CN106479982A - Anti- PD 1 monoclonal antibody Cells for production culture medium and its optimization method - Google Patents
Anti- PD 1 monoclonal antibody Cells for production culture medium and its optimization method Download PDFInfo
- Publication number
- CN106479982A CN106479982A CN201610902925.4A CN201610902925A CN106479982A CN 106479982 A CN106479982 A CN 106479982A CN 201610902925 A CN201610902925 A CN 201610902925A CN 106479982 A CN106479982 A CN 106479982A
- Authority
- CN
- China
- Prior art keywords
- medium
- culture
- cell
- monoclonal antibody
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides producing the cell culture medium of anti-PD 1 monoclonal antibody and its selecting optimization method, using Analytical Technology of Amino Acid, determine the key amino acid of impact cell growth and antibody expression, determine kinds of culture medium based on this and culture medium is carried out with the optimization of amino acid classes and addition, make cell growth, maintenance and antibody expression effect optimization.
Description
Technical field
The present invention relates to Monoclonal Antibody Cell culture process field, particular content is related to produce anti-PD-1 monoclonal antibody
Cell culture medium and its optimization method.
Background technology
Programmed death receptor 1 (Programmed Death-1), also referred to as PD-1 and CD279, is initially in 1992
Obtain and name from the mouse Tcell hybridoma 2B4.11 clone of apoptosis.PD-1 is that immunoglobulin superfamily CD28 family becomes
Member, relative molecular weight is the I type transmembrane glycoprotein of 50～55KD.PD-1 is T cell immunosuppressant receptor, in the T cell of activation
The high expression with B cell, in tumor cell can restricted T cells effector function, simultaneously modulate tumor immunologic escape with
And autoimmune response aspect plays critical effect.
As receptor, its part has two PD-L1 (B7-H1) and PD-L2 (B7-DC) to PD-1.PD-1 is combined with PD-L1
TCR and BCR can be activated, promote the Tyr phosphorylation in the ITSM domain of PD-1, and then make SH2 domain (include SHP-2
And SHP-1) it is attached to the Bao Jiang area of PD-1, cause the PI3K dephosphorylation of CD28 regulation and control, the path such as suppression downstream AKT/ERK
Activation, leads to the gene needed for T cell activation and cytokine transcription and translation to be suppressed.PD-1 can also suppress other simultaneously
The phosphorylation of signaling molecule, such as CD3, ZAP70 and PCK.The immunosuppressive action of PD-1/PD-L1 signal path is exempted to multiple
The generation of epidemic disease dysfunctional disease and development have important function, and therefore humanization anti-PD-1 monoclonal antibody is presently the most popular
Monoclonal antibody.
Chinese hamster ovary (CHO) cell is extensively applied and field of biological pharmacy, the cell culture side that bio-pharmaceuticals adopts
Formula be fed-batch culture, will certain volume basal medium load biological reactor for cell culture in, cell is inoculated
Carry out cell culture, with the continuous growth of cell in incubation, required nutrient substance is continuous after in cultivation reactor
Consume, need the supplemented medium constantly added rich in nutrient substance to maintain the nutritional need of cell, so that cell is secreted more
Many antibody.Different cell culture mediums can express high yield and high-quality antibody through screening.The method of Optimal Medium
There are Single factor screening, orthogonal experiment method, Responds Surface Methodology, uniform design, total divisor laboratory method, fractional factorial design
Method, Plackett-Burman design, central. set is legal and Box-Behnken designs etc..Analytical Technology of Amino Acid is commonly used to
Ammonia in amino acid analysises in amino acid analysises in amino acid analysises in proteins and peptides, body fluid, food, crops
The aspects such as the chiral separation of base acid analysis and aminoacid.In terms of Chinese hamster ovary celI is used for monoclonal antibody expression, not yet there is combination
Amino acid analysises carry out the relevant report of medium optimization.
Content of the invention
The invention provides producing the cell culture medium of anti-PD-1 monoclonal antibody and its selecting optimization method, using amino
Acid analysis technology, determines the key amino acid of impact cell growth and antibody expression, determines kinds of culture medium simultaneously based on this
Culture medium is carried out with the optimization of amino acid classes and addition, makes cell growth, maintenance and antibody expression effect optimization.
The cell culture medium of the anti-PD-1 monoclonal antibody of production that the present invention provides includes basal medium and feed-batch culture
Base it is characterised in that described basal medium contains 23.0g/L～28.0g/L Hycell CHO Medium culture medium,
0.1mmol/L～1.0mmol/L L-Glutamine, 0.5mmol/L～1.5mmol/L altheine；Described feed-batch culture
Base contains 98.0g/L～103.0g/L OPM CHOCD Feed 1 culture medium, 35.0mmol/L～40.0mmol/L L- group ammonia
Acid, 70.0mmol/L～80.0mmol/L L-Tyrosine, 55.0mmol/L～65.0mmol/L L-Serine, 40.0mmol/L
～50.0mmol/L L-Glutamine, 15.0mmol/L～20.0mmol/L L-Cysteine.
Preferably, 25.4g/L Hycell CHO Medium culture medium, 0.76mmol/L are contained in described basal medium
L-Glutamine, 1.04mmol/L altheine；101.25g/L OPM CHOCD Feed is contained in described supplemented medium
1 culture medium, 38.7mmol/L L-Histidine, 75.0mmol/L L-Tyrosine, 64.0mmol/L L-Serine, 49.2mmol/
L L-Glutamine, 18.7mmol/L L-Cysteine.
Cell strain of the present invention adopts the anti-PD-1 monoclonal antibody of dihydrofolate reductase (DHFR) deficiency system expression
Gene recombinaton Chinese hamster ovary celI.
Basal medium of the present invention and the dissolving of supplemented medium water for injection, pH is 7.2 ± 0.2,0.22 μm of filter membrane
Filtration sterilization.
Present invention also offers a kind of optimization method of anti-PD-1 monoclonal antibody Cells for production culture medium, its feature exists
In the method comprises the following steps：
Step 1)：The cell strain expressing anti-PD-1 monoclonal antibody is cultivated in multiple basal mediums, according to
Cell growth condition screens from 20 kinds of basal mediums and obtains 2～4 kinds of optimum bases of anti-PD-1 Monoclonal Antibody Cell culture
Basal culture medium；
Step 2)：By detecting step 1) the consumption feelings of aminoacid in 2～4 kinds of basal medium Cell saps filtering out
Condition, determines the key amino acid species of impact cell growth；
Step 3)：According in step 1) add the amino acid assays of variable concentrations in the basal medium of optimum that filters out
Determine the interpolation concentration of aminoacid in basal medium and the basal medium formulation of anti-PD-1 monoclonal antibody；
Step 4)：Will expression PD-1 monoclonal antibody cell strain in optional step 3) carry out in the basal medium that determines
Fed-batch is cultivated, and is screened according to antibody expression amount situation and obtain anti-PD-1 monoclonal antibody expression from 10 kinds of supplemented mediums
Preferably 2～4 kinds supplemented mediums；
Step 5)：By detecting step 4) the consumption feelings of aminoacid in 2～4 kinds of supplemented medium Cell saps filtering out
Condition, determines the key amino acid species of impact antibody expression；
Step 6)：According in step 4) add the amino acid assays of variable concentrations in the supplemented medium of optimum that filters out
Determine the interpolation concentration of aminoacid in supplemented medium and the feed-batch culture based formulas of anti-PD-1 monoclonal antibody.
The selected basal medium for screening experiment of the present invention is selected from the Hycell CHO Medium of Hyclone company
Culture medium, CDM4CHO culture medium and CDM4PERMAB culture medium etc., the Ex-cell CHO CD culture medium of Sigma company, also
There are totally 20 kinds of basal mediums (being shown in Table 1) of the companies such as Millipore, Shanghai Ao Pumai bio tech ltd；Feed supplement is trained
Foster base is selected from Cell Boost 6 culture medium of Hyclone company, Cellwento Feed-200 of Millipore company etc. altogether
10 kinds of supplemented mediums (being shown in Table 2).
1 20 kinds of basal medium information of table
2 10 kinds of supplemented medium information of table
Amino acid analysis method of the present invention adopts the UPLC H-class-PDA detection of Waters company, can be accurate
Separate 27 kinds of aminoacid, carry out quantitative analyses.The method of specific amino acid analysises screening culture medium is shown in embodiment.
The present invention provides the cell culture medium producing anti-PD-1 monoclonal antibody and its selects optimization method, using aminoacid
Analytical technology, by resist PD-1 monoclonal antibody production with gene recombinaton Chinese hamster ovary celI the growth feelings in multiple culture medium
Condition is analyzed, and determines the key amino acid of impact cell growth and antibody expression, determines kinds of culture medium simultaneously based on this
Culture medium is carried out with the optimization of amino acid classes and addition, the culture medium after optimization can make anti-PD-1 Monoclonal Antibody Cell life
Length, maintenance and antibody expression effect optimization.Carry out the optimization of culture medium in conjunction with Analytical Technology of Amino Acid, can be more accurately
Analysis cytotrophy metabolic condition, carries out adding appropriate aminoacid according to the amino acid spending rate in culture medium, is protecting
On the premise of card cell growth condition is good, improve antibody expression amount, provide ripe processing step for amplifying to produce, can subtract
Few resource and energy resource consumption, reduce production cost significantly.
With reference to the embodiment of specific embodiment and Figure of description, the present invention will be further described.
Figure of description
Attached Figure 120 plants basal medium viable cell density change curve；
2 20 kinds of basal medium Cell viability change curves of accompanying drawing；
Amino acid concentration in accompanying drawing 3 MH15 basal medium cell culture fluid；
Amino acid concentration in accompanying drawing 4 MA06 basal medium cell culture fluid；
Amino acid concentration in accompanying drawing 5 MG03 basal medium cell culture fluid；
After accompanying drawing 6 interpolation aminoacid, MH15 culture medium carries out the cell growth curve of batch cultivation；
7 10 kinds of supplemented mediums of accompanying drawing carry out the antibody expression amount change curve of fed-batch culture；
Aminoacid consumption data in accompanying drawing 8 FA04 culture medium cell culture fluid；
Aminoacid consumption data in accompanying drawing 9 FM09 culture medium cell culture fluid；
Aminoacid consumption data in accompanying drawing 10 FH02 culture medium cell culture fluid；
The antibody expression amount change curve of the FA04 culture medium fed-batch culture after accompanying drawing 11 interpolation aminoacid；
Accompanying drawing 12 FA04 adds the checking antibody expression amount change curve of aminoacid；
Accompanying drawing 13 is not optimised and the cell growth change curve optimizing basis and supplemented medium；
Accompanying drawing 14 is not optimised and the antibody expression amount change curve optimizing basis and supplemented medium；
Accompanying drawing 15 is not optimised the anti-PD-1 monoclonal antibody biologic activity data with cell expression in Optimal Medium.
Specific embodiment
The present invention relates to multiple terms, it is not limit the scope of the invention using these terms.
As used herein, term " cell culture fluid " includes all of cell and cell debriss.
As used herein, not supplemented medium during term " batch cultivation " generally refers to cell culture, only adds appropriate
The glucose of concentration.
As used herein, term " fed-batch type culture " generally refers to, during cell culture, be entered according to cell growth
Row supplemented medium, glucose and aminoacid etc..
As used herein, the work that term " viable cell density " and " VCD " generally refer to included in every milliliter of Cell sap is thin
Born of the same parents' sum, is represented using unit " cells/mL ".
As used herein, term " Cell viability " and " VIA " generally refer to total viable cell in every milliliter of Cell sap and account for always
The ratio of cell number, is represented using unit " % ".
As used herein, term " antibody expression amount " and " Titer " generally refer to cell in every milliliter of Cell sap and are produced
Antibody total amount, represented using unit " mg/mL " or " g/L ".
As used herein, term " highest viable cell density " and " PVCD " generally refer to cell and criticize culture and fed-batch type
In incubation, the maximum total viable cell that cell reaches, represented using unit " cells/mL ".
The optimization of embodiment 1 basal medium
By 20 kinds of basal mediums (being shown in Table 1) to the gene using the anti-PD-1 monoclonal antibody of DHFR deficiency system expression
Recombinaant CHO cell carries out 125mL shaking flask batch cultivation and (after batch cultivation phalangeal cell is seeded to basal medium, only adds Fructus Vitis viniferae
Sugar meets the demand of carbon source, does not add supplemented medium, until cell enters decline phase), condition of culture is：Inoculum density 5 ×
105Cells/mL, volume of culture is 30mL, and shaking speed is 110rpm, and cultivation temperature is 37 DEG C, CO2Concentration is 5%, in culture
0th, 3,5 days terminate (Cell viability is less than 70%) sampling detection viable cell density (VCD) and Cell viability (VIA) to culture,
20 kinds of basal medium viable cell densities and Cell viability change curve are shown in accompanying drawing 1 and accompanying drawing 2.
As illustrated in fig. 1 and 2, in 20 kinds of basal mediums, highest viable cell density (PVCD) comes being followed successively by of front three：
MH15 (Hycell), MA06 (OPM CHOCD07) and MG03 (CD FortiCHO)；The PVCD of three kinds of basal mediums is respectively
1.64×107Cells/mL, 1.55 × 107Cells/mL and 1.50 × 107Cells/mL, confirms that MH15 is 20 kinds of basis cultures
The optimum culture medium of cell growth in base；
During 20 kinds of basal medium shaking flask batch cultivations, respectively at the 0th day (D0), the 3rd day (D3) and culture terminate
When keep sample cell culture fluid 1mL, take the cell culture fluid of three kinds of basal mediums of PVCD highest to carry out supernatant amino acid analysises,
Result is as in Figure 3-5.It can be seen that amino acid concentration variation tendency is more greatly in preferably three kinds basal mediums of cell growth
Altheine (Asn) and L-Glutamine (Gln)：Amino acid concentration in MH15, MA06 and MG03 for the Asn divides from D0 to D3
Do not decrease 2.05mmol/L, 2.35mmol/L and 2.65mmol/L, amino acid concentration in MH15, MA06 and MG03 for the Gln
Decrease 1.60mmol/L, 1.92mmol/L and 2.60mmol/L from D0 to D3 respectively, the change of other amino acid concentrations is little, because
This can determine the key amino acid that Asn and Gln is impact cell growth.
Carry out amino acid concentration gradient experiment further on the basis of optimum basal medium MH15, add variable concentrations
Asn and Gln carries out shaking flask batch cultivation experiment：
Experiment condition：According to the Expenditure Levels of Asn and Gln D0 to D3 in MH15, it is added different aminoacids concentration
Experimental design, using DOE (Design of Experiment) design two factor two hydraulic test, containing
In the MH15 basal medium of 4.96mmol/L Asn and 3.24mmol/L Gln, add respectively Asn 1.04mmol/L and
3.04mmol/L, extremely final concentration of：6mmol/L and 8mmol/L, adds Gln 0.76mmol/L and 2.76mmol/L, dense to end
Spend and be：4mmol/L and 6mmol/L, experimental design such as table 3.
Add different aminoacids concentration experiment scheme in table 3 MH15
Cell culture condition：Inoculum density is 5 × 105Cells/mL, volume of culture is 30mL, and culture rotating speed is
110rpm, cultivation temperature is 37 DEG C, CO2Concentration be 5%, culture the 0th day, 3 days, 5 days to culture terminate sampling detection VCD and
VIA.
Cell growth condition such as Fig. 6 after MH15 interpolation different aminoacids is it is seen that adding Asn concentration is 1.04mmol/L
Optimal with adding the 6# test group cell growth condition that Gln concentration is 0.76mmol/L, reached highest living cells at the 7th day close
Degree, PVCD is 1.82 × 107Cells/mL, and motility rate remains also preferable, at the end of the 10th day cultivates, still has 68.3%, because
When this can determine culture medium based on MH15, the optium concentration adding Asn in culture medium is 1.04mmol/L, and Gln is
Good interpolation concentration is 0.76mmol/L.
The optimization of embodiment 2 supplemented medium
The basal medium being determined using embodiment 1,10 kinds of supplemented mediums (being shown in Table 2) carry out 125mL shaking flask batch and mend
Material culture, condition of culture：Inoculum density 8 × 105Cells/mL, volume of culture 30mL, cultivate rotating speed 110rpm, the initial temperature of culture
37 DEG C of degree, lowers the temperature as 33 DEG C when culture was to the 5th day, CO2Concentration 5%, respectively the 3rd day (D3), 5 days (D5), 7 days (D7), 9
My god (D9), 11 days (D11) add the supplemented medium of initial volume of culture 6%, the primary reference standard of screening supplemented medium
It is the expression of anti-PD-1 monoclonal antibody, so sampling detection antibody expression (Titer), 10 kinds of feed supplements at the end of culture
Culture medium antibody expression situation is shown in Fig. 7.
As shown in fig. 7, antibody expression amount comes being followed successively by of front three in 10 kinds of supplemented mediums：FA04(OPM CHOCD
Feed1), FH02 (Cell Boost 6) and FM09 (Cellvento Feed-210)；Antibody expression amount difference in three kinds of feed supplements
For 3.03g/L, 2.87g/L and 2.62g/L.Confirm that FA04 is to be beneficial to PD-1 monoclonal antibody in 10 kinds of supplemented mediums to express
Optimum supplemented medium；
During 10 kinds of supplemented mediums carry out shaking flask fed-batch culture, respectively before and after feed supplement in the 3rd, 5,7,9,11 days
Respectively leave and take cell culture fluid 1mL；The cell culture fluid taking three kinds of supplemented mediums of expression highest carries out supernatant Amino acid score
Analysis, result is as seen in figs. 8-10.It can be seen that there being some aminoacid can accumulate in the cell growth later stage in three kinds of supplemented mediums,
As L-Isoleucine (Ile), L-Alanine (Ala) and L-Valine (Val) etc., some aminoacid consume in early stage less, with
Cell density increases, and later stage amino acid consumption also continues to increase, and leads to partial amino-acid in feed supplement depleted, is not enough to
Needed for supply cell continued growth and antibody expression.According to the Expenditure Levels of these aminoacid, determine and affect anti-PD-1 monoclonal
The key amino acid of antibodies Antibodies expression is L-Histidine (His), L-Tyrosine (Tyr), L-Serine (Ser), L- glutamy
Amine (Gln) and this five kinds of L-Cysteine (Cys).
Carry out amino acid concentration gradient experiment further on the basis of optimum supplemented medium FA04, add variable concentrations five
Plant aminoacid and carry out shaking flask fed-batch culture experiment：
Experiment condition：According to His in 6% initial volume of culture feed supplement (computing formula such as formula 1) afterwards Cell sap, Tyr,
Ser, Gln and Cys Expenditure Levels in FA04, are added the experimental design of different aminoacids concentration, design five using DOE
The factor two hydraulic test, is containing 25.0mmol/L His, 50.0mmol/L Tyr, 42.0mmol/L Ser, 50.0mmol/
In the FA04 supplemented medium of L Gln and 28.0mmol/L Cys, add His 25.0mmol/L and 41.7mmol/L respectively,
Tyr 33.3mmol/L and 83.3mmol/L, Ser 41.7mmol/L and 66.7mmol/L, Gln 16.7mmol/L and
50.0mmol/L, Cys 22.0mmol/L and 55.3mmol/L, makes each aminoacid final concentration in culture medium be respectively：His
50.0mmol/L and 66.7mmol/L, Tyr 83.3mmol/L and 133.3mmol/L, Ser 83.7mmol/L and 108.7mmol/
L, Gln 66.7mmol/L and 100.0mmol/L, Cys 50.0mmol/L and 83.3mmol/L, specific experimental program such as table 4.
Formula 1：
For example：His concentration in FA04 is 25.0mmol/L, then the His concentration in culture fluid after feed supplement is 1.5mmol/
L；If requiring His concentration in culture fluid after feed supplement to be 2.5mmol/L, the His concentration added in FA04, is needed to be
41.7mmol/L.
Add the experimental program of different aminoacids concentration in table 4 FA04
Cell culture condition：Inoculum density 8 × 105Cells/mL, volume of culture 30mL, cultivate rotating speed 110rpm, CO2Dense
Spend for 5%, cultivate initial temperature and be 37 DEG C, be cooled within the 5th day 33 DEG C in culture and continue culture, respectively the 3rd day, 5 days, 7 days,
The supplemented medium adding initial volume of culture 6% in 9 days, 11 days, samples detection antibody expression at the end of culture.
Antibody expression amount situation after FA04 adds variety classes and concentration aminoacid is as shown in figure 11, intuitively analyzes antibody
Expression is up to 15# test group, and expression is 3.65g/L, predicts each aminoacid in FA04 through DOE interpretation of result optimum
Interpolation concentration is His：38.7mmol/L, Tyr：75.0mmol/L, Ser：64.0mmol/L, Gln：49.2mmol/L, Cys：
18.7mmol/L, highest expression is predicted up to 3.89g/L.On the basis of DOE prediction of result optimum combination of amino acids, in conjunction with
Preferable first three groups of DoE experimental result and be not added with the supplemented medium of aminoacid and carried out a collection of checking test, experiment side
Case such as table 5, as shown in figure 12, the experimental group average expression amount of DOE prediction is 3.84g/L to experimental result, reaches prediction, and is higher than
Highest 3.65g/L in DOE test.
Accordingly, it is determined that during using FA04 as supplemented medium, adding each aminoacid final concentration in culture medium and be respectively：L- group
Propylhomoserin 38.7mmol/L, L-Tyrosine 75.0mmol/L, L-Serine 64.0mmol/L, L-Glutamine 49.2mmol/L, L-
Cysteine 18.7mmol/L.
Table 5 FA04 adds the confirmatory experiment scheme of different aminoacids concentration
Embodiment 3
Improve cell growth for verifying after optimization basis and supplemented medium further with respect to being not optimised culture medium and having
Situation and the advantage improving antibody expression amount, have carried out cell batch feed-batch culture contrast test on 3L bioreactor.
Experimental condition as shown in table 6, co-cultures 4 batches, and 2 batches adopt basis and supplemented medium after optimization, and 2 batches of employings are not excellent
Change culture medium, in addition to culture medium difference, other condition of culture are consistent, and result of the test is shown in Figure 13-15 respectively.
Contrast test condition before and after table 6 medium optimization
The upgrowth situation of cell before and after basis optimizes with supplemented medium as seen from Figure 13, the culture medium after optimization more can promote
Enter cell growth, and maintain beneficial to Cell viability, the PVCD average out to 3.84 × 10 after Optimal Medium7Cells/mL, optimizes
PVCD average out to 2.31 × 10 before culture medium7Cells/mL, after optimization, PVCD improves 66.2%；At the end of culture, cell is lived
Rate by 61.9% before optimizing improve to optimize after 81.5%, Cell viability improve after cell state be constantly in preferably raw
Long status, the more conducively expression of PD-1 antibody.
Optimize the antibody expression amount situation before and after basis and supplemented medium as seen from Figure 14, anti-in the culture medium after optimization
Body expression average out to 3.88g/L, with respect to the 2.35g/L before optimizing, improves about 65.1%, and the culture medium after optimizing is described
The expression of PD-1 antibody more can be promoted.
Before and after taking Optimal Medium respectively, each a collection of Cell sap carries out Protein A purification, and sample after purification is resisted
The biologic activity detection of PD-1 monoclonal antibody, detection method adopts the bioluminescence test side based on transgenic cell
Method, result is shown in Figure 15.It can be seen that, it is 1.5 × 10 with the maximum luminous value before optimization after medium optimization5RTU, before illustrating to optimize
The biologic activity result of the anti-PD-1 monoclonal antibody of cell expression is consistent afterwards, does not have obvious difference.
In sum, by the basis and supplemented medium optimizing in shaking flask after amplification is applied on 3L bioreactor,
Not only improve cell growth condition compared with before optimizing, and more can promote the expression of anti-PD-1 monoclonal antibody, institute after optimization
The biologic activity expressing anti-PD-1 monoclonal antibody is consistent with before optimization.Basal medium and supplemented medium are excellent as can be seen here
Chemical conversion work(, and can be amplified further, apply and not only can ensure product quality after anti-PD-1 monoclonal antibody produces, and
Antibody production can be greatly improved, reduce production cost.
Claims (5)
1. produce anti-PD-1 monoclonal antibody cell culture medium, including basal medium and supplemented medium it is characterised in that
Described basal medium contains 23.0g/L～28.0g/L Hycell CHO Medium culture medium, and 0.1mmol/L～
The L-Glutamine of 1.0mmol/L, the altheine of 0.5mmol/L～1.5mmol/L；Described supplemented medium contains
98.0g/L～103.0g/L OPM CHOCD Feed 1 culture medium, 35.0mmol/L～40.0mmol/L L-Histidine,
70.0mmol/L～80.0mmol/L L-Tyrosine：, 55.0mmol/L～65.0mmol/L L-Serine：, 40.0mmol/L
～50.0mmol/L L-Glutamine, 15.0mmol/L～20.0mmol/L L-Cysteine.
2. culture medium according to claim 1 is it is characterised in that contain 25.4g/L Hycell in described basal medium
CHO Medium culture medium, 0.76mmol/L L-Glutamine, 1.04mmol/L altheine；In described supplemented medium
Containing 101.25g/L OPM CHOCD Feed 1 culture medium, 38.7mmol/L L-Histidine, 75.0mmol/L L-Tyrosine,
64.0mmol/L L-Serine, 49.2mmol/L L-Glutamine, 18.7mmol/L L-Cysteine.
3. culture medium according to claim 1 is it is characterised in that the cell strain that described culture medium is used for culture is to adopt two
The gene recombinaton Chinese hamster ovary celI of the anti-PD-1 monoclonal antibody of hydrogen reduction of folates deficient system expression.
4. culture medium according to claim 1 is it is characterised in that described basal medium and supplemented medium are using injection
With water dissolution, pH is that 7.2 ± 0.2,0.22 μm of membrane filtration is degerming.
5. a kind of optimization method of anti-PD-1 monoclonal antibody Cells for production culture medium it is characterised in that the method include with
Lower step：
Step 1)：The cell strain expressing anti-PD-1 monoclonal antibody is carried out batch cultivation in multiple basal mediums, according to
Cell growth condition screens from 20 kinds of basal mediums and obtains 2～4 kinds of optimum bases of anti-PD-1 Monoclonal Antibody Cell culture
Basal culture medium；
Step 2)：By detecting step 1) Expenditure Levels of aminoacid in 2～4 kinds of basal medium Cell saps filtering out, really
The fixing key amino acid species ringing cell growth；
Step 3)：According in step 1) amino acid assays that add variable concentrations in the basal medium of optimum that filters out determine
The basal medium formulation of the interpolation concentration of aminoacid and anti-PD-1 monoclonal antibody in basal medium；
Step 4)：Will expression PD-1 monoclonal antibody cell strain in step 3) carry out fed-batch in the basal medium that determines
Culture, screens from 10 kinds of supplemented mediums according to antibody expression amount situation and obtains anti-PD-1 monoclonal antibody expression preferably 2
～4 kinds of supplemented mediums；
Step 5)：By detecting step 4) Expenditure Levels of aminoacid in 2～4 kinds of supplemented medium Cell saps filtering out, really
The fixing key amino acid species ringing antibody expression；
Step 6)：According in step 4) amino acid assays that add variable concentrations in the supplemented medium of optimum that filters out determine
The feed-batch culture based formulas of the interpolation concentration of aminoacid and anti-PD-1 monoclonal antibody in supplemented medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610902925.4A CN106479982A (en) | 2016-10-17 | 2016-10-17 | Anti- PD 1 monoclonal antibody Cells for production culture medium and its optimization method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610902925.4A CN106479982A (en) | 2016-10-17 | 2016-10-17 | Anti- PD 1 monoclonal antibody Cells for production culture medium and its optimization method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106479982A true CN106479982A (en) | 2017-03-08 |
Family
ID=58270142
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610902925.4A Withdrawn CN106479982A (en) | 2016-10-17 | 2016-10-17 | Anti- PD 1 monoclonal antibody Cells for production culture medium and its optimization method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106479982A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624549A (en) * | 2017-03-23 | 2018-10-09 | 上海奥浦迈生物科技有限公司 | A kind of CHO DG44 culture mediums and its application |
| CN111009291A (en) * | 2019-07-02 | 2020-04-14 | 上海中乔新舟生物科技有限公司 | Cell culture medium optimization method |
| CN114196610A (en) * | 2021-12-28 | 2022-03-18 | 方坦思(上海)生物医药有限公司 | Cell culture medium suitable for producing monoclonal antibody against systemic lupus erythematosus and optimization method thereof |
| CN114230669A (en) * | 2021-12-24 | 2022-03-25 | 天士力生物医药股份有限公司 | Production method of bispecific antibody |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224239A (en) * | 2008-09-26 | 2011-10-19 | 先灵公司 | High titer antibody production |
| CN102533658A (en) * | 2011-12-08 | 2012-07-04 | 深圳新鹏生物工程有限公司 | Cell culture method and medium for preparing human interferon betala and application of cell culture medium |
| CN102994441A (en) * | 2012-09-19 | 2013-03-27 | 上海瀚康生物医药科技有限公司 | Cell culture medium, and preparation method and use thereof |
| CN104479020A (en) * | 2014-12-26 | 2015-04-01 | 上海复宏汉霖生物技术有限公司 | Anti-PD-1 (programmed cell death-1) humanized antibody |
| CN104894055A (en) * | 2015-06-15 | 2015-09-09 | 成都金凯生物技术有限公司 | Optimized cell culture medium and cell culture method, and application of optimized cell culture medium and cell culture method to preparation of protein and antibody |
| CN106008714A (en) * | 2016-05-24 | 2016-10-12 | 瑞阳(苏州)生物科技有限公司 | Anti-human pd-1 humanized monoclonal antibody and application thereof |
-
2016
- 2016-10-17 CN CN201610902925.4A patent/CN106479982A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224239A (en) * | 2008-09-26 | 2011-10-19 | 先灵公司 | High titer antibody production |
| CN102533658A (en) * | 2011-12-08 | 2012-07-04 | 深圳新鹏生物工程有限公司 | Cell culture method and medium for preparing human interferon betala and application of cell culture medium |
| CN102994441A (en) * | 2012-09-19 | 2013-03-27 | 上海瀚康生物医药科技有限公司 | Cell culture medium, and preparation method and use thereof |
| CN104479020A (en) * | 2014-12-26 | 2015-04-01 | 上海复宏汉霖生物技术有限公司 | Anti-PD-1 (programmed cell death-1) humanized antibody |
| CN104894055A (en) * | 2015-06-15 | 2015-09-09 | 成都金凯生物技术有限公司 | Optimized cell culture medium and cell culture method, and application of optimized cell culture medium and cell culture method to preparation of protein and antibody |
| CN106008714A (en) * | 2016-05-24 | 2016-10-12 | 瑞阳(苏州)生物科技有限公司 | Anti-human pd-1 humanized monoclonal antibody and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张慧娟等: "运用DOE法优化表达抗人类表皮生长因子受体2单克隆抗体细胞用培养基", 《中国生物制品学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624549A (en) * | 2017-03-23 | 2018-10-09 | 上海奥浦迈生物科技有限公司 | A kind of CHO DG44 culture mediums and its application |
| CN108624549B (en) * | 2017-03-23 | 2020-11-20 | 上海奥浦迈生物科技有限公司 | CHO DG44 culture medium and application thereof |
| CN111009291A (en) * | 2019-07-02 | 2020-04-14 | 上海中乔新舟生物科技有限公司 | Cell culture medium optimization method |
| CN114230669A (en) * | 2021-12-24 | 2022-03-25 | 天士力生物医药股份有限公司 | Production method of bispecific antibody |
| CN114230669B (en) * | 2021-12-24 | 2024-01-30 | 天士力生物医药股份有限公司 | Production method of bispecific antibody |
| CN114196610A (en) * | 2021-12-28 | 2022-03-18 | 方坦思(上海)生物医药有限公司 | Cell culture medium suitable for producing monoclonal antibody against systemic lupus erythematosus and optimization method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11827692B2 (en) | Mammalian cell culture | |
| JP7377316B2 (en) | Methods for harvesting mammalian cell cultures | |
| CN106479982A (en) | Anti- PD 1 monoclonal antibody Cells for production culture medium and its optimization method | |
| Yang et al. | Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity | |
| CN103080300B (en) | Increase the productive rate of cell culture and the dipeptides of vigor | |
| Yongky et al. | Process intensification in fed-batch production bioreactors using non-perfusion seed cultures | |
| EP3207132B1 (en) | Methods of shifting an isoelectric profile of a protein product and uses thereof | |
| CN102596995A (en) | Method for the production of a glycosylated immunoglobulin | |
| TWI832345B (en) | Methods for increasing mannose content of recombinant proteins | |
| Abu-Absi et al. | Cell culture process operations for recombinant protein production | |
| Kreye et al. | A novel scale‐down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM) | |
| CN107760651A (en) | A kind of cell culture medium and production method of protein | |
| CN108350075A (en) | The method for improving the galactose content of recombinant protein | |
| CN106170554A (en) | Process LAN N glycosylation approach regulatory gene is to regulate the glycosylation of recombinant protein | |
| CN103298925B (en) | For cultivating human medullary leukaemia and the method by its derivative cell | |
| Matanguihan et al. | Upstream continuous processing: recent advances in production of biopharmaceuticals and challenges in manufacturing | |
| JP7207643B2 (en) | Screening method for continuous culture conditions | |
| CN103320388A (en) | Cell culture method capable of improving antibody expression levels and improving glycosylation levels | |
| Gramer et al. | Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors, and in an industrial scale hollow fiber bioreactor system | |
| CN105838736A (en) | Screening method of cell strain of GS expression system | |
| EA037238B1 (en) | Perfusion media | |
| Reger et al. | Boosting productivity for advanced biomanufacturing by Re-using viable cells | |
| Goodman et al. | Pooled screening of CAR T cells identifies non-native signaling domains for next-generation immunotherapies | |
| McKinney et al. | Optimizing antibody production in batch hybridoma cell culture | |
| CN108823267A (en) | The method for adjusting the acid peak content of antibody secreted by CHO-K1 expression system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20170308 |