH 7.5 was used as the eluent for chromatography. The results showed that proteinase K only penetrated 69.3%, so the anion penetration mode was abandoned to purify proteinase K. The chromatogram is attached. Figure 2 .

[0069] 2.2 Cation exchange chromatography

[0070] 2.2.1 Exploration of cation chromatography conditions

[0071] The conditions of cation exchange chromatography were explored using the Boglund SP Bestarose FF chromatography column. The ultrafiltration treated proteinase K fermentation broth was taken and the pH was adjusted to 7.5 and the conductivity was 0.8 mS / cm before loading. 10 mM Tris-HCl, 1 mM CaCl2, pH 7.5 was used as the equilibrium solution; 10 mM Tris-HCl, 0-1 M NaCl, 1 mM CaCl2, pH 7.5 was used as the eluent for chromatography. The results showed that proteinase K was mainly eluted under a gradient of 0-100 mM NaCl, with a protein recovery of 92.7% and an activity of more than 30 U / mg. See the attached chromatogram for the attached Figure 3 .

[0072] 2.2.2 Cationic chromatography scale-up

[0073] Based on the exploration of cationic chromatography conditions, the chromatography parameters of SP Bestarose FF were optimized and confirmed. The sample conductivity was 0.5-2mS / cm, the sample loading capacity was 56-90mg protein / ml filler, and the elution conditions were 10mM Tris-HCl, 0-100mM NaCl, 1-5mM CaCl2, pH 7.5-9.0. In order to verify the stability of the chromatography process, under the optimized chromatography parameters, the filler volume was enlarged from 1ml in the exploration stage to 8.6ml, 28ml, and 84ml in turn. The results showed that the protein recovery of the step-by-step chromatography was greater than 70%. The results of the step-by-step chromatography are shown in Table 4. The chromatogram of SP Bestarose FF enlarged to 84ml is shown in the attached figure. Figure 4 .

[0074] Table 4 SP Bestarose FF step-by-step chromatography results

[0075]

[0076] 3. Proteinase K solid-liquid separation

[0077] Proteinase K is easy to crystallize and precipitate in a low-salt environment with a high protein concentration. The present invention utilizes this characteristic of proteinase K, adjusts the pH of the SP Bestarose FF chromatography eluent to 7.5-9.0, the Cond to 0-10mS / cm, and the protein concentration to 40-50mg / ml, stirs and mixes, and then stands at 20-25°C for 4-6h. Under the above conditions, proteinase K crystallizes and stratifies. The crystal precipitation and stratification results are shown in the attached Figure 5 .

[0078] Using our solid-liquid separation technology platform, we selected filter paperboards of appropriate sizes and positive pressure filtration devices to collect the precipitated precipitates. The filter paperboard is Shenyang Great Wall filter paperboard with a pore size of 0.2 to 5 μm. When the filter paperboard is used for filtration, the proteinase K precipitate is retained on the filter paperboard to form a filter cake, while the filtrate is clear and transparent. 30 ml of the suspension after precipitation was filtered, and three experiments were performed in total. The filtrate and the filter cake re-solution were taken for protein content determination. The results showed that the protein recovery after crystallization precipitation was greater than 90%. Collecting the precipitate in this way can achieve the purpose of removing impurities and concentrating. The determination results are shown in Table 5.

[0079] Table 5 Proteinase K crystal precipitation protein recovery results

[0080]

[0081] 4. Proteinase K freeze-drying process

[0082] The obtained filter cake containing recombinant proteinase K is added with 10-50mM Tris, 0-5mM CaCl2, pH7.5-9.0 or 10-50mM NaAc, 0-5mM CaCl2, pH 4.5-5.5 for redissolution. Mannitol is added in a ratio of W / V=(1:20) and mixed evenly. The mixed solution is poured into a freeze-drying plate, and the pre-freezing temperature is set to -40-45°C, the cooling time is 30min, and it is maintained for 3-6h. The temperature of the first stage of primary drying is set to -35-40°C, the heating time is 30min, and it is maintained for 1-2h. The vacuum degree is set to 10-20Pa. The temperature of the second stage is 0-10°C, the heating time is 4-6h, and it is maintained for 40-45h.

[0083] The preferred freeze-drying process has freeze-drying parameters controlled as follows: -45°C, cooling time of 30 minutes, and maintenance of 6 hours; the temperature of the first stage of primary drying is set to -40°C, the heating time is 30 minutes, and maintenance is 1 hour, and the vacuum degree is set to 20Pa; the temperature of the second stage is 0°C, the heating time is 6 hours, and maintenance is 45 hours. Figure 6 .

[0084] The freeze-dried powder obtained by the freeze-drying process was subjected to activity assay (casein as substrate), and the results showed that the activity values ​​of the freeze-dried powder at different sampling points were all greater than 30 U / mg, and were equivalent to the activity of Merck proteinase K. The activity assay results are shown in Table 6.

[0085] Table 6 Activity determination results of proteinase K lyophilized powder at different sampling points

[0086]

[0087] 5. Proteinase K solubility study

[0088] 5.1 Proteinase K Lyophilized Powder Dissolution Buffer

[0089] In order to explore the optimal dissolution conditions of proteinase K lyophilized powder, the present invention uses 20mM Tris-HCl, pH 9.68-7.15; 20mM PB, pH 8.02-5.60; 20mM NaAc, pH 7.60-5.20 to dissolve proteinase K lyophilized powder and observe the dissolution state. The results show that proteinase K dissolves well in the range of 20mM NaAc, pH 5.20-5.60.

[0090] To verify the result, 7 portions of proteinase K lyophilized powder were accurately weighed, 100 mg each, in 10 ml EP tubes, and 20 mM NaAc, pH 5.58, 5.48, 5.40, 5.28, 5.21 buffers were prepared, 100 ml each, and then 5 ml of the buffers with different pH values ​​were added to 5 of the tubes, and the dissolution state was observed after shaking and mixing. 5 ml of 50% glycerol and 5 ml of ultrapure water were added to two other tubes as controls. The activity test results are shown in Table 7, and the dissolution results are shown in Appendix. Figure 7 .

[0091] Table 7 The results of the determination of the solubility activity of proteinase K in sodium acetate-acetic acid buffer at different pH

[0092]

[0093]

[0094] Example 2: Proteinase K process scale-up

[0095] 2.1 Production raw materials and equipment

[0096] Proteinase K fermentation broth was purchased from Jinan Baisijie Bioengineering Co., Ltd.;

[0097] Tris(hydroxymethyl)aminomethane was purchased from Nanjing Chemical Reagent Co., Ltd., analytical grade;

[0098] Anhydrous calcium chloride, purchased from Hebei Huachen Pharmaceutical Co., Ltd., pharmaceutical grade;

[0099] Sodium chloride, purchased from Hebei Huachen Pharmaceutical Co., Ltd., pharmaceutical grade;

[0100] Sodium hydroxide, purchased from Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd., pharmaceutical grade;

[0101] Ethanol, purchased from Nanjing Chemical Reagent Co., Ltd., pharmaceutical grade;

[0102] Mannitol was purchased from Shijiazhuang Huaxu Pharmaceutical Co., Ltd., pharmaceutical grade;

[0103] Sodium thimerosal was purchased from Sinopharm Chemical Reagent Co., Ltd., analytical grade;

[0104] The ultrafiltration system was purchased from Merck Millipore, model CUF 100;

[0105] Ultrafiltration membrane package, purchased from Sartorius, model HYDRO, 5KD;

[0106] Industrial-grade chromatography column, purchased from General Electric Company, USA, model Chromflow 800;

[0107] Chromatographic filler, purchased from Shanghai Bogeron, model SP Bestarose FF;

[0108] Freeze dryer, purchased from Beijing Suyuan Zhongtian Technology Co., Ltd., model: YLDZ-0.5

[0109] 2.2 Stock solution production

[0110] Step (1): pretreatment of raw materials.

[0111] Filter 10 barrels of fermentation liquid (25L / barrel) through a 10-inch, 0.22μm sterilizing filter and divide into two barrels, each barrel is about 125L;

[0112] Step (2): initial concentration.

[0113] Each barrel of fermentation liquid in step (1) is first dialyzed 1-2 times with an equal volume of 5KD membrane package and dialyzate 1 (formula: 0.111 g / L CaCl2 aqueous solution), and then each barrel of fermentation liquid is concentrated to 75 L and the two barrels are combined into one barrel of 150 L, and then dialyzed 3-4 times with an equal volume of dialysis 1, and then dialyzed 3-5 times with an equal volume of dialysis liquid 2 (formula: 1.21 g / L Tris, 0.111 g / L CaCl2, pH 7.5±0.1, Cond0.8-1 mS / cm);

[0114] Step (3): Preparation of sample solution.

[0115] The pH of the concentrated solution in step (2) was adjusted to 7.5±0.1 with Tris, and the conductivity was adjusted to 0.8-1.2 mS / cm. The protein content of the solution was then diluted to 10-15 mg / ml with dialysate 2 (formula: 1.21 g / L Tris, 0.111 g / L CaCl2, pH 7.5±0.1, Cond 0.8-1 mS / cm), thus forming a chromatography loading solution.

[0116] Step (4) chromatography purification.

[0117] Perform chromatography purification according to the following steps 1) to 7), and collect the chromatography eluate as the target component.

[0118] 1) Column packing: GE Chromflow 800 chromatography column, SP Bestarose FF filler, column packing height 16-18 cm, packed chromatography filler volume 80-86 L, detection column efficiency greater than 3000.

[0119] 2) Filler water washing: Use purified water to wash the chromatography column 350-420L, the flow rate is about 145cm / h, and the washing time is longer than 30min.

[0120] 3) Balance: Use balance buffer (1.21g / L Tris, 0.111g / L CaCl2, pH 7.5±0.1, Cond

[0121] 0.8-1mS / cm) equilibrate the chromatography column 600-700L, with a flow rate of about 145cm / h, until the pH and conductivity baselines of the permeate at the outlet of the chromatography column are stable.

[0122] 4) Loading: Loading the sample using the chromatography loading solution of step (3), with the loading amount being less than 67.5 mg total protein / ml filler, the loading flow rate being about 145 cm / h, and the loading chromatography column inlet pressure being less than 2.0 bar.

[0123] 5) Re-equilibration: Use the above-mentioned equilibration buffer to equilibrate the column again for 550-650 L, with a flow rate of about 145 cm / h. Re-equilibrate until the UV absorption value of the permeate at the chromatography outlet is less than 20 mv.

[0124] 6) Elution and collection of target components: Use elution buffer (1.21 g / L Tris, 0.555 g / L CaCl 2， 5.844g / L NaCl, pH 7.5±0.1, Cond 12±1mS / cm) was used to elute the chromatography column at a flow rate of about 145cm / h. When the chromatographic peak appeared and the UV absorption value was greater than 30mv, collection began. Stirring was performed while collection. When the UV absorption value was less than 30mv, collection was stopped, which was the target component.

[0125] 7) Regeneration and storage: After the elution is completed, the column is regenerated with 2M NaCl and 0.5M NaOH, respectively, with a regeneration flow rate of 145cm / h and a regeneration volume of 300-420L. After the regeneration is completed,

[0126] The chromatography column was preserved in 20% ethanol.

[0127] Step (5): Separation by precipitation and filtration.

[0128] The target component collected by chromatography purification in step (4) was allowed to settle for more than 5 hours, and then positive pressure filtration was performed using a 75 μm filter paper to collect the precipitate (filter cake) and the clarified supernatant. The inlet pressure during the filtration process did not exceed 4 bar.

[0129] Step (6): Preparation of stock solution.

[0130] First, 20 L of the clarified supernatant from step (5) was added to the filter cake to dissolve it, and then 10 L of the stock solution (2.5 kg of mannitol and 5 g of thimerosal, dissolved in the clarified supernatant) was added to the filter cake. Finally, the volume was fixed to 50 L with the clarified supernatant to form the proteinase K stock solution.

[0131] 2.3 Freeze-drying

[0132] Step (1) pre-freezing: after the feed solution is shaken evenly, the solution is poured into a stainless steel freeze-drying tray and pre-frozen at -45°C for 4 hours;

[0133] Step (2) cooling the rear box: after the pre-freezing is completed, the rear box starts to cool down, the product temperature is maintained at -45°C, and the rear box is cooled for 30 minutes;

[0134] Step (3) Vacuuming: After the rear box is cooled down, the box is evacuated and the vacuum is controlled below 25 Pa;

[0135] Step (4) Heating: The board layer starts to heat up, and the temperature rises to 0°C in 6 hours, and the vacuum degree is controlled at 20-30 Pa;

[0136] Step (5) primary sublimation: maintain the plate temperature at 0°C, control the vacuum degree at 15-30pa, and enter the next section after the product temperature reaches above -1.5°C;

[0137] Step (6) Heating: Raise the temperature of the plate layer to 10°C in 30 minutes, and control the vacuum degree to 10-15 Pa;

[0138] Step (7) secondary sublimation: maintain the plate temperature at 10°C and control the vacuum degree at 5-10 Pa;

[0139] Step (8) endpoint determination: After the vacuum degree is less than 5 Pa, close the front and rear box valves. When the vacuum degree of the front box is less than 5 Pa for 3 minutes, freeze-drying is terminated.

[0140] 2.4 Finished product packaging:

[0141] Step (1) pulverizing freeze-dried powder: After freeze-drying is completed, proteinase K is pulverized and collected into a double-layer sterile bag to obtain freeze-dried fine powder.

[0142] Step (2) subpackaging: After the proteinase K freeze-dried fine powder is weighed and subpacked in the clean room isolator, the recombinant proteinase K finished product is obtained.

[0143] According to the above embodiment, the process is scaled up for production. The process is stable and reliable. The yield results of multiple batches of prepared products are shown in Table 8:

[0144] Table 8 Proteinase K production and purification yields in different preparation batches

[0145] batch Yield (%) Preparation of Batch 1 70.5％ Preparation of Batch 2 73.3％ Preparation of Batch 3 70.5％ Preparation of Batch 4 70.0％ Preparation of Batch 5 70.5％ Preparation of Batch 6 70.5％

[0146] Example 3: Proteinase K activity assay

[0147] 3.1 Experimental Principle: This method uses bovine hemoglobin as a substrate for proteinase K activity detection. Bovine hemoglobin solution is hydrolyzed into peptides and amino acids soluble in trichloroacetic acid under the action of proteinase K. Peptides and amino acids reduce phenol reagents under alkaline conditions to produce blue compounds. The color depth of the compound at a wavelength of 750nm is proportional to the concentration of the enzymatic hydrolysis product.

[0148] Activity unit definition: At 37℃ and pH 7.5, the color produced by the reaction of 1 unit of proteinase K per minute with the product of hydrolysis of urea-denatured bovine hemoglobin and the Folin-phenol reagent is equivalent to the color produced by the reaction of 1.0 μmol of tyrosine with the Folin-phenol reagent.

[0149] 3.2 Main instruments and equipment

[0150] pH meter (Sartorius PB-10)

[0151] Microplate reader (Molecular Device Versamax)

[0152] 3.3 Preparation of reagents and solutions

[0153] Potassium dihydrogen phosphate, bovine hemoglobin, sodium hydroxide, urea, calcium chloride, trichloroacetic acid, phenol reagent (Folin phenol), L-tyrosine, hydrochloric acid

[0154] 3.3.1 Preparation of reagents

[0155] Reagents are prepared using high-purity water (≥18MΩxcm resistivity at 25℃)

[0156] (1) 1 M potassium dihydrogen phosphate solution: Weigh 13.61 g of potassium dihydrogen phosphate and dissolve it in 50 ml of water. Then adjust the pH to 7.5 with 1 M KOH at 37°C and make up to 100 ml.

[0157] (2) Substrate (100 mM potassium dihydrogen phosphate-2.0% (W / V) bovine hemoglobin-6 M urea): Weigh 2.0 g of bovine hemoglobin and dissolve it in 40 ml of water at 37°C and stir for 30 min. Add 8.0 ml of 1 M NaOH and stir at 37°C for 20 min. Then add 36.0 g of urea and stir at 37°C for 60 min. Add 10 ml of 1 M potassium dihydrogen phosphate and adjust the pH to 7.5 with 5 M HCl at 37°C. Make up to 100 ml.

[0158] (3) 20 mM CaCl2 solution: Weigh 0.295 g of calcium chloride and dissolve it in water to make up to 100 ml.

[0159] (4) 305 mM TCA solution (trichloroacetic acid solution): Weigh 24.9 g of trichloroacetic acid and dissolve it in water to make up to 500 ml.

[0160] (5) NaOH solution:

[0161] a. 1M NaOH solution: weigh 20g of sodium hydroxide and dissolve it in water to make up to 500ml.

[0162] b.0.5M NaOH solution: dilute 1M NaOH solution with an equal volume of water.

[0163] (6) Hydrochloric acid solution:

[0164] a. 1M hydrochloric acid: dilute 8.33 ml of hydrochloric acid with water and make up to 100 ml.

[0165] b. 0.5M hydrochloric acid: dilute 1M hydrochloric acid with an equal volume of water.

[0166] 3.3.2 Preparation of standard curve

[0167] Weigh 20 mg of L-tyrosine, heat 80 ml of water to 70-80°C to completely dissolve it, cool to room temperature and dilute to 100 ml with water. It can be stored in a refrigerator at 2-8°C for 6 months.

[0168] The L-tyrosine standard curve was prepared according to the preparation steps in Table 9. The contents of each standard in the final reaction system were 0.0088, 0.0264, 0.044, 0.0616, 0.0792, 0.0968, 0.1144, and 0 μmol.

[0169] Table 9 L-Tyrosine standard curve preparation table

[0170]

[0171] 3.3.3 Proteinase K sample preparation

[0172] a Solid sample: Weigh 0.5g of solid powder, dissolve it in high-purity water and make up to 25ml, then dilute it with CaCl2 solution to an activity range of 0.075-0.175U / ml for measurement.

[0173] bLiquid sample: dilute the protein concentration to 20 mg / ml with high-purity water, and then dilute it with CaCl2 solution to an activity range of 0.075-0.175 U / ml for measurement.

[0174] 3.4 Detection steps

[0175] 3.4.1 Protein concentration determination of proteinase K and Merck proteinase K (BCA method)

[0176] Accurately weigh 200 mg of proteinase K lyophilized powder, add it to 10 ml of high-purity water, shake it to fully dissolve it, then dilute it 40 times with high-purity water and dilute Merck proteinase K to within the standard curve range.

[0177] Take 25 μl of the diluted sample and BSA standard and add them to the ELISA plate. Then add 200 μl of BCA working solution and incubate at 37°C for 30 min. Set the wavelength of the ELISA reader to 562 nm for reading. The concentration determination process is described in “Reference BCAPlus TM The assay was performed using the protocol in the Protein Assay Reagent protol for measurement, prod#1856210, Thermo” kit.

[0178] The concentration of Merck proteinase K was determined by the same method.

[0179] 3.4.2 Proteinase K activity assay

[0180] 3.4.2.1 Add 0.50 ml of substrate to each tube according to the table below.

[0181]

[0182] 3.4.2.2 After equilibration at 37℃ for 10 minutes, add 0.10ml of diluted enzyme solution and add 0.10ml of 20mM CaCl2 solution to the blank control tube.

[0183]

[0184] 3.4.2.3 Vortex to mix, incubate at 37℃ for 10 min, and then add 1.00 ml of 305 mM TCA solution.

[0185]

[0186] 3.4.2.4 Vortex to mix and let stand at room temperature for 20 min.

[0187] 3.4.2.5 Centrifuge at 13000rpm for 1min, remove all supernatant and vortex to mix.

[0188] 3.4.2.6 After centrifugation, take 0.20 ml of supernatant from each tube and add 0.40 ml of 0.5 M sodium hydroxide solution. Cover the tube and vortex to mix.

[0189]

[0190]

[0191] 3.4.2.7 Vortex mix each concentration of L-tyrosine standard and standard blank.

[0192] 3.4.2.8 Take 0.6 mL of the L-tyrosine standard prepared in 3.3.2 and put it into a 2 mL EP tube. Then take 0.12 mL of Folin phenol reagent and add it to the tube and shake to mix. Simultaneously add 0.12 mL of Folin phenol reagent to the sample treated in 3.4.2.6 and shake to mix. React at room temperature for 30 minutes.

[0193] 3.4.2.9 Take 300 μl of the standard and the sample after the reaction and add them to each well of the ELISA plate. Set the wavelength of the ELISA reader to 750 nm for reading.

[0194] Note: If the solution is turbid, filter it through a 0.45 μm filter before reading.

[0195] 3.4.3 Data processing

[0196] 3.4.3.1 Standard curve

[0197] ΔA 750 Standard product = A 750 Standard-A 750 blank

[0198] The linear fitting was performed with tyrosine content (μmol) as the X-axis and average OD value as the Y-axis. The formula is as follows: Y = A + BX, R 2 ≥0.99, the standard curve of the fitting is attached Figure 8 .

[0199] 3.4.4 Result analysis

[0200] 3.4.4.1 The test results of multiple batches are shown in Table 10 below:

[0201] Table 10 Proteinase K activity detection of homemade batches and commercially available controls

[0202]

[0203] 3.4.4.2 Sample calculation formula:

[0204] Sample ΔA 750 =A 750 Sample-A 750 blank

[0205] Tyrosine content (sample) = (sample ΔA 750 -A) / B

[0206] Sample enzyme activity = tyrosine content (sample) × 1.6 × dilution factor / (0.1 × 10 × 0.2)

[0207] 1.6 = Volume to completely stop the reaction (ml)

[0208] 0.2 = Volume of the F&C reagent to stop the reaction (ml)

[0209] df = dilution factor

[0210] 0.10 = enzyme solution added (mL)

[0211] 10 = Experimental incubation time (minutes)

[0212] m = weight of the enzyme to be tested (mg)

[0213] V = dissolved volume of enzyme

[0214] Total activity = sample enzyme activity / (m / V)

[0215] Specific activity = sample enzyme activity / protein content measured by BCA method

[0216] Therefore, the specific activities of the two batches of homemade proteinase K tested by the above method were 40U / mgP and 43U / mgP, respectively, which were comparable to the specific activity of the commercially available Merck proteinase K product (41U / mgP). This detection method is reliable and stable.

Claims

1. An industrial purification and freeze-drying method for recombinant proteinase K, comprising the following steps in sequence: 1) The supernatant of the fermentation broth of the recombinant proteinase K expressed by yeast is clarified, and then an equal volume of diluent is added to dilute and mix; the diluted fermentation supernatant is concentrated using an ultrafiltration membrane package with a cutoff pore size of 5 to 10 kD, and then an equal volume of dialysate is added to perform multiple dialysis to obtain a proteinase K dialysate; 2) adjusting the pH and conductivity of the proteinase K dialyzate, subjecting it to cation exchange chromatography, and eluting it using a pH or salinity gradient to obtain a high-concentration proteinase K eluate; The filler of the cation exchange chromatography is selected from SP Bestarose FF; In the step 2), the chromatography step is: 2a) equilibrating the chromatography column with 5 to 15 column volumes of an equilibration buffer having a composition of 10 to 50 mM Tris, 0 to 5 mM CaCl2, pH 7.5 to 9.0 at a flow rate of 50 to 200 cm / hour; 2b) using the proteinase K dialyzate from step 1) as a sample to be loaded at a flow rate of 50 to 200 cm / hour; wherein the sample conductivity is 0.5 to 2 mS / cm, the pH is 7.5 to 9.0, and the loading capacity is 56 to 90 mg protein / ml filler; 2c) re-equilibrate the column with an equilibration buffer of 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0 at a flow rate of 50-200 cm / hour; 2d) using an elution buffer having a composition of 10-50 mM Tris, 0-5 mM CaCl2, 0-100 mM NaCl, and a pH of 7.5-9.0, to elute the chromatography column at a flow rate of 50-200 cm / hour to finally obtain a proteinase K eluate; The protein recovery of the cationic chromatography step-by-step amplification chromatography is greater than 70%; 3) subjecting the proteinase K eluate to crystallization precipitation at room temperature, and after the precipitation is completed, obtaining a proteinase K precipitation filter cake using a positive pressure filtration device; The step 3) is: 3a) cutting filter paper with a pore size of 2.5 μm to 5 μm, loading it into a positive pressure filter, and washing the positive pressure filter device and the filter paper with an elution buffer having a composition of 10 to 50 mM Tris, 0 to 5 mM CaCl2, 0 to 100 mM NaCl, and a pH of 7.5 to 9.0; 3b) The proteinase K eluate of step 2) is adjusted to a pH of 7.5 to 9.0, a conductivity of 0 to 10 mS / cm, and a protein concentration of 40 to 50 mg / ml, and poured into a stainless steel barrel, and the ambient temperature is controlled at 20 to 25° C., and allowed to stand for more than 4 hours until the proteinase K crystals are completely precipitated; 3c) Pour the completely precipitated proteinase K into the positive pressure filter tank in batches, connect the compressed air to the filter press tank, control the pressure gauge of the filter press tank to 0-0.15MPa to start filter pressing, and finally obtain the proteinase K filter cake; The protein recovery in the proteinase K filter cake is greater than 90%; 4) adding the proteinase K precipitate filter cake to a reconstitution buffer for reconstitution, and lyophilizing to finally obtain a proteinase K lyophilized powder product; The ultrafiltration membrane package is selected from Millipore 5kD, 10kD, Sartorius 5kD, 10kD; the reconstitution buffer in step 4) is 10-50mM Tris, 0-5mM CaCl2, pH 7.5-9.0 or 10-50mM NaAc, 0-5mM CaCl2, pH 4.5-5.5; The specific steps of step 1) are: 5a) the supernatant of the recombinant proteinase K fermentation broth is clarified, the pH and conductivity of the clarified supernatant are measured, and then an equal volume of diluent is added to dilute and mix; the diluent is: 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0; 5b) installing the ultrafiltration membrane package on the ultrafiltration system, controlling the inlet pressure and the reflux pressure, adjusting the transmembrane pressure and measuring the water flux, and performing ultrafiltration concentration after the membrane package is installed; the ultrafiltration membrane package has a retention pore size of 5 to 10 kD, and the transmembrane pressure is 1 to 1.25 bar or 14.5 to 18 psi or 0.1 to 0.125 MPa; 5c) adding an equal volume of dialysate to the concentrate concentrated to the endpoint and dialyzing the concentrate for multiple times until the dialysis endpoint is reached; the dialysate comprises: 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0; the effluent at the dialysis endpoint is colorless, has a conductivity of 0.5-2 mS / cm, and a pH of 7.5-9.

0.

2. The method according to claim 1, characterized in that The specific activity of the recombinant proteinase K obtained by the method is 30-40 U / mgP.

3. The method according to claim 1, characterized in that The step 4) is: 6a) adding 10-50 mM Tris, 0-5 mM CaCl2, pH 7.5-9.0 or 10-50 mM NaAc, 0-5 mM CaCl2, pH 4.5-5.5 to the filter cake containing recombinant proteinase K obtained in step 3) to redissolve, adding mannitol at a ratio of W / V=1:20 and mixing evenly to obtain a mixed solution; 6b) pouring the mixture into a freeze-drying dish, setting the pre-freezing temperature to -40 to -45°C, cooling time to 30 minutes, and maintaining for 3 to 6 hours; 6c) Set the temperature of the first stage of primary drying to -35 to -40°C, the heating time to 30 minutes, maintained for 1 to 2 hours, and the vacuum degree to 10 to 20 Pa; the temperature of the second stage is 0 to 10°C, the heating time to 4 to 6 hours, maintained for 40 to 45 hours.

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