Method for purifying recombinant human serum albumin

Abstract

The invention relates to a purification method of recombinant human serum albumin, and belongs to the technical field of protein separation and purification. A strong cation exchange filler and a weak anion exchange filler are adopted and mixed according to a specific proportion for chromatography of a mixed bed for fine purification, preferably, high-efficiency flow-through recovery is realized under the condition that the pH is 4.5-5.5, and acidic impurities and alkaline impurities are synchronously and deeply removed by the strong cation exchange filler and the weak anion exchange filler respectively; according to the present invention, the host cell protein is used as the raw material, and the composite additive system containing sodium chloride, glycine and sorbitol is matched, such that the high recovery rate of the target protein is ensured, the aggregation tendency of the target protein in the near isoelectric point environment is effectively inhibited, and the host cell protein, the endotoxin, the aggregate, the charge isomer and other impurities can be efficiently removed, the final product purity is gt; the key impurity residue is lower than the detection limit, the process robustness is high, and the method is suitable for large-scale industrial production.

Description

Technical Field

[0001] This application relates to a purification method for recombinant human serum albumin, belonging to the field of protein separation and purification technology. Background Technology

[0002] Human serum albumin (HSA), the most abundant functional protein in human plasma, plays an irreplaceable role in physiological processes such as maintaining colloid osmotic pressure, substance transport, and antioxidant defense. Due to its excellent biocompatibility and stability, HSA has been widely used in clinical infusions, cell culture, vaccine adjuvants, and drug delivery systems. Currently, clinical HSA mainly relies on human plasma extraction, a method limited by plasma supply shortages, potential pathogen contamination risks, and ethical controversies. The preparation of recombinant human serum albumin (rHSA) using recombinant DNA technology is considered a fundamental solution to these problems.

[0003] However, rHSA is typically administered at high doses in clinical treatment, with each injection reaching 5 to 30 grams, far exceeding that of conventional recombinant drugs. Therefore, trace impurities remaining in the product may pose significant immunogenicity risks or other side effects. Thus, it is essential that the total host protein (HCP) residue and contaminants from the manufacturing process not exceed 1 ng / ml per injection dose to meet pharmaceutical safety requirements.

[0004] Existing technologies typically employ multi-step chromatographic combinations to achieve initial purification of rHSA. For example, patent CN202011105562.4 obtains rHSA with a purity of 99.97% through a five-step chromatographic process. However, such processes generally face a common bottleneck: trace amounts of HCP and charge-heterogeneous albumin remain after the main purification, forming a complex impurity system containing acidic, basic components, and other charge variants. If a single ion-exchange chromatography is used, it can only selectively remove impurities of a certain charge attribute, failing to achieve comprehensive purification. While simply cascading multiple chromatography steps may improve purity to some extent, it inevitably leads to lengthy processes, decreased yields, and increased costs, making it difficult to meet the dual demands of efficiency and economy in industrial production.

[0005] Mixed-bed ion exchange chromatography theoretically possesses the ability to simultaneously remove both anionic and cationic impurities. Since its early exploration by Rassi and Horváth et al. (J. Chromatogr. 1986, 359, 255-264), this technique has recently regained attention in specific separation scenarios. However, existing mixed-bed technologies are mostly used for analytical detection or small molecule purification. Applying mixed-bed chromatography to the industrial purification of rHSA requires solving a key technical challenge: how to construct a mixed-bed system capable of achieving highly selective separation, ensuring efficient adsorption of various charged impurities such as HCP and heterogeneous albumin while guaranteeing near-complete recovery of the target rHSA monomer. Existing mixed-bed technologies generally lack the ability to finely control this selective separation mechanism, preventing their application in rHSA purification.

[0006] Therefore, how to simultaneously remove trace amounts of 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 below 5 ng / g, without sacrificing the recovery rate of the final product, is a topic worthy of in-depth research. A search revealed that there are few studies on the use of mixed-bed ion exchange chromatography in the purification of recombinant human serum albumin, and existing purification strategies and mixed-bed technologies have failed to effectively solve this dual challenge. Summary of the Invention

[0007] To address the aforementioned issues, a purification method for recombinant human serum albumin is provided. This application employs mixed-bed chromatography with strong cation exchange and weak anion exchange packing materials mixed in a specific ratio for purification. Highly efficient flow-through recovery is achieved preferably at pH 4.5–5.5. Basic and acidic impurities are simultaneously and deeply removed by the strong cation exchange and weak anion exchange packing materials, respectively. Furthermore, a composite additive system containing sodium chloride, glycine, and sorbitol works synergistically to ensure high recovery of the target protein while effectively inhibiting its aggregation tendency in near-isoelectric point environments. This method can efficiently remove various impurities such as host cell proteins, endotoxins, aggregates, and charge isomers, resulting in a final product purity >99.99%, with key impurity residues below the detection limit. The process is robust and suitable for large-scale industrial production.

[0008] This application provides a method for purifying recombinant human serum albumin, characterized in that the purification method includes the following steps: 1) Pretreatment of fermentation broth containing recombinant human serum albumin; 2) Cation exchange chromatography; 3) Anion exchange chromatography; 4) Hydrophobic chromatography; 5) The product solution obtained in step 4) is subjected to ultrafiltration. The buffer solution used in ultrafiltration is a weak ionic strength buffer solution with pH 5.0~5.5. Then, a mixed bed ion exchange purification step is performed. In the mixed-bed ion exchange purification step: the mixed-bed chromatography is carried out in a buffer system with a pH of 4.5-5.5; the mixed-bed chromatography uses WAX packing material and SCX packing material, and the mass ratio of WAX packing material to SCX packing material is 1:(0.67-1.5); the buffer system contains a composite additive, which includes 50-150 mM sodium chloride, 10-20 mM glycine and 5-10% (w / v) sorbitol.

[0009] Optionally, the mixed-bed chromatography is carried out in a buffer system with a pH of 4.7 to 5.3.

[0010] Optionally, the mixed-bed chromatography is performed in a buffer system at pH 5.0.

[0011] Optionally, the mass ratio of the WAX ​​packing to the SCX packing is 1:(0.8~1.2).

[0012] Optionally, the mass ratio of the WAX ​​packing to the SCX packing is 1:1.

[0013] Optionally, the WAX ​​packing is a diethylaminoethyl DEAE group packing; The SCX packing is a sulfopropyl SP group packing.

[0014] Optionally, in step 5), an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa is used, and the volume of the buffer solution used in ultrafiltration is 5-10 times the volume of the product solution.

[0015] Optionally, step 1) includes heating the fermentation broth and then centrifuging it. The heating conditions are 60~80℃ for 10~30 min, the centrifugation speed is 8000~10000 rpm, and the centrifugation time is 10~20 min.

[0016] Optionally, the packing material for cation exchange chromatography in step 2) is a sulfopropyl SP group packing material; The packing material for the anion exchange chromatography in step 3) is a quaternary ammonium group Q group packing material; In step 4), the ligand for hydrophobic chromatography is selected from one or more of phenyl, aliphatic, and heterocyclic compounds.

[0017] Optionally, the weak ionic strength buffer is an acetate-sodium acetate buffer.

[0018] Optionally, the purified product has a purity >99.99%, host cell protein residue <2 ng / g rHSA, and endotoxin <0.5 EU / mL.

[0019] The beneficial effects of this application include, but are not limited to: 1. According to the purification method of recombinant human serum albumin in this application, the scheme of this application adopts mixed bed chromatography with strong cation exchange and weak anion exchange packing materials mixed in a specific ratio for purification. It is preferred to achieve efficient flow-through recovery under pH 4.5~5.5 conditions, and acidic impurities and basic impurities are simultaneously and deeply removed by strong cation exchange and weak anion exchange packing materials, respectively.

[0020] 2. According to the purification method of recombinant human serum albumin of this application, combined with a compound additive system containing sodium chloride, glycine and sorbitol, the three work synergistically to ensure a high recovery rate of the target protein while effectively inhibiting its aggregation tendency in a near isoelectric point environment.

[0021] 3. The purification method for recombinant human serum albumin according to this application provides a complete mixed bed chromatography strategy, which is particularly aimed at solving the problem of deep removal of charge isoform HCP and charge heterogeneous albumin in the purification stage. It can achieve a leap in product purity without increasing the loss of target protein.

[0022] 4. The purification method for recombinant human serum albumin according to this application can efficiently remove various impurities such as host cell proteins, endotoxins, aggregates and charge isomers, with the final product purity >99.99%, key impurity residues below the detection limit, and the process is robust and suitable for large-scale industrial production.

[0023] 5. According to the purification method of recombinant human serum albumin of this application, the acidic and basic charge isomers can be removed simultaneously and deeply through the synergistic effect of the mixed bed purification step and the composite additive system, so that the purity of the final product is >99.99% and the residual amount of key impurities such as host cell protein (HCP) is less than 2 ng / g rHSA.

[0024] 6. The purification method for recombinant human serum albumin according to this application has mild and precisely optimized process conditions. In particular, in the core purification step, by precisely controlling the pH at the isoelectric point and using composite additives, the non-specific adsorption and loss of target protein are minimized while achieving deep removal of impurities. The recovery rate of the mixed bed step is greater than 95%, and the overall process recovery rate is industry-leading.

[0025] 7. The purification method for recombinant human serum albumin according to this application has a scientifically designed process flow with good repeatability. The "SCX+WAX" packing material combination and composite additive system effectively improve the operating window and error tolerance of the purification step, reduce the performance risk caused by small fluctuations in materials and parameters, and make the process exhibit excellent repeatability and reliability, which is very suitable for large-scale industrial production.

[0026] 8. The purification method for recombinant human serum albumin according to this application integrates 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, resulting in products of excellent quality that meet stringent pharmaceutical standards. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a flowchart of the purification process for recombinant human serum albumin (rHSA) involved in this application; Figure 2 This is the HPLC purity detection chromatogram of the final rHSA product involved in Example 1 of this application; Figure 3 The following is a contour plot showing the effect of the composite additive system involved in Example 3 of this application on the recovery rate of rHSA (A is the recovery rate contour plot when Sorb is fixed at 6.2%, and B is the recovery rate contour plot when NaCl is fixed at 100.0 mM). Figure 4 The graphs shown are the response surface and linear fitting analysis of the actual and predicted values ​​of the composite additive system involved in Example 3 of this application on the recovery rate of rHSA (A is the three-dimensional surface plot of the response surface of the recovery rate, and B is the linear fitting verification graph of the actual and predicted values ​​of the recovery rate). Detailed Implementation

[0028] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0029] In this invention, the method for producing recombinant human serum albumin by Pichia pastoris fermentation can be a conventional method in the art, and will not be described in detail here. For example, you can refer to "Qian Lisheng, Jin Guangming. Comprehensive Experimental Training Tutorial for Bioengineering [M], Anhui Science and Technology Press, 2018: 150-152" or patent CN102190722A.

[0030] Example 1 This embodiment provides a method for purifying rHSA from Pichia pastoris fermentation broth, the flowchart of which is shown below. Figure 1 As shown, the specific steps include: S1. Fermentation broth pretreatment: The yeast fermentation broth expressing recombinant human albumin was centrifuged at 4℃ and 8000 rpm for 15 min, and the supernatant was collected. Sodium caprylate was added to the supernatant to a final concentration of 10 mM, and 1M HCl solution was slowly added while stirring to adjust the pH to 4.5. The mixture was heated at 65℃ for 30 min, rapidly cooled to room temperature, and centrifuged at 10000 rpm for 15 min. This centrifugation was repeated twice, and the supernatant was collected and filtered through a 0.45 µm filter membrane for sample loading. Subsequently, ultrapure water was added to dilute the solution until the conductivity was ≤15 mS / cm to obtain the pretreated sample.

[0031] S2, cation exchange trapping: An XK 50 / 60 chromatography column (Cytiva) packed with 500 mL SP Sepharose Fast Flow packing material was used. First, the column bed was equilibrated with 5 CV of 20 mM sodium acetate buffer (pH 4.5) at a flow rate of 250 cm / h. The pretreated clarified sample was pumped in with the loading direction aligned with the equilibration direction for fixed-bed adsorption. After adsorption, the column was washed directly with equilibration buffer for 5 CV until the UV baseline stabilized. Elution was then performed using a linear gradient of 0–500 mM NaCl (20 mM sodium acetate, pH 4.5) at a gradient volume of 20 CV and a flow rate of 200 cm / h. The main rHSA elution peak was collected by UV monitoring.

[0032] S3, First Ultrafiltration Fluid Replacement: The eluent was concentrated and replaced using a Pellicon 3 ultrafiltration membrane pack (Merck Millipore) with a molecular weight cutoff of 10 kDa. The sample was first concentrated to approximately 200 mL, and then dialyzed 8 times its volume by continuously adding 20 mM Tris-HCl buffer (pH 8.0) until the conductivity of the effluent was consistent with that of the added buffer.

[0033] S4, Anion Exchange Chromatography: After adjusting the conductivity of the ultrafiltered sample to 5 mS / cm, it was loaded onto a HiScale 50 / 40 chromatography column (Cytiva) packed with Q Sepharose Fast Flow packing material, with a column height of 15 cm. The column was first equilibrated and washed to baseline with 5 CV of 20 mM Tris-HCl (pH 8.0) at a flow rate of 150 cm / h. Elution was then performed using a linear gradient of 0-500 mM NaCl (20 mM Tris-HCl, pH 8.0), with a gradient volume of 15 CV, and the rHSA elution peak was collected.

[0034] S5, Second Ultrafiltration Fluid Replacement: The ultrafiltration system was used again to replace the anion exchange elution peak with 8 times the volume of 20 mM sodium phosphate buffer (pH 7.0) containing 2.0 M ammonium sulfate, and then concentrated to approximately 50 mL.

[0035] S6. Hydrophobic chromatography: High-salt samples were loaded onto an XK 26 / 40 (Cytiva) chromatography column packed with Phenyl Sepharose High Performance packing material, with a column height of 10 cm. Equilibration was performed using 5 CV of 20 mM sodium phosphate and 2.0 M ammonium sulfate (pH 7.0) at a flow rate of 150 cm / h. Elution was carried out 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 and a flow rate of 150 cm / h, and the rHSA elution peak was collected.

[0036] S7, Third Ultrafiltration Fluid Replacement: The hydrophobic chromatography eluent was ultrafiltered and replaced with 20 mM acetate-sodium acetate buffer (pH 5.0). The dialysis volume was 8 times the sample volume, and the sample volume was concentrated to approximately 25 mL.

[0037] S8, Mixed-bed ion exchange purification: Mixed-bed chromatography was performed in a 20 mM acetate-sodium acetate buffer system at pH 5.0, with the addition of a composite additive system consisting of 107 mM sodium chloride, 11 mM glycine, and 7% (w / v) sorbitol. The required masses of SP Sepharose FF (particle size 45–165 μm) and DEAE Sepharose FF (particle size 45–165 μm) dry powder packing materials were mixed in a 1:1 ratio, and 5 volumes of buffer containing the composite additive were added to prepare the slurry. The mixture was then sonicated for 3 minutes to ensure thorough dispersion. The slurry was then packed into an HR50 / 20 (Cytiva) column at 200 psi, with a column height of 15 cm, ensuring a homogeneous and compact bed. Equilibration was performed with 5 CV of 20 mM acetate-sodium acetate (pH 5.0) buffer containing the composite additive at a flow rate of 100 cm / h. After loading the sample obtained in S7, continue elution with the same equilibration buffer containing composite additives for 5 CV (flow rate 100 cm / h). Collect all flow-through and eluent (to the UV280 baseline), and combine them to obtain a high-purity rHSA product with a purity >99.99%. Figure 2 The image shows the HPLC purity detection chromatogram of the rHSA product. The first detected target component peak is rHSA, with a retention time of 13.963 min.

[0038] Test Example 1 In Example 1, the final product showed a purity >99.99% as determined by HPLC (Tosoh column, TSKgel G3000SWXL). Host protein residue (HCP) was detected using ELISA (Cygnus Pichia pastoris HCP ELISA Kit, Cat# F140), with a detection limit of less than 0.4 ng / mL, indicating no HCP was detected. Endotoxin was detected using the horseshoe crab reagent dynamic turbidimetric assay, with an endotoxin concentration <0.5 EU / mL. The purity remained relatively stable during ultrafiltration. The overall recovery rate was 50.39%, calculated by multiplying the recoveries of each step starting from the cation exchange capture step (S2). This is higher than typical purification methods, such as patent CN202011105562.4, which showed a recovery rate of 30.7% and a purity of 99.97% after five steps of chromatography.

[0039] The specific testing methods and results analysis are as follows.

[0040] Purity determination: Gel filtration HPLC was performed using a Tosoh TSKgel G3000SWXL column (7.8 mm × 300 mm), a mobile phase of 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0), a flow rate of 1.0 mL / min, a column temperature of 30℃, a detection wavelength of 280 nm, and an injection volume of 20 μL. The sample was diluted to 1 mg / mL with the mobile phase, and the chromatogram was recorded after injection. Purity was calculated as the ratio of main peak area to total peak area × 100%. Based on the gel filtration HPLC analysis, no dimers or high molecular weight polymers were detected in the sample at levels exceeding the method detection limit (0.01%). The chromatographic purity of the target monomer, calculated using the main peak area normalization method, was greater than 99.99%. This indicates that the product has extremely high purity, and the content of potential size-related impurities is below the method's limit of quantitation.

[0041] HCP detection (ELISA method): The Cygnus Technologies Pichia pastoris HCP ELISA kit (Cat# F140) was used to detect Pichia pastoris-derived host cell protein (HCP) residues using a two-site sandwich immunoenzyme assay. The procedure was strictly followed according to the standard protocol in the kit instructions. The limit of quantitation (LOQ) of this protocol is 0.4 ng / mL, and the limit of detection (LOD) is less than 0.3 ng / mL.

[0042] The simplified steps are as follows: Equilibrate the reagents to room temperature and dilute the 20× wash buffer with distilled water. Dilute the provided HCP standard in a gradient of 0, 1, 4, 20, and 75 ng / mL (two replicates per concentration). Dilute the test sample 100-fold with the purification intermediate after step S7 in the accompanying SampleDiluent (Cat# I028), and leave the sample undiluted after step S8. Add 25 μL of standard / sample / blank control and 100 μL of HRP-labeled detection antibody to each well, seal the plate, and incubate at room temperature with shaking at 400–600 rpm for 3 hours. Wash manually four times (350 μL of wash buffer per well each time), pat dry, add 100 μL of TMB chromogenic solution, incubate at room temperature in the dark for 30 minutes, then add 100 μL of stop solution. Measure the absorbance at 450 nm / 650 nm dual wavelengths within 30 minutes.

[0043] The standard curve y = 1.376404 + (0.08657905 - 1.376404) / (1 + (x / 5.268261)^1.050758) was fitted using four-parameter logistic regression (4-PL). 2=0.9992). The HCP concentration in the sample was calculated based on the fitted curve and multiplied by a dilution factor of 100 (intermediate purified after step S7). The calculated concentration corresponding to the absorbance value of the purified rHSA product was lower than the limit of quantitation (LOQ < 0.4 ng / mL) of this method. Based on the concentration of the rHSA product (200 mg / mL), the residual HCP was < 2.0 ng / g, indicating that the host protein residue was efficiently removed.

[0044] Endotoxin detection (Limulus Amebocyte Lysate (LIL) colorimetric method): Endotoxin was detected using an endotoxin detection kit manufactured by Beyotime (Beyotime, Cat#C0276S). According to the instructions, the rHSA sample obtained in Example 1 was dissolved in endotoxin test water and diluted until its expected endotoxin value was within the range of the standard curve. Background absorbance was 0.18 (<0.5), with only sample blank tubes used for background subtraction. During reagent preparation, the 20 EU / vial endotoxin standard from the kit was dissolved in 1 mL of BET water and vortexed for 3 minutes to prepare a 20 EU / ml stock solution. This stock solution was then gradually diluted to 0.100, 0.050, 0.025, and 0.010 EU / ml gradient standard solutions. Simultaneously, the endotoxin detection reagent and chromogenic agent were dissolved, and the reaction stop solution and Buffer A and Buffer B / C were prepared. In setting up the detection system, 10 μL of standard solution, sample solution, or BET water (negative control) was added to each well of a 96-well plate, followed by 10 μL of detection reagent. The plate was incubated at 37°C in the dark for 25 minutes (T1), then 10 μL of chromogenic agent was added and incubated for 6 minutes (T2). 50 μL of Buffer A, B, and C were added sequentially, and the absorbance was read at 545 nm after standing for 5 minutes. Data processing yielded the standard curve equation y = 5.6385x + 0.1829, R0. 2 =0.9932, the recovery rate of the interference experiment was 68% (50%~200%, no interference). Due to the high endotoxin content in the samples during the early purification stage, samples in steps S2~S5 were diluted 100-fold during detection to ensure the detection values ​​fell within the linear range of the standard curve, and samples in steps S6~S8 were diluted 10-fold. The endotoxin concentrations listed in Table 1 below are all original solution concentrations converted from the corresponding dilution factors. The absorbance of the diluted sample S8 was 0.210, corresponding to a concentration of 0.0046 EU / ml, which translates to an original sample endotoxin concentration of 0.050 EU / ml, consistent with the Pharmacopoeia of the People's Republic of China (2020 edition).

[0045] Recovery rate calculation: Starting from the cation exchange capture step (S2), the recovery rate for each step is calculated as (total rHSA in this step / total rHSA in the previous step) × 100%. The total recovery rate is the product of the recovery rates of each step, which is 50.39%.

[0046] The test results for each step of Example 1 in this test case are shown in Table 1 below.

[0047] Table 1 Detection results at each step

[0048] Example 2: Screening of pH conditions in a mixed bed To investigate the optimal pH for the mixed-bed purification step, three sets of comparative experiments were set up. To ensure sufficient buffer capacity over a wide pH range, the buffer pH was 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 and 5.5 (all buffers were 50 mM acetate-sodium acetate and contained a composite additive system consisting of 110 mM NaCl, 12 mM glycine and 7.5% (w / v) sorbitol). The remaining steps were the same as in Example 1. The results are shown in Table 2 below.

[0049] Table 2. Effect of buffer pH on the purification efficiency of mixed-bed chromatography (n=3)

[0050] The results showed that the process of this invention could achieve effective purification within the pH range of 4.5-5.5. However, the optimal balance between recovery and purity was achieved within the pH range of 4.7-5.3, which can be considered the robust operating window of this process. Specifically, within the core pH range of 4.9-5.1, the single-step recovery rate of rHSA was the highest (>95%), and the product purity remained stable at >99.99%. As the pH deviated to either side, the increased net charge carried by rHSA enhanced the non-specific adsorption of the oppositely charged packing material in the mixed bed, leading to a regular and symmetrical decrease in recovery rate. Simultaneously, the adsorption efficiency of the packing material for impurities with specific charge properties changed, resulting in a gradual decrease in product purity. In summary, the preferred pH range for the mixed bed purification step is 5.0 ± 0.3, and the optimal range is 5.0 ± 0.1.

[0051] Example 3: Optimization of Composite Additive Ratio Researchers continued to focus on resolving a key challenge in mixed-bed chromatography: rHSA requires efficient flow-through at a pH close to its isoelectric point (pI ~ 4.7), but this condition easily induces target protein aggregation, leading to a loss of recovery. To address this, this application introduces a composite additive system consisting of sodium chloride, glycine, and sorbitol. The optimal ratio of these three components was precisely optimized using a central composite design (CCD) approach combined with response surface methodology (RSM), while simultaneously verifying the robustness of the process.

[0052] The experimental design included 20 sets of data, as shown in Table 3. Specifically, it consisted of 8 factorial sites (covering high and low levels of sodium chloride 50–150 mM, glycine 5–20 mM, and sorbitol 2.5–10%), 6 pivot points (extending to boundary conditions, such as sodium chloride 29–171 mM, glycine 1.6–23.4 mM, and sorbitol 0–12.5%), and 6 center points (fixed at 100 mM sodium chloride, 12.5 mM glycine, and 6.25% sorbitol, repeated 6 times to assess error). All chromatographic operating conditions (column packing, pH, loading volume, etc.) were consistent with step S8 of Example 1. The final recoveries ranged from 90.85% to 95.60%, with purity maintained above 99.96%. For ease of subsequent response surface methodology, data points with purity >99.99% were uniformly counted as 100%.

[0053] Some of the experimental results are shown in Table 3 below (where X1 is NaCl, X2 is glycine, and X3 is sorbitol).

[0054] Table 3 Experimental Results

[0055] Based on experimental data, the predicted recovery rate (%) of the multiple quadratic regression equation was obtained: Y = 82.59 + 0.1289X1 + 0.3444X2 + 0.9657X3 - 0.0006X1 2 -0.0163X2 2 -0.0696X3 2 -0.0001X1X2-0.0001X1X3+0.0142X2X3 (where Y is the recovery rate, X1 is the sodium chloride concentration, X2 is the glycine concentration, and X3 is the sorbitol concentration). ANOVA analysis showed that this model had P < 0.0001 and R < 0.0142X2X3. 2 =0.9885, indicating excellent goodness of fit and effective quantification of the influence of various factors. The equation reveals that sorbitol has the largest first-order coefficient, indicating its most significant contribution to improving the recovery rate; the interaction coefficient between glycine and sorbitol is positive (0.0142), confirming a synergistic effect between the two; the quadratic coefficient of sorbitol is negative (-0.0696), indicating that the recovery rate improvement trend slows down when its concentration is too high. By solving the system of equations with zero first-order partial derivatives, the optimal conditions can be obtained: NaCl approximately 107.4 mM, Gly approximately 10.6 mM, and sorbitol approximately 6.9%, predicting a maximum recovery rate of 95.6%. like Figure 3 and Figure 4As shown in the linear fitting verification graph, the actual recovery rate scatter points of the 20 experimental groups closely follow the model's predicted fitted line distribution, with no significant deviation, indicating excellent fitting and proving that the model can reliably describe the correlation between the concentration of the composite additive and the recovery rate. In the recovery rate contour plot, the contour lines are clearly elliptical, directly reflecting the significant interaction between sorbitol and glycine. The contour lines for glycine and sodium chloride are nearly circular, indicating a weaker interaction between sodium chloride and glycine. The three-dimensional response surface plot presents a three-dimensional view. The surface rises rapidly with increasing sorbitol concentration, then the upward trend slows down after exceeding 10%, and a stable high-value plateau is formed in the range of glycine 10~18mM and sorbitol 6~10%, further verifying that the recovery rate can be stably maintained above 95% under this combination. Combining the regression equation with back-calculation, the optimal process window for a recovery rate ≥95% is finally determined to be sodium chloride 95~115mM, glycine 10~13mM, and sorbitol 6.0%~8.0% (w / v).

[0056] In summary, mathematical modeling clearly quantifies the impact of each component of the composite additive on the recovery rate of rHSA, clarifies the core role of sorbitol and its synergistic effect with glycine, resolves the technical contradiction of easy aggregation during near-isoelectric point operation, and ensures process reliability, laying a scientific foundation for the efficient implementation of mixed bed chromatography.

[0057] Example 4: Optimization of Mixed Bed Ion Exchange Packing Material Ratio To determine the optimal mass ratio of SCX to WAX exchange packing material in the mixed-bed purification step, the following five comparative experiments were conducted. The remaining purification steps were the same as in Example 1. The detection and result analysis were identical to Example 1, except that the HCP detection sample was diluted 10-fold. Group 1 (SCX:WAX=0:1): Using only DEAE Sepharose FF packing material to simulate a single WAX ​​environment; Group 2 (SCX:WAX=1.5:1): The mass ratio of SP to DEAE in the mixed packing is 1.5:1; Group 3 (SCX:WAX = 1:1): The mass ratio of SP to DEAE in the mixed packing is 1:1; Group 4 (SCX:WAX=1:1.5): The mass ratio of SP to DEAE in the mixed packing is 1:1.5; Group 5 (SCX:WAX=1:0): Using only SP Sepharose FF packing material to simulate a single SCX environment.

[0058] All groups of packing materials were packed into chromatography columns of the same size (column height 15 cm) and treated under the same conditions (buffer was 20 mM acetate-sodium acetate with a composite additive system consisting of 115 mM NaCl, 13 mM glycine and 7.5% (w / v) sorbitol, pH 5.1). The same batch of rHSA samples purified in the first seven steps (S1-S7) were collected and analyzed. The results are shown in Table 4 below.

[0059] Table 4. Effect of different mixed bed packing material ratios on purification efficiency (n=3)

[0060] The results in Table 4 show 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%, with no HCP detected, proving that this ratio is the optimal choice.

[0061] Example 5 Pilot-scale verification To verify the scalability of the purification method of the present invention, a pilot-scale purification experiment was conducted using 50 L of Pichia pastoris fermentation broth. The operation steps were scaled up based on Example 1, with key equipment and parameters adjusted as follows. The detection indicators and methods were the same as in Example 1: S1 Pretreatment: 50 L of fermentation broth was centrifuged at 8000 rpm for 15 min at 4℃ using a disc centrifuge (Shanghai Kaimaidi Separation Technology Co., Ltd.). Sodium caprylate was added to the supernatant to a final concentration of 10 mM, and the pH was adjusted to 4.5. The broth was then transferred to a plate heat exchanger (Jiangsu Maiande Group Co., Ltd., M3 type) with a heat exchange area of ​​0.5 m². 2 (Heat exchange area 0.5m²) 2 Heating at 65℃ for 30 min, cooling, and centrifuging at 10000 rpm for 15 min using a tubular centrifuge (Shanghai Fichar Separation Engineering Technology Co., Ltd., GF105 model) resulted in the supernatant being filtered through a 0.45 μm filter cartridge (Hangzhou Kebote Filter Material Co., Ltd.) and diluted to a conductivity of 12 mS / cm to obtain 80 L of clear liquid.

[0062] S2 cation exchange chromatography: Lissui BC-200-1600 column, packed with 5 L SP Sepharose Fast Flow packing material, column height approximately 16 cm. Equilibrate with 5 CV equilibration buffer (20 mM sodium acetate, pH 4.5) at a flow rate of 250 cm / h. After sample loading, gradient elution was performed, and approximately 12 L of elution peak was collected, with a recovery rate of 89.26%.

[0063] S3 First Ultrafiltration: Employs the Lishui UltraFlo-1000 system and a 10 kDa molecular weight cutoff PES membrane pack (membrane area 1m²). 2The S2 cation exchange eluent was concentrated 6 times to 2.2 L at 20±2℃, with the transmembrane pressure controlled at 1.0 bar and the flow rate at 2 L / min. It was then dialyzed with 8 volumes (16 L) of 20 mM Tris-HCl buffer (pH 7.5) until the conductivity of the permeate was consistent with that of the buffer (±0.5 mS / cm). The final recovery rate was 90.31%, and the buffer replacement rate was >99.5%, which provided a qualified feed solution for S4 anion exchange chromatography.

[0064] S4 Anion Exchange Chromatography: A Lissho BC-150-1700 column was used, packed with 3 LQ Sepharose FastFlow packing material, with a column height of approximately 17 cm. After equilibration, the sample was loaded, and gradient elution was performed. The elution peak was collected at approximately 8 L, with a recovery rate of 83.61%.

[0065] S5 Second Ultrafiltration: Using the same equipment as S3, the membrane was pretreated with 0.1 M NaOH. The S4 anion exchange eluent was concentrated to 0.5 L at 20±2℃, with the transmembrane pressure controlled at 1.2 bar and the flow rate at 1.5 L / min. Dialysis was performed with 8 times the volume (4 L) of 20 mM sodium phosphate buffer (pH 7.0) containing 2.0 M ammonium sulfate. The displacement endpoint was confirmed by the refractive index (1.3580±0.0005). The final recovery rate was 89.05%, and the ammonium sulfate concentration was stable at 1.9~2.1 M, which met the high salt binding requirements of S6 hydrophobic chromatography.

[0066] S6 hydrophobic chromatography: A Lishui BC-100-0760 chromatography column was used, packed with 0.6 L of Phenyl Sepharose HighPerformance packing material, with a column height of approximately 7.6 cm. After equilibration, the sample was loaded, and gradient elution was performed. Approximately 3 L of elution peaks were collected, with a recovery rate of 83.32%.

[0067] S7 Third Ultrafiltration: The same ultrafiltration system as above was used. After the membrane was equilibrated with a low ionic strength buffer, the S6 hydrophobic chromatography eluent was concentrated to 0.25 L at 20±2℃, with the transmembrane pressure controlled at 0.8 bar and the flow rate at 1 L / min. Dialysis was performed with 8 times the volume of 20 mM acetate-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, with a final recovery rate of 90.33%.

[0068] S8 Mixed-bed chromatography: A Lissho BC-100-1900 multi-column tandem chromatography column was used, packed with 1.5 L of a 1:1 mixture of SP+DEAE packing material, with a column height of approximately 19 cm. A 20 mM acetate-sodium acetate buffer (pH 4.9) containing a composite additive of 100 mM sodium chloride, 12 mM glycine, and 7% (w / v) sorbitol was used as the equilibration and elution buffer. After equilibration at 5 CV, the sample was loaded, and elution was continued for 5 CV (flow rate 100 cm / h) with the same buffer containing the composite additive. All flow-through and eluent were collected (to the UV280 baseline), combined to approximately 1 L. The recovery rate was 93.50%.

[0069] Pilot-scale results: The final product was tested and found to have a purity >99.99%, with no dimers detected, HCP <2 ng / g, and endotoxin <0.5 EU / mL. The total recovery rate calculated from S2 was 42.24%, proving that the process of this invention still has stable purification effect and recovery rate at the pilot-scale level and is suitable for large-scale production.

[0070] Comparative Example 1 (mixed bed step omitted) To verify the necessity and superiority of the mixed-bed purification step (S8) in this scheme, the same batch of rHSA intermediates purified by steps S1-S7 of Example 1 was used. Instead of mixed-bed chromatography, conventional gel filtration chromatography (GFC, also known as size exclusion chromatography) was used as the final purification step. This comparative experiment ensured that all preliminary purification steps were completely consistent, except for the final purification unit. The detection and result analysis were identical to those in Test Example 1, except that the HCP detection sample was diluted 10-fold. Gel filtration chromatography was performed using Cytiva Sephacryl S-200 HR packing material, packed into a chromatographic column with a 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 baselines stabilized. After loading the above rHSA intermediate, constant flow elution was continued with the same PBS at a flow rate of 100 cm / h, and the rHSA monomer elution peak within 10 column volumes was collected as the final product.

[0071] The comparison results are shown in Table 5 below.

[0072] Table 5 Comparison Results

[0073] Final product test 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 indicate that omitting the mixed-bed step cannot yield a high-purity product, and the recovery rate is significantly reduced.

[0074] Comparative Example 2: Tandem Anion / Cation Chromatography The same batch of intermediates purified in steps S1-S7 of Example 1 was used. Instead of mixed-bed purification, two sets of conventional ion-exchange chromatography tandem steps were designed for comparison. The detection and result analysis were the same as in Example 1, except that the HCP detection sample was diluted 10-fold. Comparative Example 2a (Cation-Anion Tandem): The intermediate was first loaded onto an SP Sepharose FF strong cation exchange column under the same operating conditions as in Step S2 of Example 1, with a recovery rate of 90.50%. After collecting the elution peak, it was replaced by ultrafiltration with a buffer system suitable for anion exchange chromatography (20 mM Tris-HCl, pH 8.0), with a recovery rate of 95% for this ultrafiltration step. Subsequently, the sample after buffer replacement was loaded onto a Q Sepharose FF strong anion exchange column under the same operating conditions as in Step S4 of Example 1, with a recovery rate of 85.23%. The cumulative recovery rate of the three steps was 73.28%.

[0075] Comparative Example 2b (Anion-Cation Tandem): The same intermediate was first loaded onto a Q Sepharose FF strong anion exchange chromatography column under the same operating conditions as in Step S4 of Example 1, with a recovery rate of 88.71%. After collecting the elution peak, it was replaced by ultrafiltration with a buffer system suitable for cation exchange chromatography (20 mM sodium acetate, pH 4.5), with a recovery rate of 95% for this ultrafiltration step. Subsequently, the sample after buffer replacement was loaded onto an SP Sepharose FF strong cation exchange chromatography column under the same operating conditions as in Step S2 of Example 1, with a recovery rate of 86.15%. The cumulative recovery rate of the three steps was 72.60%. The purification effect and recovery rate of the final product in both comparative examples were significantly lower than those in Example 1, as shown in Table 6 below.

[0076] Table 6 Comparison Results

[0077] The results showed that even with the same two packing materials, the traditional series mode could not remove acidic and basic impurities simultaneously in one step, and the process was more lengthy. The loss of the target protein increased during multiple pH adjustments and adsorption-elution processes. In fact, it was not as efficient and gentle as the one-step flow-through mode of the mixed bed proposed in this scheme.

[0078] Comparative Example 3 The same batch of intermediates purified in steps S1-S7 of Example 1 was divided into two portions and subjected to mixed-bed purification. The detection and result analysis were the same as in Example 1, except that the HCP detection sample was diluted 10-fold. Comparative Example 3a: pH deviation from 4.5-5.5: Mixed-bed operation was performed using 20 mM Tris-HCl buffer at pH 7.0. Under these conditions, rHSA is negatively charged and adsorbs onto the anion exchange packing material in the mixed bed, preventing flow-through and causing a sharp drop in single-step recovery to 35.14%.

[0079] Comparative Example 3b without added composite additives: In a buffer system at pH 5.0, no composite additive consisting of sodium chloride, glycine, and sorbitol was added. Under these conditions, rHSA showed increased aggregation tendency in an environment close to its isoelectric point, resulting in a decrease in single-step recovery to 72.14%, and a significant decrease in the calculated total recovery to 38.02%, with an increased aggregate content in the product.

[0080] The comparison results are shown in Table 7 below.

[0081] Table 7 Comparison Results

[0082] The results showed that the purification efficiency of the mixed bed decreased significantly under pH conditions deviating from the rHSA isoelectric point (pI ~ 4.7). The final product purities of Comparative Examples 3a and 3b were 98.25% and 99.50%, respectively, with HCP residues of 334.48 and 280.68 ng / g, respectively, and the total recovery rates also decreased significantly to 18.52% and 38.02%.

[0083] Comparative Example 4 To investigate the applicability of different strength combinations of ion exchange packing materials in mixed beds, this comparative example systematically evaluated four different packing material combinations in rHSA isoelectric point flow-through mode. All experiments used the same batch of rHSA intermediate purified in steps S1-S7 of Example 1. Except for the type of mixed bed packing material, the column packing, equilibration, sample loading, and elution conditions were identical to step S8 of Example 1. The detection and result analysis were the same as in Test Example 1, except that the HCP detection sample was diluted 10-fold.

[0084] Comparative Example 4a: Strong anion exchange packing material (Q Sepharose FF) + weak cation exchange packing material (CM Sepharose FF), mass ratio 1:1.

[0085] Comparative Example 4b: Weak anion exchange packing material (DEAE Sepharose FF) + weak cation exchange packing material (CM Sepharose FF), mass ratio 1:1.

[0086] Comparative Example 4c: Strong anion exchange packing material (Q Sepharose FF) + strong cation exchange packing material (SP Sepharose FF), mass ratio 1:1.

[0087] The final product testing results are shown in Table 8.

[0088] Table 8 Comparison Results

[0089] 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.

[0090] 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.

[0091] Comparative Example 5 The researchers explored the optimal purification sequence for mixed-bed chromatography, and the following three sets of comparative experiments are used as examples. All experiments used the same batch of fermentation broth as in Example 1, and except for the change in the position of the mixed-bed step, the operating conditions of this step, including the packing material, pH, buffer solution, and compound additives, were consistent with step S8 of Example 1.

[0092] Comparative Example 5a: The mixed bed step is placed at the very beginning of the purification process: The pretreated clarified fermentation broth was directly loaded onto the sample. Due to the extremely complex composition of the initial sample, containing a large amount of charged impurities such as host cell proteins (HCP) and nucleic acids, far exceeding the adsorption capacity of the mixed-bed packing material, the chromatography column quickly became saturated. The results contrasted sharply with Example 1: both the purity and recovery rate of rHSA decreased dramatically. Comparative Example 5a terminated at this step, with a single-step recovery rate of only 30.16% for the mixed bed. The results indicate that using a mixed bed in the initial purification stage not only fails to achieve effective purification but also leads to severe loss of the target product and waste of packing material due to impurity overload, proving this approach is not feasible.

[0093] Comparative Example 5b: The mixed-bed step is placed after cation exchange chromatography (step S2): After cation exchange capture, the elution product was ultrafiltered to a pH 5.0 buffer system and then directly purified using a mixed bed chromatography system. Subsequent purification steps, including anion exchange and hydrophobic chromatography, were then performed to complete the purification process. This route yielded the final product. However, during the mixed bed chromatography step, the sample still contained a large amount of host proteins, aggregates, and hydrophobic impurities. These impurities not only affected the recovery rate of the mixed bed chromatography step itself, but more importantly, some steps performed after the mixed bed chromatography, such as hydrophobic chromatography, might introduce new charge isomers or aggregates, which could not be removed by subsequent purification steps. Therefore, the purity and overall recovery rate of the final product were significantly lower than in Example 1.

[0094] Comparative Example 5c: The mixed-bed step is placed after anion exchange chromatography (step S4): After anion exchange chromatography, the elution product was ultrafiltered to a pH 5.0 buffer system, followed by mixed-bed purification, and then hydrophobic chromatography to complete the purification process. This route also yielded the final product. At this point, most of the strong acid and strong basic impurities had been removed from the sample, so the mixed-bed single-step recovery and the purity of the final product were higher than those of Comparative Example 5b. However, since the hydrophobic chromatography step was performed after the mixed-bed step, its operation may cause some rHSA to undergo conformational changes or aggregate, generating new charge isomers. These newly generated impurities could not be removed, thus limiting further improvement in the final purity. The overall recovery was also lower than that of Example 1.

[0095] The results are shown in Table 9.

[0096] Table 9 Comparison Results

[0097] As shown in Table 9, placing mixed-bed chromatography at the end of the process flow as the final purification step is the optimal and irreplaceable solution for achieving ultra-high purity and high total recovery of rHSA. Only at this location can the mixed bed effectively remove all charge isomers generated and remaining in the preceding steps, while avoiding secondary contamination of the purified product by subsequent steps.

[0098] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for purifying recombinant human serum albumin, characterized in that, The purification method includes the following steps: 1) Pretreatment of fermentation broth containing recombinant human serum albumin; 2) Cation exchange chromatography; 3) Anion exchange chromatography; 4) Hydrophobic chromatography; 5) The product solution obtained in step 4) is subjected to ultrafiltration. The buffer solution used in ultrafiltration is a weak ionic strength buffer solution with pH 5.0~5.

5. Then, a mixed bed ion exchange purification step is performed. In the mixed-bed ion exchange purification step: the mixed-bed chromatography is carried out in a buffer system with a pH of 4.5-5.5; the mixed-bed chromatography uses WAX packing material and SCX packing material, and the mass ratio of WAX packing material to SCX packing material is 1:(0.67-1.5); the buffer system contains a composite additive, which includes 50-150 mM sodium chloride, 10-20 mM glycine and 5-10% (w / v) sorbitol.

2. The purification method for recombinant human serum albumin according to claim 1, characterized in that, The mixed-bed chromatography was carried out in a buffer system with a pH of 4.7–5.

3.

3. The purification method for recombinant human serum albumin according to claim 2, characterized in that, The mixed-bed chromatography was performed in a buffer system at pH 5.0 ± 0.

1.

4. The purification method for recombinant human serum albumin according to claim 1, characterized in that, The mass ratio of WAX packing to SCX packing is 1:(0.8~1.2).

5. The purification method for recombinant human serum albumin according to claim 4, characterized in that, The mass ratio of WAX packing to SCX packing is 1:

1.

6. The purification method for recombinant human serum albumin according to claim 1, characterized in that, The WAX ​​packing is a diethylaminoethyl DEAE group packing; The SCX packing is a sulfopropyl SP group packing.

7. The purification method for recombinant human serum albumin according to claim 1, characterized in that, In step 5), an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa is used, and the volume of the buffer solution used in ultrafiltration is 5-10 times the volume of the product solution.

8. The purification method for recombinant human serum albumin according to claim 1, characterized in that, Step 1) includes heating the fermentation broth and then centrifuging it. The heating conditions are 60~80℃ for 10~30 min, the centrifugation speed is 8000~10000 rpm, and the centrifugation time is 10~20 min.

9. The purification method for recombinant human serum albumin according to claim 1, characterized in that, The packing material for cation exchange chromatography in step 2) is sulfopropyl SP group packing material; The packing material for the anion exchange chromatography in step 3) is a quaternary ammonium group Q group packing material; In step 4), the ligand for hydrophobic chromatography is selected from one or more of phenyl, aliphatic, and heterocyclic compounds.

10. The purification method for recombinant human serum albumin according to claim 1, characterized in that, The purified product had a purity >99.99%, host cell protein residue <2 ng / g rHSA, and endotoxin <0.5 EU / mL.

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