CN108220334A - The method of serum-free cell culture medium and highly effective expressing recombinant protein matter - Google Patents

The method of serum-free cell culture medium and highly effective expressing recombinant protein matter Download PDF

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CN108220334A
CN108220334A CN201611137227.6A CN201611137227A CN108220334A CN 108220334 A CN108220334 A CN 108220334A CN 201611137227 A CN201611137227 A CN 201611137227A CN 108220334 A CN108220334 A CN 108220334A
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郭亚军
寇庚
王延兵
张小萌
徐进
张存超
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Shanghai Metech Junao Biological Technology Co Ltd
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Abstract

The present invention provides a kind of serum-free cell culture medium for Chinese hamster ovary celI culture, the culture medium is made of basal medium and supplementing culture medium, cytidine, uridine, guanosine, thymidine and adenosine are added in basal medium, arginine, phenylalanine and methionine are added in supplementing culture medium.Using the serum free medium culture Chinese hamster ovary celI, make its high efficient expression CD47/PD L1 bifunctional fusion proteins, the CD47/PD L1 bifunctional fusion proteins obtained through cultural method culture of the present invention, sugar-type is modified based on G0F.

Description

The method of serum-free cell culture medium and highly effective expressing recombinant protein matter
Technical field
It is on December 09th, 2016 that the application, which is the applying date, application No. is CN2016111298564, entitled " nothing The divisional application of the method for serum cell culture medium and highly effective expressing recombinant protein matter " China application.The present invention relates to biological skills Art field, more particularly it relates to the method for serum-free cell culture medium and high efficient expression recombination hop protein.
Background technology
In modern medicine industry, production protein is prepared with recombinant technique and has developed into standardized way, passes through clone Encode the gene of each albumen, with the suitable expressive host of the genetic transformation to be expressed, finally generate and purify obtained weight Histone matter.Its protein includes monoclonal antibody, cell factor, fusion protein etc., this proteinoid be chiefly used in treating tumour, The plurality of medical field such as autoimmune disease, inflammatory disorder, immunosupress.
The application of protein drug needs to provide a large amount of recombinant protein to industrialize large-scale production.It is preferred that Expression system be mammalian cell, better than most of other eukaryotic expression systems based on insect cell, yeast etc. or Traditional prokaryotic expression system.
When industrialization mass produces protein, the condition of cell culture is to influence the key factor of yield, therefore, right In cell culture culture medium prescription and condition of culture determine it is most important.
Serum supplement Zooblast culture medium can usually improve the yield of cell viability and recombinant protein, but in the past In nearly 20 years, the gene recombinant protein that is obtained of the culture medium containing serum is gradually found, it can there are exogenous factors to pollute Risk.Serum is the mixture for the substance not characterized, may be with batch variation.And serum from animal or people source or Other supplements also have the risk that exogenous factor pollutes, such as mycoplasma, prion and virus pollution.
In order to overcome the risk of the cause of disease contact scar related with serum, serum free medium has been developed in the past.Without blood Clear culture medium is usually supplemented with serum substitute, such as growth factor, cell factor, albumin, insulin and turns note albumen.These Albumen is generally separated from animal origin, and therefore, culture medium is still remained by the potential risk of pathogen contamination.For example, using dividing From the bovine serum albumin(BSA) from animal or people source（BSA）, human serum albumins（HAS）Or transferrins can be convenient for cell culture （US6733746）.This method still has the danger that foreign pathogen is introduced into cell culture, for example, from HAS's HIV, the creutzfeldt-Jacob disease factor（Creutzfeld Jakob agent）Or the risk of hepatitis virus.The risk of pathogen contamination is unfavorable Application in production of the culture medium in animal and people's therapeutic agent.It has been disclosed for automatic comprising recombinant albumin and other source The culture medium of object ingredient（WO2008009642）With the culture medium containing recombinant albumin and Recombulin （US20060115901）.
For the considerations of adjusting and is safe and about buying, serum is eliminated in the commodity production of protein （Grillberger et al., Biotechnol. J. 2009,4,186-201）However, what is grown in serum free medium is dynamic Object cell may be very sensitive to nutritional deficiency, and nutritional deficiency can induce Apoptosis.Apoptosis negatively affects recombination The quality and titre of protein（Franek and Eussenegger, 2005, Biotechnol. Prig. 21,96-98）.From big In the hydrolysate of the vegetable proteins such as beans and wheat, the nutritional ingredient needed for cell of the addition in serum free medium is obtained. Franek et al. prepares the vegetable protein additive in serum free medium from phytoprotein hydrolysis, and tests them and prop up Hold the ability of growth and the secretion of mouse hybridoma（Franek et al., 2000, Biotechnol. Prog. 16,688- 692）.
Target the bifunctional antibody fusion protein of CD47 and PD-L1（Abbreviation bifunctional antibody fusion protein）It is a kind of double special Specific immunological checkpoint drug（Belong to a kind of bispecific antibody of specific type）（Number of patent application 2016103729544）. The albumen includes two parts of disulfide bond link, and a part is by SIRP α（The ligand of CD47）The Fc of mutant and antibody sections （IgG1 types）It merges, can be combined with CD47；Another part by anti-PD-L1 antibody light chain and heavy chain（IgG1 types）Composition, It can be combined with PD-L1（Structure is shown in Fig. 1）.This bifunctional antibody fusion protein differs greatly with general antibody structure, therefore is training It is also different with conventional antibody in the condition of supporting.Therefore it needs to develop to improve it for the personalized culture medium of this fusion protein Expressing quantity.
Glycosylation is a kind of important posttranslational modification of protein.The sugar chain on protein molecule surface can be to protein point The structure and function of son generates far-reaching influence, glycosylates as important post translational processing process, to the correct folding of protein Folded, positioning, immunogenicity and biological activity have a great impact.
It is glycosylation modified to be highly dependent on cell expression system and subclone selects, in cell cultivation process it is many because Element, such as the ingredient of culture medium, condition of culture can influence to glycosylate, and then influence the biological activity of human cytokines, curative effect, Immunogenicity and pharmacokinetics.
In clinical treatment, it will appear using protein formulation due to drug-induced hypersensitivity, these hypersensitivity Patients serum detection in, it may appear that the IgE antibody of specific anti alpha-galactolipin, the degree of glycosylation and glycosyl of pharmaceutical grade protein Change type to play an important role for the therapeutic effect of drug and the side reaction of drug.
Invention content
The present invention provides a kind of serum-free cell culture medium, is made of basal medium and supplementing culture medium, wherein described Cytidine, uridine, guanosine, thymidine and adenosine are added in basal medium, arginine, phenylalanine and first are added in supplementing culture medium Methyllanthionine.
The present invention also provides a kind of serum free mediums for expressing cho cell CD47/PD-L1 bifunctional fusion proteins And in Chinese hamster ovary celI high efficient expression CD47/PD-L1 bifunctional fusion proteins cultural method, use improved serum-free After the Chinese hamster ovary celI of medium culture expression CD47/PD-L1 bifunctional fusion proteins, the CD47/PD-L1 bifunctional fusions of acquisition The galactolipin that can cause immune response is not contained in the polymerization sugar chain of albumen.
The invention discloses：
A kind of serum-free cell culture medium, is made of basal medium and supplementing culture medium, which is characterized in that the basis culture Cytidine, uridine, guanosine, thymidine and adenosine are added in base, arginine, phenylalanine and methionine are added in supplementing culture medium.
Serum-free cell culture medium as described above, which is characterized in that add 1-1000mg/l in the basal medium Cytidine, 1-1000mg/l uridines, 1-1000mg/l guanosines, 1-1000mg/l thymidines and 1-1000mg/l adenosines.
Serum-free cell culture medium as described above, which is characterized in that 100mg/l born of the same parents are added in the basal medium Glycosides, 150mg/l uridines, 80mg/l guanosines, 200mg/l thymidines and 300mg/l adenosines.
Serum-free cell culture medium as described above, which is characterized in that add 1-1000mg/l in the supplementing culture medium Arginine, 1-4000mg/l phenylalanines and 1-100mg/l methionines.
Serum-free cell culture medium as described above, which is characterized in that 200mg/l essence ammonia is added in the supplementing culture medium Acid, 3000mg/l phenylalanines and 50mg/l methionines.
A kind of method using serum-free cell culture medium culture protein described in claim 1, which is characterized in that will The protein gene sequence of expression is cultivated after vector construction, transfection, colony screening using the basal medium and supplement Culture cell simultaneously expresses protein.
The method as described above for using serum free medium culture protein described in claim 1, which is characterized in that institute Protein is stated as bifunctional fusion proteins.
The method as described above for using serum free medium culture protein described in claim 1, which is characterized in that institute Bifunctional fusion proteins are stated as CD47/PD-L1：CD47 bound fractions have SEQ ID NO:2 amino acid sequence；PD-L1 is tied The light chain for closing part has SEQ ID NO:4 amino acid sequence, heavy chain have SEQ ID NO:6 amino acid sequence.
The preferred clone of CD47/PD-L1 is cultivated and expressed using above-mentioned serum-free cell culture medium, can be obtained Efficiently, stablize the CD47/PD-L1 bifunctional fusion proteins of expression, and improve the degree of glycosylation of bifunctional fusion proteins.
The invention also discloses a kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins, including following step Suddenly：a）A kind of novel anti-CD47/PD-L1 bifunctional fusion proteins：CD47 bound fractions have SEQ ID NO:2 amino acid Sequence；The light chain of PD-L1 bound fractions has SEQ ID NO:4 amino acid sequence, heavy chain have SEQ ID NO:6 ammonia Base acid sequence.b）Utilize step a）Gained nucleotide fragment construction recombination plasmid, transfection host cell screen high-expression clone； c）Using improved serum-free cell culture medium large-scale culture, anti-CD47/PD-L1 bifunctional fusion proteins are obtained, separation is pure Change；It is characterized in that, the improved serum-free cell culture medium is made of basal medium and supplementing culture medium, basis training Addition cytidine, uridine, guanosine, thymidine and adenosine in base are supported, arginine, phenylalanine and first sulphur ammonia are added in supplementing culture medium Acid.
A kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins as described above, which is characterized in that described Temperature using serum-free cell culture medium culture is 35 DEG C ~ 37 DEG C.
A kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins as described above, which is characterized in that described Temperature using serum-free cell culture medium culture is 36 DEG C.
A kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins as described above, which is characterized in that described PH value using serum-free cell culture medium culture is 6.5 ~ 7.
A kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins as described above, which is characterized in that described PH value using serum-free cell culture medium culture is 6.9.
By cultural method culture of the present invention obtain CD47/PD-L1 bifunctional fusion proteins, sugar-type modification with Based on G0F, content is that more than 50%, G1F and G2F ratios are taken second place, high mannose（Man5）Ratio is 1% or so.Due to sugar-type with Based on G0F, reduce the adverse reaction as caused by galactolipin.
The present invention is the base of basal medium CHOM-B02 sold in Shanghai Mai Tai Jun Ao Bioisystech Co., Ltd Increase cytidine, uridine, guanosine, thymidine and adenosine on plinth, as basic culture medium, in the limited public affairs of Shanghai Mai Tai monarch Austria biotechnology Addition arginine, phenylalanine and methionine, are trained as supplement on the basis of the sold supplementing culture medium CHOM-S01 of department Support base.
Description of the drawings
Fig. 1, target CD47 and PD-L1 bifunctional antibody fusion protein structure diagram；
Viable cell density test map after Fig. 2, different formulations base culture base（*106cells/ml）；
Cell viability test map after Fig. 3, different formulations base culture base；
Viable cell density test map after Fig. 4, different formulations supplementing culture medium culture（*106cells/ml）；
Cell viability test map after Fig. 5, different formulations supplementing culture medium culture；
Viable cell density test map is cultivated after Fig. 6, preferably basal medium and supplementing culture medium combination（*106cells/ml）；
Cell viability test map is cultivated after Fig. 7, preferably basal medium and supplementing culture medium combination；
Fig. 8, CD47/PD-L1 bifunctional fusion proteins sugar-type mass spectral analysis collection of illustrative plates.
Specific embodiment
Embodiment 1, CD47/PD-L1 vector constructions and Chinese hamster ovary celI transfection
According to SEQ ID NO: 1（Coding by SIRP alpha-mutants merges peptide fragment with what antibody Fc section was formed by connecting, with Y349C, T366S, L368A, Y407V are mutated the Hole structures to be formed）、SEQ ID NO: 3（Encode anti-PD-L1 antibody light chains）、SEQ ID NO: 5（Anti- PD-L1 heavy chain of antibody is encoded, the Knob structures to be formed are mutated with T366W, S354C）Nucleotide sequence, synthesis Corresponding DNA fragmentation, respectively be inserted into pcDNA3.1 carriers in, be built into expression vector pcDNA3.1-SIRP α-m-Fc, PcDNA3.1-antiPDL1-L and pcDNA3.1-antiPDL1-H, cotransfection use dihydro to CHO-dhfr- expression host cells Folic acid reductase inhibitor methotrexate (MTX) pressurization screening high-expression clone.
Obtained high-expression clone will be screened under conditions of basal medium provided by the present invention and supplementing culture medium Mass propgation, induced expression are carried out, separation, purifying obtain bifunctional fusion proteins CD47/PD-L1：CD47 bound fractions have SEQ ID NO:2 amino acid sequence；The light chain of PD-L1 bound fractions has SEQ ID NO:4 amino acid sequence, heavy chain With SEQ ID NO:6 amino acid sequence.
Experimental example 1, serum free medium basic media components determine
The culture medium C HOM-B02 that purchase Shanghai Mai Tai Jun Ao Bioisystech Co., Ltd is sold, culture medium C HOM-B02's On the basis of increase cytidine, uridine, guanosine, thymidine and adenosine, as basic culture medium.Formula 1 is on the basis of former culture medium Add 100mg/l cytidines, 150mg/l uridines, 80mg/l guanosines, 200mg/l thymidines and 300mg/l adenosines；Formula 2 is to be trained in original Addition 150mg/l cytidines, 225mg/l uridines, 120mg/l guanosines, 300mg/l thymidines and 450mg/l adenosines on the basis of foster base.
The clone of high expression CD47/PD-L1 bifunctional fusion proteins that screening obtains in selection example 1, use are above-mentioned The formula culture medium of nucleosides and original formulation culture medium are added, high-expression clone is cultivated, 36 DEG C, is cultivated under the conditions of pH6.9. Its viable cell density and the Cell viability after the culture of 5 days are detected, comparative experimental data is shown in Fig. 2 and Fig. 3.
Experimental result shows, after the Basal serum-free media culture of formula 1, living cell rate reaches 5.375 × 106Cells/ml, hence it is evident that higher than the culture medium of formula 2, also above original formulation culture medium, three groups of Cell viability is approximate.
It is therefore preferable that 1 serum free medium of formula is as basic culture medium, addition 100mg/l cytidines, 150mg/l uridines, 80mg/l guanosines, 200mg/l thymidines and 300mg/l adenosines.
Experimental example 2, serum free medium supplementing culture medium ingredient determine
The culture medium C HOM-S01 that purchase Shanghai Mai Tai Jun Ao Bioisystech Co., Ltd is sold, culture medium C HOM-S01's On the basis of addition arginine, phenylalanine and methionine, as supplementing culture medium.Formula 1 is the base in original formulation culture medium 200mg/l arginine, 3000mg/l phenylalanines and 50mg/l methionines are added on plinth；Formula 2 is in original formulation culture medium On the basis of addition 300mg/l arginine, 4500mg/l phenylalanines and 75mg/l methionines；Formula 3 is to be trained in original formulation Addition 360mg/l arginine, 5400mg/l phenylalanines and 90mg/l methionines on the basis of foster base.
Preferably go out basal medium by experimental example 1, the clone of height expression CD47/PD-L1 bifunctional fusion proteins passes through It is preferred that after base culture base, add in supplementing culture medium and continue to cultivate.It 36 DEG C, is cultivated under the conditions of pH6.9.By the training of 12 days It supports, detects the viable cell density and Cell viability of different formulations culture medium, as a result see Fig. 4 and Fig. 5.
Experimental result shows, after the supplement serum free medium culture of formula 1, living cell rate reaches 5.535 × 106Cells/ml, hence it is evident that higher than formula 2, the slightly below culture medium of formula 3, original formulation culture medium.Cell viability is equal for 0.8856 Higher than other 3 groups.
After the supplementing culture medium culture of above-mentioned different formulations, separation, purifying obtain bifunctional fusion proteins CD47/ PD-L1, the expressing quantity for measuring different formulations culture medium are respectively：Original formulation expressing quantity 0.9g/l is formulated 1 albumen table Up to amount 1.2g/l, 2 expressing quantity 1.18g/l are formulated, are formulated 3 expressing quantity 0.9g/l.
Therefore, amid all these factors consider that 1 serum free medium of optimization formula as supplementing culture medium, i.e., is trained in original formulation Addition 200mg/l arginine, 3000mg/l phenylalanines and 50mg/l methionines on the basis of foster base.
Experimental example 3, optimization formula culture are compared with original formulation culture medium
CD47/PD-L1 bifunctional fusion proteins are expressed using determining preferred basal medium and supplementing culture medium culture height Clone, is compared with original formulation culture medium, detects viable cell density and Cell viability after two kinds of medium cultures, ties Fruit sees Fig. 6 and Fig. 7.
As a result show the viable cell density after preferred basal medium and supplementing culture medium culture for 4.62 × 106Cells/ml, slightly above control group, Cell viability are higher than control group.
Experimental example 4, the detection of CD47/PD-L1 bifunctional fusion proteins sugar-type
Using LC/MS, MS/MS technologies carry out sugar chain analysis to CD47/PD-L1 bifunctional fusion proteins.
Sample preparation：CD47/PD-L1 bifunctional fusion proteins prepare the oligosaccharides on Fc sections and Fab by glycosidase digestion； Oligosaccharides on the circumscribed enzymatic treatment Fab of oligosaccharides；2-AB fluorescent marker oligosaccharides；By HILIC Solid Phase Extraction, excess 2-AB is removed, is obtained The sugar chain with fluorescent marker is obtained, carries out LC/MS and MS/MS chromatography.
CD47/PD-L1 bifunctional fusion proteins obtain free sugar chain under glucosides enzyme effect, lead to respectively after fluorescent marker Cross LC/MS, MS/MS and oligosaccharides exonuclease assay.
Through to the analysis of the sugar-type of CD47/PD-L1 bifunctional fusion proteins, the results are shown in Figure 8, sugar-type modification using G0F as Main, content is that more than 50%, G1F and G2F ratios are taken second place, high mannose（Man5）Ratio is classified in sugar-type 1% or so, Based on two antenna oligosaccharides of fucosylation, 90% or so is accounted for, goes core fucosylated oligosaccharide less, only accounts for 5% or so, in addition There are the modifications of a small amount of sialic acid.
SEQUENCE LISTING
<110>Shanghai Mai Tai Jun Ao Bioisystech Co., Ltd
<120>The method of serum-free cell culture medium and highly effective expressing recombinant protein matter
<130> 2016
<160> 6
<170> PatentIn version 3.5
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<211> 1053
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gaagaagaac tgcagatcat ccagccggac aaatctgttt ctgttgcggc gggtgaatct 60
gcgatcctgc actgcaccat cacctctctg ttcccggttg gtccgatcca gtggttccgt 120
ggtgcgggtc cggcgcgtgt tctgatctac aaccagcgtc agggtccgtt cccgcgtgtt 180
accaccgttt ctgaaaccac caaacgtgaa aacatggact tctctatctc tatctctaac 240
atcaccccgg cggacgcggg cacctactac tgcatcaaat tccgtaaagg ttctccggac 300
accgaattta aatctggtgc gggcaccgaa ctgtctgttc gtgcgaaacc gtctgagccc 360
aaatcttctg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 420
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 480
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 540
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 600
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 660
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 720
aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc ccgggatgag 780
ctgaccaaga accaggtcag cctgtcctgc gccgtcaaag gcttctatcc cagcgacatc 840
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 900
ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 960
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1020
cagaagagcc tctccctgtc cccgggtaaa tga 1053
<210> 2
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<212> PRT
<213>Artificial sequence
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Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
245 250 255
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 3
<211> 645
<212> DNA
<213>Artificial sequence
<400> 3
gatatacaga tgacccagag cccatcttct ctcagcgcat ctgtgggcga ccgtgtcacc 60
atcacttgtc gtgccagtca ggacgtctcc actgccgtcg cctggtacca acagaagcct 120
gggaaggcgc caaaactgtt aatctactcc gctagttttc tctacagcgg tgtgccatcc 180
aggttcagcg gttctgggtc gggcacagat tttaccctga ccatcagctc tctccagcct 240
gaggacttcg ctacctacta ttgtcagcag tacttgtacc accctgctac cttcggccag 300
gggacaaagg tggagatcaa gcgcacagtc gctgcccctt ccgtgttcat ttttcctccc 360
tctgacgagc agctaaagag cggtaccgct tcagtcgtgt gtttactgaa caacttttac 420
cctcgggaag ccaaggtcca gtggaaggtt gacaacgcac tccagtccgg caattcgcag 480
gagtctgtta ccgagcaaga ctccaaggac agcacatact ccctttcatc aactttgacg 540
ttgtctaagg ctgactacga gaagcataaa gtgtatgctt gcgaggtgac acatcagggc 600
ctgagttccc cagttaccaa atcattcaat cggggggaat gctga 645
<210> 4
<211> 214
<212> PRT
<213>Artificial sequence
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 5
<211> 1347
<212> DNA
<213>Artificial sequence
<400> 5
gaagtacagc tggtcgagag tggcgggggg ttggtgcagc ccggagggtc cctgcggctg 60
agttgcgccg cctccgggtt taccttctcc gactcttgga ttcactgggt gcgacaggcc 120
cctgggaaag gtctcgaatg ggtggcttgg atcagcccct acggcggatc aacctattat 180
gctgattccg tgaaggggcg cttcaccata tctgccgaca ccagtaaaaa cacagcttac 240
ctccagatga actccctgag agcagaggat actgccgtct attattgtgc cagaaggcac 300
tggccgggcg gctttgatta ctgggggcag ggcaccctcg tgaccgtgtc atctgctagc 360
accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420
gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660
tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aagagcagta caacagcacg 900
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
aaagggcagc cccgagaacc acaggtgtac accctgcccc catgccggga tgagctgacc 1080
aagaaccagg tcagcctgtg gtgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
agcctctccc tgtctcccgg taaatga 1347
<210> 6
<211> 448
<212> PRT
<213>Artificial sequence
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445

Claims (5)

1. a kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins, includes the following steps：a）It is a kind of novel anti- CD47/PD-L1 bifunctional fusion proteins：CD47 bound fractions have SEQ ID NO:2 amino acid sequence；PD-L1 engaging portions The light chain divided has SEQ ID NO:4 amino acid sequence, heavy chain have SEQ ID NO:6 amino acid sequence；b）It utilizes Step a）Gained nucleotide fragment construction recombination plasmid, transfection host cell screen high-expression clone；c）Use improved no blood Clear cell culture medium large-scale culture, obtains anti-CD47/PD-L1 bifunctional fusion proteins, isolates and purifies；It is characterized in that, institute The improved serum-free cell culture medium stated is made of basal medium and supplementing culture medium, in basal medium add cytidine, Uridine, guanosine, thymidine and adenosine, addition arginine, phenylalanine and methionine in supplementing culture medium.
2. a kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins according to claim 1, feature exist In the temperature using serum-free cell culture medium culture is 35 DEG C ~ 37 DEG C.
3. a kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins according to claim 2, feature exist In the temperature using serum-free cell culture medium culture is 36 DEG C.
4. a kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins according to claim 1, feature exist In the pH value using serum-free cell culture medium culture is 6.5 ~ 7.
5. a kind of preparation method of novel C D47/PD-L1 bifunctional fusion proteins according to claim 1, feature exist In the pH value using serum-free cell culture medium culture is 6.9.
CN201611137227.6A 2016-12-09 2016-12-09 The method of serum-free cell culture medium and highly effective expressing recombinant protein matter Pending CN108220334A (en)

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CN113480652A (en) * 2021-07-30 2021-10-08 成都景泽生物制药有限公司 Method for producing recombinant EGFR antibody active molecules by fermentation culture of recombinant CHO cells
CN114990129A (en) * 2022-05-11 2022-09-02 北京贝来生物科技有限公司 Preparation and application of mesenchymal stem cells expressing alpha PDL1 Fc fusion protein

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WO2023045140A1 (en) * 2021-09-26 2023-03-30 上海迈泰君奥生物技术有限公司 Method for increasing expression quantity of antibody in host cell
CN114480492B (en) * 2022-01-28 2023-04-21 景泽生物医药(合肥)股份有限公司 Preparation method of recombinant human antibody fusion protein

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CN104513805A (en) * 2013-10-07 2015-04-15 鲁南制药集团股份有限公司 Method for producing anti CD20 antibody
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CN113480652A (en) * 2021-07-30 2021-10-08 成都景泽生物制药有限公司 Method for producing recombinant EGFR antibody active molecules by fermentation culture of recombinant CHO cells
CN113480652B (en) * 2021-07-30 2023-04-07 成都景泽生物制药有限公司 Method for producing recombinant EGFR antibody active molecules by fermentation culture of recombinant CHO cells
CN114990129A (en) * 2022-05-11 2022-09-02 北京贝来生物科技有限公司 Preparation and application of mesenchymal stem cells expressing alpha PDL1 Fc fusion protein
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