A fermentation method for improving the structural stability and yield of recombinant human serum albumin

Abstract

The application discloses a fermentation method for improving structural stability and yield of recombinant human serum albumin, and belongs to the technical field of biological fermentation. 600 ≥180, and the living cell rate is greater than or equal to 90%; (2) during a methanol induction period, L-cysteine and L-methionine are added in batches in pulse mode with a mass ratio (0.5-2.0):1, and the final concentration is 0.1%-0.3%. The application promotes correct formation of disulfide bonds by optimizing seed quality and protein folding microenvironment. By using the method, the expression amount of rHSA reaches 24.3 g / L, the monomer purity is greater than or equal to 92%, the free sulfhydryl content is less than or equal to 3%, and the monomer purity reduction rate is less than or equal to 4% after 14 days of accelerated storage at 37 DEG C. The application significantly improves product stability while maintaining high yield, and provides an innovative process for industrialized production of high-quality rHSA.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation engineering, specifically to a fermentation method for improving the structural stability and yield of recombinant human serum albumin (rHSA). The method utilizes a Pichia pastoris fermentation process that optimizes seed culture and induction period nutrition strategies to enhance the stability of the recombinant human serum albumin (rHSA) product. Background Technology

[0002] Human serum albumin (HSA) is the most important functional protein in plasma, playing a crucial physiological role in maintaining colloid osmotic pressure, substance transport, and in vivo antioxidant activity. rHSA, as a safe alternative to plasma-derived albumin, is widely used in clinical treatment and biopharmaceuticals. The Pichia pastoris expression system, due to its strong protein secretion capacity and ease of high-density culture, has become the mainstream platform for the industrial production of rHSA.

[0003] However, rHSA molecules contain 17 pairs of disulfide bonds, and their correct formation and maintenance are crucial in determining the product's native conformation, biological activity, and long-term stability. In actual Pichia pastoris fermentation production, especially during the methanol-induced high-intensity expression stage, this molecular characteristic presents severe quality challenges: on the one hand, the rapid synthesis and secretion of rHSA overloads the endoplasmic reticulum, easily triggering unfolded protein responses; on the other hand, intense metabolic activity leads to intracellular redox imbalance and the accumulation of reactive oxygen species (ROS). This stressful microenvironment easily interferes with the correct pairing and formation of disulfide bonds, resulting in rHSA misfolding, abnormal intermolecular aggregation, or oxidative damage. The final result is a low proportion of active monomers, high content of aggregate impurities, batch-to-batch quality inhomogeneity, and potential impact on the product's long-term storage stability. This not only increases the difficulty and cost of downstream purification but also introduces potential application safety risks.

[0004] To address these challenges, existing technologies mainly focus on two directions: First, they concentrate on improving expression efficiency, such as optimizing carbon and nitrogen feedstock strategies or genetically modifying production strains to increase the synthesis rate and total yield of rHSA. However, these methods offer limited improvement over the inherent folding quality issues of the product. Second, they rely on refining downstream purification processes to remove aggregates or optimizing formulations to stabilize the final product, without addressing the instability at the fermentation stage. Furthermore, in the seed preparation stage, traditional batch or fed-batch culture methods struggle to consistently provide optimal growth environments, resulting in insufficient seed viability and uniformity. This further exacerbates cell instability during production, amplifying product quality fluctuations.

[0005] However, in existing technologies, there is no publicly reported technical approach that uses sulfur-containing amino acids as a targeted means of regulating the fermentation process to form a complete process chain of "high-quality seed preparation" and "precise nutritional intervention during the induction period" specifically to solve the product stability bottleneck of Pichia pastoris in the production of rHSA.

[0006] Therefore, there is an urgent need in this field for a novel and systematic fermentation process that can obtain highly active and uniform cells from the seed source and, through direct nutritional intervention on the protein folding microenvironment during the critical production period, fundamentally improve the intrinsic structural stability and long-term storage stability of rHSA while achieving efficient expression, thereby breaking through the limitations of existing technologies. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical problems existing in the production of rHSA using Pichia pastoris, such as insufficient cell activity, high mispairing rate of disulfide bonds in the product, easy aggregation and degradation, poor storage stability, and low fermentation production efficiency, and to provide a fermentation method that improves the structural stability and yield of recombinant human serum albumin.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: This invention provides a fermentation method for improving the structural stability and yield of recombinant human serum albumin, comprising the following steps: S1. Seed perfusion culture stage: Pichia pastoris engineered strains expressing rHSA are inoculated into the N-1 stage seed bioreactor for perfusion culture and seed liquid is harvested; S2. Production Fermentation and Induction Period Feeding: The seed liquid obtained in step S1 is inoculated into the production fermenter for fermentation culture; after the fermentation enters the methanol-induced expression period, sulfur-containing amino acid feed solution is added to the fermentation system to regulate the redox environment of the fermentation system.

[0009] Optionally, in step S1, the perfusion culture is performed at a cell density OD 600 Start when the concentration reaches 30-50, and maintain the injection rate at 0.8-2.0 reactor volumes / day.

[0010] Optionally, in step S1, the OD of the harvested seed liquid is... 600 The wet weight is not less than 180 g / L, and the viable cell rate is not less than 90%.

[0011] As a preferred technical solution, in step S1, the duration of the perfusion culture is 24 to 48 hours.

[0012] Optionally, in step S2, the sulfur-containing amino acid is selected from at least one of L-cysteine ​​and L-methionine; preferably, it includes L-cysteine ​​and L-methionine, and the mass ratio of the two is (0.5 ~ 2.0):1.

[0013] In one specific embodiment of the present invention, the mass ratio of L-cysteine ​​to L-methionine is 1:1.

[0014] Optionally, in step S2, the total concentration of the sulfur-containing amino acids in the fermentation broth is 0.1 to 0.3% (w / v).

[0015] Optionally, in step S2, the replenishment strategy for the sulfur-containing amino acid feed solution is as follows: within 48 hours after the start of the induction period, it is replenished in a pulsed manner, with the number of replenishments being 2 to 4 times.

[0016] In a specific embodiment of the present invention, the number of replenishment times is 3, and the time points of the multiple pulse replenishment are the 12th hour, 24th hour and 36th hour after the start of the induction.

[0017] Optionally, methanol is used as an inducer during the induction expression period of the fermentation culture, with the methanol concentration maintained at 2.0~5.0 g / L, the pH of the fermentation broth controlled between 5.0 and 6.0, and the temperature maintained at 28~30℃.

[0018] The present invention provides recombinant human serum albumin (rHSA) prepared by the method described herein.

[0019] Furthermore, the monomer purity of the rHSA is ≥90%, preferably, the monomer purity of the rHSA is ≥92.3%.

[0020] Furthermore, the free thiol content of the rHSA is ≤5%, preferably, the free thiol content of the rHSA is ≤2.9%.

[0021] In one specific embodiment of the present invention, after the rHSA is stored at 37°C for 14 days, the monomer purity decrease rate is ≤4%, and more preferably, the monomer purity decrease rate is ≤3.5%.

[0022] In a specific embodiment of the present invention, the rHSA prepared by the method has an expression level of 24.3 g / L, a monomer purity of 92.3%, a free thiol content of 2.8%, and a monomer purity decrease rate of only 3.47% after accelerated storage at 37°C for 14 days.

[0023] Thirdly, the present invention provides the application of the method or the rHSA in the preparation of pharmaceutical formulations, biological products or cell culture medium additives.

[0024] The beneficial effects of the present invention include, but are not limited to: (1) By systematically applying the perfusion culture technology to the N-1 seed preparation stage, this invention obtains high-quality seed liquid with highly uniform physiological state and vigorous metabolic activity from the source, laying a solid foundation for subsequent efficient and stable production.

[0025] (2) In the induction expression period of production fermentation, this invention innovatively adopts a feeding strategy with sulfur-containing amino acids as the core. By supplementing L-cysteine ​​(a direct precursor of disulfide bonds) and L-methionine (an important amino acid involved in the synthesis of intracellular antioxidant systems), the redox microenvironment of intracellular protein folding is directly and specifically optimized, effectively promoting the correct and rapid formation of rHSA disulfide bonds, and ensuring the natural conformation and high stability of the product from the post-translational modification level.

[0026] (3) Through the synergy of the above-mentioned high-quality seeds and precise folding regulation, this invention not only significantly improves the intrinsic quality of the product, but also achieves efficient expression of rHSA by maintaining good cell state, shortening the production cycle and comprehensively improving production efficiency and economic benefits.

[0027] (4) The process design of the present invention enhances the robustness of the production process and batch consistency. The feeding strategy is simple and clear, and it is easy to implement and scale up on existing industrial fermentation platforms, providing a reliable and competitive technical solution for the large-scale production of high-quality rHSA. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 The OD of Pichia pastoris during the N-1 stage perfusion culture in this embodiment of the invention. 600 Change curve; Figure 2 These are the monomer purity change curves of rHSA prepared by different fermentation processes during accelerated storage in embodiments of the present invention. Detailed Implementation

[0029] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0030] Product analysis methods: 1. Wet weight (g / L): Take a certain volume of fermentation broth, centrifuge it at 8000 rpm for 20 minutes in a pre-weighed centrifuge tube, discard the supernatant, and weigh the wet weight of the cells to calculate the wet weight.

[0031] 2. rHSA expression level (g / L): The fermentation supernatant was used for quantitative detection using a human serum albumin-linked immunosorbent assay (ELISA) kit (Yisheng Biotechnology). The specific procedure was performed according to the kit instructions, and a standard curve was plotted using the kit standards.

[0032] 3. Cell viability (%): After sampling, the percentage of viable cells was calculated by directly counting them under a microscope using the methylene blue staining method.

[0033] 4. Average expression rate (g / L / h): The induction time was recorded from the start of methanol induction to the plateau phase (an increase of ≤5% over 12 consecutive hours) of rHSA expression. Average expression rate = endpoint rHSA expression level / T.

[0034] 5. rHSA monomer purity analysis: The fermentation supernatant was filtered through a 0.22 μm filter membrane and analyzed using a high-performance liquid chromatography (HPLC) system with a size exclusion column (SEC-HPLC, such as TSKgel G3000SWxl). The mobile phase was phosphate buffer at pH 6.8, the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. Using rHSA standards as a reference, the proportion of monomer peak area to the total protein peak area was calculated.

[0035] 6. Determination of Free Thiol Group (-SH) Content: The content of free thiol groups in the sample was determined using the Ellman's reagent method. The specific method and operating procedures were explained in the instructions for the Free Thiol Group Detection Kit (Catalog No.: BC5890) from Solarbio Biotechnology Co., Ltd.

[0036] Take an appropriate amount of fermentation supernatant and, using an ultrafiltration centrifuge tube or dialysis bag with a molecular weight cutoff of 10 kDa, thoroughly replace the supernatant with the buffer provided in this kit or pH 8.0 phosphate buffer (0.1 M, containing 1 mM EDTA) at 4°C to completely remove small molecule reducing impurities. Adjust the protein concentration of the treated sample to 2 mg / mL.

[0037] Following the kit instructions, a standard curve was constructed using reduced glutathione (GSH), and the absorbance of the sample was measured at 412 nm. In a typical measurement, the linear regression equation of the standard curve was y = 0.0132x + 0.0107, with a correlation coefficient R² = 0.9903. First, based on this standard curve equation and the measured absorbance value (y) of the sample, the molar concentration of free thiol groups in the reaction system, C_SH (μM), can be calculated as (y - 0.0107) / 0.0132. Second, the molar concentration of rHSA in the sample is calculated as: C_rHSA (μM) = [rHSA concentration in sample (g / L) / 66500 (g / mol)] × 106 Next, calculate the number of free thiol groups per molecule of rHSA: n = C_SH / C_rHSA. Finally, the percentage of free thiol groups in the total thiol groups is calculated as follows: Free thiol percentage (%) = (n / 35) × 100%.

[0038] 7. Accelerated Stability Test: The purified rHSA sample (concentration 10 mg / mL, dissolved in PBS buffer, pH 7.4) was aliquoted and stored in a 37℃ incubator protected from light. Samples were taken on days 0, 7, and 14, and monomer purity was analyzed by SEC-HPLC. The rate of decrease in monomer purity was calculated as the degradation rate. The chromatographic column used for SEC-HPLC analysis was a TSKgel G3000SWxl (7.8 mm × 300 mm), and the mobile phase was 0.1 M sodium phosphate and 0.1 M sodium sulfate buffer (pH 6.8).

[0039] Unless otherwise specified, all culture media and other reagents used in this invention are conventionally prepared or commercially available. The preparation methods for the culture media used in the following examples are as follows: YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and deionized water to the required volume. Autoclave at 121°C for 20 minutes. If preparing a solid medium, add 20 g / L agar powder.

[0040] BSM basal salt medium (per 20L): 534 mL of 85% (w / w) phosphate, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, 800 g of glycerol, and deionized water to a final volume of 20 L. Sterilize at 121°C for 30 minutes.

[0041] PTM1 trace element solution (per 500 mL): 3 g copper sulfate pentahydrate, 0.044 g potassium iodide, 1.5 g manganese sulfate monohydrate, 0.1 g sodium molybdate dihydrate, 0.01 g boric acid, 0.25 g cobalt chloride, 10 g zinc chloride, 32.5 g ferrous sulfate heptahydrate, 0.1 g biotin, 2.5 mL concentrated sulfuric acid, diluted to 500 mL with deionized water, and filtered through a 0.22 μm membrane for sterilization.

[0042] Sulfur-containing amino acid feed solution: Weigh L-cysteine ​​and L-methionine and mix them in a 1:1 mass ratio. Take 100g of this mixture, dissolve it in sterile deionized water, and bring the volume to 1 L. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4℃ for later use. All the sulfur-containing amino acids mentioned above were purchased from Wuxi Bicon Biotechnology Co., Ltd.

[0043] Glycerin replenishment solution (50%, w / v): Dissolve 500 g of glycerin in deionized water and bring the volume to 1 L. Sterilize at 121°C for 20 minutes. Add PTM1 solution at 12 mL / L before use.

[0044] Methanol feed solution (100%, containing PTM1): Methanol, add PTM1 solution at 12 mL / L before use.

[0045] The present invention will be further described in detail below with reference to specific embodiments.

[0046] Example 1: Preparation of highly active seed culture (N-1 perfusion culture) 1.1 Strain Activation: A cryopreserved tube of *Pichia pastoris* strain CBS7435-rHSA (patent: CN118166053B, constructed and preserved in the laboratory) expressing rHSA was streaked onto a YPD agar plate and incubated at 30°C for 2-3 days. A single colony was picked and inoculated into a shake flask containing 100 mL of YPD liquid medium, and incubated at 30°C and 220 rpm for approximately 12 hours with shaking. This was used as the primary seed culture (OD). 600 Approximately 12).

[0047] 1.2 Inoculation and Batch Culture in N-1 Reactor: The activated seed culture was inoculated into a 5L bioreactor at a rate of 5% (v / v), with an initial working volume of 2L. Culture parameters were set as follows: temperature 30℃, pH adjusted to 5.0 with ammonia, and dissolved oxygen (DO) maintained above 20% (maintained by adjusting the stirring speed to 300-1000 rpm and the aeration rate to 1-3 vvm). Cultured until OD... 600 It reached approximately 40.

[0048] 1.3. Perfusion culture: When monitoring the OD of the culture medium online... 600 When the concentration reaches 40±2, the perfusion mode is initiated. Fresh, sterile BSM culture medium is continuously infused into the reactor at a constant rate of 1.2 reactor volumes / day (VVD) using a peristaltic pump. Simultaneously, the reactor outlet is connected to an external settling tank. Utilizing the natural gravity settling of the Pichia pastoris cells in the settling tank, the cells are concentrated and returned to the reactor, while an equal volume of clarified supernatant rich in metabolic waste is discharged, thus maintaining a high working volume and cell density within the reactor throughout the process. The ethanol concentration is monitored and controlled to remain below 0.5 g / L throughout the process.

[0049] 1.4 Seed culture harvest: After 36 hours of continuous perfusion culture, OD was monitored online. 600 The concentration was stable at 200±10, and the viable cell rate was ≥97% when samples were taken and tested using methylene blue staining. Figure 1 As shown, using the perfusion culture technique of the present invention, the cell density (OD) 600The cells exhibited rapid linear growth throughout the perfusion period, indicating vigorous cell growth and healthy metabolism. Within 36 hours, the wet weight rapidly increased to approximately 200 g / L, successfully yielding a high-density, highly active, high-quality seed culture. Perfusion was then stopped, and the entire culture medium (approximately 2 L) was used as the highly active seed culture. The seed culture was briefly stored at the culture temperature (30°C) or room temperature and used for inoculation into the production tank within one hour.

[0050] Example 2: Production Fermentation and Feeding During Induction Period 2.1 Fermentation Inoculation and Batch Culture: 2.0 L of the seed culture prepared in Example 1 was inoculated into a 50 L fermenter containing 18.0 LBSM medium, with an initial working volume of 20.0 L. Parameters were set as follows: temperature 29°C, pH 5.0, and DO > 20% maintained by adjusting the fermentation speed and aeration. Batch culture was carried out until the initial glycerol was depleted.

[0051] 2.2. Calcium-fed culture: 50% (w / v) glycerol solution (containing 12 mL / L™) was continuously added at a rate of 10 g / L / h to maintain DO ≥ 20% for 18 h. The bacterial OD was then measured at the end of the culture. 600 The concentration reached 150±10. Glycerol feeding was then stopped, and the cells were starved for 2 hours to ensure that residual glycerol was depleted and DO stabilized above 80%.

[0052] 2.3 Methanol Induction and Feeding Stage During Induction: Induction start-up: Switch to the methanol-induced expression stage. Initially, 100% methanol (containing 12 mL / L PTM1) was added at a rate of 1 g / L / h. The methanol concentration was gradually increased to a stable value of 3.0 ± 0.2 g / L, the pH was adjusted to 5.6, and the temperature was maintained at 29℃.

[0053] Supplementation of sulfur-containing amino acids: At 12h, 24h and 36h after the start of induction, the sulfur-containing amino acid feed solution (L-cysteine:L-methionine = 1:1) was added to the fermenter in a pulse manner. Based on the initial fermentation volume of 20 L, 0.16 L was added each time, and a total of 0.48 L was added after three additions, so that the final concentration of sulfur-containing amino acids in the fermentation system reached 0.24% (w / v).

[0054] 2.4 Fermentation Termination and Sampling Detection: Samples were taken every 12 hours after induction began to determine the wet weight and protein content of the fermentation supernatant. Induction was terminated when the rHSA concentration in the fermentation supernatant no longer significantly increased (increase ≤ 5%) for 12 consecutive hours, and the total induction time (T) was recorded. In this example, at the end of fermentation, the total induction time T was 80 hours, the wet weight of the fermentation broth was approximately 375 g / L, the rHSA expression level reached 24.3 g / L, and the average expression rate was 0.304 g / L / h. Analysis of the fermentation endpoint sample: SEC-HPLC showed that the rHSA monomer purity was 92.3%; the free thiol content determined by Ellman's method was 2.8% (based on total thiol).

[0055] Example 3: Optimization Experiment of Sulfur-Containing Amino Acid Ratio This embodiment, based on the process of Example 2, only changed the mass ratio of L-cysteine ​​(Cys) to L-methionine (Met) in the sulfur-containing amino acid feed solution added during the induction period to examine its synergistic effect. All experiments used the same batch of high-activity seed culture from Example 1 and were conducted in the same 50L fermenter system. Except for the feed solution ratio, all other steps, parameters, and control strategies were exactly the same as in Example 2. The control group used the same N-1 infusion seed preparation process as the experimental group, but no sulfur-containing amino acids were added during the induction period (Cys:Met=0:0), and all other process parameters were completely consistent with Example 2. The different mass ratios of sulfur-containing amino acids and the experimental results are shown in Table 1. The control group did not add any sulfur-containing amino acids. In experimental group A, the mass ratio of Cys to Met was 0.5:1; in experimental group B, the mass ratio of Cys to Met was 0.8:1; in experimental group C, the mass ratio of Cys to Met was 1:1; in experimental group D, the mass ratio of Cys to Met was 1.2:1; in experimental group E, the mass ratio of Cys to Met was 1.5:1; in experimental group F, the mass ratio of Cys to Met was 1:0; and in experimental group G, the mass ratio of Cys to Met was 0:1.

[0056] Table 1. Effects of different ratios of sulfur-containing amino acids added during the induction period on rHSA fermentation

[0057] As shown in Table 1, supplementation with Cys and Met significantly shortened the induction time, increased rHSA yield and average expression rate, and effectively maintained high cell viability. Notably, the free thiol content in the product decreased significantly with specific supplementation ratios, which typically indicates reduced protein oxidative damage and improved product quality. In summary, experimental group C (Cys:Met = 1:1) was the optimal ratio, achieving the highest yield (24.3 g / L) and the highest average production rate (0.304 g / L / h, an 87.7% increase compared to the control group) within the shortest induction time (80 h), while maintaining a high endpoint cell viability of 86% and the lowest free thiol content (2.9%). This demonstrates that Cys and Met at a 1:1 mass ratio have the best synergistic effect, efficiently supporting rHSA synthesis and proper folding, alleviating secretory stress, and simultaneously optimizing production efficiency and product quality.

[0058] Comparative Example 1: Traditional fed-batch seed culture and fermentation process To verify the advantages of the process of the present invention, a control group of traditional processes was set up. Except for the seed culture method and the feeding strategy during the induction period, the other materials, equipment and basic parameters were completely consistent with those of Example 1 and Example 2.

[0059] 1.1 Traditional Seed Preparation: The inoculum activation was the same as in Example 1. The activated seed culture was inoculated into a 5L reactor (containing 2.0L YPD medium) at a 5% inoculation rate. The culture parameters were the same as in Example 1.2. An intermittent feeding method was used: when DO rose sharply, 50% glycerol solution was added (5% of the volume each time, at 6-hour intervals, for a total of 4 times). After 36 hours of culture, the seed culture was harvested, and its OD... 600 Approximately 85, with a viable cell rate of 88%.

[0060] 1.2 Traditional Fermentation Process: The above-mentioned conventional seed culture was inoculated into a 50L production tank at a 10% inoculation rate. The BSM medium and glycerol feeding stages were performed as in Examples 2.1 and 2.2. After entering the methanol induction phase, only routine methanol feeding (concentration controlled at 3.0 g / L) and pH adjustment were performed; no amino acid feed solution was added. Fermentation was terminated when rHSA expression reached a plateau. The total induction time was 120 hours, the final wet weight of the fermented cells was approximately 214 g / L, and the viable cell rate was approximately 60%. The rHSA expression level was 6.8 g / L, and the average expression rate was 0.056 g / L / h. Product analysis: monomer purity 72.1%, free thiol content 12.5%.

[0061] Comparative Example 2: N-1 perfusion culture of seeds combined with traditional induced fermentation process 2.1 Seed preparation (N-1 perfusion culture): The operating procedures and control parameters were performed exactly as described in Example 1. The final product was a highly active seed liquid with an OD value of [missing value]. 600 The result was 205, with a viable cell rate of 97.3%.

[0062] 2.2 Traditional Induced Fermentation: The above seed culture was inoculated at 10% for production fermentation. The glycerol stage was the same as in Example 2. The methanol induction stage involved only conventional methanol feeding (same as Comparative Example 1), without the addition of sulfur-containing amino acid feed solution. Fermentation was terminated when rHSA expression reached a plateau. The total induction time was 108 hours, the wet weight of the fermentation broth was approximately 318 g / L, the rHSA expression level was 20.1 g / L, and the average expression rate was 0.186 g / L / h. Product analysis showed a monomer purity of 78.4% and a free thiol content of 8.6%.

[0063] Comparative Example 3: The effect of different feeding times during the induction period on fermentation efficiency 3.1 Seed preparation (N-1 perfusion culture): The operating procedures and control parameters were performed exactly as described in Example 1. The final product was a highly active seed liquid with an OD value of [missing value]. 600 Approximately 200, with a viable cell rate of 97%, and ethanol was not detected.

[0064] 3.2 Production Fermentation: The inoculation and glycerol feeding stages of the fermentation process strictly followed the procedures outlined in Examples 2.1 and 2.2. During the methanol induction stage, two experimental groups were set up, with only the addition time of the sulfur-containing amino acid feed solution being varied: Comparative Example 3a (Premature Feeding): Seeds and initial processes were the same as in Example 2. Simultaneously with the start of methanol induction (0 hours), a single pulse of the same total amount of sulfur-containing amino acid feed solution (Cys:Met = 1:1) as in Example 2 was added. The total induction time was 104 hours, the endpoint rHSA expression level was 20.5 g / L, the average expression rate was 0.197 g / L / h, the monomer purity was 81.6%, and the free thiol content was 7.2%.

[0065] Comparative Example 3b (late feed): Seeds and initial processes were the same as in Example 2. 72 hours after the start of methanol induction, a single pulse of the same total amount of sulfur-containing amino acid feed solution (Cys:Met = 1:1) as in Example 2 was added. The total induction time was 110 hours, the endpoint rHSA expression level was 17.8 g / L, the average expression rate was 0.162 g / L / h, the monomer purity was 77.2%, and the free thiol content was 9.5%.

[0066] The results are shown in Table 2. Based on the experimental results, Example 2 significantly outperformed Comparative Examples 3a and 3b in all key indicators. Example 2 had the shortest total induction time (80 hours), but the highest endpoint rHSA expression level (24.3 g / L) and the fastest average expression rate (0.304 g / L / h), indicating the highest production efficiency. Simultaneously, Example 2 had the highest monomer purity (92.30%) and the lowest free thiol content (2.80%), indicating the best product quality and the lowest degree of oxidative degradation. In contrast, Comparative Examples 3a and 3b had longer induction times (104 and 110 hours), but significantly lower expression levels and rates, and significantly decreased monomer purity and increased free thiol content (reaching 7.20% and 9.50%, respectively), showing that their process conditions were unfavorable for the efficient and stable production of high-quality rHSA. This demonstrates that feeding within the 12-36 hour time window after the start of induction is crucial to achieving the superior effects of this invention.

[0067] Table 2. Effects of different feeding times during the induction period on fermentation efficiency

[0068] Comparative Example 4: Traditional fed-batch seed preparation process with extended culture time This comparative example aims to investigate whether maximizing the culture time under the traditional fed-batch culture mode can achieve OD (Organic Degree) growth. 600 Seed culture that is stable at 200±10 with a viable cell rate of ≥97%.

[0069] The activation, inoculation, and initial culture parameters were performed exactly as described in section 1.1 of Comparative Example 1. The activated seed culture was inoculated into a 5L reactor (containing 2.0L YPD medium) at a 5% inoculation rate. Culture parameters were the same as in Example 1.2 (temperature 30°C, pH 5.0, DO > 20%). An intermittent fed-batch method was used: when DO rose sharply (indicating carbon source depletion), 50% glycerol solution was added, with each addition being 5% of the current culture volume. Unlike Comparative Example 1 (cultured for only 36 hours), this comparative example underwent continuous fed-batch culture, extending the total culture time to 48 hours. Samples were taken at 24, 36, 42, and 48 hours of culture to determine OD600, wet weight, viable cell rate, and ethanol concentration in the fermentation broth.

[0070] The limiting growth data of traditional fed-batch culture are shown in Table 3 below. The results show that the OD of the seed culture decreases with increasing culture time. 600 Gradually increasing in size and wet weight, but with a gradual decrease in cell viability. Furthermore, even with extended culture time to 48 hours under conventional methods, the OD of the seed culture... 600 The concentration can only reach about 110, with a wet weight of about 112 g / L. Moreover, as the culture time is extended, the viable cell rate decreases significantly from 91% to 78%.

[0071] Table 3. Limiting growth data of conventional fed-batch culture

[0072] Example 4: Accelerated stability test of the product This embodiment aims to verify the stability differences of rHSA prepared using different fermentation processes under long-term storage conditions, and further demonstrate the advantages of the process of the present invention in improving the intrinsic quality of the product.

[0073] 4.1 Experimental Materials and Equipment: Experimental samples: Fermentation supernatants from Example 2 (the optimal process of this invention), Comparative Example 1, Comparative Example 2, Comparative Example 3a, and Comparative Example 3b were selected and treated with the same downstream purification process (cation exchange chromatography and gel filtration) to obtain purified rHSA samples.

[0074] Reagents: PBS buffer (pH 7.4, formulation: 0.01 mol / L potassium dihydrogen phosphate, 0.01 mol / L disodium hydrogen phosphate, 0.137 mol / L sodium chloride, 0.0027 mol / L potassium chloride, diluted to volume with deionized water, autoclaved at 121℃ for 20 minutes, cooled before use); chromatographic grade acetonitrile, trifluoroacetic acid (purchased from Thermo Fisher).

[0075] Instruments: High performance liquid chromatograph (equipped with TSKgel G3000SWxl column), constant temperature incubator.

[0076] 4.2 Sample Preparation: Dilute all batches of purified rHSA stock solution to a concentration of 10 mg / mL using PBS buffer (pH 7.4). Filter each sample solution through a 0.22 μm filter and aliquot 1 mL into multiple sterile vials, then seal.

[0077] 4.3 Accelerated Stability Test: All aliquoted samples were stored in a 37°C incubator in the dark to simulate harsh long-term storage conditions and accelerate product degradation and aggregation. Samples were taken on day 0 (initial), day 7, and day 14 of storage. Each time a sample was taken, the corresponding time-point sample vials were removed and allowed to return to room temperature. Size exclusion chromatography (SEC-HPLC) was used to analyze the samples to accurately quantify the proportion of rHSA monomers. Chromatographic conditions: mobile phase: 0.1M sodium phosphate, 0.1M sodium sulfate buffer (pH 6.8); flow rate: 0.5 mL / min; detection wavelength: 280 nm. The percentage of the monomer peak area to the total chromatogram area (monomer peak and polymer peak) was calculated from the chromatogram to represent the monomer purity at that time point.

[0078] 4.4 Experimental Results: The accelerated stability test data of each group of samples in this embodiment are shown in Table 4 and... Figure 2 As shown in Table 4, the degradation rate is calculated as (monomer purity on day 0 - monomer purity on day n) / monomer purity on day 0 × 100% (n = 7, 14). The average of three parallel samples is taken for each data set, and the relative standard deviation (RSD) is calculated. The accelerated stability test data for the five samples in this example are shown in Table 4. The relative standard deviation (RSD) for all groups is 1.2% ~ 2.3%, all ≤ 2.5%. According to the table data, Example 2 showed significantly higher initial monomer purity (92.3%) than all comparative examples, and its purity decreased by only 3.47% after 14 days of storage, exhibiting the lowest degree of degradation and demonstrating the best monomer stability. In contrast, Comparative Example 1 had the lowest initial purity (72.1%), and its purity decreased by as much as 19.56% within 14 days, showing the worst stability. The initial purities of Comparative Examples 2, 3a, and 3b ranged from 78.4% to 81.6%, with purity decrease rates of 13.39%, 10.54%, and 12.56% respectively after 14 days, all showing significantly worse stability than Example 2. Overall, the formulation or process conditions of Example 2 were most favorable for inhibiting monomer degradation and maintaining high purity.

[0079] Table 4. Accelerated stability test results of rHSA prepared by different fermentation processes

[0080] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation method for improving the structural stability and yield of recombinant human serum albumin (rHSA), characterized in that, The method includes the following steps: S1. Seed perfusion culture stage: Pichia pastoris engineered strains expressing rHSA were inoculated into the N-1 stage seed bioreactor for perfusion culture to obtain seed liquid; S2. Production Fermentation and Induction Feeding Stage: The seed liquid obtained in step S1 is inoculated into the production fermenter for fermentation culture. During the methanol-induced expression period, sulfur-containing amino acid feed solution is added to the fermentation system.

2. The fermentation method according to claim 1, characterized in that, In step S1, the perfusion culture is carried out at a cell density OD 600 is initiated when it reaches 30 ~ 50, the perfusion rate is maintained at 0.8 ~ 2.0 reactor volumes / day; the OD 600 is not less than 180, and the viable cell rate is not less than 90%.

3. The fermentation method according to claim 2, characterized in that, In step S1, the duration of the perfusion culture is 24 to 48 hours.

4. The fermentation method according to claim 1, characterized in that, In step S2, the sulfur-containing amino acid feed solution contains L-cysteine ​​and L-methionine.

5. The fermentation method according to claim 4, characterized in that, In the sulfur-containing amino acid feed solution, the mass ratio of L-cysteine ​​to L-methionine is (0.5 ~ 2.0):

1.

6. The fermentation method according to claim 4 or 5, characterized in that, After the sulfur-containing amino acid feed solution is added to the fermentation broth, the total concentration of sulfur-containing amino acids is 0.1% to 0.3% (w / v).

7. The fermentation method according to claim 1, characterized in that, In step S2, the addition of the sulfur-containing amino acid feed solution is completed within 48 hours after the start of the induction period.

8. The fermentation method according to claim 7, characterized in that, The sulfur-containing amino acid feed solution is added in a pulsed manner, with 2 to 4 additions, and the amount added each time is equal.

9. The fermentation method according to claim 1, characterized in that, In step S2, the parameters for the methanol-induced expression period are controlled as follows: methanol concentration 2.0 ~ 5.0 g / L, fermentation broth pH 5.0 ~ 6.0, and temperature 28 ~ 30℃.

10. The recombinant human serum albumin prepared by the fermentation method according to any one of claims 1 to 9, characterized in that, The monomer purity of the rHSA is ≥90%, and the free thiol content is ≤5%.

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