DE19541345C2 - Process for the cultivation of eukaryotic cells - Google Patents

Description

The invention relates to a method for the cultivation of eukaryotic cells in a serum culture medium or high molecular weight serum substitutes with the addition of Protein cleavage products. The invention further relates to a method for carrying out the method suitable supplement.

In the cultivation of eukaryotic cells, for example to obtain monoclonal antibodies per, cell culture media containing amino acids, vitamins, sugar, Contain salts and a pH indicator. This cell culture media is called Supplement Serum or high molecular serum substitutes added. The use of animal serum as Supplement is disadvantageous for various reasons. First, the used ones Sera, - often fetal calf serum is used for example - a considerable knock-out On the other hand, the serum components complicate the purification of the obtained NEN cell products, and because of their undefined composition and the possible in it wise contained pathogenic components is their application for the production example wise pharmaceutical preparations problematic. It has therefore been tried many times, tieri to replace all or part of the serum with substitutes. Such a serum-free me dium has become known under the name "ISCOVE's", in which Rin serum albumin, transferrin, soybean lipids, selenite and possibly insulin are included. These are defined high moles produced under controlled conditions kular components.

In the publication "Peptone, a low-cost growth-promoting nutrient for intensive animal cell cultu right "by D. G-H. Jan et al., Cytotechnology 16: 17-26, 1994, pages 17-26, becomes a cell culture rungsverfahren described according to the generic type, being a substitute for the Serum so-called "peptones" can be used. A mixture is created under peptons Low molecular components understood by enzymatic digestion of extracts  of cells and / or tissues and / or products of plant or animal origin stood. Similar products can also be obtained by chemical "hydrolysis" of proteins in Extracts of cells and / or tissues and / or products of plant or animal origin jump.

In the investigations listed by the authors mentioned above, one part show replacement of the serum with different peptones achieved a higher cell density than in peptone-free culture media. At relatively high peptone concentrations, in one execution example 0.5%, but again became a lower cell growth rate and a lower one Cell density reached.

For optimal cell growth, a nutrient medium was found that was in addition to the peptone-Zu has a minimum serum concentration of 5%. It will explicitly depend on it showed that the peptones used in the tests are not suitable, the serum to replace completely. The maximum cell densities that can be achieved with the known nutrient medium with a stand culture with approx. 15 × 10⁵ cells / ml and with a perfusion culture with approx. 30 × 10⁵ cells / ml indicated.

In "Hormones and Factors That Stimulate Growth of a Rat Islet Tumor Cell Line in Serum-Free Medium "by Henry K.W. Fong et al., Diabetes, Vol. 30, pp. 1022-1028, 1981 becomes a serum free it described medium for cell cultivation. The medium contains amino acids, vitamins, Sugar, salts and additives such as insulin, prolactin, hormones and peptones. This medium will placed on a culture substrate coated with serum, so that it in the course of cell culture tion does not remain serum-free. Therefore, the medium, in contrast to the present inven only be understood as a serum-free intermediate. Since it is a unicameral cell acts as a culture vessel, the medium fulfills two functions simultaneously, namely as a nutrient medium and as a cell cultivation medium. The penetration of the entire medium with serum required for cell cultivation is in the described application method, the application on plates coated with serum, mandatory, since none in supply and culture chamber separate culture vessel is used.

Cell growth rates are comparable, but not greater than those found in 10% serum the media. Higher growth rates with high cell densities are given with the given medi so as not to achieve.

The invention has for its object a method for the cultivation of eukaryotic cells len in high density, at which the serum consumption is further reduced.

Starting from the above-mentioned method, this object is achieved in that the cells in a ge from a supply medium through a semipermeable membrane separate production chamber, where the serum or the high molecular weight Serum substitutes only the culture medium in the production chamber and the cleavage products be added to the supply medium, and that the pore size of the semipermeable Membrane is selected so that the cells and cell products, as well as serum proteins or high molecular substitutes are retained in the production chamber and the Ver protein cleavage products added to the care medium can pass through.

The volume of the supply medium can be reduced by suitable design measures be created many times larger than the volume inside the production chamber. The for the nutrition of the cell culture required low-molecular components come from the Supply medium through the dialysis membrane into the production chamber, cellular degradation Conversely, products of cell metabolism get from the production chamber via the se mipermeable membrane in the supply medium. Such, for the implementation of the Process-suitable "bioreactor" with a compartment divided into compartments is in DE-A1 42 29 325 described, which described in terms of the structural features of it benen bioreactor is hereby expressly included in the present application. On due to their compartmentalization are equally suitable as "hollow fiber reactors" cell cultivation systems. Due to the compartmentalization of the cultural area, essential higher cell densities than in the non-compartmentalized cell culture systems. Furthermore, such a compartmentalization allows a cell cultivation method in which the serum or high molecular serum substitutes only the culture containing the cells dium can be added in the production chamber. However, it has been shown that the Zuga be of serum or high molecular weight serum substitutes alone into the cells Production chamber is in most cases not sufficient to achieve high cell densities. At Such experiments were only possible after adding serum to the supply medium ho cell densities can be achieved.  

Surprisingly, it has now been found that the addition of serum to the supplier supply medium is superfluous if the supply medium cleavage products of proteins be set. The cleavage products in the sense of the invention are by enzymatic or chemical hydrolysis of proteins. They can be made from extracts of cells and / or Tissues and / or products of plant and animal origin can be obtained. Under Peptons are obtained through enzymatic digestion and under hydrolyzates Understand peptide / amino acid mixtures obtained by chemical hydrolysis. The so-called named peptones are inexpensive to produce and are therefore used for the cultivation of bacteria s that are usually much less demanding in terms of nutrition than eu cariotic cells. Depending on how they are produced, they contain peptones or hy drolysate a variety of peptides of different lengths. In addition, others can use it Biologically active additives such as vitamins, salts or trace elements can be included.

To explain the effect, it is assumed that the animal Sera usually also contain low molecular weight components necessary for cell culture tion are indispensable, but through the semipermeable membrane into the supply medium in order to be able to pass through and thereby lose the cell culture in the production chamber. By adding the low molecular weight components of the peptones or hydrolyzates these pass through the semipermeable membrane into the production chamber and never there replace the molecular components of the serum. It has been shown that with regard to cell multiplication and production of cellular components with the inventive method even better results than using Se alone around in the production chamber and in the supply medium.

Because only the relatively small volume compared to the volume of the supply medium men of the production chamber must be supplemented with serum, and on the other hand that peptone or hydrolyzate added to the supply medium compared to the serum an inexpensive cultivation process becomes available many times cheaper enables.

Such high-molecular serum substitutes in the sense of this invention include such compo nents understood that can not pass the semipermeable membrane. Crucial this is the pore size of the membrane. As a measure of the pore size of a semi-permeable Membrane becomes the "Molecular Weight Cut Off (MWCO)  scheduled. This indicates the size of molecules in kDalton that a membrane with an ent speaking MWCO value can happen.

In a preferred procedure, fission products of vegetable origin are incorporated puts. For this purpose, for example, split products made from soy flour or wheat germ or He come extracts in question. These cleavage products have the advantage that they match those in the corresponding Products of animal origin may not have possible pathogens and for their ver Therefore, there are no concerns about the use of pharmaceutical products. This The procedure has been particularly in connection with high molecular serum substitutes proven advantageous in the production chamber. By using defined seru controlled production in the production chamber in connection with Spaltpro Products of plant origin in the supply medium become the disadvantages mentioned at the beginning avoided in the known methods. First, the combination is very inexpensive, on the other hand, both the low-molecular cleavage products and the high moles can be kular serum substitutes easily due to their defined size and other properties separate from the cell products and the health concerns no longer apply visible of the possible contamination by pathogens.

In this regard, a procedure has proven particularly useful for the culture medium Substitutes defined in the production chamber instead of serum, the bovine serum albumin or comprise transferrin and / or a lipid-containing component. It is it is particularly advantageous to use fission products and serum substitutes, the gentechno have been logically won. The lipid-containing component can be, for example are soybean lipids.

A procedure in which the supply medium has a low molecular weight has proven very successful lare, hormonally active peptides are added. Such low molecular weight peptides that through the semipermeable membrane from the supply medium into the production chamber gen, can have growth-promoting properties, and they are very simple and Can be produced inexpensively by chemical or genetic engineering processes.

It has proven to be advantageous to use a semipermeable membrane with a pore size speaking of a molecular weight cut-off in the range from 8 kDalton to 14 kDalton, especially of 12.5 kDalton. Such a semipermeable membrane holds the Cells and cell products or the high molecular components of the sera or  high molecular serum substitutes in the production chamber and leaves on the other hand the low molecular weight components from the supply medium into the production chamber diffuse in.

With regard to a suitable supplement as the only or proportionate part of the be Known nutrient medium, it is appropriate that the supplement a ready-to-use Mi combination of amino acids, vitamins, sugar, salts, protein breakdown products and hormonal effective peptides with a length in the range of 2 to 100 amino acids, preferably with a length in the range of 3 to 15 amino acids, without the addition of serum.

The fact that the supply medium or the supplemented supply medium ge is ready for use, there is no need to add a suitable protein cleaving pro product and the associated dangers of contamination of the nutrient medium. There at under the term "ready to use" both one directly as a supply medium usable nutrient solution containing the supplement as well as a concentrate understood the one that is pre-cultured with a suitable liquid or a common nutrient solution, such as DMEM or RPMI 1640, must be added.

As in the supplement in the sense of the invention itself or in the supplier made therefrom medium, in contrast to the known nutrient medium, no cell cultivation takes place, but the cell culture supply medium containing the supplement only through the semipermeable membrane is provided, it is not necessary that the supplement or the supply medium serum or high molecular serum substitute produced with it contains. These are only added to the culture medium in the production chamber. Like this with allows the supply medium produced with the addition of the supplement a cell culture vation in a serum-containing culture medium, without the serum or serum substitute itself to contain.

The addition of low molecular weight, hormonally active peptides to the supplement leads to a Stimulation of cell growth. The low molecular weight peptides pass through the semiper meable membrane from the supply medium to the production chamber. They are very simple and inexpensive to produce by genetic engineering or chemical processes. Surprised it has been shown that many peptides with a length in the range from 2 to 100 amino acids, preferably one with a length in the range of 3 to 15 amino acids, not as a pure nutrient serve substances for cell culture, but also hormonal and growth-promoting  Function. The tetrapeptide, for example, comes as hormonally active peptides "Tuftsin" or thymosin peptides in question.

The hormonal effects contained in the supplement or in the supplemented supply medium seed peptides are produced synthetically. These are precise with regard to their effects definable; their use poses no problems from a hygienic point of view.

Alternatively, the hormonally active peptides are produced by fractionating peptones.

The method has proven particularly useful when the supply medium is a supplement contains a peptone from a beef extract in a concentration in the range of 0.1 wt .-% to 0.5 wt .-%. It has shown, that such a supply medium with these protein cleavage products is very inexpensive is adjustable and has cell growth-promoting properties.

In a further preferred variant, the cleavage products comprise a peptone from wheat kernel men or soy flour in a concentration in the range of 0.1 wt .-% to 0.5 wt .-%. This Protein-split products are hygienically harmless.

An embodiment of the invention is shown in the patent drawing and will follow explained in more detail. The drawings show in detail:

Fig. 1 shows a growth curve for cells of a hybridoma with the addition of serum in the medium supply,

Fig. 2 is a growth curve for cells of a hybridoma without addition of serum in the medium supply,

Fig. 3 is a growth curve for cells of a hybridoma with the addition of a peptone of animal origin as the supply medium,

Fig. 4 antibody production curves in the cell culture approaches according to Figs. 1-3 and

Fig. 5 is a growth curve for cells of a hybridoma with the addition of a vegetal origin peptone in the medium supply.

Figures 1 to 3 and 5 contain a comparison of cell growth and Figure 4 a comparison of antibody production in a "Miniperm®" bioreactor. The "Miniperm®" bioreactor is described in DE-A1 42 29 325. The bioreactor consists of a production and a supply module, which are separated from each other by a dialysis membrane. The dialysis membrane is a semi-permeable membrane made from regenerated cellulose with a nominal molecular weight cut-off (MWCO value) of 12.5 kDalton. For the cultivation, the cell line HUA I 3 B5 was used, a hybridoma cell line, the cells of which produce a monoclonal antibody of the IgG class.

Different cell batches were cultivated in this bioreactor. The culture was each started by injecting 35 ml of a cell suspension into the production module of the bioreak tors containing 2 × 10⁶ cells / ml in a serum-containing cell culture medium (DMEM). By doing Cell culture medium each contained 5% by weight of horse serum. The supply module was de each filled with 400 ml supply medium, which differed in the individual batches had compositions. The bioreactors were in a CO₂ fumigation cabinet an atmosphere with 8% CO₂ on a scooter with a speed of rotation rolled from 10 rpm.

In batch 1 ( FIG. 1), 5% horse serum had also been added to the supply medium. In batch 2 ( FIG. 2), the supply medium contained neither serum nor any other supplement. In batch 3 ( FIG. 3), no serum had been added to the supply medium, but instead a peptone was added. The peptone is a product available from Humko Sheffield Products Norwich, NY, USA, under the trade name "Primatone RL".

In the diagrams according to FIGS. 1 to 3 and 5, the course of the cell density (in 10⁶ cells / ml) is plotted against the time (in hours h) during the observation period extending over 260 hours. Clear differences in the cell growth rate can be seen from the curves of curves 1 to 3 . While in the culture mixture without the use of serum in the supply medium (curve 2 ), the cells divide only very slowly and only very low cell densities are achieved, the cells multiply relatively quickly with 5% serum in the supply medium (curve 1 ) up to high cell densities around 20 · 10⁶ cells / ml. However, the highest cell growth rate and the highest cell density are achieved in the culture batch in which "Primatone RL" is contained in the supply medium (curve 3 ). Within approximately 2.5 days, the number of cells increases from 2 × 10⁶ cells / ml to 30 106 cells / ml and then remains at an approximately constant high level of approximately 25 · 10⁶ cells / ml for a long time.

These differences in the cell density achieved are also reflected in the antibody concentrations achieved, as can be seen from FIG. 4. Compared to the "normal" approach, in which 5% serum is added to the supply medium (curve 5 ), only about half the antibody concentration is achieved in a batch in which no serum is added to the supply medium (curve 4 ). In contrast, by adding "Primatone RL" to the supply medium, almost 50% higher antibody concentrations are achieved in the same time period (curve 6 ).

The cell cultivation method described is carried out by the compartmentalization of the culture area with a production module and a supply module. By replacing Serum in the supply medium due to the low-molecular protein cleavage products is gone allows a more cost-effective procedure in terms of serum consumption. To at the above-mentioned embodiments in the supply module the same serum concentration adjustment as in the production module would require about 13 times the amount of serum been. The small and handy "Miniperm®" bioreactor, similar to a roller bottle, and the The procedure described is therefore also for experimental purposes to optimize the Ver care medium very well suited. But the pure presentation of the cell products is also there relieved that the product concentration in relation to the concentration of serum protein Due to the high cell densities that can be achieved, it is much higher than with such culture processes in which the cultural area is not compartmentalized. This particular advantage will still be there reinforced when using serum substitutes in the production module.

In a further cell culture batch, a supply medium was prepared using 0.25% by weight of a pepton made from wheat germ extract. The cell line used for the cultivation, the cell culture medium and the cell cultivation method correspond exactly to that as has been explained with reference to FIGS . 1 to 4.

The growth curve measured during cell cultivation can be seen from FIG. 5. It should be noted that the protein cleavage product used is a peptone of plant origin that meets particularly high hygienic requirements. It shows that the cell growth rate, which is characterized by the slope of curve 7 , is greater than in the case of cell cultivation in serum-containing supply medium, but somewhat less than in "Primatone RL" -containing supply medium.

Furthermore, a supply medium using a ready-to-use Supple manufactured. The supplement, which is a concentrate of a mixture of amino acids, Vit amines, sugar, salts, 0.25% by weight of a pepton made from wheat germ extract Addition of the hormonally active tripeptide Tuftsin was made using a common nutrient solution (DMEM) added.

The supply medium thus produced was filled into the supply module of the bioreactor described above. The cell line HUA I 3 B5 was also used for cultivation. 5% by weight of horse serum was contained in the cell culture medium. Otherwise, cell cultivation corresponded to the method explained with reference to FIGS. 1 to 5.

It turns out that by means of the method according to the invention and when using the ge ready-to-use supplements containing a hormonally active peptide for the production of the supply medium surprisingly high cell densities and cell growth rates can be achieved are.

Claims (11)

1. A method of culturing eukaryotic cells in a culture medium containing serum or high molecular serum substitutes with the addition of cleavage products of proteins, characterized in that the cells are cultivated in a production chamber separated from a supply medium by a semipermeable membrane, the serum or the high molecular serum substitutes only the culture medium in the production chamber and the cleavage products to be placed in the supply medium, and that the pore size of the semipermeable membrane is selected so that the cells and the cell products, as well as serum proteins or high molecular substitutes are retained in the production chamber and that Protein cleavage products added to the supply medium can pass through. 2. The method according to claim 1, characterized in that fission products of plant-based ur jump can be used. 3. The method according to claim 1 or 2, characterized in that the culture medium in the production chamber instead of serum defined substitutes, the bovine serum Al comprise bumin or transferrin and / or a lipid-containing component will.   4. The method according to any one of the preceding claims, characterized in that low-molecular, hormonally active peptides added to the supply medium will. 5. The method according to any one of the preceding claims, characterized in that egg ne semipermeable membrane with a pore size corresponding to a molecular weight Final size in the range from 8 kDalton to 14 kDalton, in particular from 12.5 kDalton is used.   6. The method according to claim 1, characterized in that the supply medium Supplement that contains a ready-to-use mixture of amino acids, vitamins, Sugars, salts, protein breakdown products and hormonally active peptides with one Length is in the range of 2 to 100 amino acids, without the addition of serum. 7. The method according to claim 6, characterized in that the hormonally active Peptides have a length in the range of 3 to 15 amino acids. 8. The method according to claim 6, characterized in that the hormonally active Peptides are made synthetically. 9. The method according to claim 6, characterized in that the hormonally active Peptides are made by fractionating peptones. 10. The method according to any one of claims 6 to 9, characterized in that the Fission products a peptone from a beef extract in a concentration in the range from 0.1% to 0.5% by weight. 11. The method according to any one of claims 6 to 10, characterized in that the Fission products a peptone from wheat germ or soy flour in a concentration in the Range from 0.1% to 0.5% by weight.