A method for shortening fermentation cycle and improving yield of recombinant human serum albumin

Abstract

This invention discloses a method for shortening the fermentation cycle and increasing the yield of recombinant human serum albumin (rHSA), belonging to the field of biofermentation technology. This invention aims to solve problems in existing technologies, such as low seed quality leading to a long fermentation lag period, decreased cell viability in the later stages of fermentation, and low protein expression efficiency and easy degradation due to product secretion stress. The core process includes: using N-1 perfusion culture technology in the seed amplification stage to prepare a high-density, high-activity, high-quality seed solution; and implementing an amino acid feeding strategy during the induction expression period in the production fermentation stage, specifically supplementing key nutrients (L-proline, L-leucine, and L-valine). Using the complete process of this invention, the fermentation expression level and product stability of rHSA can be significantly improved, the fermentation cycle can be greatly shortened, production intensity can be increased, and the overall production cost can be effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering in biotechnology, specifically relating to a method for shortening the fermentation cycle and increasing the yield of recombinant human serum albumin. More specifically, this invention relates to an optimized method for improving the yield, quality, and production efficiency of rHSA expression in Pichia pastoris by synergistically combining N-1 perfusion culture technology with an induction phase amino acid feeding strategy, applicable to the industrial-scale production of this expression system. Background Technology

[0002] Human serum albumin (HSA) is the most important functional protein in blood plasma, playing a crucial physiological role in maintaining colloid osmotic pressure, substance transport, and in vivo antioxidant activity. Recombinant human serum albumin (rHSA), as a safe alternative to plasma-derived albumin, is widely used in clinical and biopharmaceutical fields. 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, in the actual fermentation production of this system, a series of bottlenecks still exist that restrict efficiency and quality: First, at the seed level, traditional batch or fed-batch seed culture is difficult to maintain the optimal growth environment, easily leading to uneven physiological states and insufficient activity reserves in the seeds. After these seeds are inoculated into the production tank, they often exhibit long lag periods and poor adaptability, directly affecting the efficiency of the entire production cycle. Second, at the fermentation level, especially in the methanol-induced high-efficiency expression stage of exogenous proteins, cells face multiple pressures: on the one hand, the large-scale synthesis and secretion of rHSA leads to excessive endoplasmic reticulum load, easily triggering unfolded protein responses and misfolding; on the other hand, drastic changes in cell metabolism may activate extracellular proteases or lead to redox imbalance. These pressures together cause cell viability to decline rapidly during the critical period of product synthesis, and the target protein is prone to degradation and aggregation, ultimately resulting in unstable expression levels, uneven product quality, and a forced shortening of the production cycle.

[0004] To address these challenges, existing technologies largely focus on optimizing the feed-through strategies for carbon and nitrogen sources (such as glycerol, methanol, and ammonia) during the production stage, or modifying the inorganic salt composition of the basal culture medium. While these methods have some effect, their effectiveness in systematically solving the two core problems of poor seed source quality and the cell-specific nutritional needs during the induction phase is limited. Perfusion culture technology, as an efficient process intensification tool, has been applied in the production stage of mammalian cell culture to extend the culture cycle and increase yield (CN119709591A). However, the creative prepositioning and specific application of this technology to the N-1 seed preparation stage of Pichia pastoris fermentation, aiming to obtain high-density, high-activity, and physiologically homogeneous high-quality seed culture from the source, thereby laying a solid foundation for subsequent production, is a specific technical approach not disclosed in existing technologies.

[0005] Therefore, there is an urgent need for a new and systematic fermentation process that can combine precise control of upstream seed quality with precise nutritional support in the downstream production stage, in order to comprehensively solve the multiple problems in rHSA production, such as difficulty in maintaining cell activity, poor product stability, long fermentation cycle and low production efficiency. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical problems existing in the production of recombinant human serum albumin (rHSA) using Pichia pastoris, such as insufficient cell activity, easy degradation of products, long fermentation cycle and low production efficiency, and to provide a method for shortening the fermentation cycle and increasing the yield of recombinant human serum albumin.

[0007] This invention provides a method for shortening the fermentation cycle and increasing the yield of recombinant human serum albumin, comprising the following steps: S1. Seed perfusion culture stage: Pichia pastoris engineered strains expressing recombinant human serum albumin are inoculated into the N-1 stage seed bioreactor for perfusion culture to obtain seed liquid.

[0008] S2 Production Fermentation and Induction Period Feeding: The seed liquid obtained in step S1 is inoculated into the production fermenter for fermentation culture. When the induction expression period is entered, feed containing amino acids is added to the fermentation system.

[0009] Furthermore, the perfusion culture described in step S1 employs a cell retention device (such as a sedimentation device or hollow fiber column) to achieve continuous separation of cells from waste liquid. While maintaining a constant reactor volume, fresh culture medium is continuously replenished and metabolic byproducts are removed. By precisely controlling the timing of perfusion initiation, perfusion rate, and parameters such as dissolved oxygen and pH, cells proliferate rapidly under near-optimal growth conditions, ultimately yielding a high-density, highly active seed culture.

[0010] Furthermore, step S2 of the fermentation process sequentially includes a batch culture stage, a glycerol-fed amplification stage, and a methanol-induced expression stage. After fermentation enters the methanol-induced expression phase, a feed solution containing a specific amino acid combination is added to the fermentation system to alleviate the metabolic stress and endoplasmic reticulum stress faced by cells during efficient expression of exogenous proteins, thereby maintaining cell viability and product stability.

[0011] Optionally, in step S1, the perfusion culture is performed at a cell density OD 600 Initiate perfusion when the cell volume reaches 30-50, maintaining a perfusion rate of 0.8-2.0 reactor volumes / day. Under these conditions, cells remain in the logarithmic growth phase, preventing excessive accumulation of metabolic byproducts and resulting in seed cells with highly homogeneous physiological states.

[0012] Furthermore, the OD of the seed liquid harvested in step S1 600 The number of viable cells should be no less than 150, and the viability rate should be no less than 95%. This indicator shows that the seed cells have excellent inoculation activity and can significantly shorten the lag period of subsequent production fermentation.

[0013] Furthermore, in step S1, the bioreactor used for the perfusion culture is an N-1 stage bioreactor that provides seeds to the production fermenter. This invention creatively applies perfusion culture technology upfront to the seed preparation stage, rather than the production stage, thereby solving the fundamental problem of poor seed quality at its source.

[0014] Furthermore, the amino acid mentioned in step S2 includes at least one of L-proline, L-leucine, and L-valine.

[0015] Optionally, the amino acid is composed of L-proline, L-leucine, and L-valine.

[0016] Furthermore, the combination of the three amino acids can produce a significant synergistic effect: L-proline can act as a compatible solute to help cells resist osmotic pressure and oxidative stress, and stabilize the protein folding environment; L-leucine and L-valine, as branched-chain amino acids, participate in energy metabolism and tricarboxylic acid cycle replenishment, and provide key metabolic support for cells during carbon source switching and efficient protein synthesis.

[0017] Optionally, based on the mass of L-leucine 1, the mass ratio of L-proline to L-leucine is 0.4 to 1.6:1, and the mass ratio of L-valine to L-leucine is 0.5 to 1.5:1. More preferably, the mass ratio of L-proline, L-leucine, and L-valine is 1.2:1:0.8. At this preferred ratio, the synergistic effect of the three amino acids is most significant, and they can most effectively stabilize the metabolic network of Pichia pastoris under methanol-induced and rHSA-high secretion conditions.

[0018] Furthermore, the total concentration of the amino acids in the fermentation broth is 0.05~0.3% (w / v). Below the lower limit of this range, the nutritional support effect is not significant; above the upper limit of this range, it may cause excessive osmotic pressure or metabolic burden, which is detrimental to cell growth and product expression.

[0019] Further, in step S2, the amino acid supplementation is performed in a single or multiple pulsed manner between 24 and 48 hours after the start of the induction period. More preferably, it is performed in 2 to 3 pulsed manner within 24 to 36 hours after the start of the induction period, with each supplementation amount being 0.8 to 1.2% of the fermentation broth volume. Within this time window, the cells have just completed the carbon source switch and have begun to synthesize and secrete large amounts of rHSA. Supplementing key amino acids at this time can precisely meet the nutritional needs of the cells and exert the best stress relief and metabolic support effects. Supplementing too early may cause the amino acids to be used for cell proliferation rather than product synthesis, while supplementing too late will not effectively prevent the early decline in cell viability.

[0020] In one specific embodiment of the present invention, the Pichia pastoris engineered strain is a recombinant Pichia pastoris CBS7435-rHSA strain expressing recombinant human serum albumin, constructed and preserved according to the method described in patent CN118166053B.

[0021] Furthermore, methanol is used as an inducer during the induction expression period of the fermentation culture, and the pH of the fermentation broth is controlled between 5.0 and 6.0, more preferably between 5.2 and 5.8.

[0022] In a second aspect, the present invention provides recombinant human serum albumin prepared according to the method.

[0023] The beneficial effects of the present invention include, but are not limited to: (1) This invention systematically applies perfusion culture technology to the N-1 seed preparation stage, thereby obtaining high-quality seed liquid with highly uniform physiological state and vigorous metabolic activity from the source. This lays a solid foundation for subsequent production, effectively shortens the lag and adaptation period of the production tank, and significantly improves the starting point of overall fermentation efficiency.

[0024] (2) In the induction expression period of the production fermentation, the present invention innovatively adopts an amino acid feeding strategy. By supplementing L-proline, L-leucine and L-valine in a specific ratio, it directly acts on the stress response and core metabolic pathways of cells, effectively stabilizing the metabolic homeostasis and activity of cells during high-intensity expression of exogenous proteins.

[0025] (3) Through the synergistic optimization of the above-mentioned "high-quality seeds" and "precision nutrition", the present invention has achieved a significant improvement in fermentation efficiency. In practical applications, the process of the present invention can significantly improve the average expression rate (production efficiency) of rHSA, obtain higher protein yield in a shorter induction period, and maintain high cell viability and metabolic stability.

[0026] (4) The process design of the present invention enhances the robustness of the production process and improves batch-to-batch consistency. At the same time, due to the improvement of production efficiency and the optimization of product quality, the overall production cost is reduced, providing a more competitive technical solution for the industrial production of recombinant human serum albumin. Attached Figure Description

[0027] 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 cell growth curves of the embodiments and comparative examples of the present invention during the seed culture stage are shown.

[0028] Figure 2 The figures show the dynamic changes in rHSA expression levels during the induction phase for both the embodiments and the comparative examples of this invention.

[0029] Figure 3 This is a bar chart comparing the average rHSA expression rates of the embodiments and comparative examples of the present invention. Detailed Implementation

[0030] 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.

[0031] Before describing the preferred embodiments of the present invention, it is understood that the fermentation process principle provided by the present invention is applicable to the production of various secretory recombinant proteins using the Pichia pastoris expression system. In particular, for proteins with large molecular weights, susceptible to protease degradation, or under high secretory pressure (such as human serum albumin), the synergistic strategy of "N-1 perfusion culture" and "specific amino acid feeding during the induction phase" of the present invention shows unique advantages in alleviating cellular stress and maintaining product stability. The present invention is further illustrated below with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] 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, weigh the wet weight of the cells and calculate the wet weight.

[0033] 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. y=0.002+(2.235-0.002) / [1+(x / 31.25)^1.12], R²>0.99.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Amino acid supplement solution: Weigh and mix L-proline, L-leucine, and L-valine in a mass ratio of 1.2:1:0.8. Take 100g of this mixture, dissolve it in deionized water, and bring the volume to 1L. Filter through a 0.22 μm membrane for sterilization. This yields a concentrated supplement solution with a concentration of 100 g / L (10% w / v). Calculate the required supplement volume based on the desired cumulative concentration before use. All amino acids were purchased from Wuxi Bicon Biotechnology Co., Ltd.

[0040] 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.

[0041] Methanol feed solution (100%, containing PTM1): Methanol, add PTM1 solution at 12 mL / L before use.

[0042] The present invention will be further described in detail below with reference to specific embodiments.

[0043] 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: CN 118166053B, constructed and preserved by the applicant's laboratory) expressing recombinant human serum albumin 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).

[0044] 1.2 Inoculation and Batch Culture in Reactor N-1: The primary seed culture was transferred at an inoculation rate of 5% (v / v) to a 5 L bioreactor containing BSM basal salt medium (working volume 2.0 L). Culture conditions were set as follows: temperature 30℃, pH controlled to 5.0 by adding ammonia, and dissolved oxygen (DO) maintained above 20% by adjusting the stirring speed (300 ~ 1000 rpm) and aeration rate (1 ~ 3 vvm). Cultured until OD... 600 It reached approximately 40.

[0045] 1.3. Perfusion culture: When OD 600 When the volume reaches 40±2, the perfusion mode is initiated. Fresh BSM basal salt medium (with PTM1 added) is continuously infused at a constant rate of 1.2 VVD (reactor volume / day). Simultaneously, an external settling device is connected to the reactor outlet to utilize gravity sedimentation for cell retention and concentrated reflux, while simultaneously discharging an equal volume of clarified waste liquid, thereby maintaining a constant reactor volume and continuously removing metabolic byproducts. Throughout this stage, the ethanol concentration in the fermentation broth is monitored and controlled to be below 0.5 g / L. This is achieved through offline sampling and adjustments to the perfusion rate to maintain the ethanol concentration at this level.

[0046] 1.4 Seed Culture Harvest: After approximately 36 hours of perfusion culture, OD... 600The temperature stabilized at 200±10. Sampling and testing showed a viable cell rate ≥97% and a wet weight of approximately 200 g / L. At this point, perfusion was stopped, and all culture medium was harvested as a highly active N-1 seed culture, temporarily stored at 4°C, and used for downstream inoculation as soon as possible.

[0047] Example 2: Production Fermentation and Feeding During Induction Period 2.1 Fermentation Inoculation and Batch Culture: 2.0 L of the highly active seed culture prepared in Example 1 was inoculated into a 50 L production fermenter containing 18.0 L of BSM basal salt medium (with PTM1 added), resulting in an initial working volume of 20.0 L. Culture parameters were set as follows: temperature 29°C, pH 5.0, and DO > 20%. Batch culture was performed until the initial glycerol was depleted (indicated by a sharp increase in DO).

[0048] 2.2. Glycerol Fed Culture: Initiate glycerol feeding by continuously adding 50% (w / v) glycerol feed solution (containing PTM1) at a rate of 10 g / L / h, maintaining DO ≥ 20%. Continue feeding for approximately 18 hours, and at the end, OD... 600 The concentration reached 150±10. Feeding was then stopped, and the cells were starved for 2 hours to ensure that residual glycerol was depleted (DO stabilized above 80%).

[0049] 2.3 Methanol Induction and Feeding Stage: Induction start-up: Switch to the methanol-induced expression stage. Initially, add 100% methanol feed solution (containing PTM1) at a rate of 1 g / L / h. The methanol concentration is gradually increased to a stable value of 3.0±0.2 g / L, pH is adjusted to 5.6, and temperature is maintained at 29℃, with feedback control via an online methanol monitor.

[0050] Amino acid supplementation: At 24h and 36h after induction, L-proline (Pro), L-leucine (Leu), and L-valine (Val) were added to the fermenter in a pulsed manner, with a ratio of 1.2:1:0.8. 200 mL of the amino acid feed solution was added each time (based on an initial fermenter working volume of 20.0 L, each addition was 1.0% v / v). This is equivalent to adding approximately 40 g of the above amino acid mixture to the fermentation system, with a cumulative concentration of approximately 0.2% in the fermentation broth. After addition, the mixture was rapidly and evenly mixed using the fermenter's stirring system.

[0051] 2.4 Fermentation Termination and Sampling: Samples were taken every 8 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 increased significantly (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 96 hours, the wet weight of the fermentation broth was approximately 355 g / L, and the rHSA expression level reached 25.2 g / L. The average rHSA expression rate was calculated to be 0.263 g / L / h.

[0052] Example 3: Optimization Experiment of Amino Acid Ratio During Induction Period This embodiment prepared highly active seeds according to Example 1. Based on the process of Example 2, only the ratio of L-proline (Pro), L-leucine (Leu), and L-valine (Val) in the amino acid feed solution added during the induction period was changed to examine their synergistic effect. All experimental groups and comparative groups followed the same endpoint judgment criteria as in Example 2 (i.e., fermentation was terminated after the rHSA expression level reached the plateau phase), and the total induction time was recorded. Except for the amino acid feed solution formulation, all other steps, parameters, and control strategies were exactly the same as in Example 2.

[0053] 3.1 Experimental Grouping and Feed Solution Formulation All experiments were conducted in parallel using the same batch of highly active seed culture in the same 50L fermenter system. The specific experimental groups and the mass ratios of the three amino acids in the feed solution are shown in Table 1. 3.2 Fermentation Results: The effects of different amino acid ratios on key indicators of rHSA fermentation are shown in Table 1 below. The control group received no amino acid feed solution; in experimental group A, the mass ratio of Pro, Leu, and Val in the amino acid feed solution was 0.2:1:0.5; in experimental group B, the mass ratio was 0.4:1:1.0; in experimental group C, the mass ratio was 0.8:1:1.5; in experimental group D, the mass ratio was 1:1:1; in experimental group E, the mass ratio was 1.2:1:0.8; in experimental group F, the mass ratio was 1.6:1:1.2; and in experimental group G, the mass ratio was 2.0:1:0.4.

[0054] Table 1. Effects of different amino acid ratios on rHSA fermentation

[0055] Experimental group E (the preferred ratio of this invention) achieved the highest rHSA protein expression level, the most stable cell viability maintenance, and the shortest fermentation cycle, resulting in the best overall fermentation effect. This demonstrates that L-proline, L-leucine, and L-valine have a significant synergistic effect at a specific mass ratio, which can most effectively stabilize the metabolic network of Pichia pastoris under methanol-induced and high-rHSA secretion conditions, thus alleviating stress.

[0056] 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 Examples 1 and 2.

[0057] 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 BSM basal salt 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%. Seed growth curve comparison, for example... Figure 1 As shown, the cell growth rate of traditional fed-batch culture is significantly slowed down, with a wet weight of only about 85 g / L during the same period, and the seed viability and uniformity are inferior to those of the present invention. This highlights the fundamental role of the perfusion culture technology of the present invention in improving the physiological state of cells.

[0058] 1.2 Traditional Fermentation Process The above-mentioned traditional seed culture was inoculated into a 50L production tank at an inoculation rate of 23.5%. The initial OD after inoculation was... 600 Approximately 20, compared to the initial OD after inoculation in Example 2. 600 The procedures were largely the same. The BSM medium and glycerol feeding phase were performed as described in Examples 2.1-2.2. 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 was approximately 220 g / L, the rHSA expression level was 7.5 g / L, and the average expression rate was 0.063 g / L / h.

[0059] 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]. 600The result was 203, the viable cell rate was 96.5%, and ethanol was not detected.

[0060] 2.2 Traditional induction, without amino acid supplementation: 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 was carried out with conventional methanol feeding (same as Comparative Example 1), without the addition of amino acid feed solution. Fermentation was terminated when the rHSA expression level reached the plateau phase. The total induction time was 110 hours, the wet weight of the fermentation broth was approximately 310 g / L, the rHSA expression level was 18.2 g / L, and the average expression rate was 0.165 g / L / h.

[0061] Comparative Example 3: Traditional seed culture combined with amino acid feeding during the induction period 3.1 Seed preparation (traditional fed-batch culture): The operating procedures and control parameters were performed exactly as described in section 1.1 of Comparative Example 1. The final product was a traditional seed liquor with an OD of [missing information]. 600 The cell count was 83, the viable cell rate was 87%, and the ethanol concentration was 0.75 g / L.

[0062] 3.2 Production Fermentation (implementation of amino acid feeding): The conventional seed culture was inoculated at 10% for production fermentation. The glycerol stage was the same as in Example 2. The methanol induction stage was carried out entirely according to the operation of Example 2.3, implementing an amino acid feeding strategy. Fermentation was terminated when the rHSA expression level reached a plateau, with a total induction time of 112 hours. The final fermentation broth wet weight was approximately 255 g / L, the rHSA expression level was 10.5 g / L, and the average expression rate was 0.094 g / L / h. Although this is an improvement over the conventional process, the average expression rate of 0.094 g / L / h is still far lower than the complete process of this invention (0.263 g / L / h), and also lower than Comparative Example 2 which only used high-quality seeds (0.165 g / L / h).

[0063] Comparative Example 4: Effect of supplementing other types of amino acids during the induction period on fermentation efficiency 4.1 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 198, with a viable cell rate of 97%, and ethanol was not detected.

[0064] 4.2 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. At the same feeding times as in Example 2 (hours 24 and 36), different types of nutrient solutions were pulsed in each group (each addition being 1.0% of the fermentation broth volume). Comparative Example 4a: 50% (w / v) glucose solution (containing PTM1) was added. 1.0% of the fermentation broth volume was added each time, in two separate additions. This group served as a general carbon / energy source control.

[0065] Comparative Example 4b: Supplementation with a mixture of L-glutamine and L-asparagine (Gln:Asn = 1:1). These two amino acids are commonly used nitrogen sources and energy substrates in cell culture. L-glutamine and L-asparagine were weighed and mixed at a 1:1 mass ratio (both purchased from Wuxi Bicon Biotechnology Co., Ltd.). 100 g of this mixture was dissolved in deionized water and brought to a final volume of 1 L. The mixture was then filtered through a 0.22 μm filter to obtain a concentrated feed solution with a concentration of 10% (w / v). Each time the feed solution was added, it was added in a pulse manner, with each addition being 0.6% of the current total fermentation broth volume. This was done in two separate additions, accumulating to approximately 23 g of the amino acid mixture in the fermentation system, resulting in a final cumulative concentration of approximately 0.115% (w / v) in the fermentation broth. This addition amount is calculated to ensure that the total nitrogen content of the supplemented solution is approximately equivalent to that of the feed solution used in Example 2 of this invention (Pro:Leu:Val=1.2:1:0.8, addition concentration 0.2%), in order to eliminate the influence of differences in nitrogen content and specifically examine the specificity of amino acid types. After addition, the solution is rapidly and uniformly mixed using the stirring system of the fermenter.

[0066] Comparative Example 4c: A mixture of L-alanine, L-glycine, and L-serine (Ala:Gly:Ser = 1:1:1) was added. L-alanine, L-glycine, and L-serine (all purchased from Wuxi Bicon Biotechnology Co., Ltd.) were weighed and mixed in a mass ratio of 1:1:1. 100 g of this mixture was dissolved in deionized water and brought to a final volume of 1 L. The mixture was then filtered through a 0.22 μm filter to obtain a 10% (w / v) concentrated feed solution. Each time the feed solution was added, it was taken and added in a pulse manner. The amount added each time was 0.73% of the total volume of the current fermentation broth (calculated based on a total addition of 29.2 g, added in two batches of 14.6 g each, corresponding to a volume of 146 mL, representing 0.73% of 20 L of fermentation broth). The amino acid mixture was added in two batches (24h and 36h after induction), totaling approximately 29.2 g, bringing its final cumulative concentration in the fermentation broth to approximately 0.146% (w / v). This addition was calculated to ensure that the total nitrogen content of the added amino acid mixture was approximately equivalent to the total nitrogen content of the feed solution used in Example 2 of this invention (Pro:Leu:Val = 1.2:1:0.8, addition concentration 0.2%) (approximately 4.645 g), thus eliminating the influence of differences in nitrogen content and specifically examining the amino acid specificity. This group aimed to test whether small molecule non-essential amino acids (Ala, Gly, Ser) could replace the specific combination of this invention. After addition, the mixture was rapidly and uniformly mixed using the fermenter's stirring system.

[0067] Comparative Example 4d: A mixture of L-isoleucine and L-valine (Ile:Val) was added, excluding L-proline. L-isoleucine and L-valine (both purchased from Wuxi Bicon Biotechnology Co., Ltd.) were weighed and mixed at a mass ratio of 1:1. 100 g of this mixture was dissolved in deionized water and brought to a final volume of 1 L. The mixture was then filtered through a 0.22 μm filter to obtain a 10% (w / v) concentrated feed solution. Each time the feed solution was added, it was taken and added in a pulse manner. Each addition was 1.025% of the total volume of the fermentation broth (calculated based on a total addition of 41.0 g, added in two batches of 20.5 g each, corresponding to a volume of 205 mL, representing 1.025% of 20 L of fermentation broth). The amino acid mixture was added in two batches (at 24 h and 36 h after induction), totaling approximately 41.0 g, bringing the final cumulative concentration in the fermentation broth to approximately 0.205% (w / v). This addition was calculated to ensure that the total nitrogen content added was approximately equivalent to that of Example 2. This group aimed to test whether the effects of the present invention could be achieved by supplementing only branched-chain amino acids (Ile and Val) in the absence of L-proline, in order to verify the indispensability of proline.

[0068] Comparative Example 4e: L-proline (Pro) was added separately. L-proline (purchased from Wuxi Bicon Biotechnology Co., Ltd.) was weighed, dissolved in deionized water, and brought to a final volume of 100 g / L (10% w / v). The solution was then filtered through a 0.22 μm filter for sterilization. Each time, the concentrated feed solution was used, calculated based on a total addition of 38.2 g, and added in two batches of 19.1 g each time, corresponding to a volume of 191 mL, representing 0.955% of the 20 L fermentation broth volume. Therefore, each addition was 0.955% of the current total fermentation broth volume, added in two pulses (24h and 36h after induction), accumulating approximately 38.2 g of L-proline to achieve a final cumulative concentration of approximately 0.191% (w / v) in the fermentation broth. This addition amount, calculated, ensures that the total nitrogen content added is approximately equivalent to that of Example 2. The aim of this group is to test whether supplementing with proline alone is sufficient to achieve the technical effects of the present invention, and to verify the synergistic necessity of leucine and valine.

[0069] Comparative Example 4f: L-Leucine (Leu) was added separately. L-Leucine (purchased from Wuxi Bicon Biotechnology Co., Ltd.) was weighed, dissolved in deionized water, and brought to a final volume of 100 g / L (10% w / v). The solution was then filtered through a 0.22 μm filter for sterilization. Each time, the concentrated feed solution was used, calculated based on a total addition of 43.5 g, and added in two batches of 21.75 g each time, corresponding to a volume of 217.5 mL, representing 1.0875% of the 20 L fermentation broth volume. Therefore, each addition was 1.0875% of the current total fermentation broth volume, added in two pulses (24h and 36h after induction), accumulating approximately 43.5 g of L-leucine to achieve a final cumulative concentration of approximately 0.2175% (w / v) in the fermentation broth. This addition amount, calculated, ensures that the total nitrogen content added is approximately equivalent to that of Example 2. This group aims to test whether supplementing with leucine alone is sufficient to achieve the technical effects of the present invention, and to verify the synergistic necessity of proline and valine.

[0070] As shown in Table 2, compared with Example 2 (Pro:Leu:Val = 1.2:1:0.8), the endpoint rHSA expression level, average expression rate, and induced endpoint cell viability of all comparative groups were significantly reduced. Among them, the expression levels of comparative example 4c (Ala:Gly:Ser) and comparative example 4e (Pro alone) were only 16.8 g / L and 14.5 g / L, respectively, which were much lower than 25.2 g / L in Example 2; the expression level of comparative example 4d (Ile:Val, without Pro) was 19.1 g / L, which was slightly higher than glucose control (4a, 19.3 g / L) but there was no substantial difference, and it was significantly lower than Example 2; the expression level of comparative example 4f was 19.8 g / L, which was the highest among all comparative examples, but it was still 5.4 g / L lower than Example 2, and the monomer purity of its product decreased to 79%. The above results demonstrate that the specific combination of L-proline, L-leucine, and L-valine described in this invention has an irreplaceable synergistic effect, and other amino acids or single amino acids cannot achieve the same level of expression and cell viability.

[0071] Table 2. Comparative Example 4: Effect of Feed Components During Induction Period on Fermentation Efficiency

[0072] Comparative Example 5: 5.1 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 200, the viable cell rate was 97%, and ethanol was not detected.

[0073] 5.2 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 amino acid feed solution (formulation same as experimental group E:Pro:Leu:Val = 1.2:1:0.8 in Example 3) being changed. Comparative Example 5a (Premature Feeding): At the 0th hour of methanol induction start-up (i.e., at the start of induction), the amino acid feed solution was added in a single pulse, with the amount added being 2.0% of the fermentation liquid volume.

[0074] Comparative Example 5b (late feed): At the 72nd hour of methanol-induced start-up, the same total amount of amino acid feed solution was added in a single pulse.

[0075] The remaining fermentation control strategies (methanol feed, pH, temperature, etc.) for both groups were the same as in Example 2.

[0076] The results are shown in Table 3. Compared with Example 2 of this invention, although the expression levels of groups 5a and 5b, supplemented with the same amino acid mixture, were somewhat increased compared with Comparative Example 2 without supplementation, both resulted in prolonged total induction time, decreased average expression rate, and cell viability failing to reach optimal levels. The dynamic changes in rHSA expression during the induction period are shown in Table 3. Figure 2 For comparison of average expression rates, see [link to relevant documentation]. Figure 3 .

[0077] Table 3. Comparative Example 5: The effect of feeding timing during the induction period on fermentation efficiency

[0078] 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 method for shortening the fermentation cycle and increasing the yield of recombinant human serum albumin, characterized in that, Includes the following steps: S1. Seed perfusion culture stage: Pichia pastoris engineered strains expressing recombinant human serum albumin are inoculated into a 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. When the induction expression period is entered, feed containing amino acids is added to the fermentation system.

2. The method according to claim 1, characterized in that, In step S1, the initiation condition for the perfusion culture is the cell density OD. 600 The injection rate is maintained at 0.8-2.0 reactor volumes / day, reaching 30-50.

3. The method according to claim 1 or 2, characterized in that, OD of the seed solution obtained in step S1 600 The number of cells should be no less than 150, and the viable cell rate should be no less than 95%.

4. The method according to claim 1, characterized in that, In step S1, the bioreactor used for the perfusion culture is an N-1 stage bioreactor that provides seeds for the production fermenter.

5. The method according to claim 1, characterized in that, The amino acid mentioned in step S2 includes at least one of L-proline, L-leucine, and L-valine.

6. The method according to claim 5, characterized in that, Based on the mass of L-leucine 1, the mass ratio of L-proline to L-leucine is 0.4-1.6:1, and the mass ratio of L-valine to L-leucine is 0.5-1.5:

1.

7. The method according to claim 5, characterized in that, The total concentration of the amino acids in the fermentation broth is 0.05~0.3% (w / v).

8. The method according to claim 1, characterized in that, In step S2, the amino acid supplementation is performed in a single or multiple pulsed manner between 24 and 48 hours after the start of the induction period.

9. The method according to claim 1, characterized in that, Methanol was used as an inducer during the induction expression period of the fermentation culture, and the pH of the fermentation broth was controlled between 5.0 and 6.

0.

10. Recombinant human serum albumin prepared by the method according to any one of claims 1-9.

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