CN116359315A

CN116359315A - Method for evaluating biological extraction enzyme product section product and application thereof

Info

Publication number: CN116359315A
Application number: CN202310360960.8A
Authority: CN
Inventors: 胡懿俊; 汪晨曦; 朱俊; 余奕超; 徐晴晔
Original assignee: Sph No1 Biochemical & Pharmaceutical Co ltd
Current assignee: Sph No1 Biochemical & Pharmaceutical Co ltd
Priority date: 2023-04-06
Filing date: 2023-04-06
Publication date: 2023-06-30

Abstract

The invention discloses a method for evaluating a biological extraction enzyme product section product and application thereof. The method comprises the following steps: (1) Taking a product of a certain section in the biological enzyme product extraction process as a sample to be evaluated; (2) Diluting, dialyzing and freeze-drying the sample to be evaluated, preparing a solution to be tested, and detecting by a polyacrylamide gel electrophoresis method to obtain the content of the target protein to be tested; (3) Preparing a sample to be evaluated into a solution to be tested, and detecting by a BCA method to obtain the total protein mass; (4) Multiplying the content of the target protein to be detected by the total protein mass to obtain the mass of the target protein to be detected; (5) repeating steps (1) to (4) at least 1 time; the sequence of the step (2) and the step (3) is not limited. The method can evaluate the loss of the target protein to be detected in different working sections or different batches of products in the same working section in the biological enzyme product extraction process, and effectively monitor the process.

Description

Method for evaluating biological extraction enzyme product section product and application thereof

Technical Field

The invention relates to a method for evaluating a biological extraction enzyme product section product and application thereof.

Background

The biological extraction is an important technical method in the industrial production of enzyme products, and has the advantages of high yield, low cost and the like. In chemical pharmacy, biological extraction methods are widely used for producing enzyme product raw materials such as trypsin, chymotrypsin, hyaluronidase, thrombin and the like. The starting materials of the method are often biological tissues, zymogen and the like, and the purity and quality of the starting materials can influence the technological indexes such as the product yield and quality of enzyme products. Meanwhile, the product components in each working section are complex, so that qualitative and quantitative analysis cannot be effectively carried out by commonly adopted character judgment methods and the like. The whole extraction process is in an unstable and uncontrollable state, the stability of the production process is affected, and the process optimization and improvement are also performed.

Therefore, how to evaluate the content of the target protein to be detected in the section sample by a detection method which is easy to operate and low in cost, and to strengthen the monitoring of the section product of the biological extraction enzyme product, is a current problem to be solved.

Disclosure of Invention

The invention aims to overcome the defects that the components of enzyme product working section products are complex, the extraction process is uncontrollable, and the process cannot be effectively monitored and the quality of enzyme products can not be effectively controlled in the prior art, and provides a method for evaluating the enzyme product working section products by biological extraction and application thereof. The testing method of the invention combines gel electrophoresis and BCA testing method to detect the enzyme product working section products, can obtain working section product detection data qualitatively and quantitatively respectively, and evaluates the loss of target proteins to be tested in the products of different working sections in the biological extraction enzyme product technology or the loss of target proteins to be tested in the products of different batches in the same working section, thereby effectively monitoring the technological process.

The invention solves the technical problems by the following technical scheme:

the invention provides a method for evaluating the products of a biological extraction enzyme product section, which comprises the following steps:

(1) Taking a product of a certain section in the biological enzyme product extraction process as a sample to be evaluated;

(2) Diluting, dialyzing and freeze-drying the sample to be evaluated, preparing a solution to be tested, and then detecting by a polyacrylamide gel electrophoresis method according to the type of the target protein to be tested to obtain the content of the target protein to be tested in the section sample;

(3) Preparing the sample to be evaluated into a solution to be tested, and detecting by a BCA method to obtain the total protein mass in the section sample;

(4) Multiplying the content of the target protein to be detected by the total protein mass to obtain the mass of the target protein to be detected in the product of a certain section;

(5) Repeating the steps (1) - (4) at least 1 time to obtain the quality of the target protein to be detected in the products of different working sections in the biological extraction enzyme product process, thereby comparing the loss of the target protein to be detected in the products of different working sections;

or, obtaining the quality of the target protein to be detected in different batches of products in the same working section, so as to compare and obtain the loss of the target protein to be detected in different batches of products in the same working section;

the sequence of the step (2) and the step (3) is not limited.

In the present invention, the enzyme preparation is preferably one or more of trypsin, chymotrypsin, thrombin, hyaluronidase and corticotropin, more preferably trypsin or chymotrypsin. In the invention, the sample to be evaluated can be a working section product, working section waste liquid or working section waste solid.

In the present invention, when the sample to be evaluated is a section product, the section product may be in the form of a solid, a damp solid, a liquid or a suspension.

In the present invention, when the sample to be evaluated is a solid, the mass of the sample is generally 0.1 to 1.0g, for example, 0.5g.

The solids generally refer to solids having a moisture content of less than 20% by weight of water as a percentage of the total mass of the wet solids.

In the present invention, when the sample to be evaluated is a wet solid, the mass of the sample is generally 0.2 to 10g, for example, 0.5g or 5g.

The wet solids generally refer to solids having a moisture content of 20% to 50%, the% being the mass of water as a percentage of the total mass of the wet solids.

In the present invention, when the sample to be evaluated is a liquid, the volume of the sample is generally 1 to 50mL, for example, 2mL or 20mL.

The liquid generally refers to a liquid having a solids content of less than 20%.

In the present invention, when the sample to be evaluated is a suspension, the volume of the sample is generally 10 to 50mL, for example, 20mL.

The suspension generally refers to a liquid having a solids content of greater than 20%.

In the invention, when the BCA method is used for detection, the total mass or total volume of the sample to be evaluated needs to be accurately recorded in the sampling process.

In the step (2), the operations and conditions of dilution, dialysis and freeze-drying may be adjusted conventionally according to the kind of the target protein.

In step (2), the solvent used in the dilution is preferably water.

In the dilution step (2), when the sample to be evaluated is a wet solid, the mass of the sample to be evaluated is preferably 0.03 to 0.07g/mL, for example, 0.05g/mL, relative to the total volume of the diluted solution.

In the step (2), when the sample to be evaluated is a liquid, the ratio of the volume of the sample to be evaluated to the total volume of the diluted solution is preferably 0.1 to 0.3, for example, 0.2.

In step (2), during the dialysis, the sample is typically packaged in a dialysis bag.

In step (2), the dialysis fluid is preferably water, more preferably purified water.

In step (2), during the dialysis, the state of the dialysate is preferably a flowing state or a stationary non-flowing state.

Preferably, when the state of the dialysate is a flowing state, the flow rate of the dialysate is 300 to 500mL/h, for example 400mL/h. Preferably, when the state of the dialysate is a stationary non-flowing state, the number of times of substitution of the dialysate is 3 to 5, and the dialysate is substituted every 10 to 14 hours.

In step (2), the dialysis time is preferably 24 to 48 hours, for example 24 hours.

In step (2), the time of the freeze-drying is preferably 12 to 24 hours, for example 18 hours.

In step (2), the temperature of the freeze-drying is preferably-50 to-10 ℃, for example-20 ℃, -30 ℃ or-37 ℃.

In the invention, hydrochloric acid or sodium hydroxide can be added for auxiliary dissolution when the working section sample is not easy to dissolve in the process of preparing the solution to be tested.

In the step (2) and the step (3), the solvent used in the preparation process of the solution to be tested is preferably phosphate buffer solution.

Wherein the concentration of the phosphate buffer is preferably 0.08 to 0.12mol/L, for example 0.1mol/L.

Wherein the pH of the phosphate buffer solution is preferably 6.9-7.1, e.g. 7.0.

In step (2), the concentration of the solution to be tested in the detection by the polypropylene gel electrophoresis method may be conventional in the art, preferably 0.5-10mg/mL, for example 1mg/mL, 2mg/mL, 5mg/mL or 8mg/mL.

In the step (2), the polypropylene gel electrophoresis is preferably a non-reducing polypropylene gel electrophoresis.

In step (2), the polypropylene gel electrophoresis method can be performed as usual in the art. Preferably, the operation steps of the polypropylene gel electrophoresis method comprise gel preparation, electrophoresis, dyeing and decoloring.

Wherein, the concentration of the prepared gel can be adjusted conventionally according to the kind of the target protein, preferably 8-20%, such as 10%, 15% or 18%.

Wherein, during the electrophoresis, the loading amount of the solution to be tested is preferably 10-100 mug, for example 20 mug.

Wherein, before the electrophoresis, a non-reducing loading buffer solution is preferably added into the solution to be tested.

Wherein, prior to said electrophoresis, said test solution is preferably denatured by boiling at 95 ℃.

Preferably, the test solution is denatured by boiling at 95 ℃ after adding the non-reducing loading buffer and before the electrophoresis.

Wherein, in the dyeing and decoloring process, a coomassie brilliant blue dyeing method is preferably adopted.

Wherein, in the dyeing and decoloring process, the reaction condition is preferably that a shaker is used for dyeing for 1h at 37 ℃ and 180rpm, and decoloring is carried out for 2h by using a decoloring solution.

In the BCA method, when the sample to be evaluated is a wet solid, the ratio of the mass of the sample to be evaluated to the volume of the sample solution in the sample solution is preferably 3 to 7mg/mL, for example 5mg/mL.

In the BCA method in step (3), when the sample to be evaluated is a liquid, the ratio of the volume of the sample to be evaluated to the total volume of the solution to be measured is preferably 0.01 to 0.03, for example 0.02.

In the step (3), the operation steps of the BCA method may be performed as usual in the art.

Wherein, the BCA assay is performed generally following BCA kit operating specifications with 0.1M phosphate buffer (ph=7.0) as the assay background.

In step (3), the BCA method generally employs a kit conventional in the art.

In the step (3), the BCA method is generally performed by using an enzyme-labeled instrument.

In a preferred embodiment, when the enzyme preparation is trypsin, the conditions for dialysis are as follows: the dialysis time is 20-30 h, the dialyzate of the dialysis is purified water in a flowing state, and the flow rate of the dialyzate is 350-450 mL/h; the freeze drying is carried out for 12-24 hours at the temperature of minus 50-minus 10 ℃; the concentration of the solution to be measured is 0.5-2 mg/mL; the polypropylene gel electrophoresis method comprises gel preparation, electrophoresis, dyeing and decoloring, wherein the concentration of the prepared gel is 10-18% in the gel preparation process; before the electrophoresis, adding a non-reducing loading buffer solution into the solution to be detected, and boiling and denaturing at 95 ℃; in the dyeing and decoloring process, a coomassie brilliant blue dyeing method is adopted, a shaking table is used for dyeing for 1h at 37 ℃ and 180rpm, and decoloring liquid is adopted for decoloring for 2h.

In a preferred embodiment, when the enzyme preparation is chymotrypsin, the conditions for the dialysis are as follows: the dialysis time is 20-30 h, the dialyzate of the dialysis is purified water in a flowing state, and the flow rate of the dialyzate is 350-450 mL/h; the freeze drying is carried out for 12-24 hours at the temperature of minus 50-minus 10 ℃; the concentration of the solution to be measured is 0.5-2 mg/mL; the polypropylene gel electrophoresis method comprises gel preparation, electrophoresis, dyeing and decoloring, wherein the concentration of the prepared gel is 10-18% in the gel preparation process; before the electrophoresis, adding a non-reducing loading buffer solution into the solution to be detected, and boiling and denaturing at 95 ℃; in the dyeing and decoloring process, a coomassie brilliant blue dyeing method is adopted, a shaking table is used for dyeing for 1h at 37 ℃ and 180rpm, and decoloring liquid is adopted for decoloring for 2h.

The invention also provides application of the method for evaluating the products of the biological extraction enzyme product section in enzyme product quality control.

The invention applies the method for detecting the content of the target product in the product of the enzyme product section in the quality control of the enzyme product, and the analysis of the protein content of the product is carried out by analyzing the component of the product, so that the product changes along with the process steps, the qualitative and quantitative data of the process product makes a change curve, and the analysis process flow is pushed along with the process steps.

The invention uses gel electrophoresis and BCA quantitative test to identify the originally unknown intermediate product in the biological extraction process to obtain the physicochemical parameters, and then uses data processing software to form the biological extraction enzyme product process flow evaluation method with qualitative and quantitative characteristics.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

(1) The invention provides a process product detection method for the middle process of the biological enzyme product extraction process flow, is applied to the whole process flow analysis, is beneficial to analyzing production deviation, understanding the process operation purpose, searching the process optimization direction and providing theoretical basis and production data support for process improvement.

(2) The gel electrophoresis method and the BCA method adopted by the invention are mature biological methods widely applied to protein characterization, have low cost, do not need expensive instruments, are simple to operate and have no complex technical threshold, can be synchronously carried out during industrial production, and feed back data results in real time;

(3) The testing method can obtain the product detection data of the working section qualitatively and quantitatively respectively, evaluate the loss of the target protein to be tested in the products of different working sections in the biological enzyme product extraction process or the loss of the target protein to be tested in the products of different batches in the same working section, and effectively monitor the process.

Drawings

FIG. 1 is a chart of gel electrophoresis gray scale analysis of the products of section 1-5 of example 1.

FIG. 2 is a plot of the ratio of the gray scale values of section 1-5 trypsin in example 1 versus the percent change in section product.

FIG. 3 is a graph showing the protein content of section 1-5 versus the product variation content of section in example 1.

FIG. 4 is a graph showing the variation of trypsin content of sections 1-5 versus the product content of sections in example 1.

FIG. 5 shows the gray scale of the product and waste liquid gel electrophoresis of sections 1-3 of example 1.

FIG. 6 is a graph of the change in section 1-5 chymotrypsin content versus section product content for example 2.

FIG. 7 is a schematic flow diagram of trypsin process in example 1, corresponding to sections 1-5.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention.

The gel electrophoresis preparation kit (C631100-0200) and the BCA protein quantitative kit (C503021-0500) used in the following examples are purchased from Shanghai, and the raw material reagents used for preparing the solution are purchased from national medicine reagents. All purchased reagents were used directly after purchase without further purification, unless otherwise indicated. The trypsin station product used in the examples was taken from the station product of the trypsin biological extraction enzyme preparation process, as shown in fig. 7; the chymotrypsin stage products used in the examples were taken from the products of stages in the chymotrypsin bio-extraction enzyme preparation process.

Example 1

Detection of trypsin section product by gel electrophoresis

Gel electrophoresis detection method for trypsin section product as liquid

After the trypsin extraction process dialyzes the working section 5, the working section product is obtained as liquid. 20mL of the sample was sampled, diluted with water to 100mL, packed in a dialysis bag, and dialyzed against flowing purified water (flow rate 400 mL/h) for 24h. After the sample was freeze-dried at-37℃for 18 hours, an intermediate was obtained and accurately weighed to obtain 100mg, and 0.1M phosphate buffer (pH 7.0) was added to dissolve to 1mg/mL to prepare a sample to be measured.

Gel with 15% concentration was prepared according to the kit instructions and assembled into a gel electrophoresis apparatus. And adding a non-reducing loading buffer solution into the sample to be tested, boiling and denaturing at 95 ℃, and performing gel electrophoresis analysis, wherein the loading amount is 20 mug. The obtained adhesive tape is dyed by coomassie brilliant blue, the dyeing is carried out for 1h at the temperature of 37 ℃ by 180rpm of a shaker, and the decolorization is carried out for 2h by a decolorization solution. After completion, the adhesive tape was photographed under a white background, bright white light, and the pictures were processed into gray scale using ImageJ software.

Gel electrophoresis detection method for product of trypsin section as damp solid

After the trypsin extraction process crystallizes the section 4, the section product is obtained as a damp solid. After the product was prepared, a quick sample was taken and the wet solid product mass was 5g, and the sample was dissolved with water to 100mL. After the packaging of the dialysis bag, it was dialyzed with flowing purified water (flow rate 400 mL/h) for 24 hours. After the sample was freeze-dried at-37℃for 18 hours, an intermediate was obtained and accurately weighed to obtain 1g, and the sample was taken and dissolved in 0.1M phosphate buffer (1 mg/mL) to obtain a sample to be measured.

The gel electrophoresis operation and ImageJ image processing were the same as the procedure in which the process product was solid.

Gel electrophoresis detection method for products of different sections of trypsin in same batch

After obtaining the target section product samples, the target section product samples are dissolved to 100mL by water respectively, and the target section product samples are dialyzed for 24h by flowing purified water (the flow rate is 400 mL/h) after being packaged by a dialysis bag. After the sample was freeze-dried at-37℃for 18 hours, it was dissolved in 0.1M phosphate buffer to prepare a sample to be measured at the same concentration (1 mg/mL).

Gel electrophoresis detection method for products in same section of trypsin in different batches

After obtaining the target section product sample, the sample is dissolved to 100mL by water, and the sample is dialyzed for 24h by flowing purified water (flow rate 400 mL/h) after being packaged by a dialysis bag. The samples were freeze-dried at-37℃for 18h and stored at-20 ℃. After the freeze-dried products of all target section product samples are obtained, the products are dissolved by using 0.1M phosphate buffer solution to prepare samples to be measured with the same concentration (1 mg/mL).

The gel electrophoresis loading amounts were kept consistent for each set of samples, and other procedures and ImageJ image processing were the same as for the procedure in which the process product was solid.

Gel electrophoresis detection method for trypsin section waste liquid

After trypsin extraction crystallization section 4, the filtrate is produced as waste liquid. 20mL of the sample was sampled, diluted with water to 100mL, packed in a dialysis bag, and dialyzed against flowing purified water (flow rate 400 mL/h) for 24h. After the sample was freeze-dried at-37℃for 18 hours, a solid sample was obtained and accurately weighed to obtain 0.5g, and the sample was taken and dissolved in 0.1M phosphate buffer (1 mg/mL) to prepare a sample to be measured.

The gel electrophoresis operation and the image J image processing section product are solid operation steps are the same.

Trypsin section product BCA quantitative test method

BCA quantitative test method for detecting trypsin section product as damp solid

After the trypsin extraction process crystallizes the section 4, the section product is obtained as a damp solid. After the preparation of the product, 500mg (a) was rapidly sampled, dissolved in 0.1M phosphate buffer to prepare a solution (100 mL) as a sample to be tested, and the total mass of the wet solid product of this section was found to be 5000g (b).

Trypsin was used as BCA standard curve

y＝0.5237x+0.1481

Wherein x is the concentration (mg/mL) of the sample to be detected, and y is the ultraviolet absorbance.

The results of the data were measured using an enzyme-labeled instrument and an absorbance-protein concentration curve was made with Excel. The absorbance at 562nm is measured according to the operation of BCA kit instruction, and the absorbance is brought into a standard curve to obtain the corresponding protein concentration of 2mg/mL (c).

The amount of protein in the tidal solid product was calculated by the following equation for the corresponding protein concentration c and the corresponding sample configuration concentration (a/100 mL).

Section product protein mass= (protein concentration/deployment concentration) section product mass

The calculated amount of protein in the wet solids of the section product was 2000g.

BCA quantitative test method for detecting trypsin section product as liquid

After the trypsin extraction process dialyzes the working section 5, the working section product is obtained as liquid. After the product was prepared, 2mL (a) was sampled, dissolved with 0.1M phosphate buffer to prepare a solution (100 mL) as a sample to be tested, and the total volume of the liquid product of this section was measured to be 10L (b).

And (3) obtaining the protein concentration of the sample to be detected to be 1mg/mL according to the experimental step in the detection of the BCA quantitative test method with the trypsin section product being a damp solid, and calculating the protein content in the liquid product according to the following formula.

Liquid product protein content = protein concentration (100 mL/sample volume) the liquid product volume was calculated to give a protein mass in the process product liquid of 500g.

BCA quantitative test method for trypsin section waste liquid

After trypsin extraction crystallization section 4, the filtrate is produced as waste liquid. 2mL (a) was sampled, dissolved in 0.1M phosphate buffer solution to prepare a solution (100 mL) as a sample to be measured, and the section waste liquid volume was measured to be 10L (b).

And (3) obtaining the concentration of the protein corresponding to the sample to be detected by the experimental step in the detection of the BCA quantitative test method with the trypsin section product as a damp solid, and calculating the protein quantity in the waste liquid according to the following formula.

Waste liquid protein content = protein concentration (100 mL/sample volume) liquid product volume

The protein content in the waste liquid is 1000g.

Analytical extraction process flow based on gel electrophoresis and BCA quantitative method

Gel patterns of products of different working sections of the same batch of trypsin are obtained according to the gel electrophoresis detection operation of the products of different working sections of the same batch of trypsin, as shown in figure 1, and lane numbers represent the working sections of the different products. Gel grayscale pictures were analyzed using ImageJ software, corresponding to a trypsin target band at a molecular weight of 25 KDa. Gray analysis was performed on the same lane, and the proportion of the weight content of trypsin in the corresponding sample was represented by the proportion of the gray value of the band. The pancreatic protein gray scale ratio-percent change in the process product is plotted as shown in fig. 2.

And (3) obtaining the protein content of the corresponding section product in the detection operation according to the gel electrophoresis method of the trypsin same batch of different section products, and making a protein content-section product change content curve, as shown in figure 3.

Trypsin content was estimated using the following:

trypsin content = protein content gray scale fraction.

Example 2

After a certain step in the chymotrypsin extraction process, the procedure of example 1 is followed.

Effect example 1

FIG. 1 is a chart of gel electrophoresis gray scale analysis of the products of the multiple sections of example 1. It can be seen from fig. 1 that the components of the sample system to be tested are relatively simple.

FIG. 2 is a graph of trypsin gray scale ratio versus percent change in section product for example 1. As can be seen from FIG. 2, the trypsin content ratio in the sample gradually increases along with the progress of the working section, and the purity of the trypsin product gradually increases, which indicates that the purification process achieves the expected purpose.

FIG. 3 is a graph showing the protein content versus the variation content of the section product in example 1. As can be seen from fig. 3, the protein content drops drastically after section 1 product, indicating that the protein is largely removed at this step.

FIG. 4 is a graph showing the variation of trypsin content versus section product content. As can be seen from fig. 4, the trypsin content drops sharply from 2223g to 1021g after section 3 product, indicating a large loss in this process step.

By analyzing the data and combining the trypsin process flow diagram of FIG. 7, the system can be seen that the system is largely removed from the mixed protein after the working section product 1 is obtained by the salting-out working section; after the section product 3 is obtained through the crystallization section, the target trypsin of the system is largely lost. Further analysis of the process found that the crystallization section used dialysis bags, which were tested for a molecular weight cut-off of about 40kDa, greater than 23kDa of trypsin, had the potential to leak trypsin during dialysis.

Effect example 2

Taking the process step with larger loss in effect example 1 as an example, in order to further study the loss condition of the process step, the starting material of the working section and the generated waste liquid or waste solid and the like are sampled, and the product of the crystallization working section and the gel electrophoresis gray level chart of the waste liquid are obtained according to the operation of example 1, and are shown in figure 5.

As can be seen from FIG. 5, the clear trypsin band was seen in the sample at 25kDa in waste liquid lane 3, with a gray scale fraction of 30.5%, while the BCA test showed a total protein content of 1560g in the waste liquid and a trypsin content of 476g in the waste liquid was estimated as in example 1. The gray scale fraction in waste solids lane 2 was 95.6%, while the BCA test showed 680g total protein content in the waste solids, which was estimated to be 650g trypsin content in the waste solids as in example 1. Whereas lane 1 of the crystallization section product had an initial trypsin content of 2175g, the loss of trypsin was 51.8%. Therefore, it is judged that a large amount of products remain in the waste liquid and the waste solid during the crystallization process. Combining effects the crystallization section uses dialysis bags with too high a molecular weight cut-off as described in example 1, but trypsin is also found in the waste liquid, waste solids, further indicating that in the crystallization section there is leakage of trypsin, which process is worth further optimization improvement.

Effect example 3

For trypsin preparation process, collecting samples of products of each working section in the preparation process, and measuring relevant data of the products of each working section by using the method disclosed by the application, wherein the relevant data are shown in tables 1-3.

TABLE 1 Gray scale fraction of trypsin in the products of each section in trypsin preparation Process

As shown in Table 1, in the trypsin production process, the RSD of the gray scale fraction of trypsin in each section product is less than or equal to 2.33%, which indicates that the repeatability of the trypsin production process measured by using the polyacrylamide gel electrophoresis method is good.

TABLE 2 protein content in the products of each section in trypsin preparation Process

As is clear from Table 2, the RSD of the protein content in each section product of trypsin is less than or equal to 2.11%, which shows that the reproducibility of the protein content in the same section in the trypsin preparation process is good by using the BCA method.

TABLE 3 trypsin content in the products of each section in trypsin preparation Process

As shown in Table 3, the RSD of the trypsin content in the products of each section in the trypsin preparation process is less than or equal to 2.75% by using the polypropylene gel electrophoresis method and the BCA method, which proves that the repeatability of the testing method is better.

Effect example 4

Example 2 chymotrypsin content change data are summarized in tables 4-6, and chymotrypsin content-section product content change curves are shown in fig. 6.

TABLE 4 Gray scale fraction of chymotrypsin in products of each section in chymotrypsin preparation Process

As shown in Table 4, in the chymotrypsin production process, the RSD of the trypsin gray scale fraction in the products of each section is less than or equal to 2.20%, which indicates that the reproducibility of the chymotrypsin production process measured by using the polyacrylamide gel electrophoresis method is better.

TABLE 5 protein content in the products of each section in chymotrypsin preparation Process

As is clear from Table 5, the RSD of the protein content in each section of chymotrypsin is less than or equal to 4.27%, which indicates that the reproducibility of the protein content in the same section in the chymotrypsin preparation process is good by using the BCA method.

TABLE 6 chymotrypsin content in products of each section in chymotrypsin preparation Process

As shown in Table 6, the RSD of the trypsin content in each section product in the trypsin preparation process is less than or equal to 3.29% by using the polypropylene gel electrophoresis method and the BCA method, which shows that the reproducibility of the test method is better.

It can be seen from FIG. 6 that the loss of chymotrypsin at each step was maintained at a uniform level as the process was advanced, indicating that the process was stable and that no abnormal production of the batch was occurring.

Effect example 5

To further illustrate the application of this method in trypsin process flow, a summary of data obtained for trypsin section 5 between different batches of product is shown in table 7.

TABLE 7 protein content in different batches of trypsin Process at the same stage

As shown in Table 7, in the trypsin preparation process by combining the polypropylene gel electrophoresis method and the BCA method, the RSD of the trypsin content of different batches in the same process is less than or equal to 1.75%, which indicates that the trypsin process flow has high stability and good repeatability.

Effect example 6

To further illustrate the application of this method in chymotrypsin process flows, summary of data obtained for different batches of product for chymotrypsin section 1 is shown in table 8.

TABLE 8 protein content in different batches of chymotrypsin Process at the same stage

As can be seen from Table 8, in the chymotrypsin preparation process by combining the polypropylene gel electrophoresis method and the BCA method, the RSD of the chymotrypsin content between different batches at the same section is less than or equal to 1.86%, which indicates that the chymotrypsin process flow has high stability and good repeatability.

Effect example 7

Referring to patent CN 111220720A, the variation of trypsin content in sections 1-5 was calculated by standard curve method using HPLC. The trypsin content measured by HPLC method is compared with the trypsin content measured by the method of the present application as shown in Table 9.

TABLE 9 comparison of the method with HPLC method

As can be seen from Table 9, the method has no significant difference from the HPLC method, which shows that the method has higher accuracy in determining the target product content of the working section product.

The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.

Claims

1. A method for evaluating the production of a biological extraction enzyme preparation process, characterized in that it comprises the following steps:

the sequence of the step (2) and the step (3) is not limited.

2. A method for evaluating the production of a biological extraction enzyme preparation process according to claim 1, wherein the enzyme preparation is one or more of trypsin, chymotrypsin, thrombin, hyaluronidase and corticotropin, preferably trypsin or chymotrypsin;

and/or the sample to be evaluated is a working section product, working section waste liquid or working section waste solid;

wherein when the sample to be evaluated is a process product, the process product is preferably in the form of a solid, a damp solid, a liquid or a suspension.

3. The method for evaluating a biological extraction enzyme preparation process product according to claim 1, wherein the solvent used in the dilution is water;

and/or, in the course of the dilution, when the sample to be evaluated is a damp solid, the ratio of the mass of the sample to be evaluated to the total volume of the diluted solution is 0.03 to 0.07g/mL, for example 0.05g/mL; when the sample to be evaluated is a liquid, the ratio of the volume of the sample to be evaluated to the total volume of the diluted solution is 0.1 to 0.3, for example, 0.2.

4. The method for evaluating a biological extraction enzyme preparation process product according to claim 1, wherein in step (2), the dialysis meets one or more of the following conditions:

(a) The dialysis time is 24-48 hours, for example 24 hours;

(b) The dialysis fluid is water, preferably purified water;

(c) During the dialysis, the state of the dialysate is a flowing state or a standing non-flowing state;

preferably, when the state of the dialysate is a flowing state, the flow rate of the dialysate is 300-500 mL/h, for example 400mL/h;

preferably, when the state of the dialysate is a stationary non-flowing state, the number of times of substitution of the dialysate is 3 to 5, and the dialysate is substituted every 10 to 14 hours.

5. A method for evaluating a biological extraction enzyme preparation process product according to claim 1, wherein in step (2) the freeze-drying time is 12-24 hours, such as 18 hours;

and/or in step (2), the freeze-drying temperature is-50 to-10 ℃, e.g. -20 ℃, -30 ℃ or-37 ℃.

6. The method for evaluating a biological extraction enzyme preparation process product according to claim 1, wherein in the step (2) and the step (3), the solvent used in the preparation process of the solution to be tested is phosphate buffer;

wherein the concentration of the phosphate buffer is preferably 0.08 to 0.12mol/L, for example 0.1mol/L;

7. The method for evaluating a biological extraction enzyme preparation process product according to claim 1, wherein the concentration of the solution to be tested is 0.5-10mg/mL, such as 1mg/mL, 2mg/mL, 5mg/mL or 8mg/mL, when the polypropylene gel electrophoresis detection is performed;

and/or, the polypropylene gel electrophoresis is a non-reducing polypropylene gel electrophoresis.

8. The method for evaluating the products of the biological extraction enzyme preparation process according to claim 1, wherein the operation steps of the polypropylene gel electrophoresis method comprise gel preparation, electrophoresis, staining and decoloring;

wherein the concentration of the formulated gel is preferably 8-20%, such as 10%, 15% or 18%, during the gel preparation;

wherein, during the electrophoresis, the loading amount of the solution to be tested is preferably 10-100 mug, for example 20 mug;

wherein, before the electrophoresis, a non-reducing loading buffer solution is preferably added into the solution to be detected;

wherein, prior to said electrophoresis, said test solution is preferably denatured by boiling at 95 ℃;

wherein, in the dyeing and decoloring process, a coomassie brilliant blue dyeing method is preferably adopted;

9. A method for evaluating a biological extraction enzyme preparation process product according to claim 1,

the BCA method adopts an enzyme-labeled instrument for detection;

and/or, when the sample to be evaluated is a wet solid, the ratio of the mass of the sample to be evaluated to the volume of the solution to be evaluated in the solution to be measured is 3 to 7mg/mL, for example 5mg/mL, when the BCA method is performed;

or, when the sample to be evaluated is a liquid, the ratio of the volume of the sample to be evaluated to the total volume of the solution to be measured is 0.01 to 0.03, for example, 0.02.

10. Use of a method according to any one of claims 1 to 9 for evaluating the quality control of an enzyme preparation in a biological extraction of the product of an enzyme preparation section.