WO2015062175A1 - 一种用于细胞培养基的玉米活性肽添加剂 - Google Patents

一种用于细胞培养基的玉米活性肽添加剂 Download PDF

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WO2015062175A1
WO2015062175A1 PCT/CN2014/071394 CN2014071394W WO2015062175A1 WO 2015062175 A1 WO2015062175 A1 WO 2015062175A1 CN 2014071394 W CN2014071394 W CN 2014071394W WO 2015062175 A1 WO2015062175 A1 WO 2015062175A1
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corn
active peptide
cells
protein
protease
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PCT/CN2014/071394
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English (en)
French (fr)
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蔡木易
林峰
刘艳
张海欣
谷瑞增
鲁军
魏颖
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中国食品发酵工业研究院
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Priority to JP2015544345A priority Critical patent/JP6077672B2/ja
Priority to US14/583,058 priority patent/US9534026B2/en
Publication of WO2015062175A1 publication Critical patent/WO2015062175A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to a cell culture medium additive, and more particularly to a corn active peptide additive for use in a cell culture medium. Background technique
  • the medium is the most direct and important environmental factor that determines the growth and metabolism of cells in vitro.
  • Animal cell culture media usually contain animal-derived additives such as serum, and with the global outbreak of mad cow disease, the use of animal-derived additives such as serum brings infectious spongiform encephalopathies (TSEs) and other dangerous substances to the biopharmaceutical industry. The security risks have suddenly increased.
  • Drug regulators around the world require drug manufacturers to reduce the use of bovine components in pharmaceutical production, and the legislation is increasingly opposed to the use of animal-derived components such as bovine serum in the pharmaceutical industry.
  • the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) strictly control the use of animal source ingredients in commercial production media to ensure the patient's ultimate medication safety.
  • serum and other animal-derived medium supplements contain macromolecular proteins such as growth factors, transporters, adhesion factors, hormones, etc., which put pressure on the separation and purification of biological products such as antibody drugs and vaccines, and ultimately affect product yield. , increase production costs. Therefore, the development and application of non-animal cell culture supplements are in line with current development directions, and have become an important topic in the field of animal cell culture and biopharmaceuticals.
  • Corn yellow powder is a crude by-product of corn obtained by wet-grinding process, and then obtained by protein separation to obtain gluten water, which is then concentrated and dried. It is the main by-product of corn wet processing of starch, containing more than 60% protein. It also contains inorganic salts and multivitamins.
  • Maize active peptide is a mixture of small molecular peptides prepared from corn gluten meal by pretreatment, enzymatic hydrolysis, multi-stage separation and purification, concentration and drying. It is a cutting-edge in the field of bioprocessing and transformation of natural zein resources. product.
  • the corn active peptide is derived from plant protein, avoiding the problems of potential viral microbial contamination in animal components such as serum; the quality of the batch is consistent, which is beneficial to improve the stability of cell production; it does not contain macromolecular protein, which is beneficial to The cell product is isolated and purified downstream.
  • the corn active peptide is rich in nutrients, and is susceptible to bacterial microbial contamination during its processing and storage, which in turn produces a large amount of bacterial endotoxin.
  • When used as a cell culture additive it changes cell morphology, destroys cell membrane structure, induces apoptosis, etc. It has an adverse effect on the growth of cells cultured in vitro.
  • the animal serum components are more complex, many components It is still unclear, so it is impossible to determine whether the presence or absence of a certain component or a certain component adversely affects the growth of cells, thereby causing certain difficulties in the application of the corn active peptide to the serum-free medium additive. Summary of the invention
  • the invention provides a corn active peptide additive and the use thereof, the corn active peptide has low endotoxin content, and can be compounded with various basic media when used as a cell culture medium additive, and is used for a plurality of cell lines. Serum-free culture is carried out, and cell proliferation, cell viability and cell product expression are also promoted.
  • the invention also provides a preparation method of corn active peptide for cell culture medium, which has simple operation, high enzymatic hydrolysis efficiency, and can carry out small molecule active peptide with macromolecular protein, bacterial endotoxin and small molecular salt. Efficient separation, suitable for large-scale industrial production.
  • the invention provides a corn active peptide additive for cell culture medium, wherein an oligopeptide having a molecular weight of less than 1000 Dalton accounts for ⁇ 90% by mass of total protein, and the oligopeptide comprises at least AP, SAP, One or more of PAL, VNAP, PSSQ, TQPGPQ.
  • the oligopeptide further comprises one or more of AY, NAP, PVIN, AYPQ.
  • endotoxin in the corn active peptide additive is ⁇ 200 EU/g, especially endotoxin content
  • the corn active peptide additive according to the present invention is obtained by separating and purifying corn gluten by enzymatic hydrolysis using a non-specific protease and a specific protease in sequence.
  • the non-specific protease of the present invention refers to a type of protease having low specificity to a substrate to be applied and having a broad substrate specificity, such as an alkaline protease, which can hydrolyze a peptide chain of a plurality of protein molecules.
  • an alkaline protease which can hydrolyze a peptide chain of a plurality of protein molecules.
  • Producing a polypeptide or amino acid; as well as bromelain it can decompose a variety of proteins as well as peptides, lipids and amides.
  • the specific protease of the present invention refers to a type of protease which has relatively strict substrate specificity and can only act on certain specific peptide bonds in a protein molecule, such as a peptide formed by carboxypeptidase catalyzed hydrolysis of a terminal amino acid having a carboxyl group. Keys, etc.
  • the non-specific protease is selected from one or more of papain, alkaline protease, neutral protease and bromelain, and the specific protease is selected from the group consisting of carboxypeptidase and flavor protease.
  • the non-specific protease is selected from two or more of papain, alkaline protease, neutral protease, and bromelain.
  • the invention first adopts a specific protease to enzymatically hydrolyze corn gluten powder, and then further enzymatically digests the enzymatic hydrolyzed product by using a non-specific protease, and the combination of the two can achieve higher enzymatic hydrolysis efficiency, and the active peptide in the enzymatic hydrolysate, In particular, the content of the small molecular weight active peptide is high. Further, the amount of the non-specific protease is 2000 to 6000 units per gram of protein, including 0 to 1000 units of enzyme amount of papain, 500 to 2000 units of enzyme amount of alkaline protease, and 500 to 2000 units of enzyme amount.
  • the specific protease has an enzyme amount of 2000 to 3000 units per gram of protein, including 500 to 2000 units of enzyme amount of carboxypeptidase And one or both of 1000 to 2000 units of enzyme amount of flavor protease.
  • the enzyme activity unit of each protease is defined as: under the test conditions, the amount of enzyme which produces lg tyrosine per minute by hydrolysis of casein is defined as an enzyme activity unit; and the protein in each gram of protein specifically refers to The protein in the corn yellow meal, that is, each protease containing the corresponding unit enzyme amount per 1 gram of protein in the corn yellow powder.
  • the enzymatic hydrolysis of the zeaxanthin using the non-specific protease and the specific protease of the present invention can produce a plurality of oligopeptides having a molecular weight of less than 1000 Daltons, and those skilled in the art can use the enzymes according to conventional techniques in the prior art.
  • the oligopeptide is isolated and purified from the solution, such as centrifugation, microfiltration, ultrafiltration, and the like.
  • the separation and purification comprises one or more of centrifugation, microfiltration, cation exchange, nanofiltration and ultrafiltration; in particular, the separation and purification according to the present invention is specifically: Performing centrifugation, microfiltration, cation exchange, nanofiltration and ultrafiltration, wherein the microfiltration is carried out using a membrane having a pore size of 50 to 500 nm, and the nanofiltration is carried out using a membrane having a molecular weight cut off of 100 to 300 Daltons. The ultrafiltration is carried out using a filter membrane having a molecular weight cut off of 5,000 to 13,000 daltons.
  • the invention also provides the use of the above corn active peptide additive in cell serum free culture.
  • the cells include one or more of CHO cells, Vera cells, HEK-293 cells, BHK-21 cells, MARC-145 cells, hybridoma cells, MDCK cells.
  • the present invention also provides the use of the above-described corn active peptide additive for promoting cell proliferation, enhancing cell viability, and/or enhancing expression of a cell product.
  • the cell is selected from one or more of CHO cells, Vera cells, HEK-293 cells, BHK-21 cells, MARC-145 cells, hybridoma cells, MDCK cells.
  • the present invention also provides a serum-free medium comprising the corn active peptide additive according to any one of the above, wherein the concentration of the corn active peptide additive in the serum-free medium is 0.01 to 20 g/L, for example, ⁇ 10g / L, further can be 2 ⁇ 4g / L.
  • the present invention also provides a method for preparing a corn active peptide additive according to any of the above, comprising the steps of:
  • the first enzymatic hydrolysate is prepared by first enzymatic treatment of the corn yellow powder by using a non-specific protease; then the second enzymatic hydrolysis is performed on the first enzymatic hydrolysate by using a specific protease to obtain a corn proteolytic solution.
  • the amount of the non-specific protease is 2000 to 6000 units per gram of protein, including 0 to 1000 units of enzyme amount of papain, 500 to 2000 units of enzyme amount of alkaline protease, 500 ⁇ 2000 unit enzyme amount of neutral protease and 0 ⁇ 1500 unit enzyme amount of two or more of bromelain;
  • the specific protease enzyme amount is 2000 ⁇ 3000 units per gram of protein, including 500 ⁇ 2000 units Enzyme amount of carboxypeptidase and one or both of 1000 to 2000 units of enzyme amount of flavor protease.
  • the conditions of the first enzymatic treatment and the second enzymatic treatment can be adjusted by those skilled in the art depending on the composition of the enzyme to be employed.
  • the step 1) specifically includes: after mixing the corn yellow powder with water to form a protein solution, adding the non-specific protease to the protein solution in an amount of 2000 to 6000 units per gram of protein, at 40 ⁇
  • the first enzymatic treatment is carried out at 60 ° C for 1.5 to 4.5 hours to prepare a first enzymatic hydrolyzate; and the specific enzymatic protease is added to the first enzymatic hydrolysate in an amount of from 2000 to 3000 units per gram of protein.
  • the enzyme was killed to obtain a corn proteolytic solution.
  • the enzyme can be specifically: the second enzymatic hydrolyzed solution is heated to 85 ⁇ 130 ° C for 10-20 min.
  • the method further comprises: heating the protein solution to 90 to 95 ° C and holding for 15 to 60 minutes to denature the protein in the protein solution, which is beneficial to subsequent enzymatic treatment.
  • the centrifugal separator may be used for dish.
  • the microfiltration in the step 2) specifically comprises: firstly performing the first microfiltration on the centrifugal supernatant by using a filter having a pore diameter of 200 to 500 nm to obtain a first microfiltrate. Then, the first microfiltrate is subjected to a second microfiltration using a filter having a pore diameter of 50 to 200 nm to prepare a second microfiltrate.
  • the filter membrane used in the first microfiltration and the second microfiltration is a ceramic membrane; the operating pressure (overpressure) is 10 to 30 psi; the protein in the first microfiltrate and the second microfiltrate
  • the content of the protein in the present invention is a mass content. If the protein content is too low, the time of microfiltration is increased, the production efficiency is lowered, and if the protein content is too high, the filtration of the membrane is increased. The burden and the service life of the filter are reduced.
  • the inventors have found that the protein content is suitable in the range of 1 to 5%.
  • the present invention uses a two-step ceramic membrane filtration, that is, preliminary filtration and depth filtration, and preliminary filtration for removing soluble macromolecular proteins that are not removed by centrifugation.
  • the main purpose of the depth filtration is to further purify to remove substances such as impurities, and the two-step filtration not only improves the filtration efficiency, but also has a good filtration effect.
  • the cation exchange in the step 2) may specifically include: loading the cation exchange resin into the chromatography column, washing the filtrate with distilled water for 3 to 5 times, and then filtering the filtrate to 0.5.
  • a linear flow rate of ⁇ 3 C m/min was added to the column, washed with distilled water for 2 to 5 column volumes, and then eluted with a 1 M NaCl solution at a linear flow rate of 1 to 5 cm/min to collect the eluate.
  • a purification liquid is prepared; wherein the cation exchange resin may be a hydrogen type cation exchange resin such as a D001 type cation exchange resin, and a D001-FD type cation exchange resin is used in the embodiment of the present invention.
  • the invention adopts ion exchange to further separate and purify the above filtrate, which can further separate the active peptide in the filtrate from other weakly charged, oppositely charged, uncharged impurities (such as starch), thereby being better. The purity and quality of the active peptide product are guaranteed.
  • the nanofiltration in the step 2) specifically comprises: concentrating the centrifuged supernatant after the cation exchange to a protein content by using a roll film having a molecular weight cut off of 100 to 300 daltons. After washing for 5 to 10%, the content of the inorganic salt in the centrifuged supernatant after washing is ⁇ 5%; and the centrifuged supernatant after washing is concentrated to a protein content of 20 to 40%.
  • the ultrafiltration in the step 2) is carried out by using a roll film having a molecular weight cut off of 5000 to 13000 daltons, wherein the operating pressure is 10 to 30 psi, to further remove the treatment liquid. Endotoxin.
  • drying is carried out after the ultrafiltration is carried out to obtain a corn active peptide having a moisture content of 1.5 to 5%.
  • the corn active peptide of the invention has high oligopeptide content and low endotoxin content, and can be flexibly combined with various basic media when used as a cell culture medium additive, and no additional serum is needed. It can be used for serum-free culture of various animal cells.
  • the preparation method of the invention has the advantages of simple operation, high enzymatic hydrolysis efficiency, high content of small molecule active peptide in the enzymatic hydrolysate, and high molecular protein, bacterial endotoxin and small molecule in the small molecule active peptide and enzymatic hydrolysate. Salts are efficiently separated for large-scale industrial production.
  • the serum-free medium of the present invention is simple in preparation and low in cost, and can be used for culturing a plurality of cell lines, and can also promote cell proliferation, increase cell viability, and enhance expression of cell products.
  • Figure 1 is a high performance liquid chromatogram of the corn active peptide of Example 1;
  • Figure 3 is a graph showing the amount of antibody expression of CHO cells cultured in Test Example 1;
  • Figure 4 is a graph showing the cell viability of CHO cells cultured in Test Example 2;
  • Figure 5 is a cell density diagram of Vero cells cultured in Test Example 3.
  • Figure 6 is a graph showing the amount of antibody expression of hybridoma cells cultured in Test Example 4.
  • Figure 7 is a graph showing the cell viability of HEK-293 cells cultured in Test Example 5;
  • Figure 8 is a cell density diagram of BHK-21 cells cultured in Test Example 6;
  • Figure 9 is a cell density map of MARC-145 cells cultured in Test Example 7.
  • Figure 10 is a graph showing the cell viability of MDCK cells cultured in Test Example 8. detailed description
  • the composite non-specific protease (wherein the neutral protease NEUTRASE OD ⁇ and the alkaline protease ALKALINE PROTEASE CONCENTRATE have an enzyme amount of about 2000 units), and the first enzymatic treatment is carried out at about 55 ° C for 2.5 hours to prepare the first enzyme.
  • the corn proteolytic solution is placed in a disc separator and centrifuged at a speed of 4000 to 12000 r/min to separate the liquid residue from the corn proteolytic solution, and the centrifuge supernatant is collected for use;
  • the first microfiltration (primary filtration) of the centrifugation supernatant is performed by using a ceramic membrane having a pore diameter of 200 to 500 nm, and the first microfiltration time is controlled to be 1 to 3 hours, and the operating pressure is 10 to 30 psi, and the protein content is 1 ⁇ 5% of the first microfiltrate; the first microfiltrate is subjected to a second microfiltration (depth filtration) using a ceramic membrane having a pore diameter of 50 to 200 nm, and the time for controlling the second microfiltration is 1 to 3 hours, and the operating pressure is 10 ⁇ At 30 psi, a second microfiltrate having a protein content of 1 to 5% was obtained.
  • the D001-FD type cation exchange resin (Zhejiang Zhengguang Industrial Co., Ltd.) dedicated to the food industry is loaded into a chromatography column, and after washing 3 to 5 times the column volume with distilled water, the second microfiltrate is 0.5 to 3 C.
  • the linear flow rate of m/min is added to the column to control the injection volume to be 20 ⁇ 100% of the column volume, 2 ⁇ 5 times column volume with distilled water, and 1 ⁇ 5cm/min with 1M NaCl solution. The linear flow rate was eluted, and the eluate was collected to obtain a purified liquid.
  • the purified liquid is concentrated to a protein content of 5 to 10% by using a roll film having a molecular weight cut off of 100 to 300 Daltons, and then washed with distilled water, and washed for 3 to 5 times of the initial volume to be washed.
  • the content of the inorganic salt in the purified liquid after filtration is less than 5%, and further concentrated to a protein content of 20 to 40%, the concentrated liquid is collected;
  • the concentrate was subjected to ultrafiltration using a roll film having a molecular weight cut off of 5,000 to 13,000 Daltons to remove endotoxin in the concentrate to prepare a corn active peptide solution having an endotoxin content of less than 50 EU/g.
  • corn active peptide sample lg prepared above, dilute to 100 mL with mobile phase A, and ultrasonically oscillate lOmin to fully dissolve the sample, then prepare it into O.lmg/mL corn active peptide solution with a pore size of 0.2.
  • the mass spectrometry conditions are: ionization mode: Nanoliter electrospray positive ion; atomizing gas: ⁇ 2; collision gas: Ar ; source temperature: 80 °C ; cone voltage: 50V; TOF acceleration voltage: 9.1kV; MCP detector voltage: 2150V; capillary voltage: 800V; Mass accuracy of MS and MS/MS: O.lDa; After obtaining the ESI-MS mass spectrum by first-order mass spectrometry, the ions to be measured are selected from the ESI-MS mass spectrum, and then subjected to ESI-MS/MS analysis, mass spectrometry. After the MaxEnt 3 conversion of the mass spectrometer, the peptide sequence was deduced from Peptide Sequencing. The primary structure of the main components of the corn active peptide
  • the total protein content of the corn active peptide prepared in the present example is 91% or more, and the oligopeptide having a molecular weight of less than 100 ODA accounts for 98% or more of the total protein.
  • the main components of the corn active peptide include: AP, SAP, PAL, VNAP, PSSQ, AY, NAP, etc.
  • the peptides identified for the first time in the corn active peptide include AP (molecular weight 186.21 Da), SAP (molecular weight 273.29 Da), PAL (molecular weight 299.37 Da), VNAP (molecular weight 399.45 Da), PSSQ (molecular weight 417.19 Da), TQPGPQ (molecular weight 626.27 Da).
  • AP molecular weight 186.21 Da
  • SAP molecular weight 273.29 Da
  • PAL molecular weight 299.37 Da
  • VNAP molecular weight 399.45 Da
  • PSSQ molecular weight 417.19 Da
  • TQPGPQ molecular weight 626.27 Da
  • Bo Bio-Engineering Co., Ltd. a mixed composite non-specific protease (in which the amount of papain and bromelain is about 1000 units and about 1500 units, respectively), and the first enzymatic treatment is about 1.5 at 50 ⁇ 55 °C.
  • the first enzymatic hydrolysate is prepared; after adjusting the pH of the first enzymatic hydrolysate to 8 ⁇ 9, the enzyme is added to the first enzymatic hydrolyzate at an enzyme amount of about 3000 units per gram of protein.
  • a complex-specific protease consisting of carboxypeptidase Validase FP Cone.
  • DSM flavor protease FLAVOURZYME 500MG
  • Novozymes wherein the carboxypeptidase ACCELERZYME CPG and the flavor protease FLAVOURZYME 500MG have an enzyme content of 1500 units.
  • the enzymatic hydrolysate is heated to 90 ⁇ 100 ° C, and the enzyme is inactivated for 15-20 min to obtain the corn proteolytic solution.
  • a pale yellow powdery corn active peptide having a moisture content of 1.5% to 5% was obtained.
  • the oligopeptides having a molecular weight of less than 100 Oa in the corn active peptide prepared in this example account for more than 95% by mass of the total protein, and the endotoxin content is less than 50 EU/g, and contain 18 kinds of first-class in Table 3.
  • the structure of the polypeptide, its basic physical and chemical components and the molecular weight distribution results of each component are shown in Table 1 and Table 2, respectively.
  • a composite non-specific protease such as neutral protease Validase BNP-L, alkali
  • L novozymes
  • papain Nanning Pangbo Bioengineering Co., Ltd.
  • bromelain Nanning Pangbo Bioengineering Co., Ltd.
  • the enzyme enzymes of Alcalase 2.4L, papain and bromelain are about 2000 units, about 2000 units, about 1000 units and about 1000 units, and the first enzymatic treatment is carried out at 45 ⁇ 50 °C for about 3 hours.
  • the first enzymatic hydrolysate is prepared; after adjusting the pH of the first enzymatic hydrolysate to about 5.5, the amount of the enzyme per gram of protein is about 2000 units to the first enzymatic hydrolysate.
  • Add the flavor protease MAXZPRO XF (DSM) heat the second enzymatic treatment for about 2.5 hours at about 55 °C, then heat the enzymatic hydrolyzate to 120 ⁇ 130 °C, keep the enzyme for 10 ⁇ 15min, and obtain the corn.
  • DSM flavor protease MAXZPRO XF
  • the oligopeptides having a molecular weight of less than 100 Oa in the corn active peptide prepared in the present example account for more than 98% by mass of the total protein, and the endotoxin content is less than 50 EU/g, and contain 18 kinds of first grades in Table 3.
  • the structure of the polypeptide, its basic physical and chemical components and the molecular weight distribution results of each component are shown in Table 1 and Table 2, respectively.
  • Table 1 Basic physical and chemical components of the corn active peptide of the present invention
  • the corn oligopeptide powder prepared according to the method described in Chinese Patent Application No. 200810084992.5 is used as Comparative Example 1, and specifically includes:
  • the medium is dissolved in pyrogen-free ultrapure water, and the concentration is 4 times of the conventional concentration; the prepared peptide solution is mixed with DMEM/F12 medium, and ultrapure water is added to 95% of the final volume. Adjust the pH to 7.2 ⁇ 7.3, dilute to the final volume, and sterilize with 0.22 ⁇ filter to prepare the corresponding serum-free medium, wherein the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1
  • the concentration of their respective serum-free medium was 0.5 g/L (for cell density detection), the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 in their respective serum-free medium.
  • the concentrations were all 6 g/L or 10 g/L (for antibody expression detection), which were used as test groups 1 to 3 and the control group, respectively; and DMEM/F12 medium without addition of corn active peptide was used as a blank group.
  • the culture medium of the above test group 1 ⁇ 3, the control group and the blank group were separately added to the 2L bioreactor, and CHO cells having a viability higher than 95% were inoculated into the reactor for suspension culture, wherein the culture condition was: 37°. C, 5% carbon dioxide; timed sampling, and cell density and antibody expression were detected by flow cytometry and reversed-phase high performance liquid chromatography.
  • the results are shown in Figure 2 and Figure 3.
  • the medium density of the medium added with 0.5 g/L of the test group 1 to 3 was 135%, 133%, and 135% of the blank group when cultured to the second day, and the cell density was increased from the fifth day to the fifth day. They were 125%, 129%, and 127% of the blank group, respectively, while the cell density of the control group was 120% and 96%, respectively, on the 2nd and 5th day.
  • the amount of cell antibody expression in the medium of 6g/L test group 1 ⁇ 3 was 154%%, 156%, 154% of the blank group when cultured to the 6th day, and 10g/ was added.
  • the corn active peptide prepared in Examples 1 to 3 of the present invention is used as a cell culture medium.
  • the cells grow well, which can provide comprehensive nutrition for CHO cells, and the peptides of some components may also act as a functional factor for cell growth and metabolism, which is beneficial to promote cell proliferation and enhance cell product expression;
  • the effect may be related to its concentration.
  • the concentration of 0.5g/L is more suitable for cell proliferation; and the concentration range of 6g/L ⁇ 10g/L can significantly increase the expression of antibody.
  • the invention adopts the corn proteolytic product to prepare the cell culture medium additive, and the cost thereof is far lower than that of the serum used; and the medium additive is derived from the plant component and has high safety.
  • Test example 2 Test example 2
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and IMDM medium were respectively dissolved in pyrogen-free ultrapure water, and the concentration was prepared to be 4 times of the conventional concentration; the prepared peptide solution was separately prepared.
  • Mix with IMDM medium add ultrapure water to 95% of the final volume, adjust the pH to 7.2 ⁇ 7.3, dilute to the final volume, and sterilize with 0.22 ⁇ filter to prepare the corresponding serum-free medium.
  • the corn oligopeptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 each had a concentration of 4 g/L in each serum-free medium, which were used as test groups 1 to 3 and a control group, respectively, and were not added at the same time.
  • the IMDM medium of the corn active peptide was used as a blank group.
  • the culture medium of the above test group 1 ⁇ 3, the control group and the blank group were separately added to the shake flask, and the CHO cells with good growth condition were inoculated into a shake flask for culture, and the culture conditions were: 37 ° C, 5% carbon dioxide, shaking The bottle rotation speed was 100 rpm; time sampling was performed, and cell viability was detected by flow cytometry AO/PI staining. The results are shown in Fig. 4.
  • the medium of the test group 1 to 3 can obtain higher cell viability in the same culture time, especially in the case of the culture to the 10th day, the test group 1 to 3
  • the cell viability was 81%, 80%, and 79%, respectively.
  • the cell viability of the control group was 65%, and the cell viability of the blank group was significantly decreased, only 43%. Therefore, the corn active peptides prepared in Examples 1 to 3 of the present invention can provide comprehensive nutrition for CHO cells, and solve the problems of premature death and morphological unsaturation caused by lack of serum to some extent, thereby It is beneficial to increase the viability of cells and enhance the expression of cell products.
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and DMEM (high glucose) medium were respectively dissolved in pyrogen-free ultrapure water, and the concentration was prepared to be 4 times of the conventional concentration; Formulated peptide solution and DMEM (high sugar) medium is mixed, and ultrapure water is added to 95% of the final volume. The pH is adjusted to 7.2 ⁇ 7.3, and the volume is adjusted to the final volume. The bacteria are sterilized by 0.22 ⁇ filter to obtain the corresponding serum-free culture.
  • the corn oligopeptide of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 each have a concentration of 2 g/L in each serum-free medium, which are used as test groups 1 to 3 and a control group, respectively.
  • DMEM high glucose
  • the culture medium of the above test group 1 to 3, the control group and the blank group were separately added to the cell culture plate, and the African green monkey kidney cells (Vera) having a viability higher than 95% were inoculated into the cell culture plate for culture.
  • 3TC 5% carbon dioxide
  • the cell density was measured by MTT method, and the results are shown in Fig. 5.
  • the Vero cells of Test Examples 1 to 3 proliferated rapidly and were highly metabolized, and their cell densities were 120.9%, 121.8%, and 122.3%, respectively, in the blank group, while the Vera cell density in the control group was only 106.3 in the blank group. %, indicating that the medium to which the corn active peptide additive of the present invention is added can obtain a higher cell density in the same culture time when used to culture Vero cells.
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and the DMEM/F12 (volume ratio 1:1) medium were respectively dissolved in pyrogen-free ultrapure water, and the concentration was adjusted to a conventional concentration. 4 times; separately mix the prepared peptide solution with DMEM/F12 medium, and add vitamins, fatty acids and trace elements in an appropriate amount, then add ultrapure water to 95% of the final volume, adjust the pH to 7.2 ⁇ 7.3, and adjust to pH
  • the final volume was sterilized with a 0.22 ⁇ filter to prepare a corresponding serum-free medium in which the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 were each at a concentration of each serum-free medium. It was 5 g/L, which was used as the test group 1 to 3 and the control group, respectively, and the same amount of vitamins, fatty acids, and trace elements were added to the DMEM/F12 medium as a blank group.
  • the culture medium of the above test group 1 ⁇ 3, the control group and the blank group were separately added to the 2L bioreactor, and the hybridoma cells with viability higher than 95% were inoculated into the reactor for suspension culture, wherein the culture conditions were: work Volume 1L, dissolved oxygen (DO) is controlled at 50% saturated air concentration, inoculation density 2.5xl05cells/mL, temperature 37°C, stirring speed 90rpm ; antibody is harvested after 6 days of culture, and antibody expression is detected by ELISA.
  • work Volume 1L dissolved oxygen (DO) is controlled at 50% saturated air concentration, inoculation density 2.5xl05cells/mL, temperature 37°C, stirring speed 90rpm ; antibody is harvested after 6 days of culture, and antibody expression is detected by ELISA.
  • DO dissolved oxygen
  • the antibody expression levels of the test group 1 ⁇ 3 were increased by 62%, 56%, and 58%, respectively, compared with the blank group, while the antibody expression of the control group was increased by 22 compared with the blank group. %, indicating the addition of the present invention
  • the medium of the corn active peptide can obtain a higher antibody expression amount in the same culture time when used for culturing hybridoma cells.
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and the MEM medium were separately dissolved in pyrogen-free ultrapure water, and the concentration was 4 times of the conventional concentration; the prepared peptide solution was separately prepared.
  • Mix with MEM medium add appropriate amount of vitamins, fatty acids and trace elements, then add ultrapure water to 95% of the final volume, adjust the pH to 7.2 ⁇ 7.3, dilute to the final volume, and sterilize with 0.22 ⁇ filter.
  • a corresponding serum-free medium was prepared, wherein the corn oligopeptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 each had a concentration of 7 g/L in each serum-free medium, which were respectively used as test group 1 ⁇ 3 and the control group, while adding the same amount of vitamins, fatty acids, and trace elements in MEM medium as a blank group.
  • the culture medium of the above test groups 1 to 3, the control group and the blank group were separately added to the 2L bioreactor, and HEK-293 cells having a viability higher than 95% were inoculated into the reactor for suspension culture, wherein the culture conditions were: Working volume 1L, dissolved oxygen (DO) controlled at 50% saturated air concentration, inoculation density 2.0xl0 5 cells/mL, temperature 37 ° C, stirring speed 80 rpm ; timed sampling, and AO/PI staining for cell viability detection The result is shown in Figure 7.
  • HEK-293 cells cultured in the medium of the test group 1 to 3 had higher cell viability in the same culture time than the control group and the blank group, indicating the corn active peptide additive of the present invention.
  • the viability of HEK-293 cells can be significantly increased.
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and DMEM (low sugar) medium were respectively dissolved in pyrogen-free ultrapure water, and the concentration was prepared to be 4 times of the conventional concentration;
  • the peptide solution is mixed with DMEM (low sugar) medium, and the vitamins, fatty acids and trace elements are added in an appropriate amount, then ultrapure water is added to the final volume of 95%, the pH is adjusted to 7.0 ⁇ 7.2, and the volume is adjusted to the final volume, using 0.22.
  • the ⁇ filter was sterilized to prepare a corresponding serum-free medium, wherein the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 each had a concentration of 3 g/L in each serum-free medium. They were used as test groups 1 to 3 and a control group, respectively, and the same amount of vitamins, fatty acids, and trace elements were added as a blank group in DMEM (low sugar) medium.
  • DMEM low sugar
  • the medium of the above test group 1 ⁇ 3, the control group and the blank group were separately added to a 150 ml shake flask, and the vigor was high. 95% of BHK-21 cells were inoculated into a shake flask for suspension culture, wherein the culture conditions were: working volume 40 mL, inoculation density 3.0 x 105 cells/mL, temperature 37 ° C, stirring speed 100 rpm; timed sampling, and using cells The counter was tested for cell density and the results are shown in Figure 8.
  • the cell densities of the test groups 1 to 3 were 138%, 141%, and 136% of the blank group, respectively, while the cell density of the control group was only 103% of the blank group, indicating the addition of the corn active peptide additive of the present invention.
  • the medium can significantly increase its cell density and promote its proliferation when used to culture BHK-21 cells.
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and the MEM medium were separately dissolved in pyrogen-free ultrapure water, and the concentration was 4 times of the conventional concentration; the prepared peptide solution was separately prepared.
  • Mix with MEM medium add appropriate amount of vitamins, fatty acids and trace elements, then add ultrapure water to 95% of the final volume, adjust the pH to 7.0 ⁇ 7.2, dilute to final volume, and sterilize with 0.22 ⁇ filter.
  • a corresponding serum-free medium was prepared, wherein the corn oligopeptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 each had a concentration of 6.5 g/L in each serum-free medium, which were respectively used as test groups. 1 to 3 and the control group, while adding the same amount of vitamins, fatty acids, and trace elements in MEM medium as a blank group.
  • the culture medium of the above test groups 1 to 3, the control group and the blank group were separately added to the 2L bioreactor, and MARC-145 cells having a viability higher than 95% were inoculated into the reactor for suspension culture, wherein the culture conditions were: The working volume is 1L, the seeding density is 2.0x l0 5 cells/mL, the dissolved oxygen (DO) is controlled at 50% saturated air concentration, the temperature is 37, the stirring speed is 80 rpm , the sampling is timed, and the cell density is detected by the cell counter. See Figure 9.
  • the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 and DMEM (high glucose) medium were respectively dissolved in pyrogen-free ultrapure water, and the concentration was prepared to be 4 times of the conventional concentration;
  • the prepared corn active peptide solution is mixed with DMEM (high sugar) medium solution, and the vitamins, fatty acids and trace elements are added in an appropriate amount, and then ultrapure water is added to 95% of the final volume, and the pH is adjusted to 7.0 ⁇ 7.2, and the volume is adjusted to The final volume was sterilized with a 0.22 ⁇ filter to prepare a corresponding serum-free medium in which the corn active peptides of Examples 1 to 3 and the corn oligopeptide of Comparative Example 1 were each concentrated in their respective serum-free medium. It is 2.5 g/L, which is used as test group 1 ⁇ 3 and control group, respectively.
  • the same amount of vitamins, fatty acids, and trace elements were added as a blank group in DMEM (high sugar) medium.
  • the culture medium of the above test group 1 ⁇ 3, the control group and the blank group was separately added to a 150 ml shake flask, and MDCK cells having a viability higher than 95% were inoculated into a shake flask for suspension culture, wherein the culture condition was: working volume 40 mL
  • the inoculation density was 3.0 ⁇ 10 5 cells/mL
  • the temperature was 37 ° C
  • the stirring speed was 11 rpm.
  • the samples were sampled periodically, and the cell viability was detected by AO/PI staining. The results are shown in Fig. 10.
  • the MDCK cells cultured in the medium of the test group 1 to 3 had higher cell viability in the same culture time than the control group and the blank group, indicating that the corn active peptide additive of the present invention can be markedly remarkable. Increase the viability of MDCK cells.

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Abstract

本发明提供了一种用于细胞培养基的玉米活性肽添加剂，所述玉米活性肽添加剂中分子量小于1000道尔顿的低聚肽占总蛋白的质量百分比≥90%，并且所述低聚肽至少包括AP、SAP、PAL、VNAP、PSSQ、TQPGPQ中的一种或多种。本发明的玉米活性肽添加剂可以与多种基础培养基复配，用于多种动物细胞的无血清培养。

Description

一种用于细胞培养基的玉米活性肽添加剂 技术领域
本发明涉及一种细胞培养基添加剂， 特别是涉及一种用于细胞培养基的玉米活性肽 添加剂。 背景技术
培养基是决定体外培养细胞生长代谢的最直接、 最重要的环境因素。 动物细胞培养 基通常含有血清等动物源添加物， 而随着全球疯牛病的流行爆发， 血清等动物来源添加 物的使用， 给生物制药工业带来感染性海绵状脑病 （TSEs) 病毒及其它危险物质的安全 性风险骤然上升。 全世界的药品监管机构要求药物生产商在制药生产中减少牛源成分的 使用， 相关立法机关也越来越反对在制药领域使用牛血清等动物源成分。 美国食品及药 品管理局 （FDA) 和欧洲药品管理局 （EMA) 严格控制在商业化生产培养基过程中使用 动物源成分， 以保证病人最终的服药安全。 同时， 血清及其他动物源的培养基添加物中 含有生长因子、 转运蛋白、 贴壁因子、 激素等大分子蛋白质， 为抗体药物、 疫苗等生物 制品后期分离纯化带来压力， 最终影响产品得率， 增加生产成本。 因此， 非动物源细胞 培养添加物的开发及应用符合当前的发展方向， 目前已成为动物细胞体外培养及生物制 药领域的重要课题。
玉米黄粉是玉米经湿磨法工艺制得粗淀粉乳， 再经蛋白质分离得到麸质水， 然后浓 缩干燥制得的， 其是玉米湿法加工淀粉时的主要副产物， 含有 60%以上的蛋白质， 还含 有无机盐和多种维生素。 玉米活性肽是以玉米黄粉为原料， 经预处理、 生物酶水解、 多 级分离提纯、 浓缩干燥等工艺制备出的小分子肽类混合物， 其是天然玉米蛋白资源生物 加工和转化利用领域的尖端产品。 玉米活性肽来源于植物蛋白， 避免了血清等动物性成 分中潜在的病毒微生物污染等问题； 其批次间质量一致， 有利于提高细胞产品生产的稳 定性； 其不含大分子蛋白， 有益于细胞产品下游分离纯化。
玉米活性肽营养物质丰富， 在其加工及储藏过程中易受细菌微生物污染， 进而产生 大量细菌内毒素， 将其作为细胞培养添加剂使用时会改变细胞形态、 破坏细胞膜结构、 诱导细胞凋亡等， 对体外培养的细胞生长产生不利影响。 除此之外， 由于不同的加工工 艺对制备的玉米活性肽产品的组成具有较大影响， 而动物血清成分较为复杂， 很多成分 至今尚不清楚， 因此无法确定某种或某些成分的存在或者缺失是否会对细胞的生长产生 不良影响， 从而给玉米活性肽在无血清培养基添加剂上的应用造成一定的困难。 发明内容
本发明提供了一种玉米活性肽添加剂及其应用， 该玉米活性肽内毒素含量低， 在作 为细胞培养基添加剂应用时， 可与多种基础培养基进行复配， 用于对多种细胞系进行无 血清培养， 并且还能促进细胞增殖， 提高细胞活力以及增强细胞产物表达。
本发明还提供一种用于细胞培养基的玉米活性肽的制备方法， 其操作简便， 酶解效 率高， 并且可将小分子的活性肽与大分子蛋白、 细菌内毒素及小分子盐类进行高效地分 离， 适合大规模工业化生产。
本发明提供的一种用于细胞培养基的玉米活性肽添加剂，其中分子量小于 1000道尔 顿的低聚肽占总蛋白的质量百分比≥90%， 并且所述低聚肽至少包括 AP、 SAP, PAL、 VNAP、 PSSQ、 TQPGPQ中的一种或多种。
进一步地， 所述低聚肽还包括 AY、 NAP、 PVIN、 AYPQ中的一种或多种。
进一步地， 所述玉米活性肽添加剂中内毒素的含量 <200EU/g， 特别是内毒素含量
<50EU/go
根据本发明提供的玉米活性肽添加剂， 其是通过依次采用非特异性蛋白酶和特异性 蛋白酶对玉米黄粉进行酶解后， 经分离纯化制得的。
本发明所述的非特异性蛋白酶指的是对所作用的底物的特异性低、 具有较宽的底物 特异性的一类蛋白酶， 如碱性蛋白酶， 其可水解多种蛋白质分子的肽链生成多肽或氨基 酸； 又如菠萝蛋白酶， 其能分解多种蛋白质以及肽、 脂和酰胺等。 本发明所述的特异性 蛋白酶指的是底物特异性相对严格、 仅能作用于蛋白质分子中某些特定肽键的一类蛋白 酶， 如羧肽酶催化水解含羧基的末端氨基酸所形成的肽键等。
在本发明具体方案中， 所述非特异性蛋白酶选自木瓜蛋白酶、 碱性蛋白酶、 中性蛋 白酶和菠萝蛋白酶中的一种或多种， 所述特异性蛋白酶选自羧肽酶、 风味蛋白酶中的一 种或两种； 特别是， 所述非特异性蛋白酶选自木瓜蛋白酶、 碱性蛋白酶、 中性蛋白酶和 菠萝蛋白酶中的两种或两种以上。 本发明先采用特异性蛋白酶对玉米黄粉进行酶解， 然 后采用非特异性蛋白酶对酶解产物进一步进行酶解， 两者配合使用能够达到较高的酶解 效率， 并且酶解液中的活性肽、 特别是小分子量的活性肽的含量高。 进一步地， 所述非特异性蛋白酶的酶量为每克蛋白 2000〜6000单位， 其中包括 0〜 1000单位酶量的木瓜蛋白酶、 500〜2000单位酶量的碱性蛋白酶、 500〜2000单位酶量的 中性蛋白酶和 0〜1500单位酶量的菠萝蛋白酶中的两种或多种； 所述特异性蛋白酶的酶 量为每克蛋白 2000〜3000单位， 其中包括 500〜2000单位酶量的羧肽酶和 1000〜2000 单位酶量的风味蛋白酶中的一种或两种。 在本发明中， 各蛋白酶的酶活力单位定义为： 在试验条件下， 每分钟水解酪蛋白产生 lg酪氨酸的酶量定义为一个酶活力单位； 并且所 述每克蛋白中的蛋白具体指的是玉米黄粉中的蛋白， 即玉米黄粉中每含有 1 克蛋白时加 入相应单位酶量的各蛋白酶。
采用本发明所述非特异性蛋白酶和特异性蛋白酶对玉米黄粉进行酶解， 可以产生多 种分子量小于 1000道尔顿的低聚肽，本领域技术人员可以根据现有技术中的常规技术手 段从酶解液中分离纯化出所述低聚肽， 例如离心、 微滤、 超滤等。 在本发明具体方案中， 所述分离纯化包括离心、 微滤、 阳离子交换、 纳滤和超滤中的一种或多种； 特别是， 本 发明所述分离纯化具体为： 对酶解液依次进行离心、 微滤、 阳离子交换、 纳滤和超滤， 其中所述微滤采用孔径为 50〜500nm的滤膜进行，所述纳滤采用截留分子量为 100〜300 道尔顿的滤膜进行， 所述超滤采用截留分子量为 5000〜13000道尔顿的滤膜进行。
本发明还提供上述玉米活性肽添加剂在细胞无血清培养上的应用。
进一步地， 所述细胞包括 CHO细胞、 Vera细胞、 HEK-293细胞、 BHK-21细胞、 MARC-145细胞、 杂交瘤细胞、 MDCK细胞中的一种或多种。
本发明还提供上述玉米活性肽添加剂在促进细胞增殖、 提高细胞活力和 /或增强细胞 产物表达上的应用。
进一步地， 所述细胞选自 CHO细胞、 Vera细胞、 HEK-293细胞、 BHK-21细胞、 MARC-145细胞、 杂交瘤细胞、 MDCK细胞中的一种或多种。
本发明还提供一种无血清培养基， 包括上述任一所述的玉米活性肽添加剂， 所述玉 米活性肽添加剂在所述无血清培养基中的浓度为 0.01〜20g/L， 例如可以为 l〜10g/L， 进 一步可以为 2〜4g/L。
本发明还提供上述任一所述的玉米活性肽添加剂的制备方法， 包括如下步骤：
1 )首先采用非特异性蛋白酶对玉米黄粉进行第一酶解处理， 制得第一酶解液； 然后 采用特异性蛋白酶对所述第一酶解液进行第二酶解处理， 制得玉米蛋白酶解液；
2)将所述玉米蛋白酶解液进行离心， 依次对离心上清液进行微滤、 阳离子交换、 纳 滤和超滤， 制得玉米活性肽； 其中所述微滤采用孔径为 50〜500nm的滤膜进行， 所述纳 滤采用截留分子量为 100〜300道尔顿的滤膜进行， 所述超滤采用截留分子量为 5000〜 13000道尔顿的滤膜进行。
根据本发明所提供的制备方法， 所述非特异性蛋白酶的酶量为每克蛋白 2000〜6000 单位， 其中包括 0〜1000单位酶量的木瓜蛋白酶、 500〜2000单位酶量的碱性蛋白酶、 500〜2000单位酶量的中性蛋白酶和 0〜1500单位酶量的菠萝蛋白酶中的两种或多种；所 述特异性蛋白酶的酶量为每克蛋白 2000〜3000单位，其中包括 500〜2000单位酶量的羧 肽酶和 1000〜2000单位酶量的风味蛋白酶中的一种或两种。本领域技术人员可以根据所 采用的酶的组成来调节所述第一酶解处理和第二酶解处理的条件， 如酶解温度和 pH值 等。 进一步地， 所述步骤 1 )具体包括： 将玉米黄粉与水混合制成蛋白溶液后， 以每克蛋 白 2000〜6000单位的酶量向所述蛋白溶液中加入所述非特异性蛋白酶，于 40〜60°C下第 一酶解处理 1.5〜4.5小时， 制得第一酶解液； 再以每克蛋白 2000〜3000单位的酶量向所 述第一酶解液中加入所述特异性蛋白酶，于 40〜60°C下第二酶解处理 2〜3小时后，灭酶， 制得玉米蛋白酶解液。 所述灭酶可以具体为： 将第二酶解处理后的酶解液加热至 85〜 130°C保温 10〜20min。
进一步地， 在进行所述第一酶解处理之前还包括： 将所述蛋白溶液加热至 90〜95 °C 并保温 15〜60min， 使蛋白溶液中的蛋白变性， 有利于后续的酶解处理。
根据本发明所提供的制备方法， 步骤 2)所述离心的转速为 4000〜12000r/min， 所述 离心可以采用碟式分离机进行。
根据本发明所提供的制备方法， 步骤 2) 中所述微滤具体包括： 先采用孔径为 200〜 500nm的滤膜对所述离心上清液进行第一微滤， 制得第一微滤液， 然后采用孔径为 50〜 200nm的滤膜对所述第一微滤液进行第二微滤， 制得第二微滤液。
进一步地， 所述第一微滤和第二微滤所采用的滤膜为陶瓷膜； 操作压力 （过膜压力） 为 10〜30psi; 所述第一微滤液和所述第二微滤液中蛋白质的含量为 1〜5%， 本发明中所 述蛋白质的含量为质量含量， 如果所述蛋白质含量过低会增加微滤的时间， 降低生产效 率， 而蛋白质含量过高则会增加滤膜的过滤负担， 降低滤膜的使用寿命， 发明人经研究 发现， 蛋白质含量在 1〜5%的范围内较为适合。 此外， 本发明采用两步陶瓷膜过滤， 即 依次进行初步过滤和深度过滤，初步过滤用于去除离心未除去的可溶性的大分子蛋白质， 而深度过滤主要目的是进一步纯化， 以去除杂蛋白等物质， 两步过滤不仅可以提高过滤 效率， 而且过滤效果好。
根据本发明所提供的制备方法， 步骤 2)中所述阳离子交换可以具体包括： 将阳离子 交换树脂装入层析柱中， 用蒸馏水清洗 3〜5倍柱体积后， 将所述过滤液以 0.5〜3Cm/min 的线性流速加入到层析柱中，用蒸馏水清洗 2〜5倍柱体积，再用 1M的 NaCl溶液以 1〜 5cm/min 的线性流速进行洗脱， 收集洗脱液， 制得纯化液； 其中， 所述阳离子交换树脂 可以为氢型阳离子交换树脂， 例如 D001 型阳离子交换树脂， 在本发明具体方案中采用 D001-FD型阳离子交换树脂。 本发明采用离子交换对上述过滤液进行进一步分离纯化， 其可以将过滤液中的活性肽进一步与其它带弱电、 带相反电荷、 不带电的杂质 （如淀粉 等） 进行有效地分离， 从而更好地保证了活性肽产品的纯度及品质。
根据本发明所提供的制备方法， 步骤 2 ) 中所述纳滤具体包括： 采用截留分子量为 100〜300道尔顿的卷式膜将经所述阳离子交换后的离心上清液浓缩至蛋白质含量为 5〜 10%后进行洗滤，使经洗滤后的离心上清液中无机盐的含量≤5%； 再将经洗滤后的离心上 清液浓缩至蛋白质含量为 20〜40%。 发明人发现直接进行纳滤处理时效果较差， 可能是 由于纳滤膜孔径较小， 容易被活性肽分子所堵塞， 因此发明人在进行纳滤时加入了洗滤 的步骤， 并且经发明人研究发现， 洗滤之前控制溶液中的蛋白质含量为 5~10%时可以获 得较好的纳滤效果。
根据本发明所提供的制备方法， 步骤 2) 中所述超滤采用采用截留分子量为 5000〜 13000道尔顿的卷式膜进行， 其中操作压力为 10〜30psi， 用以进一步去除处理液中的内 毒素。
进一步地， 在进行所述超滤之后还包括干燥， 制得水分含量为 1.5〜5%的玉米活性 肽。
本发明方案的实施， 至少具有以下优势：
1、 本发明的玉米活性肽中低聚肽含量高， 并且内毒素含量低， 其在作为细胞培养基 添加剂应用时， 可灵活地与多种基础培养基进行复配使用， 并且无需额外添加血清， 可 用于对多种动物细胞进行无血清培养。
2、 本发明的制备方法操作简便， 酶解效率高， 酶解液中小分子活性肽的含量高， 此 外其可将小分子活性肽与酶解液中的大分子蛋白、 细菌内毒素及小分子盐类进行高效地 分离， 适合大规模工业化生产。 3、 本发明的无血清培养基配制简便， 成本低廉， 其能用于培养多种细胞系， 此外还 能够促进细胞增殖， 提高细胞活力以及增强细胞产物的表达。 附图说明
图 1为实施例 1玉米活性肽的高效液相色谱图；
图 2为试验例 1培养的 CHO细胞的细胞密度图；
图 3为试验例 1培养的 CHO细胞的抗体表达量图；
图 4为试验例 2培养的 CHO细胞的细胞活力图；
图 5为试验例 3培养的 Vero细胞的细胞密度图；
图 6为试验例 4培养的杂交瘤细胞的抗体表达量图；
图 7为试验例 5培养的 HEK-293细胞的细胞活力图；
图 8为试验例 6培养的 BHK-21细胞的细胞密度图；
图 9为试验例 7培养的 MARC-145细胞的细胞密度图；
图 10为试验例 8培养的 MDCK细胞的细胞活力图。 具体实施方式
为使本发明的目的、 技术方案和优点更加清楚， 下面将结合本发明实施例， 对本发 明实施例中的技术方案进行清楚、 完整地描述， 显然， 所描述的实施例是本发明一部分 实施例， 而不是全部的实施例。 基于本发明中的实施例， 本领域普通技术人员在没有做 出创造性劳动前提下所获得的所有其他实施例， 都属于本发明保护的范围。 实施例 1
1、 酶解处理
向蛋白质含量≥60%的玉米黄粉中加入 12倍的去离子水，混合均勾后，升温至约 90°C， 保温 15〜20分钟后， 降温至约 55°C时， 调节蛋白溶液的 pH值至 7〜8， 以每克蛋白质 4000单位左右的酶量向所述蛋白溶液中加入由中性蛋白酶 NEUTRASE O.D丄（诺维信公 司）和碱性蛋白酶 ALKALINE PROTEASE CONCENTRATE (DSM公司）混合而成的复 合非特异性蛋白酶 （其中中性蛋白酶 NEUTRASE O.D丄 和碱性蛋白酶 ALKALINE PROTEASE CONCENTRATE的酶量均为 2000单位左右）， 于约 55 °C下保温第一酶解处 理 2.5小时， 制得第一酶解液； 调节第一酶解液的 pH值为 7.5〜8.5后， 再以每克蛋白质 2000单位左右的酶量向所述第一酶解液中加入羧肽酶 ACCELERZYME CPG (DSM公 司），于约 50°C下保温第二酶解处理 2.5小时后，将酶解液加热到 110〜120°C，保温 15〜 20min进行灭酶， 制得玉米蛋白酶解液。
2、 微滤
将玉米蛋白酶解液置入碟式分离机中， 在 4000〜12000r/min的转速下进行离心， 以 对玉米蛋白酶解液进行液渣分离， 收集离心上清液备用；
采用孔径为 200〜500nm 的陶瓷膜对离心上清液进行第一微滤 （初步滤清） ， 控制 第一微滤的时间为 l〜3h，操作压力为 10〜30psi，制得蛋白含量为 1〜5%的第一微滤液； 采用孔径为 50〜200nm的陶瓷膜对第一微滤液进行第二微滤 （深度过滤） ， 控制第 二微滤的时间为 l〜3h， 操作压力为 10〜30psi， 制得蛋白含量为 1〜5%的第二微滤液。
3、 阳离子交换
将食品行业专用的 D001-FD型阳离子交换树脂 （浙江争光实业股份有限公司） 装入 层析柱中， 用蒸馏水清洗 3〜5倍柱体积后， 将所述第二微滤液以 0.5〜3Cm/min的线性 流速加入到层析柱中， 控制进样体积为层析柱体积的 20〜100%， 用蒸馏水清洗 2〜5倍 柱体积， 再用 1M的 NaCl溶液以 l〜5cm/min的线性流速进行洗脱， 收集洗脱液， 制得 纯化液。
4、 纳滤、 超滤
采用截留分子量为 100〜300道尔顿的卷式膜将所述纯化液浓缩至蛋白质含量为 5〜 10%, 然后加入蒸馏水进行洗滤， 共洗滤初始体积的 3〜5倍， 使经洗滤后的纯化液中无 机盐的含量低于 5%， 再进一步将其浓缩至蛋白含量为 20〜40%时， 收集浓缩液；
采用截留分子量为 5000〜13000道尔顿的卷式膜对所述浓缩液进行超滤， 以去除浓 缩液中的内毒素， 制得内毒素含量低于 50EU/g的玉米活性肽溶液。
5、 干燥处理
将玉米活性肽溶液进行喷雾干燥， 控制喷雾干燥净化空气进口温度为 120〜180°C， 出口温度为 65〜90°C， 制得水分含量为 1.5%〜5%的淡黄色粉末状玉米活性肽。
6、 检测
采用常规方法对制得的玉米活性肽基础理化成分及各组分的分子量分布进行分析， 结果分别见表 1和表 2。
采用 RP-HPLC (岛津 LC-20A高效液相色谱仪， 日本 SHIMADZU公司） 对制得的 玉米活性肽进行分离后， 采用 Q-TOF2质谱仪 （英国 Micromass公司） 对其主要组分进 行一级结构鉴定， 具体方法如下：
取上述制得的玉米活性肽样品 lg，用流动相 A定容至 100mL，超声振荡 lOmin使样 品充分溶解混勾后，将其配制成 O.lmg/mL的玉米活性肽溶液，用孔径为 0.2μηι的聚四氟 乙烯过滤膜过滤后，进样； 其中色谱条件为： 流动相 A: V (水）： V (三氟乙酸） =100:0.1 ; 流动相 Β: V (乙腈) ： V (水) ： V (三氟乙酸) =80:20:0.1； 检测波长： UV220nm; 流速： 0.6mL/min; 柱温： 32°C ; 进样体积： 50μί; 图 1为制得的玉米活性肽的高效液相色谱图。
分管收集各个组分峰（至少收集图 1中序号为 1〜12的组分峰）， 利用氮吹仪除去有 机溶剂， 冷冻干燥制成粉末后进行质谱检测， 其中质谱条件为： 离子化方式： 纳升电喷 雾正离子； 雾化气体： Ν2; 碰撞气体： Ar; 源温： 80°C ; 锥孔电压： 50V; TOF加速电 压： 9.1kV; MCP检测器电压： 2150V; 毛细管电压： 800V; MS和 MS/MS的质量准确 度： O.lDa; 在一级质谱扫描得到 ESI-MS质谱图后， 从 ESI-MS质谱图中选出待测离子， 然后进行 ESI-MS/MS分析， 质谱图经质谱仪的 MaxEnt 3转换后， 由 Peptide Sequencing 推导出肽段序列， 玉米活性肽主要组分的一级结构如表 3所示。
由表 1、 表 2结果可知： 本实施例制得的玉米活性肽中总蛋白含量达到 91%以上， 其中分子量在 lOOODa以下的低聚肽占总蛋白的质量百分比达到 98%以上。由表 3结果可 知： 玉米活性肽的主要成分包括： AP、 SAP、 PAL、 VNAP、 PSSQ、 AY、 NAP等， 并且 玉米活性肽中首次鉴定出的肽包括 AP (分子量 186.21 Da)、 SAP (分子量 273.29 Da)、 PAL (分子量 299.37 Da)、VNAP (分子量 399.45 Da)、PSSQ (分子量 417.19 Da)、TQPGPQ (分子量 626.27 Da)。 实施例 2
除酶解处理具体为： 向蛋白质含量≥60%的玉米黄粉中加入 9倍的去离子水， 混合均 勾后， 升温至 90〜95°C， 保温 55〜60分钟后， 降温至 50〜55°C时， 调节蛋白溶液的 pH 值为 7〜8， 以每克蛋白质 2500单位左右的酶量向所述蛋白溶液中加入由木瓜蛋白酶（南 宁庞博生物工程有限公司） 和菠萝蛋白酶 （南宁庞博生物工程有限公司） 混合而成的复 合非特异性蛋白酶 （其中木瓜蛋白酶和菠萝蛋白酶的酶量分别为 1000单位左右和 1500 单位左右）， 于 50〜55°C下保温第一酶解处理约 1.5小时， 制得第一酶解液； 调节第一酶 解液的 pH值为 8〜9后，再以每克蛋白质 3000单位左右的酶量向所述第一酶解液中加入 由羧肽酶 Validase FP Cone. (DSM公司） 和风味蛋白酶 FLAVOURZYME 500MG (诺维 信公司） 混合而成的复合特异性蛋白酶 （其中羧肽酶 ACCELERZYME CPG和风味蛋白 酶 FLAVOURZYME 500MG的酶量均为 1500单位左右），于 50〜55°C下保温第二酶解处 理约 3小时后， 将酶解液加热到 90〜100°C， 保温 15〜20min进行灭酶， 制得玉米蛋白 酶解液外， 其它同实施例 1， 制得水分含量为 1.5%〜5%的淡黄色粉末状玉米活性肽。
经检测，本实施例制得的玉米活性肽中分子量在 lOOODa以下的低聚肽占总蛋白的质 量百分比达到 95%以上， 内毒素含量低于 50EU/g， 含有表 3中的 18种一级结构的多肽， 其基础理化成分及各组分的分子量分布结果分别见表 1和表 2。 实施例 3
除酶解处理具体为： 向蛋白质含量≥60%的玉米黄粉中加入 10倍的去离子水， 混合 均勾后， 升温至约 95°C， 保温 35〜40分钟后， 降温至 45〜50°C时， 调节蛋白溶液的 pH 值为 7〜8， 并以每克蛋白质 6000单位左右的酶量向所述蛋白溶液中加入由中性蛋白酶 Validase BNP-L (DSM公司）、 碱性蛋白酶 Alcalase 2.4L (诺维信公司）、 木瓜蛋白酶（南 宁庞博生物工程有限公司） 和菠萝蛋白酶 （南宁庞博生物工程有限公司） 混合而成的复 合非特异性蛋白酶（其中中性蛋白酶 Validase BNP-L、 碱性蛋白酶 Alcalase 2.4L、 木瓜蛋 白酶和菠萝蛋白酶的酶量分别为 2000单位左右、 2000单位左右、 1000单位左右和 1000 单位左右）， 于 45〜50°C下保温第一酶解处理约 3小时， 制得第一酶解液； 调节第一酶解 液的 pH值为 5.5左右后， 再以每克蛋白质 2000单位左右的酶量向所述第一酶解液中加 入风味蛋白酶 MAXZPRO XF (DSM公司）， 于约 55 °C下保温第二酶解处理 2.5小时左右 后， 将酶解液加热到 120〜130°C， 保温 10〜15min进行灭酶， 制得玉米蛋白酶解液外， 其它同实施例 1， 制得水分含量为 1.5%〜5%的淡黄色粉末状玉米活性肽。
经检测，本实施例制得的玉米活性肽中分子量在 lOOODa以下的低聚肽占总蛋白的质 量百分比达到 98%以上， 内毒素含量低于 50EU/g， 含有表 3中的 18种一级结构的多肽， 其基础理化成分及各组分的分子量分布结果分别见表 1和表 2。 表 1 本发明玉米活性肽的基础理化成分
Figure imgf000012_0001
表 2 本发明玉米活性肽的分子量分布
Figure imgf000012_0002
表 3 实施例 1玉米活性肽主要组分的一级结构
Figure imgf000012_0003
对照例 1
按照申请号为 200810084992.5的中国发明专利中所述方法制备的玉米低聚肽粉作为 对照例 1， 其具体包括：
将 2.5kg水与 150g玉米蛋白粉混配调浆， 调节 pH值为 8， 加热至 60°C并保温水浴 搅拌 60min; 将料液离心后弃清液， 向渣料中加水 2.5kg稀释， 加热至 60°C并保温水浴 搅拌 60min， 离心， 弃清液， 加 2kg水调浆， 调节 pH值为 8后， 加热至 60°C， 加入 lg 碱性蛋白酶酶解 3h， 再调节 pH值为 7， 降温至 45°C后加入 lg中性蛋白酶酶解 5h， 高温 灭酶后离心分离， 将清液经陶瓷膜及卷式纳滤膜过滤， 经浓缩、 干燥， 制得玉米低聚肽 粉， 经检测， 玉米低聚肽粉中分子量小于 1000道尔顿的低聚肽的含量为 80%， 细菌内毒 素的含量>200(^11/§。 试验例 1
1、 无血清培养基配制
分别将实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽与 DMEM/F12(体积比 1：
1 )培养基分别用无热原的超纯水溶解， 配制浓度为常规浓度的 4倍； 分别将配制的肽溶 液与 DMEM/F12培养基进行混合， 并加超纯水至最终体积的 95%， 调整 pH到 7.2〜7.3， 定容至最终体积， 用 0.22μηι滤膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的 玉米活性肽和对照例 1 的玉米低聚肽在其各自的无血清培养基的浓度分别均 0.5g/L (用 于细胞密度检测）， 实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽在其各自的无血 清培养基的浓度均为 6g/L或 10g/L (用于抗体表达量检测） ， 其分别作为试验组 1〜3和 对照组； 同时以不添加玉米活性肽的 DMEM/F12培养基作为空白组。
2、 细胞培养
向 2L生物反应器中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活 力高于 95%的 CHO细胞接种于反应器中进行悬浮培养， 其中培养条件为： 37°C， 5%二 氧化碳； 定时取样， 并采用流式细胞术和反相高效液相色谱进行细胞密度、 抗体表达量 的检测， 结果见图 2、 图 3。
如图 2所示，添加 0.5g/L试验组 1〜3的培养基在培养至第 2天时细胞密度分别为空 白组的 135%%、 133%、 135%,在培养至第 5天时细胞密度分别为空白组的 125%、 129%、 127%, 而对照组在培养至第 2天和第 5天时细胞密度分别为空白组的 120%和 96%。 如 图 3所示， 培养 6天后， 添加 6g/L试验组 1〜3的培养基在培养至第 6天时细胞抗体表 达量分别为空白组的 154%%、 156%、 154%, 添加 10g/L时细胞抗体表达量分别为空白 组的 146%、 151%、 144%, 而对照组在添加 6g/L和 10g/L时细胞抗体表达量分别为空白 组的 131%和 113%。 由此说明： 本发明实施例 1〜3制得的玉米活性肽在作为细胞培养基 添加剂使用时细胞生长状态良好， 其可为 CHO细胞提供全面的营养， 并且部分成分的肽 还可能作为细胞生长代谢的功能因子， 有利于促进细胞增殖及增强细胞产物表达； 此外， 玉米活性肽的作用效果可能与其浓度相关， 0.5g/L的浓度更适宜细胞增殖;而 6g/L〜 10g/L 的浓度范围可显著提高抗体表达量。 本发明采用玉米蛋白酶解产物制备细胞培养基添加 剂， 其成本远远低于使用血清； 并且此培养基添加剂来源于植物成分， 安全性高。 试验例 2
1、 无血清培养基配制
分别将实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽与 IMDM培养基分别用 无热原的超纯水溶解， 配制浓度为常规浓度的 4倍； 分别将配制的肽溶液与 IMDM培养 基进行混合， 并加超纯水至最终体积的 95%， 调整 pH到 7.2〜7.3， 定容至最终体积， 用 0.22μηι滤膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的玉米活性肽和对照例 1 的玉米低聚肽在其各自的无血清培养基的浓度均为 4g/L，其分别作为试验组 1〜3和对照 组， 同时以不添加玉米活性肽的 IMDM培养基作为空白组。
2、 细胞培养
向摇瓶中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 将生长状况良好的 CHO细胞接种于摇瓶中进行培养，培养条件为： 37 °C , 5%二氧化碳，摇瓶转速为 lOOrpm; 定时取样， 采用流式细胞术 AO/PI染色法进行细胞活力检测， 结果见图 4。
如图 4所示， 相对于对照组和空白组， 试验组 1〜3的培养基在相同的培养时间内可 以获得更高的细胞活力，特别是在培养至第 10天时，试验组 1〜3的细胞活力分别为 81%、 80%、 79%, 对照组的细胞活力为 65%， 空白组的细胞活力显著下降， 仅为 43%。 由此 说明： 本发明实施例 1〜3制得的玉米活性肽可为 CHO细胞提供全面的营养， 并且在一 定程度上解决细胞因缺乏血清而造成的过早死亡及形态不饱和等问题， 从而有利于提高 细胞的活力及增强细胞产物表达。 试验例 3
1、 无血清培养基配制
分别将实施例 1〜3的玉米活性肽和对照例 1 的玉米低聚肽与 DMEM (高糖） 培养 基分别用无热原的超纯水溶解， 配制浓度为常规浓度的 4 倍； 分别将配制的肽溶液与 DMEM (高糖） 培养基进行混合， 并加超纯水至最终体积的 95%， 调整 pH到 7.2〜7.3， 定容至最终体积， 用 0.22μηι滤膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的 玉米活性肽和对照例 1的玉米低聚肽在其各自的无血清培养基的浓度均为 2g/L， 其分别 作为试验组 1〜3和对照组， 同时以不添加玉米活性肽的 DMEM (高糖） 培养基作为空 白组。
2、 细胞培养
向细胞培养板中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活力高 于 95%的非洲绿猴肾细胞 （Vera) 接种于细胞培养板中进行培养， 培养条件为： 3TC , 5%二氧化碳； 培养 24h后， 采用 MTT法进行细胞密度检测， 结果见图 5。
如图 5所示， 试验例 1〜3的 Vero细胞增殖迅速、 代谢旺盛， 其细胞密度分别为空 白组的 120.9%、 121.8%、 122.3%, 而对照例的 Vera细胞密度仅为空白组的 106.3%， 说 明添加有本发明玉米活性肽添加剂的培养基在用于培养 Vero细胞时，在相同的培养时间 内可以获得更高的细胞密度。 试验例 4
分别将实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽与 DMEM/F12(体积比 1 : 1 )培养基分别用无热原的超纯水溶解， 配制浓度为常规浓度的 4倍； 分别将配制的肽溶 液与 DMEM/F12培养基进行混合， 并适量添加维生素、 脂肪酸、 微量元素， 然后加超纯 水至最终体积的 95%， 调整 pH到 7.2〜7.3， 定容至最终体积， 用 0.22μηι滤膜除菌， 制 得相应的无血清培养基，其中实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽在其各 自的无血清培养基的浓度均为 5g/L， 其分别作为试验组 1〜3 和对照组， 同时以向 DMEM/F12培养基中添加相同量的维生素、 脂肪酸、 微量元素作为空白组。
向 2L生物反应器中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活 力高于 95%的杂交瘤细胞接种于反应器中进行悬浮培养，其中培养条件为：工作体积 1L， 溶氧 （DO) 控制在 50%饱和空气浓度， 接种密度 2.5xl05cells/mL， 温度 37°C， 搅拌速 度 90rpm; 在培养 6天后收获抗体， 并采用 ELISA方法进行抗体表达量的检测， 结果见 图 6。
如图 6所示， 相对于对照组和空白组， 试验组 1〜3抗体表达量相对空白组分别增加 了 62%、 56%、 58%, 而对照组抗体表达量相对空白组仅增加了 22%， 说明添加本发明 玉米活性肽的培养基在用于培养杂交瘤细胞时， 在相同的培养时间内可以获得更高的抗 体表达量。 试验例 5
分别将实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽与 MEM培养基分别用无 热原的超纯水溶解， 配制浓度为常规浓度的 4倍； 分别将配制的肽溶液与 MEM培养基 进行混合， 并适量添加维生素、 脂肪酸、 微量元素， 然后加超纯水至最终体积的 95%， 调整 pH到 7.2〜7.3， 定容至最终体积， 用 0.22μηι滤膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽在其各自的无血清培养基的浓度 均为 7g/L，其分别作为试验组 1〜3和对照组，同时以 MEM培养基添加相同量的维生素、 脂肪酸、 微量元素作为空白组。
向 2L生物反应器中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活 力高于 95%的 HEK-293细胞接种于反应器中进行悬浮培养， 其中培养条件为： 工作体积 1L， 溶氧 （DO) 控制在 50%饱和空气浓度， 接种密度 2.0xl05cells/mL， 温度 37°C， 搅 拌速度 80rpm; 定时取样， 并采用 AO/PI染色法进行细胞活力的检测， 结果见图 7。
如图 7所示， 相对于对照组和空白组， 试验组 1〜3的培养基所培养的 HEK-293细 胞在相同的培养时间内具有更高的细胞活力， 说明本发明的玉米活性肽添加剂可以显著 提高 HEK-293细胞的活力。 试验例 6
分别将实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽与 DMEM (低糖）培 养基分别用无热原的超纯水溶解， 配制浓度为常规浓度的 4 倍； 分别将配制的肽溶 液与 DMEM (低糖） 培养基进行混合， 并适量添加维生素、 脂肪酸、 微量元素， 然 后加超纯水至最终体积的 95%， 调整 pH到 7.0〜7.2， 定容至最终体积， 用 0.22μηι 滤膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的玉米活性肽和对照例 1的 玉米低聚肽在其各自的无血清培养基的浓度均为 3g/L， 其分别作为试验组 1〜3和对 照组， 同时以 DMEM (低糖） 培养基添加相同量的维生素、 脂肪酸、 微量元素作为 空白组。
向 150ml摇瓶中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活力高 于 95%的 BHK-21细胞接种于摇瓶中进行悬浮培养， 其中培养条件为： 工作体积 40mL， 接种密度为 3.0x l05cells/mL， 温度 37°C， 搅拌速度 lOOrpm; 定时取样， 并采用细胞计数 仪进行细胞密度的检测， 结果见图 8。
如图 8所示， 试验组 1〜3的细胞密度分别为空白组的 138%、 141%、 136%, 而对照 组的细胞密度仅为空白组的 103%，说明添加本发明玉米活性肽添加剂的培养基在用于培 养 BHK-21细胞时可以显著地提高其细胞密度， 促进其增殖。 试验例 7
分别将实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽与 MEM培养基分别用无 热原的超纯水溶解， 配制浓度为常规浓度的 4倍； 分别将配制的肽溶液与 MEM培养基 进行混合， 并适量添加维生素、 脂肪酸、 微量元素， 然后加超纯水至最终体积的 95%， 调整 pH到 7.0〜7.2， 定容至最终体积， 用 0.22μηι滤膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的玉米活性肽和对照例 1的玉米低聚肽在其各自的无血清培养基的浓度 均为 6.5g/L， 其分别作为试验组 1〜3和对照组， 同时以 MEM培养基添加相同量的维生 素、 脂肪酸、 微量元素作为空白组。
向 2L生物反应器中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活 力高于 95%的 MARC-145细胞接种于反应器中进行悬浮培养， 其中培养条件为： 工作体 积 1L， 接种密度为 2.0x l05cells/mL， 溶氧 ( DO )控制在 50%饱和空气浓度， 温度 37 ， 搅拌速度 80rpm; 定时取样， 并采用细胞计数仪进行细胞密度的检测， 结果见图 9。
如图 9所示， 试验组 1〜3的培养基所培养的 MARC-145细胞在相同的培养时间内 具有更高的细胞密度，说明本发明的玉米活性肽添加剂有利于促进 MARC-145细胞增殖。 试验例 8
分别将实施例 1〜3的玉米活性肽和对照例 1 的玉米低聚肽与 DMEM (高糖） 培养 基分别用无热原的超纯水溶解， 配制浓度为常规浓度的 4倍； 分别将配制的玉米活性肽 溶液与 DMEM (高糖） 培养基溶液进行混合， 并适量添加维生素、 脂肪酸、 微量元素， 然后加超纯水至最终体积的 95%， 调整 pH到 7.0〜7.2， 定容至最终体积， 用 0.22μηι滤 膜除菌， 制得相应的无血清培养基， 其中实施例 1〜3的玉米活性肽和对照例 1的玉米低 聚肽在其各自的无血清培养基的浓度均为 2.5g/L， 其分别作为试验组 1〜3和对照组， 同 时以 DMEM (高糖） 培养基添加相同量的维生素、 脂肪酸、 微量元素作为空白组。
向 150ml摇瓶中分别加入上述试验组 1〜3、 对照组和空白组的培养基， 并将活力高 于 95%的 MDCK细胞接种于摇瓶中进行悬浮培养， 其中培养条件为： 工作体积 40mL， 接种密度为 3.0x l05cells/mL， 温度 37°C， 搅拌速度 llOrpm; 定时取样， 并采用 AO/PI 染色法进行细胞活力的检测， 结果见图 10。
如图 10所示， 相对于对照组和空白组， 试验组 1〜3的培养基所培养的 MDCK细胞 在相同的培养时间内具有更高的细胞活力， 说明本发明的玉米活性肽添加剂能够显著提 高 MDCK细胞的活力。 最后应说明的是： 以上各实施例仅用以说明本发明的技术方案， 而非对其限制； 尽 管参照前述各实施例对本发明进行了详细的说明， 本领域的普通技术人员应当理解： 其 依然可以对前述各实施例所记载的技术方案进行修改， 或者对其中部分或者全部技术特 征进行等同替换； 而这些修改或者替换， 并不使相应技术方案的本质脱离本发明各实施 例技术方案的范围。

Claims

权利 要 求
1.一种用于细胞培养基的玉米活性肽添加剂， 其特征在于， 所述玉米活性肽添加剂 中分子量小于 1000道尔顿的低聚肽占总蛋白的质量百分比≥90%， 并且所述低聚肽至少包 括 AP、 SAP、 PAL、 VNAP、 PSSQ、 TQPGPQ中的一种或多种。
2. 根据权利要求 1所述的玉米活性肽添加剂， 其特征在于， 所述低聚肽还包括 AY、 NAP、 PVIN、 AYPQ中的一种或多种。
3. 根据权利要求 1所述的玉米活性肽添加剂， 其特征在于， 所述玉米活性肽添加剂 中内毒素的含量<20(^11/§。
4. 根据权利要求 1所述的玉米活性肽添加剂， 其特征在于， 所述玉米活性肽添加剂 是通过依次采用非特异性蛋白酶和特异性蛋白酶对玉米黄粉进行酶解后， 经分离纯化制 得的。
5. 根据权利要求 4所述的玉米活性肽添加剂， 其特征在于， 所述非特异性蛋白酶选 自木瓜蛋白酶、 碱性蛋白酶、 中性蛋白酶和菠萝蛋白酶中的一种或多种， 所述特异性蛋 白酶选自羧肽酶、 风味蛋白酶中的一种或两种。
6. 权利要求 1至 5任一所述的玉米活性肽添加剂在细胞无血清培养上的应用。
7. 根据权利要求 6所述的应用， 其特征在于， 所述细胞包括 CHO细胞、 Vera细胞、 HEK-293细胞、 BHK-21细胞、 MARC-145细胞、 杂交瘤细胞、 MDCK细胞中的一种或 多种。
8. 权利要求 1至 5任一所述的玉米活性肽添加剂在促进细胞增殖、提高细胞活力和 / 或增强细胞产物表达上的应用。
9. 根据权利要求 8所述的应用， 其特征在于， 所述细胞包括 CHO细胞、 Vera细胞、 HEK-293细胞、 BHK-21细胞、 MARC- 145细胞、 杂交瘤细胞、 MDCK细胞中的一种或 多种。
10. —种无血清培养基，其特征在于，包括权利要求 1至 5任一所述的玉米活性肽添 加剂， 所述玉米活性肽添加剂在所述无血清培养基中的浓度为 0.01〜20g/L。
11. 权利要求 1至 5中任一所述的玉米活性肽添加剂的制备方法， 包括如下步骤：
1 )首先采用非特异性蛋白酶对玉米黄粉进行第一酶解处理， 制得第一酶解液； 然后 采用特异性蛋白酶对所述第一酶解液进行第二酶解处理， 制得玉米蛋白酶解液； 2)将所述玉米蛋白酶解液进行离心， 依次对离心上清液进行微滤、 阳离子交换、 纳 滤和超滤， 制得玉米活性肽； 其中所述微滤采用孔径为 50〜500nm的滤膜进行， 所述纳 滤采用截留分子量为 100〜300道尔顿的滤膜进行， 所述超滤采用截留分子量为 5000〜 13000道尔顿的滤膜进行。
12. 根据权利要求 11所述的制备方法， 其特征在于， 所述非特异性蛋白酶的酶量为 每克蛋白 2000〜6000单位， 其中包括 0〜1000单位酶量的木瓜蛋白酶、 500〜2000单位 酶量的碱性蛋白酶、 500〜2000单位酶量的中性蛋白酶和 0〜1500单位酶量的菠萝蛋白酶 中的两种或多种；所述特异性蛋白酶的酶量为每克蛋白 2000〜3000单位，其中包括 500〜 2000单位酶量的羧肽酶和 1000〜2000单位酶量的风味蛋白酶中的一种或两种。
13. 根据权利要求 12所述的制备方法， 其特征在于， 所述步骤 1 ) 具体包括： 将玉 米黄粉与水混合制成蛋白溶液后， 以每克蛋白 2000〜6000单位的酶量向所述蛋白溶液中 加入所述非特异性蛋白酶，于 40〜60°C下第一酶解处理 1.5〜4.5小时，制得第一酶解液； 再以每克蛋白 2000〜3000 单位的酶量向所述第一酶解液中加入所述特异性蛋白酶， 于 40〜60°C下第二酶解处理 2〜3小时后， 灭酶， 制得玉米蛋白酶解液。
14. 根据权利要求 11所述的制备方法， 其特征在于， 步骤 2) 中所述微滤具体包括: 先采用孔径为 200〜500nm 的滤膜对所述离心上清液进行第一微滤， 制得第一微滤液， 然后采用孔径为 50〜200nm的滤膜对所述第一微滤液进行第二微滤， 制得第二微滤液。
15. 根据权利要求 11所述的制备方法， 其特征在于， 步骤 2) 中所述纳滤具体包括: 采用截留分子量为 100〜300道尔顿的卷式膜将经所述阳离子交换后的离心上清液浓缩至 蛋白质含量为 5〜10%后进行洗滤， 使经洗滤后的离心上清液中无机盐的含量≤5%； 再将 经洗滤后的离心上清液浓缩至蛋白质含量为 20〜40%。
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