FI96864B - Non-therapeutically useful HIV glycoprotein GP 160 and method for its preparation - Google Patents

Abstract

Proteins produced in cells collect together to form particulate aggregates. The invention relates to the reversible conversion of said aggregated proteins into morphological forms I, II and III which differ from one another, depending on the presence or absence of a non-denaturing detergent. The invention also relates to a process for the isolation and purification of said aggregated forms of proteins produced in cells.

Description

96864

Non-therapeutically useful HIV glycoprotein GP 160 and method for its preparation

The invention relates to the non-therapeutically produced HIV glycoprotein GP 160 produced in cells, to its non-therapeutic use and to a method for its isolation and purification.

All physiologically active cells produce the proteins they need to maintain their respective vital functions. The synthesized proteins have different functions. Some act as enzymes, i.e., they catalyze metabolic reactions in the cell. Their job is to tune the vital functions of the organism to which the cell belongs, and some form the structure of the cell.

However, proteins produced in these cells may have an antigenic effect on other organisms, i.e. they induce immune responses in these organisms. Viruses are also organisms that contain, for example, proteins in their envelopes that can act as antigens.

20 "Instructions" for protein synthesis are in the genes of cells that can be considered functional units of genetic material. The method for modifying the genome of a cell, for example a bacterium, by transferring a gene from, for example, an animal or human cell to another cell, which is often a bacterial cell, is well controlled. The bacterium then also produces a protein containing the corresponding information in the foreign gene. One of the best known examples is insulin produced by the human pancreas, which is genetically equivalent. the information has been transferred to Escherichia coli.

«« · '”· 30 Proteins produced both naturally and by genetic engineering of cells are either secreted into the culture medium or accumulate in the cells, and are then isolated by known methods after cell disruption and possibly dissolved. the methods generally use surfactants after certain fractionation and purification steps 2 96864, which must be quite specifically suitable for the proteins to be isolated and purified in each case.The problem of finding the right surfactants for isolating and purifying the desired proteins is still a problem.

It is therefore an object of the present invention to provide agents and methods for isolating and purifying proteins produced by physiologically active cells while maintaining or enhancing the biological activity of the proteins.

This object is achieved by providing proteins in the absence of surfactants in the first, approximately spherical, electro-optically visible, aggregated, electrophoresed stationary form I and in the presence of a first, non-denaturing surfactant in the second, lower , in the presence of an electronically detectable, aggregated, electrophoresically immobilized Form II and in the presence of a second, non-denaturing surfactant, in an electrophoresically movable Form III which is not electronically visible, said Forms I, II and III being reversibly interchangeable with each other. in the presence or absence of surfactants.

; ; ; 25 In the context of protein isolation procedures, it has surprisingly been shown that the use of non-denatured • ·. ·. ·. surfactants amphiphilic proteins are not in their monomeric form but in aggregated form. in forms with quite surprisingly advantageous properties. In the absence of a non-denaturing surfactant, the aggregated forms have a dense structure, referred to herein as the aggregated form I. When in this form, the particles are best suited to remove impurities from the surfaces of these particles. · '35 The dense shape of the particle then provides some protection against denaturation of the 3,96864 active centers, whether they are enzymatically active centers or epitopes that elicit immune responses. However, the dense particles in aggregated form I are also biologically active, albeit electrophoretically immobile.

In the presence of a non-denaturing surfactant, the dense, spherical Form I of the aggregated proteins can be converted to a more or less relaxed form, which has the advantage of removing impurities that become more readily available as Form I becomes Form II as isolation and purification procedures progress. In addition, the biological effects of the proteins are surprisingly enhanced in the form of aggregated proteins II. For example, it has been shown that the immunogenicity of isolated proteins is enhanced if the biological property of the isolated protein is the immunizing antigenicity of the protein. The biologically active proteins, or possibly also parts of these proteins which are biologically active, are thus particularly active in expanded form and are particularly suitable for purifying proteins from constituents which are enclosed in aggregated forms for the reasons mentioned here in an approximately "cloudy" form.

•:; 25 Proteins in aggregated form II are not • ·· · jV. also move in electrophoresis.

• · • ·. Particularly surprising in the case of the particulate aggregated proteins of the invention is that. that the aggregated forms I and II are reversibly interchangeable with each other depending on the absence and presence of the first non-denaturing surfactant; way. The particulate aggregated proteins described thus always provide a suitable complex with the enormous advantages of the reversible conversion of the various forms I and II of these aggregates to a facilitated purification procedure, improved shelf life and increased bioavailability of the isolated proteins. in terms of power.

When a second, non-denaturing surfactant is used, the proteins can be converted from aggregated form II to monomeric form III of the proteins, whereby this state is also, surprisingly, completely reversible when the second, non-denaturing protein is removed. Thus, in a manner dependent on the absence or presence of non-denaturing surfactants, proteins isolated from physiologically active cells can be intentionally and controllably reversibly converted to each desired and targeted form optimal for the treatment step or use.

Approximately spherical aggregated Form I is preferred for storage or controlled, potentially desired reduced biological activity of the isolated protein. The spherical shape also protects the potentially sensitive active centers during the purification steps from potentially denaturing treatments.

Aggregate Form II has an approximately cloudy structure depending on the duration of treatment and the concentration of non-denaturing enzyme. Structure opening; ; ; In a way that depends on 25 teas, • ·· * tV is used in the further cleaning steps. proteinaceous impurities which:. may be derived, for example, from host cells or vector systems used for protein production X m *.

. The biological activity of the proteins is enhanced.

»· R * · | · * 30 Both spherical, aggregated form I and directionally cloudy, aggregated form II can be made · * -? electronically visible if the individual protein produced has a molecular weight of at least 10,000 daltons. However, if, for example, only certain antigenic determinants, 5,96864, which are proteins containing only a few amino acids, are produced by genetic engineering, aggregated, multi-protein forms I and II may also be below the electron optical detection limit.

The purity of the electrophoretically immobilized proteins in Forms I and II is 80%, preferably 98%, i.e. the aggregated Forms I and II contain the desired proteins in a relatively homogeneous composition.

The aggregation of proteins within cells preferably takes place when the proteins are the expression products of genetically engineered cells, in particular when the products are produced in excess in these cells by known methods. Overproduction can be achieved either by the presence of several vectors containing the gene encoding the desired protein, or by attaching particularly strong promoters in front of the corresponding gene. If the proteins produced are proteins that are soluble in the cells, they aggregate into the described spherical particles.

Proteins produced by genetically engineered host cells preferably function antigenically. These proteins are preferred genetically engineered pro-; ; 25 teen products if the antigens are intended to .1 2 3. ·. corresponding active immunization is required. In this case, it is * · «• · ·. in particular envelope proteins of pathogenic viruses, in which case the viruses can be either human or animal. pathogenic.

The isolated proteins are preferably glycoproteins, and the invention therefore relates to non-therapeutic? The HIV glycoprotein GP 160 to be used is characterized by what is claimed in claim 1.

The gene corresponding to the protein can be expressed in a mammalian cell system, for example, through the vaccinia vector 9 96864. Mackett et al. [J. Virol. 49 (1984) 857-846] describe a method for inserting specific genes into vaccinia virus vectors. The foreign gene is first inserted into the plasmid vector 5 in a manner known per se after the vaccinia virus transcriptional regulatory element (7.5 K promoter). This chimeric gene is flanked in a recombinant plasmid by vaccinia virus sequences encoding the viral thymidine kinase (TK) gene. The plasmid is then introduced into cells pre-infected with wild-type vaccinia virus (strain WR). The fusion then occurs in a TK region that is homologous to both viral DNA and plasmid and allows insertion of a chimeric gene into the vaccinia virus genome. The recombinant virus thus obtained has a phenotype of 15 TK and is thus no longer able to produce thymidine kinase and grows in a selective medium containing 5-bromodeoxyuridine. In this way, recombinant vaccinia viruses can be prepared which in each case contain genes which cause the production of proteins, possibly in excess, which then accumulate in mammalian cells into protected, aggregated forms containing numerous individual proteins. Similar expression systems are known for other antigenic proteins. For example, •; ; 25 combinations of vaccinia virus encoding hepatitis B virus surface antigens. To achieve this, * «·, ·. sex produced hepatitis B virus surface antigens under the control of vaccinia virus regulatory mechanisms, where. a number of vaccinia virus-specific promoters were used

• t I

30 aiming to increase the expression level of a foreign gene.

• · · '· [* In addition, vaccine virus combinations have been prepared that contain more than one hepatitis B virus gene.

With regard to the preparation of recombinant expression systems for the genetic engineering of a desired protein, reference is generally made herein to Win- «« (4 7 96864 nacker, E. L., Gene und Klone, eine Einfiihrung in die Gen-technologie, VCH-Verlagsgesellschaft mbH, Weinheim 1984).

The protein produced in said cells may be in aggregated, multi-protein forms. In particular, in aggregated form II, enhanced activity is observed, which may be associated with the active centers being free in the opened aggregated form II and however, all active proteins are three-dimensionally related to each other, increasing the probability of encountering reactive proteins and active regions.

Proteins accumulated in particulate form can be advantageously bound to another substance, especially a high molecular weight protein, by conjugation to provide an improvement in protein stability, activity, in vivo recovery, and biological half-life.

The particulate aggregated proteins are preferably present in eukaryotic cells, especially animal or human cells.

The desired protein is then particularly preferably produced in primary cell cultures or in tax cells recovered from cell lines. In CHO cells or in primate; ; ; 25 in chicken embryo fibroblast cells. Said host cells are advantageous as expression systems because in them the post-translational transformations take place in a manner desirable for the end products used, such as. for example, glycosylation of expression product proteins. These • · r * ··> * 30 glycosylated proteins then accumulate into a single particulate form, which may be described as spherical forms I or cloudy forms II after treatment with the first non-denaturing surfactant and transformed into a second. , after treatment with undenatured 1,35 surfactant to the third aggressed form III.

96864 8

Tax cells are particularly preferred for the production of the HIV glycoprotein 160 described above. This cellular system was chosen because it has a relatively high growth rate. Infection of tax cells with recombinant vaccinia virus containing the gene encoding the desired protein to be isolated by the method of this invention results in normal synthesis of the desired protein, its Gly cosylation, and possible further processing steps. By using a dual infection system with two combinations, the amount of protein desired can be significantly increased. This system contains a combination that results in the production of T7 phage polymerase under the control of the vaccinia virus P7.5 promoter. This combination is used for infection in combination with a combination of the HIV T7 promoter and the glycoprotein 160 gene. The T7 promoter is a rather potent promoter and ensures a high level of expression, for example about 10 times the amount of the desired protein.

The systems described are to be considered as a preferred system. An overview of the many possibilities for using certain vectors to combine desired genes, to insert these vectors in desired cells, and to prepare proteins encoded by foreign genes is presented, for example, in Genetic Engineering 3, ed. Robert; . ; 25 Williamson, Academic Press 1982 and Spektrum der Wissen- V. schaft, Industrielle Mikrobiologie 1985.

I * *

A non-denaturing surfactant, the presence or absence of which substantially affects the presence of proteins isolated from physiologically active cells, in different aggregated forms or in monomeric form, is preferably a certain ionic, non-ionic or * * zwitterionic surfactant It is a known problem in protein chemistry or protein purification procedures that the choice of surfactant to recover the desired protein in a very pure form and always in a biologically active state is always a challenge. It has now surprisingly been found that the use of non-denaturing surfactants having a certain ionic, non-ionic or zwitterionic nature can fully meet the above-mentioned requirements for the protein purification method, so that the aggregated proteins present in these proteins in addition, unexpected configuration changes meet these requirements unexpectedly optimally. As mentioned above, especially in aggregated Form II, in an open or cloud-like configuration, the biological activity contained in a single protein, whether enzymatic or antigenic in nature, is clearly enhanced.

A preferred nonionic surfactant is octyl glucoside or a derivative of this surfactant, preferably in a concentration of 0.25 to 5%, especially 1%.

It is also preferred to use a bile acid salt as a surfactant as well as a derivative thereof, preferably deoxycholate surfactant (DOC), which is particularly suitable for isolating and purifying the spherical configuration of multiple HIV glycoproteins 160 if this protein is both purified: . . Using said surfactant in a concentration of 0.25 to 5%, preferably 1%, that deoxycholate is used in connection with the use of GP 160, for example in combination with aluminum hydroxide.

The non-denaturing surfactant, which is preferably used to convert aggregated proteins from Form II to Monomeric Form III, is a zwittergent * zwitterionic surfactant in the same concentration. 0.25 to 5%, preferably 1%. Zwitter- ®. .... .... The surfactants in the Ghent series are zwitterionic «·> 3 5 surfactants which have become known as 10 96864 sulfobetaines and have the following general structural formula: + CH 0

I ^ I

„CnH2n + 1-N- (CH2> 2-S- ° '5 i, ch2 o

Proteins in aggregated Form I, as mentioned above, are particularly well suited for maintaining proteins 10 stable. However, they can also be used for the desired biochemical modifications or reactions, since the proteins accumulated in aggregated Form I are also biologically active.

Proteins in aggregated Form II are preferably used to enhance the biological activity of the proteins.

Each aggregated form of the protein is suitable for diagnostic use both in the spherical, cloudy or monomeric forms described and in the forms coupled with other high molecular weight proteins described above.

An additional use of aggregated proteins is substitution treatment, recovery of monoclonal antibodies, and recovery of such agents; . . 25 to qualitatively detect and quantitatively determine body fluids.

«·« ·, ·, In addition to particles consisting of a number of proteins, t · ί which, depending on the presence or absence of a non-denaturing surfactant, may have different shapes in different configurations which may be reversibly converted to V, The present invention also relates to a method for isolating and purifying desired proteins produced in cells, preferably gene-engineered cells, wherein in a first purification step, the surface proteins in the first aggregated form are removed. impurities, the proteins are then converted to a second aggregated Form II using a non-denaturing surfactant and the impurities achievable in this aggregated Form II are removed in a second purification step, and the proteins are converted to monomeric Form III by the addition of a second non-denaturing surface. and reversibly back to aggregated form I or II by removing non-denaturing surfactants. The method according to the invention is characterized by what is stated in claim 13.

The enormous advantages of this method have already been discussed in substance above, and are based on the fact that the various aggregated forms of the proteins are re-version interchangeable.

The substantially spherical form I makes it possible, thanks to the tight arrangement of the proteins, for easier removal of the components of the medium from the surface of the spherical protein accumulation. After the second purification step has been carried out, in which the impurities which are particularly easily achievable due to the opening in the aggregated form II are placed, the aggregated form I can be reversibly recovered by removing the non-denaturing reagent in which the form is present. . For example, the active centers of proteins are particularly ··· · jV. well protected within the spherical protein formation », ·. so that the proteins can be stored particularly well in aggregated form I. The aggregated form II has, as already mentioned, in addition to the advantages associated with easier purification of the product from the host cell, V: in which the protein is produced. , components or vectors containing a gene encoding a secreted protein have the added advantage that the biological activity of the proteins can be enhanced substantially with a plurality of active centers in three dimensions due to the aggregated state of the proteins.

12 96864

Depending on the experimental design, the cells or cell membranes are removed by centrifugation and extracted with a buffer solution containing a non-denaturing surfactant and a protease inhibitor system or, if the desired macromolecular agent is transferred from the cells to the supernatant, to this supernatant is added to the supernatant. a protease inhibitor system wherein the non-denaturing surfactant is, for example, deoxycholate (DOC) and is used at a concentration of about 0.25 to 5%, but preferably 1%.

When the substance to be isolated and purified is glycoprotein, the solutions thus obtained are concentrated with lectins immobilized on a solid matrix and the carbohydrate-free impurities are separated. The non-denaturing surfactant used at this stage, in addition to its property of converting the substance to Form II, also has the ability to inhibit non-specific adsorption. Elution is performed with glycosides.

Another preferred purification step can be accomplished by binding the prepurified, high molecular weight substance specifically to an immune adsorption column. After sufficient washing with buffer, elution is performed with a chaotropic agent, for example urea or KSCN (2 to 8, preferably 3, mol / l). Subsequent dialysis against buffer · .. 25 returns to particulate form I. If necessary ··· · • ·. the high molecular weight substance is converted to monomeric form III with a certain zwitterionic surfactant and t · · * * is purified by ion exchange chromatography. By dialysis against a surfactant-free buffer, the aggregated Forms II and I are obtained from monomeric Form III.

♦ * * The various aggregated forms of the proteins produced by the described cells can be made visible by electron microscopy.

The invention is illustrated in the following detailed figures and examples.

13 96864

Figure 1 is an electron micrograph of glycoprotein 160 in Tris buffer, magnification 278,640-fold. The proteins are in a dense, spherical aggregated form I.

Figure 2 is an electron micrograph of HIV glycoprotein in 160 buffer containing 1% DOC. The proteins are in an open, cloudy aggregated form II.

Example 1 Isolation and Purification of HIV-glycoprotein 160 Isolation and purification of HIV-glycoprotein 160 is performed according to the following program: 1. Genetically engineered tax cells producing XIV-glycoprotein 160 are centrifuged at the desired density.

2. The pellet obtained by centrifugation is resuspended in TBS buffer (pH 7.4) containing 1 mmol / l CuSO 4 and 0.5 mmol / l ZnCl 2.

3. The resuspended pellet is extracted with 50 mM

With Tris-HCl buffer (pH 8.3) containing 1% DOC, 1 mmol / l CuSO 4 and 0.5 mM ZnCl 2. The suspension is clarified by centrifugation at 25 ° C for 30 minutes.

4. The supernatant is subjected to lens lectin chromatography in which the desired proteins are adsorbed onto the column and washed with DOC buffer. Proteins are eluted with 5% methyl • · ·. "with glycoside wash buffer.

• · · *; *. * 5. The eluate from the lectin column is diluted with • · · *; * · * Tween buffer. Absorption is then performed on an immuno-affinity chromatography column, which is washed with TBS-Tween buffer and then with TBS. Treatment with enzymes that cleave DNA and RNA. Elute with 3 M KSCN solution and dialyze against surfactant-free buffer.

6. Add 1 * to the dialyzed eluate; 35% Zwittergent and 5% betaine and adsorbed on a mono-Q matrix acting as an anion exchanger, eluted with a KSCN gradient and dialyzed against buffer.

7. Perform a second lens lectin chromatography on ab-5 by sorption and washing with surfactant-free buffer. Elution is performed with 5% methyl liglycoside solution, followed by dialysis against TBS. This step of the procedure results in the concentration of the protein.

10 Example 2

The HIV glycoprotein (GP) 160 purified by the procedure of Example 1 was subjected to an efficacy test showing the immunogenic potency of Gly-15 coprotein 160 in both dense, spherical form I and open, cloudy form II.

For each immunization, 5 dilutions were made containing 10 μg, 2.5 μg, 0.625 μg, 0.158 μg, and 0.04 μg GP 160 / ml. 10 BALBc mice were immunized subcutaneously with each dilution; thus, 50 animals were required for each immunogen species. In this experiment, four different adsorption methods were tested.

1. GP 160 + 0.2% Al (OH) 3 2. [GP 160 + 0.25% DOC] + 0.2% Al (OH) 3 3. [GP 160 + 0.25% DOC] + [ 0.2% Al (OH) 3 + 0.25% DOC] 4. [GP 160 + 0.25% DOC] + [0.2% Al (OH) 3 + washed)] «· · *; 1.1 All animals were immunized subcutaneously with 1 ml of • · · II solution. The serum of each individual animal was examined

anti-HIV ELISA (SORIN) for the presence of GP 160 antibodies and calculated the GP

The EDS0 value of # 160 in reacting animals by the known Spearman-: 1 Kaerber method.

<4 (11 · is 96864

Results 1. I - V 10 pg GP 160 / ml + 0.2% Al (OH) 3

Reacted Concentration in animals maximum I concentrated 1 100 5 II 1: 4 1 10 III 1:16 0 / IV 1:64 1 10 V 1: 256 1 10 ED ^ q: 10 pg / ml GP 160 10 2. VI - X 10 pg (GP 160 / ml + DOC + 0.2% Al (OH) 3

Reacted Concentration in animals up to VI concentrated 7 1000 15 VII 1: 4 5 200 VIII 1:16 1 10 IX 1:64 0 / X 1: 256 0 / ED5Q: 3.3 pg / ml GP 160 20 3. XI - XV 10 pg GP 160 / ml + DOC + DOC-Al (OH) 3

Reacted Concentration. : animals up to XI concentrated 7,800! 25 XII 1: 4 5 1000 • · «:; V XIII 1:16 6 100: · ** ·: XIV 1:64 2 100 • · * V .: XV 1: 256 1 100 EDj-q: 1.09 pg / ml GP 160 • · · • · · • «· 1 · · 16 96864 4. XVI - XX 10 jig GP 160 / ml + DOC + Al (OH) 3

Reacted Concentration in animals up to XVI concentrated 0/5 XVII 1: 4 1 10 XVIII 1:16 0 / XIX 1:64 0 / XX 1: 256 0 / EDc-q: 10 μg / ml GP 160 10 Results of efficacy test for HIV glycoprotein 160 show a variation in the immunogenicity of glycoprotein 160 according to the aggregated form. The ED50 value, which indicates the amount of substance used to which 50% of the immunized animals respond, is significantly reduced if the surfactant DOC is present. In the presence of surfactant DOC, glycoprotein 160 is present in aggregated form II, in which the antigenic epitopes are free on the one hand and three-dimensionally together on the other hand, so as to result in an enhanced immune response.

• · • «· • · • · • · · · · · · · · · · · · · · · · · · • • • • • • • • • • · · · · · ·

Claims (18)

96864 A non-therapeutically useful HIV glycoprotein GP 160, characterized in that it is in an opened, electronically visible, aggregated, electrophoretically immobile form (Form II) and is obtainable by treating what is produced in cells. approximately spherically aggregated, dense, electronically visible, electrophoretically immobile GP 160 (Form I) with a non-denaturing surfactant. GP 160 according to Claim 1, characterized in that it has a purity of at least 80%, preferably at least 98%. GP 160 according to Claim 1 or 2, characterized in that it is the expression product of genetically modified cells, in particular overexpressing cells. GP 160 according to Claim 3, characterized in that it has an antigenic effect. GP 160 according to one of the preceding claims, characterized in that the cells are eukaryotic, preferably animal or human cells. . ·. GP 160 according to claim 5, characterized in that. 25 n e t t that the cells are harvested from the primary cell • · ·. cultures or cell lines and are in particular Vero- • · · *. ’V cells, CHO cells or primary chicken embryo fibroblasts • · ·’ · 1; 1 cells. • · * · "· 1 GP 160 according to one of the preceding claims, characterized in that the surfactants are certain ionic, nonionic or zwitterionic surfactants. GP 160 according to Claim 7, characterized in that octyl glucoside or a derivative thereof is used as the first nonionic surfactant for the conversion of Form I into Form II, preferably in a concentration of 0.25 to 5%, in particular 1%, and as a second, zwitterionic surfactant, a Zwitter-® .... Gent series substance is used, preferably in a concentration of 0.25 to 5%, in particular 1%, for the conversion of Form II to the monomeric form (Form III). GP 160 according to Claim 7, characterized in that the surfactant is a salt of bile acid or a derivative thereof, preferably deoxycholate 10 (DOC), preferably in a concentration of 0.25 to 5%, in particular 1%. Non-therapeutic use of the HIV glycoprotein GP 160 according to any one of the preceding claims in its aggregated form II to enhance the biological effect. 11. Diagnostic use of HIV glycoprotein GP 160 Form II. 12. Use of HIV glycoprotein GP 160 Form II for the recovery of anti-HIV antibodies for the qualitative detection or quantification of said antibodies in body fluids. 13. A method of producing non-therapeutic cells: ·. isolation of GP 160 protein for human use, ·. : and for cleaning, characterized in that • · I 25 in the first cleaning step in the first aggregation • ·. “GP 160 in Form I is used to remove surface impurities in a manner known per se, then • • · * · '· * GP 160 is then modified using a non-denaturing surfactant. which is octyl glucoside or a derivative thereof, preferably in a concentration of 0.25 to 5% or a salt of bile acid or a derivative thereof, preferably in a concentration of 0.25 to 5% to the second aggregated form II and the impurities obtainable in the second purification step in this aggregated form II are removed per se. -35, and optionally the GP 160 is converted to monomeric Form 96864 by the addition of a second, non-denaturing surfactant of the Zwittergent series, preferably at a concentration of 0.25 to 5%, which form can be reversibly regenerated. convert to aggregated Form I or II by removing said non-denaturing surfactants, respectively. Method according to Claim 13, characterized in that the GP 160 is an expression product of genetically modified cells, in particular cells producing an excess of 10 proteins. Method according to Claim 13 or 14, characterized in that the cells are eukaryotic, preferably animal or human cells. The method according to claim 15, characterized in that the cells are recovered from primary cell cultures or cell lines and are in particular Vero cells, CHO cells or primary chicken embryo fibroblast cells. Process according to Claim 13, characterized in that octyl glucoside or a derivative thereof is used as the first nonionic surfactant, preferably in a concentration of 0.25 to 5%, in particular 1%, and Zwittergent is used as the second zwitterionic surfactant. series of substances, preferably in a concentration of 0.25 to 5%, in particular 1%. • ♦: · 1 Process according to Claim 13, characterized in that the surfactant is a salt of a bile acid or a derivative thereof, preferably deoxycholate (DOC), preferably in a concentration of 0.25 to 5%, in particular - 30% 1%. • · · • · · ··· 1 · 96864