A method for purifying a recombinant human serum albumin

Abstract

The application relates to a purification method of recombinant human serum albumin, and belongs to the technical field of protein separation and purification. The mixed bed chromatography of strong cation exchange and weak anion exchange fillers mixed in a specific proportion is adopted for fine purification, high-efficiency flow-through recovery is preferably realized at pH 4.5-5.5, the strong cation exchange and weak anion exchange fillers are used for synchronous deep removal of acidic impurities and basic impurities, and a composite additive system containing sodium chloride, glycine and sorbitol is used, the three synergistically act on each other, the aggregation tendency of the target protein in the environment close to the isoelectric point is effectively inhibited, the scheme can efficiently remove various impurities such as host cell proteins, endotoxins, aggregates and charge isomers, the purity of the final product is greater than 99.99%, the residual amount of key impurities is lower than the detection limit, and the process has strong robustness and is suitable for large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to a purification method of recombinant human serum albumin, belonging to the technical field of protein separation and purification. BACKGROUND

[0002] Human serum albumin (HSA) is the most abundant functional protein in human plasma, which plays an irreplaceable role in maintaining colloid osmotic pressure, material transport and in vivo antioxidant defense, etc. Due to its excellent biocompatibility and stability, HSA has been widely used in clinical transfusion, cell culture, vaccine adjuvant and drug delivery system. At present, the clinical HSA mainly depends on the extraction of human plasma, which is limited by the shortage of plasma supply, the risk of contamination by potential pathogens and ethical controversy. The preparation of recombinant human serum albumin (rHSA) by recombinant DNA technology is considered to be a fundamental way to solve the above problems.

[0003] However, rHSA usually needs to be administered at a high dose in clinical treatment, and the dose of each injection can reach 5-30 grams, which is much higher than that of conventional recombinant drugs, so the trace impurities remaining in the product may cause significant immunogenicity risk or other side effects. Therefore, the total host protein (HCP) residue of each injection dose and the contaminants in the production process must be less than 1 ng / ml to meet the safety requirements of pharmaceuticals.

[0004] The prior art usually adopts a multi-step chromatography combination to achieve the preliminary purification of rHSA. For example, patent CN202011105562.4 obtains rHSA with a purity of 99.97% through a five-step chromatography process. However, such processes generally face a common bottleneck: the micro-HCP and charge heterogeneity albumin remaining after the main purification constitute a complex impurity system containing acidic, basic components and other charge variants. If a single ion exchange chromatography is used, only impurities of a certain type of charge attribute can be selectively removed, and overall purification cannot be achieved; and if multiple chromatography is simply connected in series, although the purity may be improved to a certain extent, it will inevitably lead to a long process, reduced yield and rising costs, which is difficult to meet the dual needs of efficiency and economy of industrial production.

[0005] Mixed-bed ion exchange chromatography (Mixed-Bed Ion Exchange Chromatography) theoretically has the ability to simultaneously remove anionic and cationic impurities. Since Rassi and Horváth et al. (J. Chromatogr. 1986, 359, 255-264) early exploration, the technology has been re-focused in specific separation scenarios in recent years. However, the existing mixed bed technology is mostly used for analytical detection or small molecule purification. In the industrialized purification of rHSA, a key technical problem must be solved: how to build a mixed bed system that can achieve high selective separation, so that it can efficiently adsorb various charged impurities HCP and charge heterogeneity albumin while ensuring that the target rHSA monomer can flow through the collection with a recovery rate close to 100%. However, the existing mixed bed technology system generally lacks the ability to finely regulate the selective separation mechanism, which makes it impossible to be applied in rHSA purification.

[0006] Therefore, how to simultaneously remove trace HCP with different charge properties and albumin variants with similar physicochemical properties under mild process conditions through an efficient and integrated purification unit, while controlling HCP to be less than 5 ng / g without sacrificing the recovery rate of the final product, is a very worthwhile research topic. After searching, it was found that there were few studies on the use of mixed-bed ion exchange chromatography process in the purification of recombinant human serum albumin, and the existing purification strategies and mixed bed technology could not effectively solve this double challenge. SUMMARY

[0007] In order to solve the above problems, a purification method of recombinant human serum albumin is provided. The application adopts a mixed bed chromatography of strong cation exchange and weak anion exchange filler mixed in a specific ratio for purification, and preferably realizes efficient flow-through recovery at pH 4.5-5.5. Alkaline impurities and acidic impurities are simultaneously removed by strong cation exchange and weak anion exchange filler, and a composite additive system containing sodium chloride, glycine and sorbitol is used. The three work together to ensure high recovery rate of the target protein while effectively inhibiting its aggregation tendency in the environment near the isoelectric point. The application can efficiently remove various impurities such as host cell proteins, endotoxins, aggregates and charge isomers. The final product has a purity of >99.99%, the key impurity residue is lower than the detection limit, and the process has strong robustness and is suitable for large-scale industrial production.

[0008] The application provides a purification method of recombinant human serum albumin, characterized in that the purification method comprises the following steps:

[0009] 1) pretreating a fermentation broth containing recombinant human serum albumin;

[0010] 2) cation exchange chromatography;

[0011] 3) anion exchange chromatography;

[0012] 4) hydrophobic chromatography;

[0013] 5) subjecting the product liquid obtained in step 4) to ultrafiltration, wherein a weak ionic strength buffer at pH 5.0-5.5 is used in the ultrafiltration, and then performing a mixed bed ion exchange purification step;

[0014] In the mixed bed ion exchange purification step: the mixed bed chromatography is performed in a buffer system at pH 4.5-5.5; the mixed bed chromatography uses WAX filler and SCX filler, and the mass ratio of the WAX filler to the SCX filler is 1:(0.67-1.5); the buffer system contains a composite additive, and the composite additive comprises 50-150 mM sodium chloride, 10-20 mM glycine and 5-10% (w / v) sorbitol.

[0015] Optionally, the mixed bed chromatography is performed in a buffer system at pH 4.7-5.3.

[0016] Optionally, the mixed bed chromatography is performed in a buffer system at pH 5.0.

[0017] Optionally, the mass ratio of the WAX filler to the SCX filler is 1:(0.8-1.2).

[0018] Optionally, the mass ratio of the WAX filler to the SCX filler is 1:1.

[0019] Optionally, the WAX filler is a diethylaminoethyl DEAE group filler.

[0020] The SCX filler is a sulfopropyl SP group filler.

[0021] Optionally, the ultrafiltration in step 5) uses an ultrafiltration membrane with a molecular weight cut-off of 10-30 kDa, and the volume of the buffer used in the ultrafiltration is 5-10 times the volume of the product liquid.

[0022] Optionally, step 1) comprises a step of heating the fermentation broth and then centrifuging, and the heating treatment conditions are 60-80°C for 10-30 min, and the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10-20 min.

[0023] Optionally, the filler for the cation exchange chromatography in step 2) is a sulfopropyl SP group filler.

[0024] The filler for the anion exchange chromatography in step 3) is a quaternary ammonium Q group filler.

[0025] The ligand of the hydrophobic chromatography in step 4) is selected from one or more of phenyl, aliphatic, and heterocyclic.

[0026] Optionally, the weak ionic strength buffer is an acetic acid-sodium acetate buffer.

[0027] Optionally, the purity of the product obtained after purification is >99.99%, the host cell protein residue is <2 ng / g rHSA, and the endotoxin is <0.5 EU / mL.

[0028] The beneficial effects of the present application include but are not limited to:

[0029] 1. The purification method of recombinant human serum albumin according to the present application, the present application scheme adopts a mixed bed chromatography of strong cation exchange and weak anion exchange fillers mixed in a specific ratio for fine purification, and high-efficiency flow-through recovery is realized under the condition of pH 4.5-5.5, and acidic impurities and basic impurities are simultaneously and deeply removed by the strong cation exchange and weak anion exchange fillers.

[0030] 2. The purification method of recombinant human serum albumin according to the present application, a composite additive system containing sodium chloride, glycine and sorbitol is used, and the three work together to effectively inhibit the aggregation tendency of the target protein in the environment near the isoelectric point while ensuring high recovery rate of the target protein.

[0031] 3. The purification method of recombinant human serum albumin according to the present application, which provides a complete set of mixed bed chromatography strategies for the problem of deeply removing charge isomer HCP and charge heterogeneity albumin in the fine purification stage, and can realize a leap in product purity without increasing the loss of target protein.

[0032] 4. The purification method of recombinant human serum albumin according to the present application, which can efficiently remove various impurities such as host cell proteins, endotoxins, aggregates and charge isomers, and the final product has a purity of >99.99% and the residual amount of key impurities is lower than the detection limit, and the process has strong robustness and is suitable for large-scale industrial production.

[0033] 5. The purification method of recombinant human serum albumin according to the present application, which can simultaneously and deeply remove acidic and basic charge isomers through the synergistic effect of the mixed bed fine purification step combined with the composite additive system, so that the final product has a purity of >99.99% and the residual amount of key impurities such as host cell proteins (HCP) is lower than 2 ng / g rHSA.

[0034] 6. The purification method of recombinant human serum albumin according to the present application, the process conditions are mild and accurately optimized, especially in the core purification step, by accurately regulating the pH at the isoelectric point and using a complex additive system, the deep removal of impurities is achieved while the non-specific adsorption and loss of target protein are minimized, the recovery rate of the mixed bed step is greater than 95%, and the overall process recovery rate is industry-leading.

[0035] 7. The purification method of recombinant human serum albumin according to the present application, the whole process flow is designed scientifically and has good repeatability, the "SCX+WAX" filler combination and the complex additive system effectively improve the operation window and fault tolerance rate of the purification step, reduce the performance risk caused by small fluctuations in material and parameters, and make the process show excellent repeatability and reliability, which is very suitable for large-scale industrial production.

[0036] 8. The purification method of recombinant human serum albumin according to the present application, the integrated strategy comprehensively uses cation exchange, anion exchange, hydrophobic interaction and innovative mixed mode chromatography, which can systematically remove various impurities such as host cell proteins, endotoxins, DNA, aggregates and charge isomers, and the product quality is excellent, meeting the strict pharmaceutical standards. BRIEF DESCRIPTION OF DRAWINGS

[0037] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0038] Figure 1 The purification process flow of recombinant human serum albumin (rHSA) involved in the present application;

[0039] Figure 2 The HPLC purity detection spectrum of the final rHSA product involved in Example 1 of the present application;

[0040] Figure 3 The contour analysis graph of the influence of the complex additive system on the recovery rate of rHSA involved in Example 3 of the present application (A is the recovery rate contour graph when Sorb is fixed at 6.2%, and B is the recovery rate contour graph when NaCl is fixed at 100.0 mM);

[0041] Figure 4 The linear fitting analysis graph of the response surface and the actual value and the predicted value of the recovery rate of the complex additive system on the recovery rate of rHSA involved in Example 3 of the present application (A is the response surface three-dimensional surface graph of the recovery rate, and B is the linear fitting verification graph of the actual value and the predicted value of the recovery rate). DETAILED DESCRIPTION

[0042] The application will be described in detail below with reference to examples, but the application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the application are purchased through commercial channels.

[0043] In the present application, the method of Pichia pastoris fermentation to produce recombinant human serum albumin can be a conventional method in the art, which is not described here, for example, see "Qian Lisheng, Jin Guangming. Comprehensive experimental training tutorial of bioengineering [M], Anhui Science and Technology Press, 2018: 150-152" or patent CN102190722A.

[0044] Example 1

[0045] This example provides a method for purifying rHSA from Pichia pastoris fermentation broth, the flow chart is as shown in Figure 1 The specific steps include:

[0046] S1, fermentation broth pretreatment:

[0047] Centrifuge the yeast-expressed recombinant human albumin fermentation broth at 4℃, 8000 rpm for 15 min, and collect the supernatant. Add sodium caprylate to the supernatant to a final concentration of 10 mM, slowly add 1M HCl solution under stirring, adjust the pH to 4.5, heat at 65℃ for 30 min, quickly cool to room temperature, centrifuge at 10000 rpm for 15 min, repeat the centrifugation twice, collect the supernatant and pass through a 0.45 µm filter membrane for loading. Then dilute with ultrapure water until the solution conductivity is ≤15 mS / cm to obtain the pretreated sample.

[0048] S2, cation exchange capture:

[0049] An XK 50 / 60 chromatography column (Cytiva) packed with 500 mL SP Sepharose Fast Flow packing material is used. First, equilibrate the column bed with 5 CV of 20 mM sodium acetate buffer (pH 4.5) at a flow rate of 250 cm / h. Pump the pretreated clarified sample in the same direction as the equilibration direction, and perform fixed bed adsorption. After adsorption is complete, directly rinse with 5 CV of equilibration buffer until the UV baseline is stable. Then apply a linear gradient of 0-500 mM NaCl (20 mM sodium acetate, pH 4.5) for elution, gradient volume 20 CV, flow rate 200 cm / h, collect the main elution peak of rHSA by UV monitoring.

[0050] S3, first ultrafiltration and replacement:

[0051] Ultrafiltration concentration and buffer exchange of eluate was performed using Pellicon 3 ultrafiltration cassettes (Merck Millipore) with a molecular weight cut-off of 10 kDa. The sample was first concentrated to approximately 200 mL and then dialyzed against 8 volumes of 20 mM Tris-HCl buffer (pH 8.0) until the conductivity of the outlet was identical to the conductivity of the added buffer.

[0052] S4, anion exchange chromatography:

[0053] After adjusting the conductivity of the ultrafiltrated sample to 5 mS / cm, it was loaded onto a HiScale 50 / 40 chromatography column (Cytiva) packed with Q Sepharose Fast Flow media, column height 15 cm. The column was first equilibrated and rinsed to baseline with 5 CV of 20 mM Tris-HCl (pH 8.0) at a flow rate of 150 cm / h, followed by elution using a linear gradient of 0-500 mM NaCl (20 mM Tris-HCl, pH 8.0) with a gradient volume of 15 CV, collecting the rHSA elution peak.

[0054] S5, second ultrafiltration buffer exchange:

[0055] The anion exchange elution peak was again buffer exchanged using an ultrafiltration system, dialyzing against 8 volumes of 20 mM sodium phosphate buffer (pH 7.0) containing 2.0 M ammonium sulfate and concentrating to approximately 50 mL.

[0056] S6, hydrophobic chromatography:

[0057] The high-salt sample was loaded onto an XK 26 / 40 chromatography column (Cytiva) packed with Phenyl Sepharose High Performance media, column height 10 cm. The column was equilibrated with 5 CV of 20 mM sodium phosphate, 2.0 M ammonium sulfate (pH 7.0) at a flow rate of 150 cm / h. Elution was performed using a decreasing linear gradient of 2.0-0 M ammonium sulfate (20 mM sodium phosphate, pH 7.0) with a gradient volume of 10 CV, collecting the rHSA elution peak at a flow rate of 150 cm / h.

[0058] S7, third ultrafiltration buffer exchange:

[0059] The hydrophobic chromatography eluate was ultrafiltrated and buffer exchanged to 20 mM acetic acid-sodium acetate buffer (pH 5.0) with a dialysis volume of 8 volumes of sample and a sample volume of approximately 25 mL.

[0060] S8, mixed-bed ion exchange polishing:

[0061] Mixed bed chromatography was performed in 20 mM acetic acid-sodium acetate buffer at pH 5.0 with additional complex additive system consisting of 107 mM sodium chloride, 11 mM glycine and 7% (w / v) sorbitol. The required mass of SP Sepharose FF (particle size 45-165 pm) and DEAE Sepharose FF (particle size 45-165 pm) dry powder fillers were mixed in a 1:1 ratio, slurried with 5 volumes of the above-mentioned complex additive-containing buffer, and ultrasonically treated for 3 minutes to ensure complete dispersion. Subsequently, packing was performed under a pressure of 200 psi into an HR50 / 20 chromatography column (Cytiva) with a column height of 15 cm, ensuring a uniform and compact column bed. Equilibration was performed with 5 CV of 20 mM acetic acid-sodium acetate (pH 5.0) buffer containing complex additives at a flow rate of 100 cm / h. After loading the sample obtained in S7, elution was continued with the same complex additive-containing equilibration buffer for 5 CV (flow rate 100 cm / h), and all flow-through and eluate (to the UV280 baseline) were collected and combined to obtain the high-purity rHSA product with a purity of >99.99%, as shown in Figure 2 The HPLC purity detection spectrum of the rHSA product is shown in FIG. 1, and the first detected target component peak is rHSA, with a retention time of 13.963 min.

[0062] Test Example 1

[0063] The final product of Example 1 was detected by HPLC (column Tosoh, TSKgel G3000SWXL) to have a purity of >99.99%, and the host protein residue (HCP) content was detected by ELISA (kit purchased from Cygnus Pichia pastoris HCP ELISA Kit, Cat# F140), which has a detection limit of less than 0.4 ng / mL, and no HCP was detected. The endotoxin concentration was <0.5 EU / mL by the limulus reagent dynamic turbidity method. The purity was basically unchanged after the ultrafiltration step, and the product of the recovery rate of each step was calculated from the cation exchange capture step (S2), and the total recovery rate was 50.39%. Higher than the general purification means, for example, in patent CN202011105562.4, after five steps of chromatography, the recovery rate was 30.7%, and the purity was 99.97%.

[0064] The specific detection method and result analysis are as follows.

[0065] Purity test: Gel filtration HPLC, column: Tosoh TSKgel G3000SWXL (7.8 mm x 300 mm), mobile phase: 0.1 M potassium dihydrogen phosphate-potassium hydrogen phosphate buffer (pH 7.0), flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 280 nm, injection volume: 20 μL; sample was diluted to 1 mg / mL with mobile phase, chromatogram was recorded after injection, purity was calculated by main peak area / total peak area x 100%. Based on the analysis results of gel filtration HPLC, no dimer or high molecular weight polymer with content higher than the limit of detection (0.01%) of the method was detected in the sample. The chromatographic purity of the target monomer was greater than 99.99% calculated by the main peak area normalization method. It showed that the product had extremely high purity, and the content of potential size-related impurities was lower than the limit of quantification of the method.

[0066] HCP test (ELISA method): Cygnus Technologies Pichia pastoris HCP ELISA kit (Cat# F140) was used to detect host cell protein (HCP) residues from Pichia pastoris by double-site sandwich immunoenzyme analysis. The standard protocol in the kit instructions was strictly followed, and the lower limit of quantification (LOQ) of the protocol was 0.4 ng / mL, and the detection limit (LOD) was less than 0.3 ng / mL.

[0067] The brief steps are as follows: equilibrate the reagents to room temperature, dilute the 20x wash solution with distilled water. Dilute the provided HCP standard at 0, 1, 4, 20, 75 ng / mL gradient (2 replicate wells for each concentration). Dilute the purification intermediate after step S7 in the matching Sample Diluent (Cat# I028) 100-fold, and do not dilute the sample after step S8. Add 25 μL of standard / sample / blank control and 100 μL of HRP-labeled detection antibody to each well, and after sealing, incubate at room temperature with 400-600 rpm shaking for 3 hours. Wash by hand 4 times (add 350 μL of wash solution to each well each time), pat dry, add 100 μL of TMB color developing solution, and incubate at room temperature in the dark for 30 minutes. Add 100 μL of stop solution, and measure the absorbance at 450 nm / 650 nm within 30 minutes.

[0068] The standard curve was fitted using four-parameter logistic regression (4-PL) y = 1.376404 + (0.08657905 - 1.376404) / (1 + (x / 5.268261) ^ 1.050758) (R 2= 0.9992). The HCP concentration in the sample was calculated according to the fitting curve, and multiplied by the dilution factor 100 (purified intermediate product after step S7). The calculated concentration corresponding to the absorbance value of the rHSA finished product purified by the present application was lower than the lower limit of quantification (LOQ < 0.4 ng / mL) of the method. According to the concentration of the rHSA finished product (200 mg / mL), the HCP residual amount was < 2.0 ng / g, indicating that the host protein residues were efficiently removed.

[0069] Endotoxin detection (limulus reagent colorimetric method): Endotoxin was detected using an endotoxin detection kit produced by Biyun Tian (Biyun Tian, Cat#C0276S). According to the requirements of the instructions, the rHSA sample obtained in Example 1 was dissolved with endotoxin test water and diluted to a value within the standard curve range. The background absorbance was 0.18 (<0.5), and only the sample blank tube was deducted from the background; during the reagent preparation stage, the 20 EU / bottle endotoxin standard of the kit was dissolved with 1 mL of BET water and vortexed for 3 minutes to prepare a 20 EU / ml stock solution, which was gradually diluted to 0.100, 0.050, 0.025, 0.010 EU / ml gradient standard solution, and at the same time, the endotoxin detection reagent, color developing agent, and reaction termination liquid and Buffer A, Buffer B / C were prepared; when the detection system was set, 10 μL of standard solution, sample solution or BET water (negative control) was added to each well of the 96-well plate, then 10 μL of detection reagent was added, and incubated at 37°C for 25 minutes (T1) in the dark, then 10 μL of color developing agent was added and incubated for 6 minutes (T2), then 50 μL of Buffer A, B, C was added in turn, and after standing for 5 minutes, the absorbance was read at 545 nm; the standard curve equation y = 5.6385x + 0.1829, R 2 = 0.9932, recovery rate of interference experiment 68% (50%~200%, no interference), among them, due to the high endotoxin content in the sample before purification, in order to ensure that the detection value falls within the linear range of the standard curve, the samples in steps S2~S5 were diluted by 100 times during detection, and the samples in steps S6~S8 were diluted by 10 times. The endotoxin concentrations listed in Table 1 below are the original concentrations calculated back according to the corresponding dilution factors. The absorbance of the diluted sample S8 is 0.210, corresponding to a concentration of 0.0046 EU / ml, which is converted to an original sample endotoxin concentration of 0.050 EU / ml, which meets the requirements of the People's Republic of China Pharmacopoeia (2020 edition).

[0070] Recovery rate calculation: from the cation exchange capture step (S2), the recovery rate of each step was calculated as (the total amount of rHSA of this step / the total amount of rHSA of the previous step) x 100%, and the total recovery rate was the product of the recovery rates of each step, which was 50.39%.

[0071] The detection results of each step of Example 1 in the present test example are shown in Table 1 below.

[0072] Table 1 Detection results of each step

[0073]

[0074] Example 2 Mixed bed pH condition screening

[0075] To investigate the optimum pH in the mixed bed polishing step, three groups of comparative experiments were set up. To ensure sufficient buffer capacity in a wide pH range, in addition to the buffer pH being 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 and 5.5 (the buffer is 50 mM acetic acid-sodium acetate, and contains a composite additive system composed of 110 mM NaCl, 12 mM glycine and 7.5% (w / v) sorbitol), the rest of the steps are the same as Example 1, and the results are shown in Table 2 below.

[0076] Table 2 Effect of buffer pH on mixed bed chromatography purification (n = 3)

[0077]

[0078] The results show that within the pH range of 4.5-5.5, the process of the present application can achieve effective purification. However, the recovery rate and purity reach the best balance in the pH range of 4.7-5.3, which can be considered as the robust operating window of the process. Among them, in the core interval of pH 4.9-5.1, the single-step recovery rate of rHSA is the highest (>95%), and the product purity is stable at >99.99%. When the pH deviates to both sides, the non-specific adsorption of rHSA carrying increased net charge to the opposite charge filler in the mixed bed is enhanced, resulting in a regular and symmetrical decrease in recovery rate; at the same time, the adsorption efficiency of the filler to impurities with specific charge properties changes, resulting in a gentle decrease in product purity. In summary, the preferred pH range of the mixed bed polishing step is 5.0 ± 0.3, and the optimal range is 5.0 ± 0.1.

[0079] Example 3 Optimization of composite additive ratio

[0080] The experimenters continued to focus on solving a key contradiction in the mixed bed chromatography: rHSA needs to flow through efficiently at a pH close to its isoelectric point (pI ~ 4.7), but this condition is prone to induce aggregation of the target protein, resulting in loss of recovery rate. Therefore, in the present application, a composite additive system composed of sodium chloride, glycine and sorbitol is introduced, and the optimal ratio of the three is accurately optimized by the systematic experimental method of central composite design (CCD) combined with response surface analysis (RSM), while the robustness of the process is verified.

[0081] The experimental design included 20 groups of data, as shown in Table 3, which were specifically composed of 8 factorial points (covering high and low levels of sodium chloride 50-150 mM, glycine 5-20 mM, sorbitol 2.5-10%), 6 axial points (extended to boundary conditions, such as sodium chloride 29-171 mM, glycine 1.6-23.4 mM, sorbitol 0-12.5%), and 6 central points (fixed sodium chloride 100 mM, glycine 12.5 mM, sorbitol 6.25%, repeated 6 times to evaluate errors), all of which were consistent with the S8 step of Example 1 in terms of chromatographic operating conditions (column packing, pH, sample loading, etc.). The final recovery data range was 90.85%-95.60%, and the purity was maintained above 99.96%, of which the data points with purity > 99.99% were uniformly treated as 100% for subsequent response surface analysis.

[0082] Some experimental results are shown in Table 3 (wherein X1 is NaCl, X2 is glycine, and X3 is sorbitol).

[0083] Table 3 Experimental results

[0084]

[0085] Based on the experimental data, a multiple quadratic regression equation was obtained to predict the recovery rate (%): Y = 82.59 + 0.1289X1 + 0.3444X2 + 0.9657X3 - 0.0006X1 2 -0.0163X2 2 -0.0696X3 2 -0.0001X1X2 - 0.0001X1X3 + 0.0142X2X3 (wherein Y is the recovery rate, X1 is the concentration of sodium chloride, X2 is the concentration of glycine, and X3 is the concentration of sorbitol). The ANOVA test showed that the model P < 0.0001, R 2 = 0.9885, the goodness of fit was excellent, and the effects of each factor could be effectively quantified. The equation revealed that the coefficient of the first-order term of sorbitol was the largest, indicating that it contributed most significantly to the improvement of the recovery rate; the coefficient of the interaction term between glycine and sorbitol was positive 0.0142, confirming the synergistic effect between the two; the coefficient of the second-order term of sorbitol was negative -0.0696, indicating that the recovery rate would slow down when the concentration of sorbitol was too high. By solving the equation set with the first-order partial derivative equal to zero, the optimal conditions were obtained: NaCl about 107.4 mM, Gly about 10.6 mM, sorbitol about 6.9%, and the predicted maximum recovery rate was 95.6%

[0086] As Figure 3 and Figure 4As shown in the linear fitting verification diagram, the actual recovery rate of the 20 groups of experiments is closely distributed around the model prediction fitting line without obvious deviation, indicating excellent fitting and proving that the model can reliably describe the correlation between the complex additive concentration and the recovery rate. In the recovery rate contour map, the contour lines are obviously elliptical, directly reflecting the significant interaction between sorbitol and glycine, and the contour lines of glycine and sodium chloride are nearly circular, indicating that the interaction between sodium chloride and glycine is weak. The three-dimensional response surface graph presents that the surface first rapidly rises with the increase of sorbitol concentration, and then the rising trend slows down after 10%, and a stable high-value platform is formed in the interval of 10-18 mM of glycine and 6-10% of sorbitol, further verifying that the recovery rate can be stably maintained above 95% under this combination. Combined with the regression equation, the best process window for the recovery rate ≥95% is determined as 95-115 mM of sodium chloride, 10-13 mM of glycine, and 6.0%-8.0% (w / v) of sorbitol.

[0087] In summary, the mathematical modeling clearly quantifies the influence of each component of the complex additive on the recovery rate of rHSA, identifies the core role of sorbitol and the synergistic effect with glycine, solves the technical contradiction of easy aggregation near the isoelectric point, and also ensures the reliability of the process, laying a scientific foundation for the efficient implementation of mixed bed chromatography.

[0088] Example 4 Optimization of the ratio of mixed bed ion exchange fillers

[0089] To determine the optimal mass ratio of SCX and WAX exchange fillers in the mixed bed purification step, the following five groups of comparative experiments were set, with the remaining purification steps being the same as in Example 1, and the detection and result analysis being the same as in Test Example 1 except that the HCP detection sample was diluted 10 times:

[0090] Group 1 (SCX:WAX=0:1): only DEAE Sepharose FF filler was used to simulate a single WAX environment;

[0091] Group 2 (SCX:WAX=1.5:1): the mass ratio of SP to DEAE in the mixed filler was 1.5:1;

[0092] Group 3 (SCX:WAX=1:1): the mass ratio of SP to DEAE in the mixed filler was 1:1;

[0093] Group 4 (SCX:WAX=1:1.5): the mass ratio of SP to DEAE in the mixed filler was 1:1.5;

[0094] Group 5 (SCX:WAX=1:0): only SP Sepharose FF filler was used to simulate a single SCX environment.

[0095] All groups of fillers were packed in the same size of chromatography column (column height 15 cm), and the same batch of rHSA sample purified by the first seven steps (S1-S7 steps) was treated under the same conditions (buffer was 20 mM acetic acid-sodium acetate, and contained a complex additive system consisting of 115 mM NaCl, 13 mM glycine and 7.5% (w / v) sorbitol, pH 5.1), and the flow-through was collected and analyzed, and the results are shown in Table 4 below.

[0096] Table 4 Effect of different mixed bed filler ratios on purification (n = 3)

[0097]

[0098] Table 4 shows that when the SCX:WAX ratio is 1:1 (Group 3), the rHSA recovery rate is the highest at 95.56% and the purity is >99.99%, and no HCP is detected, proving that this ratio is the best choice.

[0099] Example 5 Pilot-scale verification

[0100] To verify the scalability of the purification method of the application, a pilot-scale purification experiment of 50 L Pichia pastoris fermentation broth was carried out, and the operation steps were scaled up based on Example 1. The key equipment and parameters were adjusted as follows, and the detection indicators and methods were the same as in Example 1:

[0101] S1 Pretreatment: 50 L of fermentation broth was centrifuged at 8000 rpm for 15 min at 4°C using a disc centrifuge (Shanghai Kaimaidi Separation Technology Co., Ltd.), the supernatant was added with sodium caprylate to 10 mM, and the pH was adjusted to 4.5 before using a plate heat exchanger (Jiangsu Maiander Group Co., Ltd., M3 type) with a heat exchange area of 0.5 m 2 (heat exchange area 0.5 m 2 ) heated at 65°C for 30 min, and then centrifuged at 10000 rpm for 15 min using a tubular centrifuge (Shanghai Ficair Separation Engineering Technology Co., Ltd., GF105 type), and the supernatant was filtered through a 0.45 μm filter (Hangzhou Kebaiter Filter Material Co., Ltd.), diluted to a conductivity of 12 mS / cm, and 80 L of clear liquid was obtained.

[0102] S2 Cation exchange chromatography: Lixie BC-200-1600 chromatography column, packed with 5 L of SP Sepharose Fast Flow filler, column height about 16 cm. Equilibrated with 5 CV of equilibration buffer (20 mM sodium acetate, pH 4.5) at a flow rate of 250 cm / h, gradient eluted after loading, and about 12 L of elution peak was collected, with a recovery rate of 89.26%.

[0103] S3 first ultrafiltration: using the UltraFlo-1000 system and 10 kDa molecular weight cut-off PES membrane package (membrane area 1 m 2 ), the S2 cation exchange eluate was concentrated 6 times to 2.2 L at 20±2℃, the transmembrane pressure was controlled at 1.0 bar and the flow rate was 2 L / min; then it was dialyzed and replaced with 8 volumes (16 L) of 20 mM Tris-HCl buffer (pH 7.5) until the conductivity of the permeate was consistent with the buffer (±0.5 mS / cm), the final recovery rate was 90.31%, the buffer replacement rate was >99.5%, and the qualified feed solution was provided for S4 anion exchange chromatography.

[0104] S4 anion exchange chromatography: a BC-150-1700 chromatography column from Life Science was selected, filled with 3 L of Q Sepharose FastFlow packing, and the column height was about 17 cm. After equilibration, the sample was loaded and eluted by gradient, and about 8 L of elution peak was collected with a recovery rate of 83.61%.

[0105] S5 second ultrafiltration: the same equipment as S3 was used, and the membrane package was pretreated with 0.1 M NaOH. The S4 anion exchange eluate was concentrated to 0.5 L at 20±2℃, the transmembrane pressure was controlled at 1.2 bar, and the flow rate was 1.5 L / min; it was dialyzed and replaced with 8 volumes (4 L) of 20 mM sodium phosphate buffer (pH 7.0) containing 2.0 M ammonium sulfate, and the replacement endpoint was confirmed by refractive index (1.3580±0.0005), the final recovery rate was 89.05%, and the ammonium sulfate concentration was stabilized at 1.9~2.1 M, meeting the high salt binding requirements of S6 hydrophobic chromatography.

[0106] S6 hydrophobic chromatography: a BC-100-0760 chromatography column from Life Science was selected, filled with 0.6 L of Phenyl Sepharose High Performance packing, and the column height was about 7.6 cm. After equilibration, the sample was loaded and eluted by gradient, and about 3 L of elution peak was collected with a recovery rate of 83.32%.

[0107] S7 third ultrafiltration: the same ultrafiltration system as above was still used, and the membrane package was equilibrated with a low ionic strength buffer. The S6 hydrophobic chromatography eluate was concentrated to 0.25 L at 20±2℃, the transmembrane pressure was controlled at 0.8 bar, and the flow rate was 1 L / min; it was dialyzed and replaced with 8 volumes (20 mM acetic acid-sodium acetate buffer (pH 5.0), until the permeate pH was 5.0±0.1 and the conductivity was 3.0±0.5 mS / cm, and the final recovery rate was 90.33%.

[0108] S8 mixed bed chromatography: a LiSorb BC-100-1900 multi-column series-parallel chromatography column, packed with 1.5 L of 1:1 mixed SP+DEAE filler, with a column height of about 19 cm. A 20 mM acetic acid-sodium acetate buffer (pH 4.9) containing 100 mM sodium chloride, 12 mM glycine and 7% (w / v) sorbitol as a complex additive was used as the equilibration and elution buffer. After equilibration with 5 CV, elution was continued with the same buffer containing the complex additive for 5 CV (flow rate 100 cm / h), and all flow-through and eluate (to the UV280 baseline) were collected, about 1 L was combined, and the recovery rate was 93.50%.

[0109] Pilot test results: The final product was detected to have a purity of >99.99%, no dimer was detected, HCP <2 ng / g, and endotoxin <0.5 EU / mL. The total recovery rate from S2 was 42.24%, proving that the process of the present application still has stable purification effect and recovery rate at the pilot scale, and is suitable for large-scale production.

[0110] Comparative Example 1: omit the mixed bed step

[0111] To verify the necessity and superiority of the mixed bed polishing step (S8) of the present scheme, the same batch of rHSA intermediate product purified by steps S1-S7 of Example 1 was taken, and instead of the mixed bed chromatography, a conventional gel filtration chromatography (GFC, also known as size exclusion chromatography) was used as the final polishing step. This comparative experiment ensured that all the previous purification bases were completely consistent except for the final polishing unit. Except that the HCP test sample was diluted 10 times, the rest was the same as Test Example 1:

[0112] The gel filtration chromatography used Sephacryl S-200 HR filler from Cytiva, which was packed in a chromatography column with a column height of 80 cm. Before use, the column bed was equilibrated with 5 column volumes (CV) of 0.01 M PBS (pH 7.0) at a flow rate of 100 cm / h until the UV280 absorbance and conductivity baseline were stable. After the above rHSA intermediate product was loaded, the same PBS was used for constant flow elution at a flow rate of 100 cm / h, and the rHSA monomer elution peak in 10 column volumes was collected as the final product.

[0113] The comparison results are shown in Table 5 below.

[0114] Table 5: Comparison results

[0115]

[0116] The final product detection results: purity 98.35%, HCP residue 1119.51 ng / g, endotoxin 0.20 EU / mL, single-step recovery rate 80%, and total recovery rate 42.16%. The results show that omitting the mixed bed step cannot obtain a high-purity product, and the recovery rate is significantly reduced.

[0117] Comparative Example 2 Anion / cation chromatography in series

[0118] The same batch of intermediates after purification by steps S1-S7 in Example 1 was taken, and instead of mixed bed polishing, two groups of conventional ion exchange chromatography in series steps were designed for comparison, wherein, except that the HCP detection sample was diluted 10 times, the rest was the same as Example 1:

[0119] Comparative Example 2a (cation first and then anion in series): the intermediates were first loaded into a SP Sepharose FF strong cation chromatography column, and the operating conditions were the same as Example 1 step S2. The recovery rate of this step was 90.50%. After collecting the elution peak, it was replaced into a buffer system suitable for anion exchange chromatography (20 mM Tris-HCl, pH 8.0) by ultrafiltration. The recovery rate of this ultrafiltration step was calculated as 95%. Then, the sample after liquid replacement was loaded into a Q Sepharose FF strong anion chromatography column, and the operating conditions were the same as Example 1 step S4. The recovery rate of this step was 85.23%. The cumulative recovery rate of the three steps was 73.28%.

[0120] Comparative Example 2b (anion first and then cation in series): the same intermediates were first loaded into a Q Sepharose FF strong anion chromatography column, and the operating conditions were the same as Example 1 step S4. The recovery rate of this step was 88.71%. After collecting the elution peak, it was replaced into a buffer system suitable for cation exchange chromatography (20 mM sodium acetate, pH 4.5) by ultrafiltration. The recovery rate of this ultrafiltration step was calculated as 95%. Then, the sample after liquid replacement was loaded into a SP Sepharose FF strong cation chromatography column, and the operating conditions were the same as Example 1 step S2. The recovery rate of this step was 86.15%. The cumulative recovery rate of the three steps was 72.60%. The final product purification effect and recovery rate of the two groups of comparative examples were significantly lower than Example 1, and the specific data are shown in Table 6.

[0121] Table 6 Comparison results

[0122]

[0123] The results show that even if the same two fillers are used, the traditional series mode cannot simultaneously remove acidic and basic impurities in one step, and the process is more lengthy, and the loss of target protein increases in the process of multiple pH adjustment and adsorption-elution. Therefore, the mixed bed one-step flow-through mode is more efficient and mild than the traditional series mode.

[0124] Comparative Example 3

[0125] The same batch of intermediates purified in steps S1-S7 of Example 1 was divided into two parts, and each part was subjected to mixed bed polishing. Except that the HCP detection sample was diluted 10 times, the rest was the same as Test Example 1.

[0126] Comparative Example 3a: pH deviated from 4.5-5.5: mixed bed operation was performed using 20 mM Tris-HCl buffer at pH 7.0. Under this condition, rHSA was negatively charged and would be adsorbed by the anion exchange filler in the mixed bed, which could not realize flow-through, resulting in a sharp decrease in single-step recovery rate to 35.14%.

[0127] Comparative Example 3b: no addition of composite additives: in the buffer system at pH 5.0, no composite additives composed of sodium chloride, glycine and sorbitol were added. Under this condition, the aggregation tendency of rHSA in the environment close to its isoelectric point was intensified, resulting in a decrease in single-step recovery rate to 72.14%, and the total recovery rate calculated therefrom was also greatly reduced to 38.02%, and the content of aggregates in the product increased.

[0128] The comparison results are shown in Table 7 below.

[0129] Table 7 Comparison results

[0130]

[0131] The results show that under the condition of pH deviated from the isoelectric point (pI ~ 4.7) of rHSA, the effect of mixed bed polishing is significantly decreased. The final product purity of Comparative Examples 3a and 3b is 98.25% and 99.50%, respectively, and the HCP residual is 334.48 and 280.68 ng / g, respectively, and the total recovery rate is also greatly reduced to 18.52% and 38.02%.

[0132] Comparative Example 4

[0133] In order to explore the applicability of the strength combination of ion exchange fillers in the mixed bed, this comparative example evaluated four different filler combinations under the isoelectric point flow-through mode of rHSA. All experiments used the same batch of rHSA intermediates after purification in steps S1-S7 of Example 1, and except for the type of mixed bed filler, the operation conditions such as column packing, equilibration, sample loading and elution were exactly the same as the step S8 of Example 1. Except that the HCP detection sample was diluted 10 times, the rest was the same as Test Example 1.

[0134] Comparative Example 4a: strong anion exchange filler (Q Sepharose FF) + weak cation exchange filler (CMSepharose FF), mass ratio 1:1.

[0135] Comparative Example 4b: Weak anion exchange packing material (DEAE Sepharose FF) + weak cation exchange packing material (CM Sepharose FF), mass ratio 1:1.

[0136] Comparative Example 4c: Strong anion exchange packing material (Q Sepharose FF) + strong cation exchange packing material (SP Sepharose FF), mass ratio 1:1.

[0137] The final product testing results are shown in Table 8.

[0138] Table 8 Comparison Results

[0139]

[0140] As shown in Table 8, combinations of packing materials with different ion exchange strengths exhibited significant performance differences in near-isoelectric point flow-through mode. Specifically, in Comparative Example 4a (weak cation + strong anion), the strong anion packing material still strongly adsorbed the slightly negatively charged rHSA at pH 5.0, resulting in a significant decrease in the target protein recovery rate to 80.24%. Simultaneously, the weak cation packing material, due to insufficient ionization at pH 5.0, had insufficient adsorption capacity for alkaline impurities, leading to an increase in the residual host cell protein content to 162.88 ng / g. While Comparative Example 4b (weak cation + weak anion) achieved a higher recovery rate of 89.55%, the functional limitations of the weak cation packing material resulted in insufficient removal efficiency for alkaline charged isomers; both the product purity (97.63%) and the residual host cell protein content (266.15 ng / g) failed to meet the optimal standards. In Comparative Example 4c (strong cation + strong anion), because both packing materials have strong electrostatic adsorption capabilities, rHSA was significantly adsorbed onto the chromatography column, resulting in a sharp drop in single-step recovery rate to 77.71%. This does not meet the requirements for industrial production.

[0141] In contrast, the "strong cation + weak anion" combination used in this scheme fully utilizes the complete ionization characteristics of the strong cation packing material in a slightly acidic environment, ensuring efficient removal of alkaline impurities. At the same time, the moderate charge characteristics of the weak anion packing material can effectively capture acidic impurities while avoiding excessive interaction with the target protein. Thus, while maintaining a high recovery rate of 95.63%, it achieves an ultra-high purity of >99.99% and a host cell protein residue of less than 2 ng / g.

[0142] Comparative Example 5

[0143] The experimenters explored the best purification step sequence of the mixed bed chromatography. The following three sets of comparative experiments are used to illustrate. All experiments use the same batch of fermentation broth as Example 1, and except for the position of the mixed bed step, the operating conditions of the step itself, including fillers, pH, buffer, composite additives, etc. are consistent with the S8 step of Example 1.

[0144] Comparative Example 5a: Mixed bed step placed at the very beginning of the purification process:

[0145] The pretreated clarified fermentation broth was directly loaded. Since the initial sample composition is extremely complex, containing a large amount of host cell proteins (HCP), nucleic acids and other charged impurities, far exceeding the adsorption capacity of the mixed bed filler, the chromatography column is quickly saturated. The results are in contrast to Example 1: the purity and recovery rate of rHSA are both sharply decreased. This comparative example 5a is terminated only at this step, and the mixed bed single-step recovery rate is as low as 30.16%. The results show that using a mixed bed at the beginning of purification not only cannot achieve effective purification, but also will cause serious loss of target product and waste of filler due to impurity overload, proving that this scheme is not feasible.

[0146] Comparative Example 5b: Mixed bed step placed after cation exchange chromatography (step S2):

[0147] After completing the cation exchange capture, the elution product is ultrafiltrated to a buffer system with pH 5.0, and then directly subjected to mixed bed purification, and then the subsequent purification steps including anion exchange and hydrophobic chromatography are continued to complete the entire purification process. This route obtains the final product. However, since the mixed bed step is performed, the sample still contains a large amount of host proteins, aggregates and hydrophobic impurities, which not only affect the recovery rate of the mixed bed step itself, but more importantly, some steps after the mixed bed, such as hydrophobic chromatography, may introduce new charge isomers or aggregates, and there are no subsequent purification steps in the process to remove them. Therefore, the purity and total recovery rate of the final product are both significantly lower than Example 1.

[0148] Comparative Example 5c: Mixed bed step placed after anion exchange chromatography (step S4):

[0149] After anion exchange chromatography, the eluted product was ultrafiltrated into a buffer system of pH 5.0, followed by mixed bed polishing, and then hydrophobic chromatography purification step was continued to complete the whole purification process. The final product was also obtained by this route. At this time, the sample has removed most of the strong acidic and strong basic impurities, so the mixed bed single step recovery rate and the purity of the final product are higher than those of Comparative Example 5b. However, since the hydrophobic chromatography step is performed after the mixed bed, the operation process can cause partial rHSA conformational changes or aggregation, resulting in the generation of new charge isomers, and these newly generated impurities cannot be removed, thereby limiting the further improvement of the final purity. The total recovery rate is also still lower than that of Example 1.

[0150] The results are shown in Table 9.

[0151] Table 9 Comparison results

[0152]

[0153] According to the results in Table 9, it can be seen that placing the mixed bed chromatography at the end of the process flow as the final polishing step is the optimal and irreplaceable solution to achieve ultra-high purity and high total recovery rate of rHSA. Only in this position, the mixed bed can effectively remove all charge isomers generated and remaining in the previous steps, while avoiding secondary contamination of the purified product in subsequent steps.

[0154] The above only describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for purifying a recombinant human serum albumin, characterized by, The purification method comprises the following steps: 1) pretreating a fermentation broth containing recombinant human serum albumin; 2) cation exchange chromatography; 3) anion exchange chromatography; 4) hydrophobic chromatography; 5) subjecting the product liquid obtained in step 4) to ultrafiltration, wherein the buffer used in the ultrafiltration is a weak ionic strength buffer at pH 5.0-5.5, and then performing a mixed-bed ion exchange purification step; In the mixed-bed ion exchange purification step: the mixed-bed chromatography is performed in a buffer system at pH 4.7-5.3; the mixed-bed chromatography uses WAX filler and SCX filler, and the mass ratio of the WAX filler to the SCX filler is 1:(0.8-1.2); the buffer system contains a composite additive, and the composite additive consists of 50-150 mM sodium chloride, 10-20 mM glycine and 5-10% (w / v) sorbitol; The purity of the product obtained after purification is >99.99%, the residual host cell protein is <2 ng / g rHSA, and the endotoxin is <0.5 EU / mL.

2. The method of purifying recombinant human serum albumin according to claim 1, characterized in that, The mixed-bed chromatography is performed in a buffer system at pH 5.0±0.

1.

3. The method of purifying recombinant human serum albumin according to claim 1, characterized in that, The mass ratio of the WAX filler to the SCX filler is 1:

1.

4. The method of purifying recombinant human serum albumin according to claim 1, wherein, The WAX filler is a diethylaminoethyl DEAE group filler; The SCX filler is a sulfopropyl SP group filler.

5. The method of purifying recombinant human serum albumin according to claim 1, characterized in that, In step 5), the ultrafiltration uses an ultrafiltration membrane with a molecular weight cut-off of 10-30 kDa, and the volume of the buffer used in the ultrafiltration is 5-10 times the volume of the product liquid.

6. The method of purifying recombinant human serum albumin according to claim 1, characterized in that, Step 1) comprises the step of centrifuging after heating the fermentation broth, and the heating treatment conditions are 60-80℃ for 10-30 min, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10-20 min.

7. The method of purifying recombinant human serum albumin according to claim 1, wherein, In step 2), the filler for cation exchange chromatography is a sulfopropyl SP group filler; In step 3), the filler for anion exchange chromatography is a quaternary ammonium Q group filler; In step 4), the ligand for hydrophobic chromatography is selected from one or more of phenyl, aliphatic and heterocyclic groups.

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