CN102876626A - Serum-free and protein-free all-chemical-component-definition culture medium for supporting CHO high-density suspension growth - Google Patents

Abstract

The invention discloses a serum-free and protein-free all-chemical-component-definition culture medium for supporting the CHO high-density suspension growth. The serum-free culture medium contains amino acids, inorganic salts, trace elements, vitamins, carbohydrates and other organic molecules. The serum-free culture medium has the advantages of no containment of any extracted or recombinant proteins, polypeptides or hydrolyzates, short cell growth time, high density, low metabolic refuse accumulation, good cell vitality maintenance, realization of the protein expression higher than that of products of same kind, and low cost because of all chemical component definition.

Description

The substratum that the serum-free of supporting the CHO high density suspension to grow defines without the full chemical composition of albumen

Technical field

The present invention relates to a kind of serum free medium of supporting animal cell culture, especially the substratum that a kind of serum-free of the CHO of support high density suspension growth defines without the full chemical composition of albumen, support the Chinese hamster ovary celI high density suspension to have the advantages that chemical composition defines when cultivating, belong to the cell engineering field.

Background technology

The expression of engineered protein was risen abroad from the eighties in last century, so far near 30 years, and was widely used in biological study, medical research industry.Especially since biological medicine industry particularly be a dark horse after the nineties take the treatment monoclonal antibody as the biotech company of product so that the sector every year is to surpass 50% compound growth rate high speed development.In the world wide, granted and just in the restructuring human cytokines in declaration, surpass 70-80% based on animal cell culture production, in all are produced based on the protein drug of animal cell culture, being most widely used of CHO (Chinese hamster ovary cell), accounted for and ratified more than 70% of protein drug, and declared more than 80% of Clinical Project.

Chinese hamster ovary celI and other zooblast ratios, foreign protein are easy to synthetic and are secreted in the substratum; Correct assembling and the albumen of modifying after translating make it to approach with native protein; Be easy to amplify, be convenient to the industrialization high-density culture; The history that is applied to clinical medicine production is long, safe; Therefore, it is very important that the cultivation of Chinese hamster ovary celI just becomes, and wherein the design and optimization of substratum becomes again the most important thing.Define substratum from the early stage chemical composition that has blood serum medium to be transitioned into serum free medium and most praise highly in the world now, none is not dangerous in the production technique, unstable, uneconomical and effective not component to be adjusted into as far as possible do not have virus/the exogenous factor pollution, batch repeatability is high, and is with low cost and support higher cell density and protein expression direction.

The external development along with animal cell culture and expression technology in recent years, the Tehnologies with Life has appearred, the companies such as Thermofisher are that some reagent companies of representative develop the substratum of cultivating for the Chinese hamster ovary celI high density suspension, comprise common serum free medium, protein-free medium and chemical composition define substratum, but owing to belonging to the core secret of the trade of each company, keep secret.

Domestic also the beginning in the last few years developed multiple serum free medium for Chinese hamster ovary celI successively, but number average is at existing minimum medium such as DMEM mostly, DMEM/F12, upper various VITAMIN, somatomedin, the Transferrins,iron complexes of adding in the basis such as IMDM, proteolysate and trace element, complicated, the nutritive equilibrium degree is poor and basically all contain albumen or its hydrolysate class is difficult to Quality Control, affects the component of downstream purification.Simultaneously cell cultures performance is general, and batch cultivation seldom surpasses the density of every milliliter in 500 ten thousand cells.

Summary of the invention

For the deficiencies in the prior art, the invention provides the substratum that the serum-free of a kind of CHO of support high density suspension growth defines without the full chemical composition of albumen, introduce the components that comprise trace element more, substitute necessary somatomedin such as Regular Insulin and Transferrins,iron complexes in the general substratum design, and by metabolic analysis and statistics experimental design, allow more balance of each nutrition composition.Finally obtain one without any the non-substratum that defines composition, support simultaneously Chinese hamster ovary celI better growth and shorter cell amplification doubling time.

The technical scheme that the present invention solves the problems of the technologies described above is as follows:

1) utilizes existing bibliographical information design simplification substratum framework formula;

2) select interpolation can substitute somatomedin, Transferrins,iron complexes and raising Growth of Cells performance component;

3) utilize the statistics experimental design to determine the optimal dose of each component;

Based on above consideration, substratum of the present invention mainly is made of the component of following several respects:

Amino acid: as protein/polypeptide synthetic precursor and the macromolecular intermediate of synthetic other biological, also produce metabolisable energy simultaneously;

Inorganic salt: NaC l keeps the osmotic pressure balance, coordinates common transhipment organic macromolecule and enters cell; K ⁺, Ca ²⁺, Mg ²⁺, Zn ²⁺Plasma is then participated in metabolism and signal transduction and is promoted adherent and cell proliferation; Various negatively charged ion (SO ₄ ^2-, NO ₃ ^-Deng) then be mainly used in regulating rotary cytolemma potential energy or as the precursor of sulfur-bearing or nitrogen element organic molecule;

VITAMIN: VITAMIN in cellular metabolism mainly as following cell function organic catalyst function.Such as the metabolism of carbon molecule, transamination, carboxylation and decarboxylate turn usefulness into, oxidative phosphorylation;

Carbohydrate: glucose is the main carbohydrate that needs in the cell cultures, as the main energy derive of cellular metabolism, reaches the macromolecular carbon sources such as synthetic DNA; Sodium.alpha.-ketopropionate and inositol also usually are used for shortening the intermediary metabolism of cell;

Trace element: a lot of trace metal ions certain concentration range have clearly to the growth of cell and promoter action and the restraining effect of protein expression.

Except in addition, also added phenol red indicator as medium pH in the substratum, and

F-68 is as the tensio-active agent of avoiding the shearing force injury in the suspension cell culture for the protection of cell.

According to the not same-action with above all kinds of material cell growth, the present invention arranges the material of above classification by following concentration:

Amino acid whose component and consumption:

The component of inorganic salt and consumption:

Component and the consumption of trace element:

The component of VITAMIN and consumption:

Component and the content of carbohydrate and other organic molecules:

Further, the phenol red consumption of indicator as medium pH that adds in the serum free medium is: 2.0～7.0mg/L; The tensio-active agent that adds The consumption of F-68 is: 330～1540mg/L.

Serum free medium provided by the invention is compared with existing Chinese hamster ovary celI serum free medium and is had the following advantages:

1. without any somatomedin and other albumen additives, chemistry defines component nothing but, and all components chemical structure is known, is convenient to preparation, stores;

2. support the growth of high-density Chinese hamster ovary celI, keep the vigor time long, define substratum for present business-like chemical composition;

3. owing to being small-molecule substance, cost is low.

The present invention will be further described below in conjunction with drawings and Examples.

Description of drawings

Fig. 1 is that serum-free of the present invention defines the growing state that substratum (T11) is supported the CHO-K1 cell without the albumen chemical composition;

Fig. 2 is that serum-free of the present invention defines the growing state that substratum (T11) is supported the CHO-S cell without the albumen chemical composition;

Fig. 3 is that serum-free of the present invention defines the growing state that substratum (T11) is supported the CHO-DG44 cell without the albumen chemical composition;

Fig. 4 is that serum-free of the present invention defines the growing state that substratum (T11) is supported the CHO-DUKX-B11 cell without the albumen chemical composition;

Fig. 5 be serum-free of the present invention without the albumen chemical composition define substratum (T11) and commercialization substratum Invitrogen CD-CHO and Hyclone CDM4CHO in the simultaneous test aspect the growing state of supporting the CHO-K1 cell.

Embodiment

Below in conjunction with accompanying drawing principle of the present invention and feature are described, institute gives an actual example and only is used for explaining the present invention, and the method among the embodiment is the routine test technology of this area if no special instructions.

Embodiment 1

The substratum preparation

The basal component of serum free medium is divided into 10 groups of concentrated solutions:

S1 is inorganic salt solution, is the 50X concentrated solution

S2 is trace element, is the 5000X concentrated solution

S3 is amino acid solution, is the 10X concentrated solution

S4 is TYR solution, is the 100X concentrated solution

S5 is carbon source and other organic molecules

S6 is L-glutaminate solution, the 40X concentrated solution

S7 is the VITAMIN concentrated solution, the 3500X concentrated solution

S8 is folic acid solution, the 200X concentrated solution

S9 is riboflavin solution, the 1500X concentrated solution

S10 is sodium hydrogen carbonate solution, the 100X concentrated solution

Above-mentioned concentrated culture is joined in the container of 2L successively, and liquid nutrient medium and the called after T11 substratum of preparation 1X are with between the NaCl of 5M adjusting osmotic pressure to 270～275mosm and use 7.5%NaHCO ₃Or then 1N HCl adjusting pH uses 0.22 micron membrane filtration degerming between 7.0～7.2.

Embodiment 2

Cell cultures

Prepare substratum, and with its insert 37 the degree water-baths in 30 minutes, take out stand-by; Get CHO-K1, CHO-S, each recovery of CHO-DG44 and CHO-DUKX-B11 is in the DMEM/F12 substratum that contains 8%FBS, and is for subsequent use after two generations of increasing;

The above various cells that recovery is good are with 5.0X10 ⁵The density that cell is every milliliter is seeded in the 125mL triangular flask that contains the 25mLT11 substratum, and the concentration of FBS (foetal calf serum) is about 4% in the maintenance substratum; Triangular flask placed contain 5% CO ₂Shaking table cultivate in 37 degree cultivate rotating speed 120rpm; Every day counting cells, when cell density greater than 1.5X10 ⁶During density that cell is every milliliter, with 5.0X10 ⁵The density that cell is every milliliter is passaged in the new 125mL triangular flask, and the concentration of FBS is about 1% in the maintenance substratum; And so forth, until after 3 generations centrifugal going down to posterity removed whole serum.

With above-mentioned domestication good cell with 3.0X10 ⁵The density that cell is every milliliter is seeded in the 500mL triangular flask that contains 150mL; All triangular flasks are placed contain 5% CO ₂Shaking table cultivate in 37 degree cultivate rotating speed 120rpm, the counting cells of taking a sample every day, and calculate Cell viability with Trypan Blue.Wherein CHO-K1 peak value cell count is 6.56X10 ⁶Every milliliter in cell is inoculated after 9 days Cell viability still at (Fig. 1) more than 60%; CHO-S peak value cell count is 6.87X10 ⁶Every milliliter in cell, inoculate rear 6 days after Cell viability still about 50% (Fig. 2); CHO-DG44 peak value cell count is 6.73X10 ⁶Every milliliter in cell, inoculate rear 6 days after Cell viability still about 60% (Fig. 3); CHO-DUKX-B11 peak value cell count is 4.23X10 ⁶Every milliliter in cell, inoculate rear 6 days after Cell viability still about 60% (Fig. 4).

Embodiment 3

Carry out cell cultures relatively with the substratum of other companies

Press the method domestication CHO-K1 cell of embodiment 2 in Life technologies/GIBCO CD-CHO substratum, and in Thermofisher/Hyclone CDM4CHO substratum;

Get the good CHO-K1 cell of separately domestication, carry out parallel laboratory test; With CHO-K1 with 3.0X10 ⁵The density that cell is every milliliter is seeded in the 500mL triangular flask that contains 150mL; All triangular flasks are placed contain 5% CO ₂Shaking table cultivate in 37 degree cultivate rotating speed 120rpm, the counting cells of taking a sample every day, and calculate Cell viability with Trypan Blue.Keep cell cultures, when motility rate descends and viable cell density is lower than 2.0X10 ⁶Stop during density that cell is every milliliter when the group experiment.The CHO-K1 that final experimental result is found the CD-CHO experimental group reached peak value 4.24X10 on the 4th day after inoculation ⁶The density that cell is every milliliter is inoculated rear 6 days cell densities and is down to 2.0X10 ⁶Below every milliliter of density of cell, the CHO-K1 of CDM4-CHO experimental group reached peak value 4.09X10 on the 5th day after inoculation ⁶The density that cell is every milliliter is inoculated rear 7 days cell densities and is down to 2.0X10 ⁶Below every milliliter of density of cell; The present invention (T11) substratum then just obviously is better than above two substratum from inoculating rear the 4th day beginning Growth of Cells, and reaches peak value 6.56X10 in rear 6 days in inoculation ⁶The density that cell is every milliliter, and be maintained to the 9th day (213.5 hours) cell density and just drop to 1.95X10 ⁶The density that cell is every milliliter.

All reagent of above example are all available from Sigma-Aldrich company, and 0.22 micron filter membrane is Millipore company product.

The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (5)

1. a serum-free of supporting the growth of CHO high density suspension is characterized in that without the substratum that the full chemical composition of albumen defines, and described serum free medium contains following component: amino acid, inorganic salt, trace element and VITAMIN,

Wherein, described amino acid whose component and consumption are:

The component of described inorganic salt and consumption are:

The component of described trace element and consumption are:

The component of described VITAMIN and consumption are:

2. a kind of serum free medium according to claim 1 is characterized in that, described serum free medium also contains carbohydrate and other organic molecules of following component and consumption:

3. a kind of serum free medium according to claim 1 and 2 is characterized in that, also contains phenol redly as indicator in the described serum free medium, and described phenol red consumption is: 2.0～7.0mg/L.

4. a kind of serum free medium according to claim 1 and 2 is characterized in that: also contain in the described serum free medium F-68 is as tensio-active agent, and is described

The consumption of F-68 is: 330～1540mg/L.

5. a kind of serum free medium according to claim 2 is characterized in that, described carbohydrate is glucose, Sodium.alpha.-ketopropionate or inositol.

Images