Method for adjusting hydroxylation level of recombinant protein

Abstract

The present invention relates to a method for adjusting the hydroxylation level of a recombinant protein produced by fermentation, said method comprising the use of a modifier comprising one or more of the following trace elements during the fermentation: iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, silicon. The methods of the present invention enable the hydroxylation modification level of the recombinant protein to be reduced by at least about 7% compared to a control group without the use of a modulator.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the field of cell culture, and in particular to a method for adjusting the hydroxylation level of a recombinant protein. Background Art

[0002] Post-translational modification plays an important role in the processing and maturation of proteins. It can change the physical and chemical properties of proteins, affect the spatial conformation, steric hindrance and stability of proteins, and then affect the biological activity of proteins, causing functional changes in proteins.

[0003] The consistency of the post-translational modification level of biosimilar drugs with that of the original drug is an important indicator for similarity evaluation. Therefore, regulating post-translational modifications has become one of the difficulties in the development of biosimilar drugs. In industry, cell culture process optimization is generally used for regulation, including the screening of culture media and feeds, adjustment of culture parameters, etc. However, so far, most of the cell culture process optimization for regulating post-translational modifications is for the regulation of glycosylation modifications, and there are few reports on the optimized fermentation process for regulating hydroxylation modifications. Therefore, it is urgent to provide a method for regulating the hydroxylation modification of recombinant proteins and a culture system for host cells expressing the recombinant proteins. Summary of the invention

[0004] To solve the above problems, the present invention provides a method for adjusting the hydroxylation level of a recombinant protein, wherein the recombinant protein is produced by fermentation, and the method comprises using a regulator containing one or more selected from the following trace elements during the fermentation process: iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, and silicon.

[0005] In some embodiments, the modulator is animal-derived component-free (ADCF) and does not contain growth factors, polypeptides and hydrolysates thereof, phenol red, poloxamer, hypoxanthine, thymidine, L-glutamine, or any combination thereof.

[0006] In some embodiments, the modulator is TE MaxA medium.

[0007] In some embodiments, during the fermentation process, when the cell density reaches 3×10 6 The regulator was then used after 10 cells / mL.

[0008] In some embodiments, using the regulator comprises adding the regulator to the cell culture, wherein the volume of the added regulator is 0.1% to 10% of the initial culture volume, such as adding it every day, preferably, adding the regulator at a volume concentration of 0.1%-5%.

[0009] In some embodiments, a feed medium is also used during the fermentation process.

[0010] In some embodiments, the post-translational modification of the recombinant protein includes hydroxylation modification, and the types of recombinant proteins include but are not limited to polypeptide hormones, cytokines, plasma protein factors, recombinant enzymes, fusion proteins, monoclonal antibodies, etc., wherein the recombinant protein is selected from tasiglutide, dulaglutide, semaglutide, teriparatide, afamelanotide, thymopentin, gastric inhibitory peptide, tyrosine tyrosine peptide, leuprorelin (LB), triptorelin, goserelin, buserelin, histrelin, deslorelin, tartrelin, futirelin, gonadorelin, nafarelin, cetrorelix (RK) , degarelix, abarelix, tevirelix, bivalirudin, atosiban, carbetocin, actidil, vapreotide, desmopressin, salmon calcitonin, ziconotide, salivary gland undecapeptide, nesiritide, eptifibatide, epifentide, enfuvirtide, glatiramer, icatibant, octreotide (OCT), lanreotide, pasireotide, pramlintide, actidil, romiplostim, caperitide, amoxicillin, liraglutide (LT), exenatide, linaclotide, collagen or a combination thereof, preferably collagen or dulaglutide.

[0011] In some embodiments, the cells used in the fermentation are mammalian cells capable of expressing the recombinant protein.

[0012] The present invention also provides a use of a regulator in reducing the hydroxylation level of a recombinant protein, wherein the recombinant protein is produced by fermentation, and the regulator contains one or more of the following trace elements: iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, and silicon.

[0013] In some embodiments, the modulator is animal-derived component-free (ADCF) and does not contain growth factors, polypeptides and hydrolysates thereof, phenol red, poloxamer, hypoxanthine, thymidine, L-glutamine, or any combination thereof.

[0014] In some embodiments, the modulator is TE MaxA medium.

[0015] In some embodiments, the post-translational modification of the recombinant protein includes hydroxylation modification, and the types of recombinant proteins include but are not limited to polypeptide hormones, cytokines, plasma protein factors, recombinant enzymes, fusion proteins, monoclonal antibodies, etc., wherein the recombinant protein is selected from tasiglutide, dulaglutide, semaglutide, teriparatide, afamelanotide, thymopentin, gastric inhibitory peptide, tyrosine tyrosine peptide, leuprorelin (LB), triptorelin, goserelin, buserelin, histrelin, deslorelin, tartrelin, futirelin, gonadorelin, nafarelin, cetrorelix (RK) , degarelix, abarelix, tevirelix, bivalirudin, atosiban, carbetocin, actidil, vapreotide, desmopressin, salmon calcitonin, ziconotide, salivary gland undecapeptide, nesiritide, eptifibatide, epifentide, enfuvirtide, glatiramer, icatibant, octreotide (OCT), lanreotide, pasireotide, pramlintide, actidil, romiplostim, caperitide, amoxicillin, liraglutide (LT), exenatide, linaclotide, collagen or a combination thereof, preferably collagen or dulaglutide.

[0016] The present invention also provides a kit for adjusting the hydroxylation level of a recombinant protein produced by fermentation, the kit comprising (1) TE MaxA medium and (2) at least one feed medium, such as CELLBOOST TM 7a and / or CELL BOOST TM 7b.

[0017] The present invention also provides a kit for adjusting the hydroxylation level of a recombinant protein produced by fermentation, the kit comprising (1) TE MaxA medium and (2) at least one feed medium, such as BalanCDfeed4. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cell growth curves at different regulator concentrations are shown, VCD (viable cell density): viable cell density; VIA (cell viability): cell viability.

[0019] Figure 2A and Figure 2B The reverse phase chromatogram of the peptide fragments of dulaglutide obtained by the method of Example 2 after trypsin digestion is shown ( Figure 2A : reference group; Figure 2B : experimental group). DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0021] definition

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0023] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0024] The term "about" refers to a range of ±10%, ±5%, ±2% or ±1% of the value it modifies.

[0025] The term "cell culture" refers to a population of cells suspended in a culture medium under conditions suitable for cell survival and / or growth, and may comprise a combination of the culture medium and the population of cells suspended therein.

[0026] The term "cell density" refers to the number of cells present in a given volume of culture medium.

[0027] The term "batch culture" refers to a method of culturing a culture in which a limited amount of nutrients is input at one time in a closed system.

[0028] The term "fed-batch culture" refers to a culture method in which additional components, such as cell nutrient supplements that are depleted during the culture process, are continuously or intermittently supplied to the culture at a certain point in time after the start of the culture process.

[0029] The term "continuous culture" refers to a culture method in which fresh culture medium is added to the fermentation vessel at a certain rate, while the culture medium is removed at the same rate, thereby maintaining a constant liquid volume in the fermentation vessel and allowing the cell culture to grow in a nearly constant state.

[0030] In a first aspect of the present invention, a method for adjusting the hydroxylation level of a recombinant protein is provided, wherein the recombinant protein is produced by fermentation. When used in the present invention, the term "hydroxylation" refers to the hydroxylation of one or more amino acid residues produced by post-translational modification or other reasons after the recombinant protein is expressed in a cell / bacteria.

[0031] The term "hydroxylation level" refers to the separation of the various peptide fragments produced by digestion (e.g., by reverse phase chromatography) after the target protein is fully digested (e.g., treated with one or more proteases, such as trypsin, and / or treated with a physical method), and then the area normalization method is used to calculate the percentage of hydroxylated and unmodified peaks in the same group of target polypeptides (the same group of target polypeptides is the same target polypeptide with different post-translational modifications or without post-translational modifications). It can be understood by those skilled in the art that the same group of target polypeptide peaks contains multiple peaks, one of which corresponds to the hydroxylated modified portion, and the rest corresponds to the unhydroxylated modified portion. The areas of the two types of peaks add up to the total area, and the hydroxylation level refers to the hydroxylated peak area divided by the total area.

[0032] The term "adjust" refers to controlling or reducing the hydroxylation level of the recombinant protein so that it reaches, approaches or is lower than a target value (such as the hydroxylation level of the corresponding original drug), or the hydroxylation level is reduced compared to a culture process that has not been adjusted.

[0033] The term "fermentation" refers to culturing host cells under conditions suitable for the growth of host cells capable of expressing a recombinant protein and expressing the recombinant protein, including, for example, inoculating host cells expressing the recombinant protein in a basal medium for fermentation, and then culturing the host cells under suitable conditions (e.g., 28-37° C.), for example, for about 3 to 20 days.

[0034] The host cells suitable for the present invention are mammalian cells, including but not limited to Chinese hamster ovary cells (including CHO-K1, CHO DG44, CHO S, CHOZN and CHO DUXB11), human myeloid leukemia cells (including GLYCOEXPRESS), HeLa (human cervical cancer cells), African green monkey kidney fibroblasts (including CV-1, COS-1 and COS-7), African green monkey kidney epithelial cells (including Vero), BHK, MDCK, C127, PC12, human embryonic kidney cells (including HEK-293, HEK-293T, HEK-293F and HEK-293E), PER C6, NS0, WI38, R1610, BALBC / 3T3, HAK, SP2 / O, P3x63-Ag8.653, BFA-1c1BPT, or RAJI cells, and the host cells can express the desired recombinant protein after gene editing. Any medium suitable for culturing corresponding host cells known in the art can be used as the basic medium of the present invention, such as commercially available Pro CHOTM 5 Protein-free CHO Medium (Lonza), Power CHOTM GS Serum-free CHO Medium (Lonza), Power CHOTM Advance Chemically Defined (Lonza), Power CHOTM 2 Serum-free Medium (Lonza), EX-Cell Advanced CHO Fed-batch medium (Sigma), Cellvento TM CHO-110AGT (Merck), EX-Cell Cellvento 4CHO (Merck), CDCHO Medium (Gibco), CD OptiCHO TMMedium (Gibco), ExpiCHO SPM (Gibco), Dynamis medium (Gibco), ActiPro cell culture media (Hyclone), LM-SFM4CHO (Hyclone), LM-CDM4PERMAb (Hyclone), LM-HyCELL CHO (Hyclone), LM-CDM4MAb (Hyclone), BalanCD growth A (Irvine), CHO Max A (Maibang Biotech), CHO Max C (Maibang Biotech), CHO Max D2 (Maibang Biotech), CHO MaxX (Maibang Biotech), CHO Max Y (Maibang Biotech), CHO Max Z (Maibang Biotech), Eden B600S (Beangene Biotech) or a combination thereof.

[0035] Thus, in some embodiments, the method of the present invention comprises inoculating host cells expressing a recombinant protein in a basal medium and then culturing the host cells under suitable conditions. Various inoculation densities can be used. In some embodiments, the inoculation density is 1.0×10 4 to 1.0×10 7 In some embodiments, the seeding density is 1.0×10 5 to 1.0×10 6 In some embodiments, the seeding density is 4.0×10 5 Up to 8.0×10 5 In some embodiments, the seeding density is 5.0×10 5 Up to 6.0×10 5 cells / mL.

[0036] The method of the present invention comprises using a regulator containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) selected from the following trace elements during the fermentation process: iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, silicon. Preferably, the regulator does not contain growth factors, polypeptides and their hydrolysates, phenol red, poloxamer (P188), hypoxanthine, thymidine, L-glutamine or any combination thereof.

[0037] The inventors of the present invention unexpectedly discovered that the hydroxylation level of recombinant proteins can be reduced by using such a regulator (e.g., TE MaxA medium (Shanghai Maibang Biotechnology Co., Ltd.)) during the fermentation process. TE MaxA is a chemically defined medium composed of a variety of trace elements and does not contain any animal-derived components, growth factors, polypeptides and their hydrolysates, phenol red, poloxamer, hypoxanthine, thymidine, or L-glutamine.

[0038] In some embodiments, the hydroxylation modification level of the recombinant protein is reduced by at least about 7%, at least about 8%, at least about 11%, at least about 13%, or even at least about 27% compared to a control group without the use of the regulator. In some embodiments, the degree of reduction in the hydroxylation modification level of the recombinant protein is positively correlated with the amount of the regulator used. That is, the higher the amount of the regulator used, the more the hydroxylation level of the recombinant protein is reduced.

[0039] In some embodiments, the method of the present invention comprises adding the modulator to the cell culture, for example, adding the modulator to the host cell culture at the same time as the host cell inoculation, or at least one day after the host cell inoculation (for example, 1 day, 2 days, 3 days, 4 days or 5 days after inoculation). In some other embodiments, during the fermentation process, when the cell density reaches 3×10 6 The regulator is used after adding cells / mL. For example, in the case of fed-batch culture fermentation, a regulator of 0.1% to 10% volume concentration (volume concentration refers to the volume concentration relative to the initial culture volume) and preferably 0.1% to 5% volume concentration is added to the cell culture (for example, added every day). Of course, the method of the present invention is not limited thereto, and those skilled in the art can select a regulator of a suitable volume concentration according to the hydroxylation level to be achieved as needed, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0% volume concentration or any value therebetween.

[0040] In some embodiments, the method of the present invention also includes using feed medium during fermentation.The feed medium is mainly used for supplementing the nutrients exhausted by cells, to support cell stationary phase growth and protein expression.When used in the present invention, feed medium does not include TE MaxA culture medium or a culture medium with a composition substantially the same as that thereof.The culture medium for the culture of any suitable corresponding host cell as known in the art can be used as the feed medium of the present invention, such as commercially available EX-Cell Advanced CHO Feed1 (Sigma), EX-Cell Cellvento 4Feed (Merck), Efficient feed A+ (Gibco), Efficient feed B+ (Gibco), Efficient feed C+ (Gibco), Cell BoostTM7a (Hyclone), Cell BoostTM7b (Hyclone), BalanCD feed4 (Irvine), CHO Max FA (Maibang Biology), CHO Max FB (Maibang Biology), CHO Max FX (Maibang Biology), CHOMax FB (Maibang Biology) or its combination. Those skilled in the art can select appropriate feed medium addition concentration and timing according to the cell growth conditions during the fermentation process.

[0041] The method of the present invention is applicable to recombinant proteins prepared by cell culture technology and requiring regulation of post-translational hydroxylation modification levels. The types of recombinant proteins include but are not limited to polypeptide hormones, cytokines, plasma protein factors, recombinant enzymes, fusion proteins, monoclonal antibodies, etc. For example, the recombinant protein can be dulaglutide, wherein the recombinant protein is selected from tasiglutide, dulaglutide, semaglutide, teriparatide, afamelanotide, thymopentin, enteric inhibitory peptide, tyrosine tyrosine, leuprorelin (LB), triptorelin, goserelin, buserelin, histrelin, deslorelin, tartrelin, futirelin, gonadrelin, Arelin, nafarelin, cetrorelix (RK), degarelix, abarelix, tevireix, bivalirudin, atosiban, carbetocin, actidil, vapreotide, desmopressin, salmon calcitonin, ziconotide, salivary gland undecapeptide, nesiritide, eptifibatide, epifentide, enfuvirtide, glatiramer, icatibant, octreotide (OCT), lanreotide, pasireotide, pramlintide, actidil, romiplostim, caperitide, amnimustine, liraglutide (LT), exenatide, linaclotide, collagen or a combination thereof, preferably collagen or dulaglutide.

[0042] In some embodiments, the method of the present invention is used to adjust the hydroxylation level of dulaglutide. Dulaglutide (trade name Trulicity) is a long-acting GLP-1 receptor agonist hypoglycemic drug developed by Eli Lilly. In addition to common glycosylation, oxidation and deamidation modifications, there are some uncommon modification types in protein biopharmaceuticals, such as phosphorylation, hydroxylation, formaldehyde and acetaldehyde modifications.

[0043] In a second aspect of the present invention, there is provided a use of a regulator in reducing the hydroxylation level of a recombinant protein, the recombinant protein being produced by fermentation, the regulator containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) selected from the following trace elements: iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, silicon. Preferably, the regulator does not contain growth factors, polypeptides and their hydrolysates, phenol red, poloxamer (P188), hypoxanthine, thymidine, L-glutamine, or any combination thereof.

[0044] In one embodiment, the regulator is TE MaxA culture medium purchased from Shanghai Maibang Biotechnology Co., Ltd.

[0045] In a third aspect of the present invention, there is provided a kit for adjusting the hydroxylation level of a recombinant protein, wherein the recombinant protein is produced by fermentation, and the kit comprises (1) a regulator, wherein the regulator contains one or more of iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, and silicon, has no animal-derived components (ADCF), and does not contain growth factors, polypeptides and their hydrolysates, phenol red, poloxamer, hypoxanthine, thymidine, L-glutamine, or any combination thereof; and (2) a feed medium.

[0046] In some embodiments, the feed medium is selected from EX-Cell Advanced CHO Feed1 (Sigma), EX-Cell Cellvento 4Feed (Merck), Efficient feed A+ (Gibco), Efficient feed B+ (Gibco), Efficient feed C+ (Gibco), Cell Boost 7a (Hyclone), Cell Boost 7b (Hyclone), BalanCD feed4 (Irvine), CHO Max FA (Maibang Bio), CHO Max FB (Maibang Bio), CHOMax FX (Maibang Bio), CHO Max FB (Maibang Bio) or a combination thereof.

[0047] In yet other embodiments, the kit of the present invention comprises (1) TE MaxA medium and (2) CELLBOOST™7a and / or CELL BOOST™7b.

[0048] In yet other embodiments, the kit of the present invention comprises (1) TE MaxA medium and (2) BalanCDfeed4.

[0049] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0050] Example

[0051] Example 1 Construction of cells expressing Dulaglutide molecules

[0052] The amino acid sequence of dulaglutide is shown in SEQ ID NO.1. The nucleotides expressing dulaglutide are gene synthesized according to conventional methods in the art. The obtained synthetic sequence is inserted into the same restriction site of the eukaryotic expression vector after double enzyme digestion to obtain an expression vector. The vector is integrated into CHO K1 cells (Chinese hamster ovary cells (CHO), purchased from Merck) by chemical reagent transfection. A stable cell pool is obtained by pressure screening, and the stable cell pool is screened by a single cell clone printer and comprehensive cell growth, expression level and product quality to obtain a suitable single cell clone cell line. The single cell clone cell line obtained by screening is divided into 1 ml aliquots and stored in liquid nitrogen for a long time after program cooling.

[0053] Example 2 Use AdvancedCHO Fed-batch as basal medium

[0054] The specific experimental plan is as follows:

[0055] 1) Experimental methods:

[0056] a. Cell recovery and passaging: Take out the single cell clone cell line A (the first batch of cells prepared according to the method of Example 1) from the liquid nitrogen tank and quickly transfer it to a 37°C water bath for thawing. After thawing, transfer it to 10 ml of fresh preheated culture medium and centrifuge it to discard the supernatant. After centrifugation, resuspend it in a 125 ml shake flask and adjust the cell density to 0.5×10 6 cells / ml. After recovery, the cells were subcultured every 3 days, and the viable cell density after subculture was 0.5×10 6 cells / mL, recovery and subculture medium is CD CHOFUSION Medium.

[0057] b. Fed-batch culture: After cell recovery, the cells were subcultured and inoculated into 50 mL culture shake tubes for fed-batch culture fermentation. The fermentation basal medium was AdvancedCHO Fed-batch medium, starting volume 18 mL, seeding density 0.5 × 10 6 On the third day of fermentation, when the cell density exceeded 3×10 6 cells / mL, and 3%+0.3% of the starting culture volume of Cell BoostTM7a+7b was added every day. The experimental groups were set as follows: the reference group (Advanced) did not add the regulator, and the experimental group (Advanced+TE Max A) added 0.5% volume concentration of TEMax A every day from the third day of fermentation.

[0058] The cell growth was detected by Vicell and the cells were harvested on the 14th day. The expression level was detected by OCTET, and the hydroxylation modification level of dulaglutide was detected by peptide mapping.

[0059] 2) Experimental results: The expression level and hydroxylation level of dulaglutide in Example 2 are shown in Table 1 and Figure 2A and 2B shown.

[0060] exist Figure 2A (reference group) and Figure 2B The four peaks shown in the chromatogram of (experimental group) are different forms of the target peptide (EFIAWLVK), of which peak 3 is the target peptide with hydroxylation modification, and peaks 1, 2, and 4 are the target peptide without hydroxylation modification. The ratio of the area of ​​peak 3 to the sum of the areas of the four peaks is the hydroxylation modification ratio.

[0061] Table 1 Example 2 Dulaglutide expression and hydroxylation results

[0062] Experimental Group Expression level (g / L) Hydroxylation level (%) Advanced 4.69 11.15 Advanced+0.5%TE Max A 4.47 8.01

[0063] 3) Experimental conclusion: In the Advanced medium fermentation, adding 0.5% TE Max A every day from the third day of culture can effectively reduce the hydroxylation level. Compared with the control group, the hydroxylation modification level was reduced by 28.2%, and the cell growth and expression level were not much different from the reference group.

[0064] Example 3 Using Eden B600S as basal medium

[0065] The specific experimental plan is as follows:

[0066] 1) Experimental methods:

[0067] a. Cell recovery and cell culture: Take out the single cell clone cell line A from the liquid nitrogen tank and quickly transfer it to a 37°C water bath for thawing. After thawing, transfer it to 10 ml of fresh preheated culture medium and centrifuge and discard the supernatant. After centrifugation, resuspend it in a 125 ml shake flask and adjust the cell density to 0.5×10 6 cells / ml. After recovery, the cells were subcultured every 3 days, and the viable cell density after subculture was 0.5×10 6 cells / mL, recovery and subculture medium is CD CHO FUSION Medium.

[0068] b. Fed-batch culture: After cell recovery, the cells were subcultured and inoculated into 250 mL culture shake flasks for fed-batch culture fermentation. The fermentation basal medium was Eden B600S (purchased from Shanghai Bio-Technology Co., Ltd.) with a starting volume of 50 mL and an inoculation density of 0.5×10 6 On the third day of fermentation, when the cell density exceeded 3×10 6 cells / mL, and 3%+0.3% of the initial culture volume of Cell BoostTM7a+7b was added every day. The experimental groups were set as shown in Table 2.

[0069] Table 2 Experimental group settings of Example 3

[0070] Experimental Group Conditioner Eden B600S N / A Eden B600S+0.25%TE Max A From the third day of fermentation, 0.25% TE Max A was added daily. Eden B600S+0.5%TE Max A From the third day of fermentation, 0.5% TE Max A was added daily. Eden B600S+0.75%TE Max A From the third day of fermentation, 0.75% TE Max A was added daily.

[0071] The cell growth was detected by Vicell, and the reference group was harvested on the 14th day, and the experimental group was harvested on the 13th day. The expression level was detected by HPLC, and the hydroxylation modification level of dulaglutide was detected by liquid phase peptide mapping method.

[0072] 2) Experimental results: The expression level and hydroxylation level of dulaglutide in Example 3 are shown in Table 3.

[0073] Table 3 Example 3 Delaglutide expression and hydroxylation results

[0074] Experimental Group Expression level (g / L) Hydroxylation level (%) Eden B600S 5.03 13.12 Eden B600S+0.25%TE Max A 4.78 11.38 Eden B600S+0.5%TE Max A 4.08 10.59 Eden B600S+0.75%TE Max A 3.95 10.17

[0075] 3) Experimental conclusion: In the Eden B600S medium fermentation, when 0.25% TE Max A was added, the expression level was close to that of the blank control group, only decreasing by 5%, and the hydroxylation level decreased by about 13.3%. When 0.75% TE Max A was added, the hydroxylation level decreased by about 22.5%.

[0076] Example 4 Using different concentrations of TE MAX A

[0077] The specific experimental plan is as follows:

[0078] 1) Experimental methods:

[0079] a. Cell recovery and passage: Take out the single cell clone cell line B (the second batch of cells prepared according to the method of Example 1) from the liquid nitrogen tank and quickly transfer it to a 37°C water bath for thawing. After thawing, transfer it to 10 ml of fresh preheated culture medium and centrifuge it to discard the supernatant. After centrifugation, resuspend it in a 125 ml shake flask and adjust the cell density to 0.5×10 6 cells / ml. After recovery, the cells were subcultured every 3 days, and the viable cell density after subculture was 0.5×10 6 cells / mL, recovery and subculture medium is AdvancedCHO Fed-batch medium.

[0080] b. Fed-batch culture: After cell recovery, the cells were subcultured and inoculated into 250 mL culture shake flasks for fed-batch culture fermentation. The fermentation basal medium was AdvancedCHO Fed-batch medium, starting volume 50 mL, inoculation density 0.5 × 10 6 On the third day of fermentation, when the cell density exceeded 3×10 6 cells / mL, and 3%+0.3% of the initial culture volume of Cell BoostTM7a+7b was added every day. The experimental groups were set as shown in Table 4.

[0081] Table 4 Experimental group settings of Example 4

[0082] Experimental Group Conditioner Advanced N / A Advanced+0.5%TE MAX A From the third day of fermentation, 0.5% TE Max A was added daily. Advanced+1%TE MAX A From the third day of fermentation, 1% TE Max A was added daily. Advanced+2%TE MAX A From the third day of fermentation, 2% TE Max A was added daily.

[0083] The cell growth was detected by Vicell, and the cells were harvested on the 12th day. The expression level was detected by HPLC, and the hydroxylation modification level of dulaglutide was detected by liquid phase peptide mapping.

[0084] 2) Experimental results: The cell growth curve in Example 4 is as follows Figure 1 The expression level and hydroxylation level of dulaglutide are shown in Table 5.

[0085] Table 5 Example 4 Degree Laglutide expression and hydroxylation results

[0086] Experimental Group Expression level (g / L) Hydroxylation level (%) Advanced 5.32 12.06 Advanced+0.5%TE MAX A 5.00 10.4 Advanced+1%TE MAX A 4.34 9.34 Advanced+2%TE MAX A 3.34 8.7

[0087] 3) Experimental conclusion: With the increase of TE Max A addition, the hydroxylation level at the time of culture harvest is getting lower and lower. When 0.5% TE Max A is added, the expression level is close to that of the blank control group, only decreasing by 6%, and the hydroxylation level decreases by about 13.8%. When 2% TE Max A is added, the hydroxylation level decreases by about 27.9%.

[0088] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for adjusting the hydroxylation level of a recombinant protein produced by fermentation, the method comprising using a regulator containing one or more of the following trace elements selected from the group consisting of iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, and silicon during the fermentation process.

2. The method of claim 1, wherein the modulator is animal-derived component-free (ADCF) and does not contain growth factors, polypeptides and their hydrolysates, phenol red, poloxamer, hypoxanthine, thymidine, L-glutamine or any combination thereof.

3. The method according to claim 1, wherein the regulator is TE MaxA medium.

4. The method according to any one of claims 1 to 3, wherein during the fermentation process, when the cell density reaches 3×10 6 The regulator was then used after 10 cells / mL.

5. The method according to claim 4, wherein using the regulator comprises adding the regulator to the cell culture, the volume of the added regulator being 0.1% to 10% of the initial culture volume, for example, adding it every day, preferably, adding a regulator at a volume concentration of 0.1%-5%.

6. The method according to any one of claims 1 to 3, further comprising using a feed medium during the fermentation process.

7. The method according to any one of claims 1 to 3, wherein the post-translational modification of the recombinant protein comprises hydroxylation modification, and the recombinant protein is any one of a polypeptide hormone, a cytokine, a plasma protein factor, a recombinase, a fusion protein, and a monoclonal antibody.

8. The method according to any one of claims 1 to 3, wherein the cells used in the fermentation are mammalian cells capable of expressing the recombinant protein.

9. Use of a regulator in reducing the hydroxylation level of a recombinant protein, wherein the recombinant protein is produced by fermentation, and the regulator contains one or more trace elements selected from the following: iron, copper, zinc, manganese, chromium, cobalt, vanadium, tin, nickel, molybdenum, iodine, fluorine, selenium, and silicon.

10. The use according to claim 9, wherein the regulator is free of animal-derived components (ADCF) and does not contain growth factors, polypeptides and hydrolysates thereof, phenol red, poloxamer, hypoxanthine, thymidine, L-glutamine or any combination thereof.

11. The use according to claim 9, wherein the regulator is TE MaxA medium.

12. The use according to any one of claims 9 to 11, wherein the post-translational modification of the recombinant protein comprises a hydroxylation modification, the post-translational modification of the recombinant protein comprises a hydroxylation modification, and the recombinant protein is any one of a polypeptide hormone, a cytokine, a plasma protein factor, a recombinase, a fusion protein, and a monoclonal antibody.

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