Method for purifying recombinant gE protein

Abstract

The invention relates to the technical field of biological medicine, and discloses a purification method of recombinant gE protein. According to the method, the recombinant gE protein expressed by CHO cells is purified through composite anion exchange chromatography, hydroxyapatite chromatography, affinity chromatography and gel filtration chromatography in sequence. Researches show that after four-step chromatographic purification, the electrophoresis purity and the liquid chromatography purity of the finally obtained gE protein can reach 95% or above, and various impurities can be well removed. The purification method provided by the invention is efficient, economical and reliable, and can meet the requirements of biopharmaceutical production.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a method for purifying recombinant gE protein. Background Art

[0002] The gE protein (glycoprotein E) is an important membrane protein on the surface of various viruses (such as members of the Herpesviridae family), and plays a key role in virus infection, immune escape and host interaction. Vaccines based on recombinant gE protein are mainly used to prevent diseases caused by specific pathogens. Through recombinant DNA technology, scientists can insert the gene encoding the gE protein into an expression system (such as bacteria, yeast or mammalian cells), so as to mass-produce the gE protein for vaccine development.

[0003] The purification of recombinant gE protein is a key step in vaccine production, aiming to isolate high-purity and high-activity gE protein from a complex expression system. Chromatography technology is widely used in protein purification due to its high separation accuracy and simple operation. Among them, ion exchange chromatography can separate according to the difference in the surface charge of proteins, affinity chromatography uses the specific binding of affinity ligands to target proteins to achieve efficient purification, and gel filtration chromatography can remove protein aggregates and small molecule impurities. However, a single chromatography method is difficult to meet the high-purity requirements, and a combination of multiple chromatography technologies is required.

[0004] In addition, there are differences in the physical and chemical properties of different proteins, and the purification process needs to be optimized specifically to ensure the activity and purity of the target protein. Therefore, establishing an efficient, economical and reliable recombinant protein purification process is crucial for the development of the biomedical industry. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the present invention creatively combines Ni affinity chromatography with composite anion exchange chromatography, hydroxyapatite (CHT) chromatography and gel filtration chromatography, overcoming the problem of diffuse bands after CHT chromatography purification. And various parameter indicators of Ni affinity chromatography are optimized, and finally an efficient, economical and reliable method for purifying recombinant gE protein is obtained.

[0006] The present invention first provides a method for purifying recombinant gE protein, which successively uses composite anion exchange chromatography, hydroxyapatite chromatography, affinity chromatography and gel filtration chromatography to purify CHO cell cultures containing gE protein; the affinity chromatography is Ni affinity chromatography.

[0007] In a specific embodiment, the medium for Ni affinity chromatography is any one of NiBestarose FF, Ni Smart Beads 6FF, Polar MC60-Ni Excel, and Agarosix MC90-Ni Excel, preferably Agarosix MC90-Ni Excel.

[0008] In a specific embodiment, the NaCl concentration in the Ni affinity chromatography equilibration buffer is 0 - 300 mM, preferably 0 - 100 mM, more preferably 0 mM, 50 mM, 100 mM, and most preferably 50 mM.

[0009] In a preferred embodiment, the equilibration buffer component further includes imidazole, and its concentration is 0 - 4 mM, preferably 2 mM imidazole.

[0010] In a more preferred embodiment, the equilibration buffer components are 40 - 60 mM PB, 0 - 100 mM NaCl, 0 - 4 mM imidazole, pH 7.5 - 8.5.

[0011] In the most preferred embodiment, the equilibration buffer components are 50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0.

[0012] In a specific embodiment, the NaCl concentration in the Ni affinity chromatography elution buffer is 0 - 300 mM, preferably 0 - 100 mM, more preferably 0 mM, 50 mM, 100 mM, and most preferably 50 mM.

[0013] In a preferred embodiment, the elution buffer component further includes imidazole, and its concentration is 60 - 250 mM, preferably 60 - 150 mM, more preferably 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, and most preferably 100 mM.

[0014] In a more preferred embodiment, the elution buffer components are 40 - 60 mM PB, 0 - 100 mM NaCl, 60 - 150 mM imidazole, pH 7.5 - 8.5.

[0015] In the most preferred embodiment, the elution buffer components are 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0.

[0016] In a specific embodiment, the Ni affinity chromatography elution is linear elution or gradient elution.

[0017] In a preferred embodiment, the elution conditions are 40 - 60 mM PB, 0 - 100 mM NaCl, 0 - 4 mM imidazole, pH 7.5 - 8.5; In the most preferred embodiment, the elution conditions are 50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0.

[0018] In a specific embodiment, the Ni affinity chromatography medium loading is 0 - 14.5 g / L.

[0019] In a preferred embodiment, the chromatography column for Ni affinity chromatography is filled with 1.0 L of affinity medium Agarosix MC90 - Ni Excel, and the chromatography column is equilibrated with no less than 6 column volumes (6 CV, 6 L) of equilibration buffer (50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0). The flow - through solution of gE protein from CHT is loaded onto the affinity chromatography column at a flow rate not greater than 196 ml / min (150 cm / h). The chromatography column is eluted with no less than 4 column volumes (4 CV, 4 L) of elution buffer (50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0), and then eluted with elution buffer (50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0), and the absorption peak of the target protein with an OD 280 not less than 150 mAU is collected to obtain the purified gE protein.

[0020] The new method for purifying gE protein provided by the present invention uses Ni affinity chromatography after CHT chromatography, which can effectively remove diffuse bands while enriching proteins. Through the four - step chromatography purification method of the present invention, the electrophoresis purity and liquid chromatography purity of the finally obtained gE protein can reach more than 95%. At the same time, the removal rate of CHO cell DNA by Ni affinity chromatography purification reaches 91%, the removal rate of CHO cell proteins reaches 75%, the residual amount of bacterial endotoxin is below 0.2 EU / mg, far less than the standard of the residual amount of bacterial endotoxin in the original solution (less than 25.0 EU / mg), and various impurities have achieved good removal effects, meeting the requirements of large - scale production. Description of the Drawings

[0021] Figure 1 It is a non - reducing electrophoresis diagram of the sample during hydrophobic chromatography.

[0022] Figure 2 It is a non - reducing electrophoresis diagram of the sample during anion - exchange chromatography.

[0023] Figure 3 It is a non - reducing electrophoresis diagram of the sample during affinity chromatography.

[0024] Figure 4 It is a non - reducing electrophoresis diagram of the sample during the affinity chromatography medium screening experiment process.

[0025] Figure 5 It is a non - reducing electrophoresis diagram of the affinity chromatography samples of each group.

[0026] Figure 6 Non-reducing electrophoresis diagrams of affinity chromatography samples for each group.

[0027] Figure 7 Linear elution chromatogram of affinity chromatography for Group A.

[0028] Figure 8 Non-reducing electrophoresis diagram of the linear elution sample of affinity chromatography for Group A.

[0029] Figure 9 Gradient elution chromatogram of affinity chromatography for Group B.

[0030] Figure 10 Non-reducing electrophoresis diagram of the gradient elution sample of affinity chromatography for Group B.

[0031] Figure 11 Reducing and non-reducing electrophoresis diagrams of the combined samples of affinity chromatography for Group A.

[0032] Figure 12 Non-reducing electrophoresis diagram of affinity chromatography samples.

[0033] Figure 13 Non-reducing electrophoresis diagram of samples during the affinity chromatography process.

[0034] Figure 14 Affinity chromatography chromatogram.

[0035] Figure 15 Non-reducing electrophoresis diagram of samples at each flow-through endpoint of affinity chromatography.

[0036] Figure 16 Scaled-up affinity chromatography chromatogram.

[0037] Figure 17 Scaled-up non-reducing electrophoresis diagram of affinity chromatography. Detailed implementation manners

[0038] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0039] In this specification, the meaning expressed by "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0040] In this specification, the "some specific / preferred implementation manners", "other specific / preferred implementation manners", "implementation manners", etc. mentioned refer to the specific elements (e.g., features, structures, properties, and / or characteristics) related to the implementation manner, which are included in at least one of the implementation manners described herein, and may or may not exist in other implementation manners. Additionally, it should be understood that the elements can be combined in various implementation manners in any suitable way.

[0041] In this specification, the numerical range expressed as "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0042] In this specification, the gE protein, also known as glycoprotein E (Glycoprotein E, gE), refers to an important membrane protein derived from the surface of a virus (such as a member of the Herpesviridae family) in the present invention, which plays a key role in virus infection, immune escape, and host interaction. In the present invention, specifically, it refers to a recombinant gE protein cultured in CHO cells. In a specific embodiment, the sequence of the recombinant gE protein can be found at 31 - 539aa in GenBank: WKR23623.1 (https: / / www.ncbi.nlm.nih.gov / protein / WKR23623.1). It has relatively complete glycosylation modification and has a certain hydrophobicity. The theoretical isoelectric point pI of the protein is about 5.3. The CHO cell culture refers to the cell culture fluid collected after separating cells and cell culture fluid by depth filtration after the extracellular expression of the gE protein in CHO cells. In a specific embodiment, the pH of the CHO cell culture material solution is about 7.0. Further research found that Ni affinity chromatography is used to further purify and enrich the target protein because the gE protein forms a His-like tag during the expression and modification process. Therefore, any gE protein that can form a His-like tag can be purified by the purification method of the present invention. Specifically, the N-terminal truncation of the gE protein with the sequence shown at 31 - 539aa in GenBank: WKR23623.1 is no more than 65 amino acids, preferably no more than 25 amino acids; the C-terminal truncation is no more than 40 amino acids. There is no continuous or regularly spaced histidine in the N-terminal truncation part, and there is no histidine in the C-terminal truncation part. And the truncations at the N-terminal and C-terminal have little impact on the molecular weight of the gE protein, so the purification method of the present invention can be used for all of them.

[0043] The recombinant gE protein can be obtained by using conventional technical means of modern molecular biology. Typical methods include, for example, the method of expressing the varicella-zoster virus (VZV) gE protein in CHO cells, which includes the following steps: (1) Clone the gE protein gene (full-length, truncated, mutated, or modified sequences, etc.) into an expression vector; (2) Transfect the expression vector obtained in step (1) into CHO-K1 cells; (3) Through the screening of a mixed clone cell population and monoclonal cell screening, obtain an engineering cell line stably expressing the gE protein; (4) Perform expression using the engineered cell line described in step (3): Amplify and culture in a carbon dioxide shaker and a 50L WAVE bioreactor, and then inoculate into a 50L WAVE bioreactor for expression culture. During the culture process, supplement nutrients to provide sufficient nutrition for the cells to grow and express proteins. After 10 - 15 days of culture, harvest the cell culture medium and perform depth filtration on it to obtain the cell culture product for the purification of recombinant gE protein.

[0044] In the specific embodiments of the present invention, "target protein" and "objective protein" have the same meaning, both referring to the gE protein obtained after chromatography purification.

[0045] In this specification, only a simple textual description of the definitions of various chromatography methods is provided, but the definitions of various chromatography methods are not limited to the textual description and also have the definitions known to those skilled in the art. For example: In this specification, ion exchange chromatography refers to the electrostatic interaction between the charged groups of proteins and the chromatography matrix. The separation of proteins depends on the charge on the protein surface, the pH of the mobile phase, and the salt concentration. The binding of proteins to the chromatography matrix depends on the interaction between the charge on the protein surface and the charge carried by the matrix. This interaction is reversible, and this interaction can be disrupted by a linear gradient of salt or by changing the pH, thereby eluting the proteins bound to the chromatography matrix. Ion exchange chromatography is divided into cation chromatography and anion chromatography. Since proteins have different charge amounts under different pH conditions. Anion exchange matrices bind proteins with negative charges, so these proteins are retained on the column, and then the proteins adsorbed on the column are eluted by the eluent. Proteins with weaker binding are eluted first. The composite anion exchange chromatography in the present invention is a chromatography technique that combines anion exchange with other interaction mechanisms (such as hydrophobic interaction, hydrogen bond, π - π interaction, etc.).

[0046] In this specification, hydroxyapatite (CHT) refers to a protein purification method based on hydroxyapatite as the chromatography medium. This chromatography medium has both ion exchange and metal affinity: Ca 2+ and PO4 3- . Among them, the PO4 3- ions bind to positively charged proteins through ionic bonds, having cation exchange characteristics; the Ca 2+ ions bind to the free carboxyl clusters of negatively charged proteins in a metal chelation manner. It has unique selectivity and resolution, and has special separation capabilities that other media do not have.

[0047] In this specification, Ni affinity chromatography is an affinity chromatography technique based on metal ion coordination, specifically used for purifying recombinant proteins with a histidine tag (His - tag). Its core is through nickel ions (Ni²+ Bind to the imidazole ring in the histidine tag to achieve specific purification of the target protein.

[0048] In this specification, gel filtration chromatography, also known as molecular sieve chromatography or size exclusion chromatography, is a chromatography technique for separating biological macromolecules based on molecular size differences. Its core principle is to use the pore size of a porous gel medium to elute substances of different molecular weights in order of volume from large to small, and it is widely used in the separation and purification of biological molecules such as proteins, nucleic acids, and polysaccharides, molecular weight determination, and buffer replacement.

[0049] In this specification, Solution 08E01 is the equilibration buffer and the elution buffer, and its components are 50 mM PB, 2 mM imidazole, 50 mM NaCl, pH 8.0. Among them, the control range of NaCl is 0 - 100 mM, the control range of imidazole is 0 - 4 mM, and the control range of pH is 7.5 - 8.5. Solution 08F01 is the elution buffer, and its components are 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0. The control range of NaCl is 0 - 100 mM, the control range of imidazole is 60 - 150 mM, and the control range of pH is 7.5 - 8.5.

[0050] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, or conditions of the present invention all belong to the scope of the present invention.

[0051] Unless otherwise specified, the experimental materials, reagents, and instruments used in the embodiments of the present invention can all be obtained commercially. Unless specifically specified, the technical means used in the embodiments are all conventional means well known to those skilled in the art.

[0052] Example 1, Purification Process of Recombinant gE Protein A method for purifying recombinant gE protein includes the following steps: (1) The gE protein is extracellularly expressed in CHO cells. Deep filtration is used to separate cells from cell culture medium, and the cell culture medium, that is, the cell culture product, is collected for protein purification. The pH of the gE protein cell culture product feed liquid is about 7.0. Because the gE protein has relatively complete glycosylation modification during expression and has a certain hydrophobicity, and the theoretical isoelectric point pI of the protein is about 5.3, a composite anion exchange chromatography is used to enrich and preliminarily purify the target protein, removing most of the impurity proteins and nucleic acids; (2) Hydroxyapatite (CHT) chromatography is used for further purification. In hydroxyapatite chromatography, the target protein passes through, and most of the impurity proteins and nucleic acids and other impurities bind to the chromatography column, thereby achieving the effect of purifying the target protein; (3) Ni affinity chromatography is used for further purification and enrichment of the target protein; (4) According to the molecular weight of the target protein being around 75 kDa, gel filtration chromatography with a separation range of 10 - 600 kDa was used to remove a small amount of multimeric proteins and small molecule impurity proteins while performing buffer exchange for the stock solution, providing a stable storage formulation for the target protein.

[0053] Since the gE protein is expressed in CHO cells and has relatively rich glycosylation modifications and certain hydrophobicity, the electrophoresis band of the feed liquid is relatively diffuse. After CHT chromatography purification, the purity of the target protein in the feed liquid is relatively high, but there are still a small amount of relatively diffuse protein bands above and below the target protein band that need to be purified and removed. Hydrophobic chromatography purification was attempted based on the hydrophobicity of the gE protein, but in hydrophobic chromatography, part of the target protein passed through the column, resulting in low protein recovery and diffuse bands ( Figure 1 ). Anion exchange chromatography purification was also attempted according to the protein charge difference. Although anion exchange chromatography can enrich the target protein, there are still diffuse bands ( Figure 2 ).

[0054] Through creative exploration, the present inventors found that after Ni affinity chromatography, the diffuse bands can be effectively removed ( Figure 3 ). After four-step chromatography purification in this application, the electrophoresis purity and liquid chromatography purity of the gE protein finally reached over 95%, meeting the requirements of the preparation.

[0055] Example 2. Screening of Affinity Chromatography Media There are certain differences in the ligand binding, particle size, binding capacity, etc. of Ni affinity chromatography media from different manufacturers, which have a great impact on the purification effect and recovery of the target protein. Therefore, it is necessary to screen the Ni affinity chromatography media. Taking the gE protein as the research object, different Ni affinity media were screened, and the information of each chromatography medium and grouping is shown in Table 1.

[0056] Table 1. Summary Table of Information of Each Ni Affinity Chromatography Medium

[0057] Equal volume of Ni dilution solution (50 mM PB, 600 mM NaCl, pH 8.0) was added to the CHT flow-through solution and mixed evenly. Then it was divided into four equal parts and respectively loaded onto each group of affinity chromatography (column volume 10 ml). After loading, it was rinsed with equilibration buffer A (50 mM PB, 300 mM NaCl, pH 8.0) for no less than 4 CV. After rinsing, Ni elution buffer B (50 mM PB, 250 mM imidazole, pH 8.0) was used for linear elution with a gradient of 0 - 40%B for 10 CV, and the elution peaks were collected. Finally, the chromatography column was washed with 100%B. The protein concentration and electrophoresis purity of each collected sample were detected, and the amount of harvested protein was calculated.

[0058] During the sample loading process of Ni affinity chromatography for each group (except Group D), the UV detection value of the breakthrough solution increased significantly at different stages and remained until the end of sample loading and elution. The results of non-reducing electrophoresis of the collected flow-through solution are shown in Figure 4 . It can be seen from Figure 4 that the amount of the target protein flowing through in Group A is the largest, followed by Group C, and there is a small amount of flow-through in Group B. During the sample loading process of Ni affinity chromatography in Group D, the UV absorption value of the breakthrough solution is relatively low and does not meet the collection requirements, so there is no electrophoresis detection. Moreover, there is no obvious upward trend in the UV spectrum, and there is no flow-through of the target protein. The purity of the target protein in the eluted samples of each group has been significantly improved and is comparable, and the effect of removing diffuse proteins is achieved.

[0059] According to the detection results of the sample protein concentration and the purity analysis results of non-reducing electrophoresis during the Ni affinity chromatography process for each group, the harvest amounts of the target proteins for each group are calculated, as shown in Table 2. It can be seen from Table 2 that the harvest amount of the target protein in Group A is the lowest, the harvest amounts in Group B and Group C are comparable, the harvest amount in Group D is the highest, and it is speculated that the target protein is not fully loaded because there is still no obvious upward trend in the UV of the flow-through solution until the end of sample loading and elution in Group D, and the relative loading capacity of the medium is the highest. In terms of purity, the purity of the eluted target proteins in each group is above 95%, and there is no obvious difference. Therefore, the affinity chromatography medium is determined to be Agarosix MC90-Ni Excel affinity medium.

[0060] Table 2. Summary Table of Experimental Data for Screening of Affinity Chromatography Medium

[0061] Example 3. Optimization of NaCl Concentration in Affinity Chromatography Equilibration Buffer Adding a certain concentration of NaCl to the equilibration buffer in Ni affinity chromatography can reduce the non-specific binding of impurity proteins and effectively improve the purification effect of the target protein. Therefore, the NaCl concentration in the affinity chromatography equilibration buffer is optimized to determine the appropriate NaCl concentration of the affinity chromatography equilibration buffer.

[0062] Using Agarosix MC90-Ni Excel affinity medium, the NaCl concentration in the equilibration buffer was optimized. The experimental groups are shown in Table 3. The CHT flow-through was divided into four parts, each about 120 ml. Equal volumes of the corresponding diluents of each group were added for dilution. After mixing, they were respectively loaded onto the affinity chromatography column. After loading, they were rinsed with the corresponding equilibration buffer of each group for no less than 3 CV. Subsequently, they were continuously rinsed with the rinse buffer (50 mM PB, 200 mM NaCl, pH 8.0) for no less than 3 CV. After the rinsing was completed, the target protein was eluted with a Ni elution buffer (50 mM PB, 200 mM NaCl, 100 mM imidazole, pH 8.0) in a gradient manner, and the elution peak was collected. Finally, the chromatography column was rinsed with a Ni cleaning solution (50 mM PB, 200 mM NaCl, 250 mM imidazole, pH 8.0). The protein concentration and electrophoretic purity of each collected sample were detected, and the amount and yield of the harvested protein were calculated.

[0063] Table 3 Optimization grouping table of NaCl concentration in the equilibration buffer for affinity chromatography

[0064] The non-reducing electrophoresis detection results of each group during the Ni affinity chromatography process are shown in Figure 5 , with the increase of the NaCl concentration, the purification of the target protein by affinity elution decreased slightly. The protein yields of each group in the affinity chromatography are shown in Table 4. There was no significant difference in the purity of the target protein in the elution main peak among groups A-C, and it decreased slightly in group D; there was no significant difference in the yield of the target protein among groups A-D, and there was no significant difference in the collected volume of the elution main peak. It shows that a lower NaCl concentration in the sample loading solution and equilibration buffer for Ni affinity chromatography has a better purification effect on the target protein. Therefore, to reduce the duration of the sample loading process, the NaCl concentration in the sample loading solution can be directly loaded without adjustment, and the NaCl concentration range of the equilibration buffer can be set to 0-100 mM, and the intermediate value of 50 mM can be taken as the target value.

[0065] Table 4 Summary table of affinity chromatography data for each group

[0066] According to the above experimental results, the sample solution was directly loaded onto the chromatography column. The equilibration buffer components were 50 mM PB, 50 mM NaCl, pH 8.0, and the NaCl control range was 0-100 mM.

[0067] Example 4. Optimization of NaCl concentration in the elution buffer for affinity chromatography Increasing a certain concentration of NaCl in the elution buffer for Ni affinity chromatography may affect the purification effect of the target protein. Therefore, the NaCl concentration in the elution buffer for affinity chromatography was optimized to determine the appropriate NaCl concentration of the elution buffer.

[0068] Optimize the NaCl concentration of the affinity chromatography elution buffer. The experimental groups are shown in Table 5. Take 482 ml of the CHT flow-through solution, add an equal volume of diluent (50 mM PB, 400 mM NaCl, pH 8.0), mix well, and divide it into four equal parts for affinity chromatography loading. After loading, wash with the equilibration buffer (50 mM PB, 200 mM NaCl, pH 8.0) for no less than 4 CV. After the washing is completed, elute the target protein with the corresponding eluent for each group, collect the elution peak, and finally wash the chromatography column with the washing solution (50 mM PB, 200 mM NaCl, 250 mM imidazole, pH 8.0). Detect the protein concentration and electrophoretic purity of each collected sample, and calculate the harvested protein amount and recovery rate.

[0069] Table 5. Experimental grouping table for optimizing the NaCl concentration of the affinity chromatography elution buffer

[0070] The non-reducing electrophoresis detection results of the affinity chromatography elution samples for each group are shown in Figure 6 . From Figure 6 it can be seen that there is no significant difference in the non-reducing electrophoretic purity of the target protein in the main peaks of groups A - D.

[0071] According to the protein concentration detection and non-reducing electrophoretic purity analysis results of the Ni affinity chromatography eluents for each group, calculate the recovery rates of the target proteins for each group as shown in Table 6. From Table 6, it can be seen that there are no significant differences in the collected volumes of the main peaks, the purity and recovery rates of the target proteins in groups A - D. The NaCl concentration of the elution buffer has no obvious effect on the purification effect of the target protein. Therefore, a low NaCl concentration can be used for elution, and it can be kept consistent with the concentration of the equilibration buffer, that is, the NaCl concentration range is 0 - 100 mM, and the intermediate value of 50 mM is taken as the target value.

[0072] Table 6. Summary table of affinity chromatography data for each group

[0073] According to the above experimental results, determine that the composition of the Ni affinity chromatography elution buffer is 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0, and the NaCl control range is 0 - 100 mM.

[0074] Example 5. Optimization of affinity chromatography washing and elution conditions

[0075] Ni affinity chromatography usually uses a high concentration of imidazole to elute the target protein, and adding a low concentration of imidazole during the washing process after sample loading can improve the purity of the target protein. Now, establish and optimize the affinity chromatography washing and elution conditions to determine the appropriate washing and elution conditions.

[0076] The elution and washing conditions of affinity chromatography were preliminarily established by linear elution and gradient elution methods. First, the concentration ranges of imidazole for washing and elution were preliminarily established by linear elution, and then the imidazole concentration for washing and elution was further confirmed by imidazole gradient elution. The experiment was divided into two groups. Group A was the linear elution method, and Group B was the gradient elution method.

[0077] Experimental process of Group A: Take the flow-through of gE protein CHT chromatography for affinity chromatography loading. Subsequently, wash with the equilibration buffer (50 mM PB, 50 mM NaCl, pH 8.0) for no less than 4 CV. The elution buffer consisted of 50 mM PB, 50 mM NaCl, 250 mM imidazole, pH 8.0, and perform a linear elution of 0 - 100% B for 20 CV. Collect the elution peak. The chromatogram is shown in Figure 7 , and the non-reducing electrophoresis patterns of each sample are shown in Figure 8 , and the protein concentration was detected to calculate the yield of the target protein.

[0078] Change to elution buffer B (50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0). From Figure 7 and Figure 8 , it can be seen that the purity of the target protein in the starting A1 - A2 of linear elution is slightly poor. To reduce protein loss while removing impurity proteins, the washing conditions were set to 2%, 4%, 5%, 8% B respectively (i.e., about 3% B in Group A). The purity of the target protein during the whole process of linear elution in Group A did not vary significantly. Combining with Figure 7 the elution peak distribution in, the elution conditions for Group B were determined to be 40% B (the peak tip, i.e., about 13% B in Group A), 60% B (between the latter half of the peaks, i.e., about 25% B in Group A), 100% B (the peak tail, i.e., about 40% B in Group A). Finally, wash the chromatography column with the elution buffer of Group A. Collect each washing, elution, and cleaning peak, detect the protein concentration and non-reducing electrophoresis purity, and calculate the yield of the target protein.

[0079] The chromatogram of Group B is shown in Figure 9 , and the non-reducing electrophoresis detection of washing and elution is shown in Figure 10 , and the detection results of the combined samples of Group A affinity chromatography by reducing and non-reducing electrophoresis are shown in Figure 11 . From Figure 9 , it can be seen that with the increase of the washing gradient, there are certain elution peaks in the chromatogram at OD 280 , but the higher the washing gradient, the more serious the tailing. Most of the target protein can be eluted at 40% B. From Figure 10 and Figure 11 , it can be seen that there is no obvious difference in the purity of the target protein in each gradient of washing and elution, and it is equivalent to the purity of the samples in linear elution.

[0080] Based on the detection results of the target protein concentration by affinity chromatography for each group and the analysis results of the electrophoretic purity, the harvested amount of the target protein was calculated as shown in Table 7. As can be seen from Table 7, the total protein in Group B only accounted for 61.72% of that in Group A, which might be due to the overly long trailing of the 8%B elution. To improve the protein recovery rate, the elution can be adjusted to 2%B (i.e., the imidazole concentration is 2 mM). The control range is 0 - 4%. The target protein eluted with 40%B accounted for 84% of the total protein, but the elution volume was relatively large, which was close to the linear elution A1 - A12 in Group A. The purity of the target protein eluted with 60% and 100% was good. To reduce the elution volume, the elution condition can be determined as 100%B. Therefore, the elution condition for the gE protein affinity chromatography is tentatively set as 50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0, and the imidazole control range is 0 - 4 mM. To simplify the solution preparation, the equilibration buffer can be adjusted to be the same as the elution buffer. The elution buffer is set as 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0.

[0081] Table 7. Summary of Affinity Chromatography Data for Each Group

[0082] Verify the affinity chromatography process parameters initially established for the above elution and elution conditions of affinity chromatography to determine that the process is stable, feasible, and has good reproducibility.

[0083] Take the flow-through of the gE protein CHT chromatography for affinity chromatography. After the sample loading of the feed solution is completed, wash it with the eluent (50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0) for no less than 4 CV. After the washing is completed, elute the target protein with the elution solution (50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0), and finally wash the chromatography column with the cleaning buffer (50 mM PB, 50 mM NaCl, 250 mM imidazole, pH 8.0). Collect each sample to detect the protein concentration and non-reducing electrophoretic purity, and calculate the harvested amount of the target protein.

[0084] The non-reducing electrophoretic detection results of affinity chromatography are shown in Figure 12 , and as can be seen from Figure 12 , the purity of the target protein in each eluted sample is quite comparable. The summary of chromatography data is shown in Table 8. As can be seen from Table 8, the electrophoretic purity of the target protein in the elution main peak can reach 94.33%, the proportion can reach 89.13%, the recovery rate reaches 68.28%, and the volume (1.5 CV) is significantly reduced, meeting the process requirements, and the affinity chromatography process is stable and feasible.

[0085] Table 8. Summary Table of Affinity Chromatography Data

[0086] In summary, the affinity chromatography process of gE protein is verified to be feasible. The equilibrium and elution conditions of the gE protein affinity chromatography are tentatively set as 50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0, and the elution buffer is set as 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0.

[0087] Example 6. Optimization of Imidazole Concentration for Affinity Chromatography Elution The target protein is eluted by imidazole in affinity chromatography. The imidazole concentration affects the purity and yield of the target protein. Therefore, it is necessary to further optimize the imidazole concentration in the established elution conditions to determine a reasonable range.

[0088] Take the CHT chromatography flow-through solution of gE protein for affinity chromatography loading. After loading, wash with the washing buffer (50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0) for no less than 4 CV. The elution buffer components are 50 mM PB, 50 mM NaCl, pH 8.0, and the imidazole concentrations are set as 60, 80, 100, 120, and 150 mM in sequence. The experiment is divided into 5 groups, namely groups A - E. Each group elutes the target protein with the corresponding elution buffer, and finally washes the chromatography column with the cleaning buffer (50 mM PB, 50 mM NaCl, 250 mM imidazole, pH 8.0). Collect the samples during the purification process to detect the protein concentration and non-reducing electrophoresis purity, and calculate the amount and yield of the target protein in each group.

[0089] The non-reducing electrophoresis detection results of the samples during the affinity chromatography process of each group are shown in Figure 13 , from Figure 13 it can be seen that the purity of the target protein eluted by the affinity chromatography of each group is quite comparable. The summary of the affinity chromatography data of each group is shown in Table 9. It can be seen from Table 9 that the elution volumes of each group are not very different, and the purity and yield of the target protein are quite comparable. Therefore, the imidazole concentration range in the elution buffer can be determined to be 60 - 150 mM, and the target value is set at 100 mM.

[0090] Table 9. Summary Table of Affinity Chromatography Data of Each Group

[0091] According to the above experimental results, the components of the affinity chromatography elution buffer are determined to be 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0, and the imidazole control range is 60 - 150 mM.

[0092] Example 7. Determination of Affinity Chromatography Medium Loading Capacity The loading capacity of the affinity chromatography medium has a certain impact on the purity and yield of the target protein. Therefore, it is necessary to determine the loading capacity of the affinity chromatography medium.

[0093] Take the CHT chromatography flow-through solution of gE protein for affinity chromatography (column volume 10 ml) loading. Along with the flow-through OD280 As the [parameter] increases, the flow-through endpoint samples are collected sequentially. After sample loading, elute with eluent (50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0) for at least 4 CV. After elution, use elution buffer (50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0) for elution and collect the eluate. Finally, wash the chromatography column with cleaning solution (50 mM PB, 50 mM NaCl, 250 mM imidazole, pH 8.0). Collect each sample to detect the non-reducing electrophoresis purity and protein concentration, and calculate the protein amount.

[0094] The Ni affinity chromatography diagram and the non-reducing electrophoresis detection results of each flow-through endpoint sample in Ni affinity chromatography are shown in Figure 14 and Figure 15 . The relative quantitative analysis results of the non-reducing electrophoresis detection of each flow-through endpoint sample are shown in Table 10. From Figure 14 and Figure 15 it can be seen that the affinity chromatography sample loading starts to slowly flow through at 157 ml, and the flow-through trend increases significantly at 217 ml of sample loading. As shown in Table 10, at 217 ml of sample loading, the relative flow-through amount of the target protein reaches 5.7%, corresponding to a sample loading protein amount of 145 mg (ignoring the flow-through protein amount), and the maximum medium loading capacity reaches 14.5 g / L, meeting the requirements of pilot-scale production.

[0095] Table 10. Summary table of relative quantification of purity of each flow-through endpoint in affinity chromatography

[0096] In summary, it is determined that the maximum dynamic loading capacity of the affinity chromatography medium is 14.5 g / L, meeting the requirements of pilot-scale production.

[0097] Example 8. Scale-up of affinity chromatography process According to the determined conditions above, verify the scale-up of the affinity chromatography process for gE protein. The model of the affinity chromatography column is Easy-Axi 100*500, the column bed volume is 1.0 L. During the chromatography process, the pressure-limiting mode is adopted, the pressure is controlled below 0.3 MPa, and the flow rate is not more than 262 ml / min (200 cm / h).

[0098] Equilibrate the chromatography column with no less than 6 column volumes (6CV, 6L) of solution 08E01 (50 mM PB, 2 mM imidazole, 50 mM sodium chloride, pH 8.0) at a flow rate not exceeding 262 ml / min. Load the flow-through of gE protein from the CHT chromatography process (derived from the scale-up of the CHT chromatography process) onto the affinity chromatography column at a flow rate not exceeding 196 ml / min (150 cm / h). After loading, wash the chromatography column with no less than 4 column volumes (4CV, 4L) of solution 08E01. Immediately after washing, elute with solution 08F01 (50 mM PB, 50 mM sodium chloride, 100 mM imidazole, pH 8.0) at a flow rate not exceeding 196 ml / min, and collect the absorption peak of the target protein with an OD 280 not less than 150 mAU (see Figure 16 ). Determine the total protein concentration of the affinity chromatography eluate by the BCA method, and determine the non-reducing electrophoresis purity of the target protein in the eluate by non-reducing SDS-PAGE (see Figure 17 ). Calculate the amount of the target protein harvested, and detect the residual amounts of CHO cell DNA, bacterial endotoxin, CHO cell protein, tributyl phosphate, Ni ions, and imidazole in the affinity chromatography eluate sample, and calculate the removal rates of impurities such as CHO cell DNA, bacterial endotoxin, and CHO cell protein, as well as tributyl phosphate.

[0099] The results show (Table 11) that the non-reducing purity and HPLC purity of the target protein of gE protein after affinity chromatography both reach over 95%, and the recovery rate of the target protein reaches 70%, meeting the requirements of pilot-scale production. The removal rate of CHO cell DNA by affinity chromatography of gE protein reaches 91%, the removal rate of CHO cell protein reaches 75%, and the residual amount of bacterial endotoxin is below 0.2 EU / mg, far less than the standard of the residual amount of bacterial endotoxin in the original solution (less than 25.0 EU / mg). All impurities have achieved good removal effects, and the affinity chromatography scale-up process is feasible.

[0100] Table 11. Summary Table of Affinity Chromatography Process Scale-up Data

[0101] According to the above research results, the gE protein affinity chromatography process is determined as follows: Use an Easy-Axi 100*500 chromatography column packed with 1.0 L of affinity medium (Agarosix MC90-Ni Excel), and equilibrate the chromatography column with no less than 6 column volumes (6 CV, 6 L) of solution 08E01 (50 mM PB, 2 mM imidazole, 50 mM sodium chloride, pH 8.0, sodium chloride control range 0 - 100 mM, imidazole control range 0 - 4 mM, pH control range 7.5 - 8.5). Load the gE protein CHT flow-through solution onto the affinity chromatography column at a flow rate not exceeding 196 ml / min (150 cm / h). After loading, wash the chromatography column with no less than 4 column volumes (4 CV, 4 L) of solution 08E01, and then elute with solution 08F01 (50 mM PB, 50 mM sodium chloride, 100 mM imidazole, pH 8.0, sodium chloride control range 0 - 100 mM, imidazole control range 60 - 150 mM, pH control range 7.5 - 8.5), and collect the target protein absorption peak with an OD280 not less than 150 mAU.

Claims

1. A purification method for recombinant gE protein, characterized in that, The CHO cell culture containing the gE protein was purified successively by mixed anion exchange chromatography, hydroxyapatite chromatography, affinity chromatography and gel filtration chromatography; the affinity chromatography was Ni affinity chromatography.

2. The purification method according to claim 1, characterized in that, The medium for the Ni affinity chromatography is any one of NiBestarose FF, Ni Smart Beads 6FF, Polar MC60-Ni Excel and Agarosix MC90-Ni Excel, preferably Agarosix MC90-Ni Excel.

3. The purification method according to claim 1 or 2, characterized in that, The NaCl concentration of the Ni affinity chromatography equilibration buffer is 0 - 300 mM, preferably 0 - 100 mM, more preferably 0 mM, 50 mM, 100 mM.

4. The purification method according to claim 3, wherein The equilibration buffer component further includes imidazole, and its concentration is 0 - 4 mM, preferably 2 mM imidazole.

5. The purification method according to claim 4, characterized in that, The equilibration buffer component is 40 - 60 mM PB, 0 - 100 mM NaCl, 0 - 4 mM imidazole, pH 7.5 - 8.5; Preferably, the equilibration buffer component is 50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.

0.

6. The purification method according to claim 1 or 2, characterized in that, The NaCl concentration of the Ni affinity chromatography elution buffer is 0 - 300 mM, preferably 0 - 100 mM, more preferably 0 mM, 50 mM, 100 mM.

7. The purification method according to claim 6, characterized in that, The elution buffer component further includes imidazole, and its concentration is 60 - 250 mM, preferably 60 - 150 mM, more preferably 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, most preferably 100 mM.

8. The purification method according to claim 7, wherein The elution buffer component is 40 - 60 mM PB, 0 - 100 mM NaCl, 60 - 150 mM imidazole, pH 7.5 - 8.5; Preferably, the elution buffer component is 50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.

0.

9. The purification method according to claim 1 or 2, characterized in that, The Ni affinity chromatography elution is linear elution or gradient elution; Preferably, the elution condition is 40 - 60 mM PB, 0 - 100 mM NaCl, 0 - 4 mM imidazole, pH 7.5 - 8.5; More preferably, the elution condition is 50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.

0.

10. The purification method according to claim 1 or 2, characterized in that, The Ni affinity chromatography medium loading capacity is not higher than 14.5 g / L; The chromatography column for Ni affinity chromatography is packed with 1.0 L of affinity medium Agarosix MC90-Ni Excel, and the chromatography column is equilibrated with no less than 6 column volumes (6 CV, 6 L) of equilibration buffer (50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0). The flow-through of gE protein CHT is loaded onto the affinity chromatography column at a flow rate of no more than 196 ml / min (150 cm / h). The chromatography column is washed with no less than 4 column volumes (4 CV, 4 L) of wash buffer (50 mM PB, 50 mM NaCl, 2 mM imidazole, pH 8.0), and then eluted with elution buffer (50 mM PB, 50 mM NaCl, 100 mM imidazole, pH 8.0). Collect the absorption peak of the target protein with an OD 280 not less than 150 mAU to obtain the purified gE protein.

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