Method for preparing recombinant human albumin with low o-glycosylation level

Abstract

Provided is a method for preparing recombinant human albumin with a low O-glycosylation level, comprising: in a fermentation process, using at least one measure to improve the growth rate of a strain and reduce a protein O-glycosylation level. The method specifically comprises: S1, preparing a seed solution; S2, putting a BSM culture medium into a fermentation tank, adjusting a pH value to 5.0-5.8, sterilizing, cooling and then calibrating dissolved oxygen, setting a temperature and a ventilation rate, adding a sterilized PTM1 solution into the culture medium, and adjusting the pH value of the culture medium to 5.0-5.8 again; S3, inoculating shake flask seeds into the fermentation tank, calibrating the dissolved oxygen, and setting a rotating speed; S4, when the dissolved oxygen suddenly increases, entering a glycerol feeding stage, and adding glycerol at a constant rate in a fed-batch manner; S5, simultaneously adding glycerol and methanol in the fed-batch manner, adjusting the pH value to 6.0-6.5, and cooling; S6, adding methanol in the fed-batch manner; and S7, when the fermentation is finished, emptying the fermentation tank, centrifuging a fermentation product, and collecting a supernatant. The method can reduce protein O-glycosylation, and the expressed recombinant protein contains an extremely low level of glycosylation modification.

Description

A method for preparing recombinant human albumin with low O-glycosylation level Technical Field

[0001] The present invention relates to a method for preparing recombinant human albumin with low O-glycosylation level, belonging to the field of medicine. Background Art

[0002] Human serum albumin is the most abundant protein in human plasma. Its non-glycosylated single-chain polypeptide consists of 585 amino acids with a molecular weight of approximately 66.5 kDa. It is widely used clinically to treat critical conditions such as shock, edema, or ascites caused by hemorrhagic trauma and burns, as well as hypoproteinemia.

[0003] Unlike traditional recombinant drugs, recombinant human albumin generally requires a total infusion dose of 10g or more to be therapeutically effective in clinical use. If the recombinant human albumin contains a certain amount of glycosylated or glycated recombinant human albumin at a high protein background, the total infusion volume of these abnormally structured proteins will be magnified during clinical use, potentially causing clinical allergic reactions and other hazards.

[0004] Albumin itself lacks N-glycosylation sites, but may exhibit some degree of O-glycosylation at its S / T sites. Recombinant products expressed in yeast, in particular, often exhibit a certain degree of mannosylation due to the inherent characteristics of the expression system. This can lead to potential immunogenicity, rapid clearance from the human body, and a short half-life. Therefore, there is an urgent need to identify methods for regulating the O-glycosylation modification of recombinant proteins expressed in yeast.

[0005] The glycosylation process is catalyzed by glycosyltransferases, which link the sugar groups on the sugar donor to the sugar acceptor via glycosidic bonds. Therefore, the study of protein glycosylation modification can be approached from three perspectives: the sugar donor, the sugar acceptor, and the glycosyltransferase. The first mannose attached to yeast proteins is provided by polyterpenoid phosphomannose (Dol-P-Man). Studies have demonstrated that knocking out the gene encoding Dol-P-Man synthase completely abolishes O-glycosylation in yeast proteins. Other commonly used methods for removing O-glycosylation from yeast proteins include replacing or deleting amino acid residues capable of O-linking and disrupting or removing genes encoding glycosyltransferases. While effective, these methods have significant technical drawbacks. Studies have shown that defects in mannosyltransferase genes may compromise cell wall integrity, leading to low cell viability. Replacement or deletion of amino acid residues capable of O-linking in target proteins can alter their properties and functions.

[0006] The present invention aims to provide a new method for reducing O-glycosylation levels, which does not involve genetic modification technology and is simple to operate, and provides a reference for the regulation of glycosylation of recombinant proteins in yeast expression systems. Summary of the Invention

[0007] The present invention provides a method for preparing recombinant human albumin with low O-glycosylation level, which can reduce protein O-glycosylation, and the expressed recombinant protein contains extremely low level of glycosylation modification.

[0008] To achieve this object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a method for preparing recombinant human albumin with low O-glycosylation level, the method comprising taking at least one measure to increase the growth rate of the strain and reduce the protein O-glycosylation level during the fermentation process;

[0010] The measures include changing at least one of the following fermentation conditions:

[0011] 1) Inoculum size;

[0012] 2) Seed liquid bacterial concentration;

[0013] 3) Initial pH of the culture medium;

[0014] 4) culture temperature;

[0015] 5) ventilation volume;

[0016] 6) Stirring speed.

[0017] Preferably, a method for preparing recombinant human albumin with low O-glycosylation level comprises the following steps:

[0018] S1. Seed solution preparation: Take the preserved bacterial strain and streak it on a plate, pick a single colony and inoculate it into YPD liquid medium for cultivation. When the OD600 of the shake flask seeds reaches 10-12, use it as the seed solution for tank filling;

[0019] S2. Add BSM medium to the fermenter, adjust the pH to 5.0-5.8, sterilize, and after cooling, calibrate the dissolved oxygen, set the temperature and ventilation rate, add the sterilized PTM1 solution to the medium, and adjust the pH of the medium to 5.0-5.8 again;

[0020] S3, discard part of the culture medium in the fermentation tank through the sampling port, connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen, set the speed, and maintain the dissolved oxygen above 20% by adjusting the speed and ventilation during fermentation;

[0021] S4, when the dissolved oxygen suddenly increases, the glycerol feeding stage is entered, and glycerol feeding is added at a uniform rate;

[0022] S5, after 10-12 hours of glycerol feed addition, enter the mixed feed stage, glycerol feed and methanol feed are added simultaneously, the pH is adjusted to 6.0-6.5, and the temperature is lowered;

[0023] S6, after 2-3 hours of feeding, enter the methanol feeding phase, add methanol feeding, and monitor various fermentation parameters in real time;

[0024] S7. After the fermentation is completed, the tank is placed, the fermentation product is centrifuged, and the supernatant is collected.

[0025] Preferably, ammonia is used to adjust the pH value.

[0026] Preferably, the strain in step S1 is CBS7435-rHSA, and the CBS7435-rHSA strain is prepared by the following method:

[0027] R1. Design and synthesize the recombinant human albumin DNA sequence, the sequence of which is shown in SEQ ID NO.1;

[0028] R2. The optimized sequence was ligated into the pPICZαA vector with restriction enzyme sites PmII and XbaI. The ligation product was transformed into E. coli DH5α competent cells and plated on LB plates containing zeocin. A single clone was picked and transferred into LB liquid medium containing zeocin. The plasmid was re-extracted and confirmed by restriction enzyme digestion and sequencing. The expression plasmid was correctly constructed and named pPICZαA-rHSA.

[0029] R3. The plasmid vector was linearized by Sac I and then electroporated into CBS7435 competent cells. The transformed strain was spread on YPD resistance plates containing zeocin, and positive single clones were picked to obtain the recombinant human albumin expression strain CBS7435-rHSA.

[0030] In the present invention, SEQ ID NO.1 is:

[0031] Preferably, in step S1, each 1 L of YPD liquid culture medium includes the following components: 5-10 g yeast extract, 10-20 g peptone, and 10-20 g glucose.

[0032] Preferably, in step S1, the culture temperature of the bacterial strain in the YPD liquid culture medium is 28-30°C.

[0033] Preferably, in step S1, the OD600 of the shake flask seeds is 10.

[0034] Preferably, in step S2, the pH value is adjusted to 5 twice.

[0035] Preferably, in step S2, each 20 L of BSM culture medium comprises the following components: 534-600 mL of 85% phosphoric acid, 18.6-20 g of calcium sulfate dihydrate, 364-400 g of potassium sulfate, 298-320 g of magnesium sulfate dihydrate, 82.6-100 g of potassium hydroxide, and 800-850 g of glycerol.

[0036] Further preferably, each 20 L of BSM culture medium includes the following components: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol, and the volume is adjusted to 20 L.

[0037] Preferably, in step S2, each 500 mL of PTM1 solution includes the following components: 3-5 g of copper sulfate pentahydrate, 0.044-0.1 g of potassium iodide, 1.5-2 g of manganese sulfate monohydrate, 0.1-0.2 g of sodium molybdate dihydrate, 0.01-0.03 g of boric acid, 0.25-0.35 g of cobalt chloride, 10-12 g of zinc chloride, 32.5-35 g of ferrous sulfate heptahydrate, 0.1-0.2 g of biotin, and 2.5-3.0 mL of sulfuric acid.

[0038] Further preferably, in step S2, each 500 mL of PTM1 solution comprises the following components: 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 sulfuric acid, the volume is adjusted to 500 mL, and filtered for sterilization.

[0039] Preferably, the glycerol feed comprises: glycerol, purified water, defoaming agent and PTM1.

[0040] More preferably, the glycerol phase feed is as follows: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 180 mL of PTM, which are mixed and placed in a 10 L storage bottle for sterilization.

[0041] Preferably, the methanol feed comprises: methanol and a defoaming agent.

[0042] Further preferably, methanol feed: 20 L of methanol, filtered and sterilized; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0043] Compared with the prior art, the beneficial effects and significant improvements of the technical solution of the present invention are:

[0044] 1. The method of the present invention is to reduce the protein O-glycosylation level by increasing the growth rate of the strain during the fermentation process, which is simple to operate and easy to control.

[0045] 2. The method of the present invention does not require the destruction or removal of any glycosyltransferase gene, thus avoiding the negative impact on cell integrity that may be caused by glycosyltransferase gene defects.

[0046] 3. The method of the present invention does not require deletion or replacement of amino acid residues containing glycosylation sites, and the expressed recombinant protein has the same amino acid sequence as the natural protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the embodiments of the present invention.

[0048] FIG1 shows the OD600 values ​​during the fermentation process of Examples 1-3 and Comparative Examples 1-3;

[0049] FIG2 shows the wet weight results during the fermentation process of Examples 1-3 and Comparative Examples 1-3. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents of the present invention, those skilled in the art may make various changes and modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0051] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0052] "Recombinant proteins" in this context are proteins obtained using recombinant DNA or recombinant RNA technologies. In vitro recombinant protein production primarily encompasses four systems: prokaryotic protein expression, mammalian cell protein expression, yeast protein expression, and insect cell protein expression. The proteins produced vary in activity and application methods. Choosing the appropriate protein expression system based on your downstream application can improve your expression success rate.

[0053] The "culture medium" of the present invention is a synthetic nutrient medium used for the growth and maintenance of microorganisms, plant tissues, and animal tissues. It generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins, and water. Depending on the specific needs of each culture medium, compounds that the culture medium cannot synthesize itself, namely growth factors, may be added.

[0054] "Fermentation" in the present invention refers to the process by which people utilize the vital activities of microorganisms under aerobic or anaerobic conditions to prepare the microorganisms themselves, or directly obtain metabolites or secondary metabolites. Fermentation is sometimes also written as "酦酵", and its definition varies depending on the usage context. Generally, the so-called fermentation mostly refers to a certain decomposition process of organic substances by organisms. Fermentation is a biochemical reaction that humans came into contact with earlier, and it is now widely used in the food industry, biological and chemical industries. It is also a basic process in bioengineering, namely fermentation engineering. The research on its mechanism and process control continues.

[0055] "Strain" in the present invention refers to microorganisms used as living cell catalysts in the fermentation process, including four categories: bacteria, actinomycetes, yeasts, and molds. A large number of microorganisms are sourced from nature, from which useful strains are isolated and screened, then improved and stored for production use.

[0056] The recombinant human albumin expression strain CBS7435-rHSA used in the present invention was self-constructed. All examples in the present invention were fermented and cultured using the above strain, and its construction method includes the following steps:

[0057] S1. Design and synthesize the recombinant human albumin DNA sequence, as shown in SEQ ID NO.1;

[0058] S2. Connect the optimized sequence to the pPICZαA vector, with the restriction enzyme sites being PmII and XbaI. Transfer the ligation product into competent Escherichia coli DH5α cells, coat it on an LB plate containing zeocin, pick monoclonal colonies and transfer them into an LB liquid medium containing zeocin, re-extract the plasmid, and after restriction enzyme digestion identification and sequencing verification, the expression plasmid is correctly constructed and named pPICZαA-rHSA;

[0059] S3. After linearizing the plasmid vector with Sac I restriction enzyme, electrotransform it into competent CBS7435 cells. Coat the transformed strain on a YPD resistant plate containing zeocin, pick positive monoclonal colonies to obtain the recombinant human albumin expression strain CBS7435-rHSA.

[0060] Among them, SEQ ID NO.1 is:

[0061] The following further elaborates on the present invention with specific examples.

[0062] Example 1

[0063] 1.1. Preparation of the culture medium

[0064] YPD: 10 g yeast extract, 20 g peptone, 20 g glucose, dilute to 1 L, and sterilize by high-pressure steam.

[0065] PTM1: 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 sulfuric acid, dilute to 500 mL, and filter sterilize.

[0066] BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

[0067] Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

[0068] Methanol feed: 20 L of methanol, sterilized by filtration; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0069] 1.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA and streak it on a plate. Pick a single colony and inoculate it into 1LYPD liquid medium. Cultivate it at 30°C and 220 rpm. When the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank filling.

[0070] 1.3. Add 20 L of BSM medium to the fermenter, adjust the pH to 5.0 with ammonia, and sterilize at 121°C for 30 min. After cooling, adjust the dissolved oxygen to 0%, set the temperature to 30°C, the ventilation rate to 20 L / min, and the tank pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 5.0 again.

[0071] 1.4. Discard approximately 1 L of culture medium from the fermenter through the sampling port, bringing the volume to approximately 19 L before inoculation. Place the shake flask seeds into the fermenter, calibrate the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. During fermentation, adjust the rotation speed (300-1000 rpm) and ventilation to maintain the dissolved oxygen above 20%.

[0072] 1.5. When the dissolved oxygen suddenly increases, enter the glycerol feeding stage and add glycerol at a uniform rate. The parameter requirements remain unchanged.

[0073] 1.6. After 10 hours of glycerol feed addition, the mixed feed stage begins. Glycerol feed and methanol feed are added simultaneously. The pH is adjusted to 6.0 and the temperature is lowered to 25°C. Other parameters remain unchanged.

[0074] 1.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and the fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0075] 1.8. After 132 hours of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0076] Example 2

[0077] 2.1 Culture medium preparation

[0078] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high-pressure steam.

[0079] PTM1: 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 sulfuric acid, dilute to 500 mL, and filter sterilize.

[0080] BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

[0081] Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

[0082] Methanol feed: 20 L of methanol, filtered and sterilized; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0083] 2.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA and streak it on a plate. Pick a single colony and inoculate it into 500 mL YPD liquid medium. Cultivate it at 30°C and 220 rpm. When the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank filling.

[0084] 2.3. Add 20 L of BSM medium to the fermenter, adjust the pH to 5.0 with ammonia, and sterilize at 121°C for 30 min. After cooling, adjust the dissolved oxygen to 0%, set the temperature to 30°C, the ventilation rate to 20 L / min, and the tank pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 5.0 again.

[0085] 2.4. Discard approximately 500 mL of culture medium from the fermenter through the sampling port, bringing the volume to approximately 19.5 L before inoculation. Place the shake flask seed into the fermenter, calibrate the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. During fermentation, adjust the rotation speed (300-1000 rpm) and ventilation to maintain the dissolved oxygen above 20%.

[0086] 2.5. When the dissolved oxygen increases suddenly, enter the glycerol feeding stage and add glycerol at a uniform rate. The parameter requirements remain unchanged.

[0087] 2.6. After 10 hours of glycerol feed addition, the mixed feed stage was entered, and glycerol feed and methanol feed were added simultaneously. The pH was adjusted to 6.0 and the temperature was lowered to 25°C.

[0088] 2.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and the fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0089] 2.8. After 132 hours of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0090] Example 3

[0091] 3.1 Culture medium preparation

[0092] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 1 L, and sterilize by high-pressure steam.

[0093] PTM1: 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 sulfuric acid, dilute to 500 mL, and filter sterilize.

[0094] BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

[0095] Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

[0096] Methanol feed: 20 L of methanol, filtered and sterilized; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0097] 3.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA and streak it on a plate. Pick a single colony and inoculate it into 1LYPD liquid medium. Cultivate it at 28°C and 220 rpm. When the OD600 of the shake flask seeds reaches 10, use it as the seed solution for tank filling.

[0098] 3.3. Add 20 L of BSM medium to the fermenter, adjust the pH to 5.0 with ammonia, and sterilize at 121°C for 30 min. After cooling, adjust the dissolved oxygen to 0%, set the temperature to 28°C, the ventilation rate to 20 L / min, and the tank pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 5.0 again.

[0099] 3.4. Discard approximately 1 L of culture medium from the fermenter through the sampling port, bringing the volume to approximately 19 L before inoculation. Place the shake flask seeds into the fermenter, calibrate the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. During fermentation, adjust the rotation speed (300-1000 rpm) and ventilation to maintain the dissolved oxygen above 20%.

[0100] 3.5. When the dissolved oxygen suddenly increases, enter the glycerol feeding stage and add glycerol at a uniform rate. The parameter requirements remain unchanged.

[0101] 3.6. After 10 hours of glycerol feed addition, the mixed feed stage was entered, and glycerol feed and methanol feed were added simultaneously. The pH was adjusted to 6.0 and the temperature was lowered to 25°C.

[0102] 3.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and the fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0103] 3.8. After 132 hours of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0104] Comparative Example 1

[0105] 4.1. Culture medium preparation:

[0106] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high-pressure steam.

[0107] PTM1: 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 sulfuric acid, dilute to 500 mL, and filter sterilize.

[0108] BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

[0109] Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

[0110] Methanol feed: 20 L of methanol, sterilized by filtration; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0111] 4.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA and streak it on a plate. Pick a single colony and inoculate it into 500 mL YPD liquid medium. Cultivate it at 30°C and 220 rpm. When the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank filling.

[0112] 4.3. Add 20 L of BSM medium to the fermenter, adjust the pH to 6.0 with ammonia, and sterilize at 121°C for 30 min. After cooling, adjust the dissolved oxygen to 0%, set the temperature to 30°C, the ventilation rate to 20 L / min, and the tank pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 6.0 again.

[0113] 4.4. Discard approximately 500 mL of culture medium from the fermenter through the sampling port, bringing the volume to approximately 19.5 L before inoculation. Place the shake flask seed into the fermenter, calibrate the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. During fermentation, adjust the rotation speed (300-1000 rpm) and ventilation to maintain the dissolved oxygen above 20%.

[0114] 4.5. When the dissolved oxygen increases suddenly, enter the glycerol feeding stage and add glycerol at a uniform rate. The parameter requirements remain unchanged.

[0115] 4.6. After 10 hours of glycerol feed addition, the mixed feed stage begins. Glycerol feed and methanol feed are added simultaneously. The temperature is lowered to 25°C. Other parameters remain unchanged.

[0116] 4.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and the fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0117] 4.8. After 132 hours of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0118] Comparative Example 2

[0119] 5.1. Culture medium preparation

[0120] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high-pressure steam.

[0121] PTM1: 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 sulfuric acid, dilute to 500 mL, and filter sterilize.

[0122] BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

[0123] Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

[0124] Methanol feed: 20 L of methanol, filtered and sterilized; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0125] 5.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA and streak it on a plate. Pick a single colony and inoculate it into 500 mL YPD liquid medium. Cultivate it at 30°C and 220 rpm. When the OD600 of the shake flask seeds reaches 10, use it as the seed solution for tank filling.

[0126] 5.3. Add 20 L of BSM medium to the fermenter, adjust the pH to 6.0 with ammonia, and sterilize at 121°C for 30 min. After cooling, adjust the dissolved oxygen to 0%. Set the temperature to 30°C, the ventilation rate to 20 L / min, and the tank pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 6.0 again.

[0127] 5.4. Discard approximately 500 mL of culture medium from the fermenter through the sampling port, bringing the volume to approximately 19.5 L before inoculation. Place the shake flask seeds into the fermenter, calibrate the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. During fermentation, adjust the rotation speed (300-1000 rpm) and ventilation to maintain the dissolved oxygen above 20%.

[0128] 5.5. When the dissolved oxygen increases suddenly, enter the glycerol feeding stage and add glycerol at a uniform rate. The parameter requirements remain unchanged.

[0129] 5.6. After 10 hours of glycerol feed addition, the mixed feed stage begins. Glycerol feed and methanol feed are added simultaneously. The temperature is lowered to 25°C. Other parameters remain unchanged.

[0130] 5.7. After 2 hours of mixed feed addition, enter the methanol feed period, add methanol feed, monitor the fermentation parameters in real time, and make specific adjustments according to the situation.

[0131] 5.8. After 132 hours of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0132] Comparative Example 3

[0133] 6.1. Culture medium preparation:

[0134] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high-pressure steam.

[0135] PTM1: 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 sulfuric acid, dilute to 500 mL, and filter sterilize.

[0136] BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

[0137] Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL / L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

[0138] Methanol feed: 20 L of methanol, sterilized by filtration; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0139] 6.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA and streak it on a plate. Pick a single colony and inoculate it into 500 mL YPD liquid medium. Cultivate it at 28°C and 220 rpm. When the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank filling.

[0140] 6.3. Add 20 L of BSM medium to the fermenter, adjust the pH to 6.0 with ammonia, and sterilize at 121°C for 30 min. After cooling, adjust the dissolved oxygen to 0%, set the temperature to 28°C, the ventilation rate to 20 L / min, and the tank pressure to 0.05 MPa. Add 87 mL of sterilized PTM1 solution to the medium and adjust the pH to 6.0 again.

[0141] 6.4. Discard approximately 500 mL of culture medium from the fermenter through the sampling port, bringing the volume to approximately 19.5 L before inoculation. Place the shake flask seed into the fermenter, calibrate the dissolved oxygen to 100%, and set the rotation speed to 300 rpm. During fermentation, adjust the rotation speed (300-1000 rpm) and ventilation to maintain the dissolved oxygen above 20%.

[0142] 6.5. When the dissolved oxygen increases suddenly, enter the glycerol feeding stage and add glycerol at a uniform rate. The parameter requirements remain unchanged.

[0143] 6.6. After 10 hours of glycerol feed addition, the mixed feed stage begins. Glycerol feed and methanol feed are added simultaneously. The temperature is lowered to 25°C. Other parameters remain unchanged.

[0144] 6.7. After 2 hours of mixed feed addition, enter the methanol feed period, add methanol feed, monitor the fermentation parameters in real time, and make specific adjustments according to the situation.

[0145] 6.8. After 132 hours of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0146] Example 4

[0147] During the fermentation of Examples 1-3 and Comparative Examples 1-3, samples were taken every 12 h to measure OD600 values ​​and wet weights.

[0148] The results are shown in Figure 1 and Figure 2. The experimental results show that Example 1 and Example 3 have the fastest growth rates, and the order of growth rate from largest to smallest is: Example 3 > Example 1 > Example 2 > Comparative Example 2 > Comparative Example 3 > Comparative Example 1.

[0149] In summary, the growth rate of bacteria in the early stage of fermentation can be promoted by changing the inoculation amount of the strain, the concentration of the seed liquid bacteria, the temperature, and the pH fermentation conditions.

[0150] Example 5

[0151] The fermentation broths obtained in Examples 1-3 and Comparative Examples 1-3 were purified and glycoprotein was quantified using the sulfuric acid phenol method. Mannose dried to a constant weight under reduced pressure was used as a reference substance, and control solutions of different concentrations were prepared. The solution was developed with the appropriately diluted test sample using the sulfuric acid phenol method and the absorbance was measured. The standard curve was fitted using the absorbance and the concentration of the reference substance to obtain the mannose content of the sample. The total protein content of the sample was determined using the Kjeldahl method. Finally, the ratio of the mannose content in the sample to the total protein was calculated [W / W, mannose mg·g(Pro) -1 ], and the results are shown in Table 1.

[0152] Table 1 Determination of mannose content in recombinant protein by sulfuric acid phenol method

[0153] The results in Table 1 verify that the method of the present invention, i.e., changing the inoculum size, seed liquid cell concentration, temperature, and pH fermentation conditions during the fermentation process, can increase the growth rate of the strain and reduce the mannosylation of the Pichia pastoris recombinant protein. Moreover, under certain conditions, the O-glycosylation modification level of the expression product is inversely proportional to the growth rate of the strain.

[0154] The applicant declares that, in the description of the above specification:

[0155] The descriptions of terms such as "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in an appropriate manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine different embodiments or examples and features of different embodiments or examples described in this specification without causing any contradiction.

[0156] Finally, it should be noted that:

[0157] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same.

[0158] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. A method for preparing recombinant human albumin with low O-glycosylation level, characterized in that: The method comprises taking at least one measure during the fermentation process to increase the growth rate of the strain and reduce the protein O-glycosylation level; The measures include changing at least one of the following fermentation conditions: 1) Inoculum size; 2) Seed liquid bacterial concentration; 3) Initial pH of the culture medium; 4) culture temperature; 5) ventilation volume; 6) Stirring speed.

2. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 1, comprising the following steps: S1. Seed solution preparation: Take the preserved bacterial strain and streak it on a plate, pick a single colony and inoculate it into YPD liquid medium for cultivation. When the OD600 of the shake flask seeds reaches 10-12, use it as the seed solution for tank filling; S2. Add BSM medium to the fermenter, adjust the pH to 5.0-5.8, sterilize, and after cooling, calibrate the dissolved oxygen, set the temperature and ventilation rate, add the sterilized PTM1 solution to the medium, and adjust the pH of the medium to 5.0-5.8 again; S3, discard part of the culture medium in the fermentation tank through the sampling port, connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen, set the speed, and maintain the dissolved oxygen above 20% by adjusting the speed and ventilation during fermentation; S4, when the dissolved oxygen suddenly increases, the glycerol feeding stage is entered, and glycerol feeding is added at a uniform rate; S5, after 10-12 hours of glycerol feed addition, enter the mixed feed stage, glycerol feed and methanol feed are added simultaneously, the pH is adjusted to 6.0-6.5, and the temperature is lowered; S6, after 2-3 hours of feeding, enter the methanol feeding phase, add methanol feeding, and monitor various fermentation parameters in real time; S7. After the fermentation is completed, the tank is placed, the fermentation product is centrifuged, and the supernatant is collected.

3. A method for preparing recombinant human albumin with low O-glycosylation level according to claim 2, characterized in that: The strain used in step S1 is CBS7435-rHSA, and the CBS7435-rHSA strain is prepared by the following method: R1. Design and synthesize the recombinant human albumin DNA sequence, the sequence of which is shown in SEQ ID NO.1; R2. Ligate the sequence to the pPICZαA vector with restriction enzyme sites PmII and XbaI. Transform the ligation product into E. coli DH5α competent cells and plate on LB plates containing zeocin. Pick a single clone and transfer it into LB liquid medium containing zeocin. Re-extract the plasmid. After restriction enzyme digestion and sequencing verification, the expression plasmid was correctly constructed and named pPICZαA-rHSA. R3. The plasmid vector was linearized by Sac I and then electroporated into CBS7435 competent cells. The transformed strain was spread on YPD resistance plates containing zeocin, and positive single clones were picked to obtain the recombinant human albumin expression strain CBS7435-rHSA.

4. A method for preparing recombinant human albumin with low O-glycosylation level as claimed in claim 2, characterized in that: In step S1, each 1 L of YPD liquid culture medium includes the following components: 5-10 g yeast extract, 10-20 g peptone, and 10-20 g glucose.

5. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 2 or 4, characterized in that: In step S1, the culture temperature of the bacteria in the YPD liquid culture medium is 28-30°C.

6. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 2, characterized in that: In step S1, the OD600 of the seeds in the shake flask was 10.

7. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 2, wherein: In step S2, the pH value is adjusted to 5 twice.

8. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 2, characterized in that: In step S2, each 20 L of BSM culture medium includes the following components: 534-600 mL of 85% phosphoric acid, 18.6-20 g of calcium sulfate dihydrate, 364-400 g of potassium sulfate, 298-320 g of magnesium sulfate dihydrate, 82.6-100 g of potassium hydroxide, and 800-850 g of glycerol.

9. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 2, wherein: In step S2, each 500 mL of PTM1 solution includes the following components: 3-5 g of copper sulfate pentahydrate, 0.044-0.1 g of potassium iodide, 1.5-2 g of manganese sulfate monohydrate, 0.1-0.2 g of sodium molybdate dihydrate, 0.01-0.03 g of boric acid, 0.25-0.35 g of cobalt chloride, 10-12 g of zinc chloride, 32.5-35 g of ferrous sulfate heptahydrate, 0.1-0.2 g of biotin, and 2.5-3.0 mL of sulfuric acid.

10. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 9, characterized in that: The glycerol feed includes: glycerol, purified water, defoamer and PTM1; the methanol feed includes: methanol and defoamer.

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