CN105255811A - Preparing method for serum-free and animal-source-free culture medium additive suitable for growth of insect cells - Google Patents
Preparing method for serum-free and animal-source-free culture medium additive suitable for growth of insect cells Download PDFInfo
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- CN105255811A CN105255811A CN201510760016.7A CN201510760016A CN105255811A CN 105255811 A CN105255811 A CN 105255811A CN 201510760016 A CN201510760016 A CN 201510760016A CN 105255811 A CN105255811 A CN 105255811A
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Abstract
The invention relates to the technical field of biological pharmacy, in particular to development of a serum-free and animal-source-free culture medium additive suitable for insect cells mainly including Sf9 and High Five cells. A traditional culture medium Grace or IPL-41 serves as a basis, the additive beneficial for the growth of the insect cells is added, in the insect cell culture process, the additive has a function of replacing serum, price is low, the insect cells can grow for 240 h continuously, and cell activity can keep over 95%.
Description
Technical field
The present invention relates to biological pharmacy technical field, being specifically related to a kind of being applicable to mainly comprises Sf9 and HighFive cell to insect cell) the development of additive of the non-animal derived cultivation of serum-free.
Background technology
At present, conventional insect cell has Sf-9 cell, that is: Spodopterafrugiperda (noctuid is coveted on meadow) cell and HighFive cell, HighFive cell (BTI-TN-5B1-4) is the clone and separate strain coming from cabbage looper (Trichopulsiani) parental cell line, is usually used in the expression of recombinant proteins of rod string design (BEVS).Baculovirus expression system (baculovirusexpressionsystem) applies more eukaryotic expression system in recent years, and it in expressed in insect cells various exogenous genes, can comprise fungi, plant, bacterium, the gene of virus.The double-stranded DNA virus of baculovirus to be a class with insect cell be natural host, has the species specificity of height, does not infect vertebrates, harmless to people, animal.That current research is more is autographa californica nuclear polyhedrosis virus (AcNPV), and its host is that noctuid is coveted on meadow.And Sf-9 cell and HighFive cell, be the host cell of baculovirus expression system.Being applied to the expression of albumen and preparing in purifying, there are many advantages, comprise: the recombinant protein of expression can correctly fold, and forms disulfide linkage; Modification processing can be carried out after translation as glycosylation, phosphorylation, amidation and signal peptide cutting etc., make recombinant protein on structure and function closer to native protein; Compared with other eukaryotic expression systems, baculovirus expression system can efficiently expressing exogenous gene, and expression amount reaches as high as 50% of infected insect cell total protein concentration; Large fragment foreign gene can be held.
At present, the calf serum that domestic production technique many employings Grace substratum or IPL-41 substratum add 5-10% is cultivated, or with commercialization serum free medium as SF900II adds suitable serum free culture system again.It is serum free medium that a lot of commodity are known as, but, be difficult to accomplish to depart from serum free culture system completely.In addition, because current domestic serum supply growing tension is constantly high along with price, obvious restriction is created to domestic biological antibody manufacturing enterprise.In addition, from the angle of producing, protein containing a large amount of complicated component in serum, this pole is unfavorable for that vaccine, viruslike particle, cytokine are separated with the downstream purification of the biological products such as monoclonal antibody, chemical composition simultaneously contained by serum is uncertain, also there is lot stability problem in serum, but also easily causes bacterium, fungi, virus and mycoplasma contamination.Therefore, use no or little the trend that serum becomes Insect cellculture as far as possible, prior art is usually through animal source compositions such as interpolation bovine serum albumin, Transferrins,iron complexes, Regular Insulin, reduce the addition of serum, but the security of the protein of animal source composition to biological products (as vaccine) has a significant impact, so in the urgent need to a kind of cell cultures additive that can replace serum function.Cell can either be made to grow fast, keep higher vigor, additionally reduce the possibility of pollution, improve the stability of cellular product, reduce the protein ingredient content of animal-origin simultaneously, be beneficial to later-period purification technique, reduce production cost.
At present, business-like Insect culture medium has Grace substratum, IPL-41 substratum, TC100 substratum, these substratum are all the substratum of classics used in conjunction with serum, also has business-like serum free medium as SF900II, SF900III, Express
sFM,
deng series product, these product prices are high and formula is secret, and are difficult to remove serum free culture system completely, the vigor of the maintenance cell that can not continue, or cost is higher, is seldom directed to insect cell, develops the additive of alternative serum.The present invention is based on above-mentioned present situation on product, develop the cell additive of the replacement serum of applicable insect cell suspension growth, in the middle of the process of cultivating insect cell, the function replacing serum can be played, and cheap.Based on conventional medium Grace or IPL-41, be added with the additive being beneficial to insect cell growth, insect cell can obtain the continuous growth of 240h, and cell viability can remain on more than 95%.
Summary of the invention
The object of the invention is, overcome defect of the prior art, a kind of serum free medium of applicable insect cell suspension growth is provided, in substratum triturating, there is with some the compound of specific function, lipid component, yeast and plant hydrolyzed thing, trace element and VITAMIN instead of the conventional animal source composition added in substratum, while reduction serum use cost, reduce serum and animal derived protein in substratum and carry the probability of exogenous pathogen fungi pollution, protein content simultaneously in substratum significantly reduces, extremely be conducive to later-period purification technique, improve the stability of cellular product.
For achieving the above object, the technical solution used in the present invention is: provide the serum free medium that a kind of applicable insect cell bottle is cultivated, it is characterized in that, on the basis of conventional Grace or IPL-41 substratum, with the addition of amino acid, lipid, trace element and hydrolyzate composition, in order to the function of alternative serum, for the addition of serum-free, added ingredients is as follows:
Glucose 3-15g/L,
L-arginine 2-9mM,
Altheine 1-8mM,
L-glycine 5-14mM,
L-Histidine 2.77mM,
ILE 8.41mM,
L-Leu 5-11mM,
1B 4-9mM,
L-Methionine 0.1-0.7mM,
L-threonine 1-9mM,
L-Trp 0.2-1.4mM,
L-ties propylhomoserin, 2-5mM
L-PROLINE 7-10mM,
Cottonseed hydrolyzate 300-3000mg/L,
Rice hydrolyzate 300-3000mg/L,
Wheat bran hydrolyzate 300-3000mg/L,
Recombulin 2-25mg/L,
Sodium.alpha.-ketopropionate 50-300mg/L,
Sodium Selenite 0.0010.04mg/L,
Ironic citrate 0.05-1mg/L,
Linolic acid 0.1-0.3mg/L,
Soybean lecithin 10-65mg/L,
Cholesterol 5-25mg/L,
Tropolone 0.1-6mg/L,
F-68100-600mg/L,
Reduced glutathion 0.04-0.5mg/L,
L-glutaminate 3-15g/L.
Wherein, F-68 is the abbreviation of pluronicF-68, and the conventional additives in cell cultivation process, has provide protection to cell.
Generally acknowledged implication is had in the art about " cottonseed hydrolyzate ", " wheat bran hydrolyzate " and " rice hydrolyzate ", be exactly by cottonseed, wheat bran, rice, through special technique, comprise after the means such as enzymic digestion, acid treatment process, extract and be applicable to Growth of Cells component, all there is commercial reagents at present, the hydrolyzate that the present invention specifically uses Difico company to produce.
Grace and IPL-41, is substratum disclosed in composition, all has commercial reagents at present, the basic medium that the present invention specifically uses LifeTechnology company to produce.
The serum-free culture medium supplement of the present invention's development, not only reduces culture medium cost but also significantly decreases the content of animal derived protein in substratum, simplifying the process of serum free medium research and development, be more conducive to the abstraction and purification of cell derived product.
For achieving the above object, another technical solution used in the present invention is the collocation method of the serum free medium that applicable insect cell bottle is cultivated, and it is characterized in that, described compound method comprises following processing step:
S1: inject water to 90% of cumulative volume in a reservoir, all pours into one bag of Grace or IPL-41 substratum in container, and to be washed down by remaining medium in bag with a small amount of water for injection and be incorporated to container, gentle agitation is dissolved;
S2: said components is joined in solution successively, gentle agitation is dissolved;
S3: gentle agitation, to dissolving completely, injects water to 100% of cumulative volume;
S4: adjust pH to 6.2 with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid soln;
S5: degerming with 0.2 μm of filter membrane positive press filtration;
S6: should airtightly at 2 DEG C ~ 8 DEG C keep in Dark Place after solution preparation completes.
Wherein preferred technical scheme is that in described S1 step, the water temperature of water for injection is 30 DEG C.
Advantage of the present invention and beneficial effect are that this substratum, by the interpolation of amino acid, lipid, trace element, hydrolyzate etc., instead of the function of serum.Compare traditional IPL-41+10%FBS and Grace+10%FBS, more can promote that cell grows fast, increases cell density, shortens the doubling time, improves cell viability, illustrates that this invention substratum is more suitable for the serum free suspension cultivation of insect cell cell.
Accompanying drawing explanation
Fig. 1: Sf9 cell is growth curve (left hurdle) and Sf9 cell growth curve (right hurdle) in Grace+10%FBS in Grace+SM.
Fig. 2: HighFive cell is growth curve (left hurdle) and HighFive cell growth curve (right hurdle) in IPL-41+10%FBS in IPL-41+SM.
Embodiment
Embodiment 1. is applicable to the configuration of the serum free medium of insect cell growth
The invention provides a kind of serum free medium and compound method thereof of applicable insect cell growth, its substratum based on Grace or IPL-41 of Gibico production adds amino acid, lipid, trace element, hydrolyzate etc., material therefor is if no special instructions all purchased from sigma company, and concrete additive is as follows:
Glucose, 8g/L;
L-arginine, 5.49mM;
Altheine, 3.9mM;
L-glycine, 12.89mM;
L-Histidine, 2.77mM;
ILE, 8.41mM;
L-Leu, 10.4mM;
1B, 7.59mM;
L-Methionine, 0.49mM;
L-threonine, 4.66mM;
L-Trp, 0.91mM;
L-ties propylhomoserin, 4.52mM;
L-PROLINE, 8.22mM;
Cottonseed hydrolyzate 800mg/L;
Rice hydrolyzate 3000mg/L;
Wheat bran hydrolyzate 300mg/L;
Recombulin 25mg/L;
Sodium.alpha.-ketopropionate 60mg/L;
Sodium Selenite 0.01mg/L;
Ironic citrate 0.5mg/L;
Linolic acid 0.2mg/L;
Soybean lecithin 45mg/L;
Cholesterol 20mg/L;
Tropolone 6mg/L;
F-68200mg/L；
Reduced glutathion 0.04mg/L;
L-glutaminate 7g/L.
By above additive from called after SM.
Wherein, " cottonseed hydrolyzate ", " wheat bran hydrolyzate " and " rice hydrolyzate " have generally acknowledged implication in the art, be exactly by cottonseed, wheat bran, rice, through special technique, comprise after the means such as enzymic digestion, acid treatment process, extract and be applicable to Growth of Cells component, all have commercial reagents at present, the hydrolyzate that the present invention specifically uses Difico company to produce.
Grace and IPL-41, is substratum disclosed in composition, all has commercial reagents at present, the basic medium that the present invention specifically uses LifeTechnology company to produce.
The compound method of substratum comprises following processing step:
S1: inject water to 90% of cumulative volume in a reservoir, all pours into one bag of Grace or IPL-41 substratum in container, and to be washed down by remaining medium in bag with a small amount of water for injection and be incorporated to container, gentle agitation is dissolved;
S2: said components is joined in solution successively, gentle agitation is dissolved;
S3: gentle agitation, to dissolving completely, injects water to 100% of cumulative volume;
S4: adjust pH to 6.2 with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid soln;
S5: degerming with 0.2 μm of filter membrane positive press filtration;
S6: should airtightly at 2 DEG C ~ 8 DEG C keep in Dark Place after solution preparation completes.
The contrast experiment contrast experiment that embodiment 2. substratum of the present invention is used for cell cultures is divided into two groups:
Test one: compare test (as shown in Figure 1) with the growing state of Sf-9 cell in Grace+SM, Grace+10%FBS;
Test two: compare test (as shown in Figure 2) at the experimental conditions of IPL-41+SM, IPL-41+10%FBS with HighFive cell.
The particular case of Growth of Cells is as shown in table 1.
Visible, the peak concentration of Sf-9 cell and the growth of HighFive cell in Grace+SM and IPL-41+SM is respectively 600*104/ml and 690*104/ml, and cell viability is all more than 95%; And the peak concentration of Sf-9 cell as a control group in Grace+10%FBS substratum is 520*104/ml, cell viability Schwellenwert is 93.2%; HighFive cell peak concentration in IPL-41+10%FBS substratum is 580*104/ml, and minimum cell viability is 90.6%.
Table 1.Sf-9 cell and HighFive cell growing state in different culture media
Test surperficial substratum of the present invention to add conventional medium, obviously facilitate Growth of Cells by the interpolation of amino acid, lipid, trace element, hydrolyzate composition, shorten the doubling time, and cell viability is also apparently higher than control group.Grace and the IPL-41 substratum increase serum of comparing traditional is cultivated, substratum of the present invention not removing only the interpolation of serum, and cell density is higher, cell viability is better, so substratum of the present invention is more suitable for insect cell, the serum free suspension comprising Sf-9 cell and HighFive cell is cultivated.
The above, be only preferred embodiment of the present invention, not any formal and substantial restriction is done to the present invention, all those skilled in the art, do not departing within the scope of technical solution of the present invention, when utilizing disclosed above technology contents, and a little change made, modify with differentiation equivalent variations, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (4)
1. a serum free medium for applicable Insect cellculture, is characterized in that, on the basis of conventional Grace or IPL-41 substratum, added ingredients is as follows:
Glucose 3-15g/L,
L-arginine 2-9mM,
Altheine 1-8mM,
L-glycine 5-14mM,
L-Histidine 2.77mM,
ILE 8.41mM,
L-Leu 5-11mM,
1B 4-9mM,
L-Methionine 0.1-0.7mM,
L-threonine 1-9mM,
L-Trp 0.2-1.4mM,
L-ties propylhomoserin, 2-5mM
L-PROLINE 7-10mM,
Cottonseed hydrolyzate 300-3000mg/L,
Rice hydrolyzate 300-3000mg/L,
Wheat bran hydrolyzate 300-3000mg/L,
Recombulin 2-25mg/L,
Sodium.alpha.-ketopropionate 50-300mg/L,
Sodium Selenite 0.0010.04mg/L,
Ironic citrate 0.05-1mg/L,
Linolic acid 0.1-0.3mg/L,
Soybean lecithin 10-65mg/L,
Cholesterol 5-25mg/L,
Tropolone 0.1-6mg/L,
F-68100-600mg/L,
Reduced glutathion 0.04-0.5mg/L,
L-glutaminate 3-15g/L.
2. according to the substratum described in claim 1, wherein, insect cell is Sf9 and HighFive cell.
3. the compound method of substratum described in claim 1, described compound method comprises following processing step:
S1: inject water to 90% of cumulative volume in a reservoir, all pours into one bag of Grace or IPL-41 substratum in container, and to be washed down by remaining medium in bag with a small amount of water for injection and be incorporated to container, gentle agitation is dissolved;
S2: said components is joined in solution successively, gentle agitation is dissolved;
S3: gentle agitation, to dissolving completely, injects water to 100% of cumulative volume;
S4: adjust pH to 6.2 with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid soln;
S5: degerming with 0.2 μm of filter membrane positive press filtration;
S6: should airtightly at 2 DEG C ~ 8 DEG C keep in Dark Place after solution preparation completes.
4. collocation method according to claim 3, in described S1 step, the water temperature of water for injection is 30 DEG C.
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Cited By (1)
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| CN106367379A (en) * | 2016-12-07 | 2017-02-01 | 天康生物股份有限公司 | Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof |
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| CN106367379A (en) * | 2016-12-07 | 2017-02-01 | 天康生物股份有限公司 | Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof |
| CN106367379B (en) * | 2016-12-07 | 2019-09-10 | 天康生物股份有限公司 | A kind of insect cell maintaining liquid and its preparation method improving swine fever virus E2 recombinant protein expression quantity |
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