Mitogens of Leukocytes and Inflamed Tissues

The destruction of tissue in inflammation caused by nonimmunological and immunological processes induces the formation of different endogenic substances (mediators and hormones). They regulate the complex steps of activation of the inflammation and tissue regeneration processes. The mediators are formed either by limited and regulated proteolysis of plasma and serum protein factors as humoral mediators; or they are liberated by active secretion and/or cell lysis from cells and tissues as cellular mediators. The mediators and hormones are especially important as specific carriers of chemical information which are formed and secreted by leukocytes in the course of cell proliferation processes (mitosis processes). They are components of the body's defence system whose systemic and local activation they regulate. The mediators contribute to the removal and detoxification of the body's own components and/or foreign components. In addition, by regulation of cell proliferation and tissue growth processes in wound-healing, they contribute to the restoration of physiological functions of the organism. As the classical hormones of endocrine glands, inflammatory mediators are trace components of tissues or blood and are present in very minute concentrations only. Experimental evidence shows that only up to 5,000 of such mediator protein molecules can be maintained in a steady state equilibrium by a cell in the mitotic cycle in its surrounding medium.

A reaction resulting in the division of a cell accompanied by doubling of the chromosome set is called "mitosis". Leukocytes comprise the mature, fully differentiated circulating type of granulocytes (segmented neutrophilic, eosinophilic and basophilic phagocytes) and the mononuclear leukocytes (monocytic phagocytes and lymphocytes) with their different sub-species (T, B-cells, etc.). Of these circulating cells, only the mature mononuclear leukocytes are capable of further division and differentiation. To the precursors of these mature leukocyte types for the development and maturation line (hematopoiesis) of the segmented granulocytes (Leukopoiesis) belong in particular the granulocytic bands. Their precursor is the mature and juvenile metamyelocyte which develops from the sequence of myelocyte, promyelocyte, myeloblast and bone marrow stem cell.

The body's own endogenous chemical substances which control and regulate the processes of this cell defferentiation and maturation line are called leukopoietins; see H. E. Whipple and M. I. Spitzer (eds.), "Leukopoiesis in Health and Disease, Ann. N.Y. Acad. Sci., 113 (1964), p. 511 to 1092. The leukopoietins include, amongst others, substances which induce the division and differentiation of the leukocytes and of their precursors (mitogens).

There exist numerous publications on the mitosis of peripheral and tissue mononuclear leukocytes and of leukocyte precursors of the bone marrow which are capable of division; see J. Lobue and S. A. Gordon, "Humoral Control of Growth and Differentiation", vol. I, Academic Press, New York, 1973. There also exist reports on different types of biological activities, for instance in cell cultures, with mitogenic effects on leukocytes or their precursors. Such an activity is for instance the "colony-stimulating factor" described by T. R. Bradley and D. Metcalf, Aust. J. Exp. Biol. Med. Sci. 44 (1966), p. 287 as well as by numerous other authors. This term comprises all activities which can, for instance, be observed in serum or urine extracts. This factor is capable of stimulating the proliferation and differentiation of granulocytes and macrophage precursors in in vitro cultures.

A similar activity is the "T cell growth factor" described by D. Y. Mochizuki et al. J. Immunol. Meth. 39 (1980), p. 185 to 201. This activity is said to originate for instance from T-cells and macrophages and is capable of maintaining T-cell clones in long-lasting cultures in vitro.

The designation LAF (lymphocyte-activating factor) applies to another biological activity described; see I. Gery and R. E. Handschuhmacher, Zell. Immunol. 11 (1974), p. 162.

To date, it has not been possible to isolate and define any of the numerous mitosis-stimulating activities as specified substance. On the contrary, all papers only deal with the experimental proof of the mitosis-stimulating activity of chemically undefined solutions or mixtures. Therefore, there is no certain knowledge on the chemical nature of the active substances and their biological specifity.

The mitosis activity is measured as "mitosis-index" of a cell population. The ratio of the number of cells in mitotic cycle to the overall number is measured by chromosome analysis. Another test is based on the incorporation of radioactive thymidine; see J. Paul, "Cell and Tissue Cultures" 5th edition, 1975, Churchill Livingstone, London.

It is therefore a primary object of this invention to provide a new class of cellular mitogens from leukocytes.

It is another object of this invention to provide a new class of cellular mitogens from leukocytes in highly purified form.

It is another of this invention to provide a new class of cellular mitogens from leukocytes in physical quantities for practical use.

It is another object of this invention to provide a new class of mitogens from leukocytes, which represent biologically specific, active and naturally acting mediators stimulating cell division and differentiation of leukocytes and/or their precursors.

It is another object of this invention to provide a new class of mitogens from leukocytes, which are suitable for specifically influencing the defense state of mammalian (e.g. human) organisms.

It is still another object of this invention to provide a process for producing and obtaining a new class of mitogens from leukocytes in an economical, biotechnically useful and relatively simple manner.

It is still another object of this invention to provide a process for producing and obtaining a new class of mitogens from leukocytes in a highly purified, molecularly homogeneous form and in physical quantities for practical use.

It is still another object of this invention to provide a pharmaceutical composition for specifically influencing the defence state of the body of mammalians.

These and other objects and advantages of the present invention will be evident from the following description of invention.

The subject matter of the invention are mitogens of leukocytes and inflamed tissue, which are characterized by the following properties:

(a) biological activities in vivo and in vitro:

selective induction of division and differentiation (mitosis) of bone marrow and/or tissue leukocytes;

they are substantially free of other biological effects;

(b) physico-chemical properties:

electrophoretic migration in acryl amide matrices at a pH of 7.40 is anodic;

soluble in aqueous media including in 10% ethanol at a pH value of at least 4.0 to 10;

they absorb reversibly in structure and biological activity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography.

The mitogens of leukocytes evaluated for the first time and obtained in highly purified form in this invention are further characterized by the fact that they are substantially free of other biological effects. More particularly, the mitogens of the invention do not show:

mobilization of adulat and juvenile leukocytes (no leucocytosis or leftward shift reaction);

capillary permeability-enhancing activity in the skin test;

spasmogenic effects on smooth muscles;

spasmogenic effects on striated muscles;

endotoxin contents and endotoxin-like or similar activities;

chemical attraction effects (chemotaxis) of leukocytes in vitro;

positive or negative chemokinetic effects on leukocytes in vitro;

phagocytosis-stimulating effects on leukocytes in vitro;

apparent shock or other systemically detrimental effects of the immediate or protracted type on the

intact organism of mammals in vivo;

lysis effects in vitro on erythrocytes, thrombocytes and leukocytes in vitro;

phlogistic activity in situ;

chemotropic mitogen effect on blood vessel cells;

chalone activity on blood vessel cells in vitro.

The mitogens of the invention have typical protein properties and protein reactions (folin and biuret reactions). Their melting point is approximately 200.degree. C. (decomposition in an air and oxygen-free atmosphere).

The mitogens of the invention are cellular inflammatory protein mediators with topochemically and biologically specific activity. It is their biological task to stimulate the division and differentiation of some specific types of peripheral leukocytes and of their precursor elements in the bone marrow (blasts). These leukopoietins are no normal, independent blood or serum components. Like many other hormones and mediators, they are formed in vitro in leukocyte cultures or in vivo upon accumulation of leukocytes at the site of inflammation.

The absence of other biological effects shows that the mitogens of the invention differ in many of their biological and chemical properties from structural and functional properties of the bacterial endotoxins. The other molecular properties of the mitogens of the invention, in particular their low blood levels necessary for their activity show a similarity of these inflammatory mediators with hormones. The active threshold doses are about 10 to 50 pmol/l. A LD.sub.50 value cannot be measured, since no lethal effects have been observed even with doses 1,000 times the amount of the physiologically active threshold dose.

The mitogens of the invention can be divided into two classes: substances inducing the mitosis of specific types of peripheral leukocytes capable of mitosis are called--according to the type of target cells on which the act--"lymphomitogens" (acting on lymphocytes) or "histiomitogens" (acting on histiocytes=macrophages). Compounds which stimulate the mitosis of leukocyte precursors of the bone marrow are called "blastogens". This nomenclature is in line with that suggested by hormone nomenclature commissions: Firstly, new substances are termed in sequence by the cell type which forms them; secondly by the cell or tissue type on which they act, i.e. the target cell and thirdly, by the action itself.

The mitogens of the invention stem from monocytes, granulocytes or lymphocytes. This different origin is also taken into consideration in their designation. Consequently, the monocyto-blastogen (MBG) is a mitogen stemming from monocytes and stimulating the mitosis of leukocyte precursors of the bone marrow. Analogously, the granulocyto-blastogen (GBG) is a mitogen stemming from granulocytes and also stimulating the mitosis of leukocyte precursors of the bone marrow. Accordingly, the monocyto-histiomitogen is a mitogen stemming from monocytes and inducing the mitosis of histiocytes (=macrophages). Finally, the lymphocyto-lymphomitogen (LLM) is a mitogen from lymphocytes and induces the mitosis of lymphocytes.

Apart from and in addition to the above-mentioned properties which mitogens have in common, the MBG has the following specific properties:

(a) biological effects:

specific stimulation of division and differentiation of bone marrow leukocytes;

effective threshold dose in vitro <50 pmol/l

(b) physico-chemical properties:

molecular weight of the native protein (primary structure); approximately 25,000 dalton;

insoluble in an ammonium sulfate solution at 90% saturation (3.6 mol/l);

absorption spectrum (UV, visible and near IR-range) as given in FIG. 1;

extinction coefficients according to the following Table I:

The MBG is secreted by monocytes. Its secretion can be stimulated for instance by the action of other mitogens, such as polyvalent lectin mitogens, for instance from Canavalia ensiformis (Concanavalin A, CON), endotoxins or cellular immune reactions.

Apart from or in addition to the above-mentioned properties which mitogens have in common, the GBG has the following specific properties:

(a) biological activities in vivo:

specific stimulation of division and differentiation of bone marrow leucocytes;

effective threshold dose in vitro: <5 nmol/l

(b) physico-chemical properties:

molecular weight of the native protein (primary structure): approximately 85,000 daltons;

insoluble in an ammonium sulfate solution at 90% saturation (3.6 mol/l);

absorption spectrum (UV, visible and near IR-range) according to FIG. 2;

extinction coefficient according to the following Table II

The GBG is secreted by granulocytes. Its formation is stimulated by the removal of the cells from the physiological environment (blood circulation), for instance by migration into the tissue. Therefore, stimulation by the addition of mitogens is not necessary in this case. Apart from or in addition to the above-mentioned properties which mitogens have in common, the MHM has the following specific properties:

(a) biological activities:

specific stimulation of mitosis of peritoneal macrophages;

effective threshold dose in vitro <1 nmol/l

(b) physico-chemical properties:

molecular weight of the native protein (primary structure): approximately 13,000 daltons;

insoluble in a 90% saturated ammonium sulfate solution (3.6 mol/l);

absorption spectrum (UV, visible and near IR-range) as given in FIG. 3;

extinction coefficient according to the following table III:

As with the MBG, the MHM is secreted by monocytes. As in the case of the MBG, its formation can be stimulated by mitogenic action on monocytes, for instance by polyvalent lectins, endotoxins or cellular immune reactions.

Apart from or in addition to the above-mentioned properties which mitogens have in common, the LLM has the following specific properties:

(a) biological activities:

specific stimulation of mitosis of peripheral lymphocytes;

effective threshold dose in vitro: <0,5 nmol/l

(b) physico-chemical properties:

molecular weight of the native protein (primary structure): approximately 17,000 dalton;

insoluble in a 90% saturated ammonium sulfate solution (3.6 mol/l);

absorption spectrum (UV, visible and near IR-range) as given in FIG. 4;

extinction coefficient according to the following Table IV

The LLM is secreted by lymphocytes. As in the case of mitogens stemming from the monocytes of the invention, its formation can be stimulated by mitogenic action on lymphocytes, for instance by polyvalent lectins, endotoxins or cellular immune reactions.

In non-physiological concentrations of up to 10 .mu.mol/l., the mitogens of the invention have neither chemotactic nor chemokinetic, nor phagocytosis and mitosis-stimulating and chalone activities on neutrophil, eosionophil and mononuclear leukocytes of man, rabbit, pig, dog, guinea pig or rat. Furthermore, they have no spasmogenic activity on smooth muscles of the guinea pig ileum and no capillary permeability enhancing activity in the guinea pig skin test using Evans blue as intravenously applied dye marker. Finally, they have no apparent shock and other systemically detrimental activity of the immediate or protracted type in guinea pigs or rabbits, even when intravenously applied as one bolus up to a 1,000 fold dose of the biologically active (blood vessel sprout-inducing) threshold. Furthermore, mitogens have no other apparent systemic biological activity when intravenously applied in a single high dose of about 1 nmol/kg to guinea pigs or rabbits. Moreover, they have no pyrogenic activity in rabbits, as shown by the standardized method by measurement of rectal temperature according to Europ. Pharmacopoeia, vol. II (1975), p. 56 to 59.

FIGS. 1 to 4 show the UV absorption spectra of the mitogens MBG, GBG, MHM and LLM in water at 20.degree. C. and an extinction scale (0-100) E=0-2 at a light path d=1 cm.

FIG. 5 schematically shows a standard-pyrogen assay according to Europ. Pharmacopoeia, vol. II (1975): The rectal temperature of 3 rabbits having an average weight of 3 kg is measured prior (V,A), during (*) and 30-180 minutes after (P) intravenous application of 30 .mu.g of MHM (corresponding to 1.3 nmolMHM/kg animal) in 1 ml 0.9 (w/v) % physiological saline.

The 1975 edition of the European Pharmacopocia, the British (1973) and the American (USP) (1975) Standards allow the designation "pyrogen-free" to be applied to preparations for which the sum of the fluctuations of the rectal temperature in a total of three experimental rabbits does not exceed the value of 2.6.degree. C. and, in particular, is below 1.15.degree. C. The experimental results given fulfills these criteria. According to these definitions, the MHM-preparation is pyrogen-free and without febrile activity. This also applies to the other highly purified mitogen preparations. This extremely sensitive criterion for contamination of proteins with bacterial endotoxins and other ubiquitous pyrogens demonstrates the great efficacy of the process of the purification of the cellular mitogens of the invention. It is an obvious parameter for the biological specificity of the mitogens.

The mitogens prepared and obtained according to the invention are valuable, endogenous substances. They can be used for specifically influencing the defence-state of the body, for instance the immune system. They are suitable to specifically influence the mitosis and differentiation of leukocytes and their precursors and thus also of leukocyte functions, for instance in inflammation reactions, mycoses and heart infarctions. Moreover, mitogens can be used for preparing their antibodies.

The mitogens of the invention are applied alone or in admixture parentally, preferably intravenously or locally to mammalians, for instance man, in the form of usual pharmaceutical compositions in a daily dose of <50 fmol in concentrations of >50 pmol/l. Another subject matter of the invention is a process for the biotechnical preparation and isolation of mitogens from leukocytes and from inflamed tissue sites. It is charcterized in that either the leukocytes or the inflamed tissue are homogenized; or that leukocytes are cultured and the mitogens formed or liberated are isolated from the homogenates of from the supernatant culture solution.

In principle, it is possible to prepare mediators from leukocytes directly without cell cultures. However, such a procedure is not economical: The leukocytes are destroyed by the process; the yields in mediators are low, since their synthesis and secretion is not stimulated prior to isolation; the mediators can be contaminated by intracellular structural constituents of leukocytes. Therefore, in the process of the invention, it is preferred to isolate the mitogens from the supernatant solution of the leukocyte culture. In principle, the leukocytes can be cultured in any leukocyte-compatible medium.

For the culture of different cell types, such as bone marrow cells, heart muscle cells or leukocytes, different culture media are known. These media normally are aqueous solutions which contain numerous different compounds. Main constituents of these culture media are salts, sugars and metabolites, amino acids and derivatives, nucleosides, vitamins, vitaminoids, coenzymes, steroids and other additives, such as tensides, heavy metal salts and indicator dyes. Special examples of known culture media are named "HAM", "MEDIUM 199" and "NCTC", see H. J. Morton, In Vitro 6 (1970) p. 89 to 108. When culturing cells for more than one hour, as in the case of leukocytes, mostly serum (e.g. fetal calf serum or horse serum) is added to the culture medium. The serum constituents are said to be favourable for the maintenance of cellular functions. However, if the serum-containing culture solution is to be subjected to processes for isolating proteins (mediators) which are formed by culturing cells, the preparation of trace protein products is difficult for reasons of the multipilicity of compounds making up the complex mixture of serum added to the culture. In addition, under such conditions, upon addition of serum to a cell culture medium, it is difficult if not at all impossible to recognize the origin of the mediators: It is then an open question whether or not a distinct mediator is of humoral (serum) or cellular (leukocyte) origin and from which species this mediator stems. Thus, the mediator may be derived from the species whose cells have been cultured; or, alternatively, it may be derived from the species from which the added (mostly heterologous) serum stems.

Besides serum-containing culture media, serum-free, synthetic media are also known; see H. J. Morton, loc. cit; I. Hayashi and G. H. Sato, Nature 259 (1976) p. 132-134; N. N. Iscove and F. Melchers, J. Exp. Med. 147 (1978) p. 923-933.

However, these known media likewise have drawbacks for both the culture of cells and for the preparation of the mediators formed from the culture supernatant. The tensides, heavy metal salts and/or dyes contained therein may damage or irreversibly contaminate the trace mediator proteins.

On the other hand, such known serum-free media are devoid of essential constituents which are necessary for maintaining the structural and functional viability of leukocytes. Therefore, none of the culture media known so far can be suitably used for the culture of leukocytes and the biotechnical preparation of cellular trace components, such as mitogens.

For the culture of leukocytes, a new, fully synthetic chemically defined culture media is preferably used. It provides favourable conditions for cell culture and facilitates the preparation and isolation of the cellular mitogen proteins from the culture supernatant.

The fully synthetic, chemically defined cell culture medium preferably used in this invention contains the normal groups of compounds, such as salts, sugars, polyols, uronic acids, and derivatives, amino acids and derivatives, nucleosides and nucleoside bases, vitamins, vitaminoids, phytyl derivatives, coenzymes and steroids in aqueous solution. It is characterized in that it additionally contains one or a mixture of several compounds which so far have not been considered for use in cell culture media. These are especially valuable for expression of the life functions, for the proliferation of leukocytes and for promoting their capability to produce mediators. These substances include unsaturated fatty acids, flavanoids, ubiquinone, vitamin U, mevalolactone and L-carnosine.

In prolonged leukocyte culturing, the cell culture medium is preferably used without addition of serum. Instead, it contains at least one defined protein.

In further preferred embodiments of the invention, the synthetic, serum-free cell culture medium used in this invention may contain additional compounds, e.g. polyhydroxy compounds and sugars, amino acids, nucleosides, anionic compounds and/or vitamins which are not common in the known culture media. These compounds are useful in culturing leukocytes. The constituents in the culture medium used in this invention are equilibrated in their ratios so that their concentrations mainly correspond to the natural concentration ranges of the plasma; see Ciba-Geigy AG (editor) (1969) in Documenta Geigy, Wissenschaftliche Tabellen seventh edition, Geigy S. A. Basle.

Preferably, the cell culture medium is free of tensides, heavy metal salts and dye indicators which can damage the cells and may have a detrimental effect on the isolation of the desired cell products.

The exact composition and the properties of the new cell culture medium are described in the patent application Ser. No. 358,045 filed simultaneously on the basis of West German Patent Application No. P 31 10 559.9.

The cell culture medium with the composition given in Table V below is especially preferred in the process of the invention for culturing leukocytes.

The medium is prepared with water of ASTM-1-quality; see ASTM D-1193-70 Standard Specification for Reagent Water 1970; Annual Book of ASTM-Standards, Easton, Md., ASTM 1970. In addition, it is freed from possible endotoxin-contaminations by ultrafiltration on tenside-free membranes with an exclusion limit of 10,000 daltons. The resulting medium is sterilized by filtration on tenside-free membrances with a port size of 0.2 .mu.m.

Dependent on the type of desired product, either mixed populations of leukocytes or homogenous leukocyte types are cultured. The preparation and culture of leukocytes must be performed under sterile conditions. Culturing is performed for a period sufficiently long to obtain a satisfactory mediator level. A suitable period of time is 10 to 50 hours. Shorter periods result in lower mediator yields and the process is thus not economical. On the other hand, the medium is used up after a culture period of 50 hours and the cells begin to die. An increase of the yield can therefore not be obtained in this case, except in the case of subculturing of cells and renewal of the culture medium.

The leukocytes are cultured at a temperature of about 30.degree. to 42.degree. C., preferably at about 37.degree. C. At lower temperatures the culture process is not satisfactory, while at temperatures of above 42.degree. C. the leukocytes are damaged.

Culturing is carried out at a concentration of about 10.sup.6 to 5.times.10.sup.8 cells/ml, preferably 10.sup.7 to 10.sup.8 cells/ml. At lower cell concentrations the mediator yield per volume unit of the culture solution is too low. With too large culture volumes, the process is not economical. At cell concentrations of above 5.times.10.sup.8 cells/ml, nutrition of the cells in the medium becomes rapidly inefficient.

Culturing can be carried out in normal atmosphere. Preferably increased carbon dioxide partial pressure is maintained during culturing. This pressure can amount to about 10 vol%. 2 vol% are preferred. The oxygen supply to the culture is of great importance. Oxygen can be supplied e.g. by bubbling air through the culture. To avoid contamination of the culture, the air is preferably sterilized and heat-decontaminated, i.e. it is freed of endotoxins and other organic constituents. The cell suspension is stirred or agitated during culturing.

Certain types of the inventive mitogens are already obtained in satisfactory yields by normal culture of leukocytes or certain leukocyte types. The GBG, for instance, is obtained in high yields by culturing mixed populations of leukocytes or homogenous populations of granulocytes under the above-indicated conditions.

Other types of mitogens of the invention are, however, only formed in small amounts by normal culture of leukocytes or certain leukocyte types. This applies for instance to the mitogens of mononuclear cells. To produce them in higher yields, it is necessary to stimulate the cells in culture to mitosis. Possible mitosis-inducing influences are the addition of polyvalent mitogens, endotoxin-mitogens and immune reactions on the cell surface of sensitized cells. Examples of suitable mitogens are lectins, in particular those of Canavalia ensiformis (Concanavalin A=CON). The mitosis-inducing factor CON is added as a solution to the culture medium.

To terminate culturing, the leukocytes are centrifuged from the supernatant culture solution which is subsequently processed for the resulting mitogens. To avoid damaging the cells and thus contamination of the culture solution with cell particles, the culture is centrifuged at relatively low speed, i.e. at about 300 to 400.times.g. After removal of the major part of the cells from the supernatant, it is expedient to centrifuge the latter again at a higher speed. In this way, the remaining floating particles are removed. The separated leukocytes can either be cultured again, cryo-preserved or used for other biotechnical purposes.

The supernatant culture solution freed from the cells contains the secretion products of the cultured leukocytes. These include the mitogens of the invention and a number of other proteins and other substances. Their concentration in the culture solution is approximately within the nanomolar range. Consequently, a yield of about 1 to 10 mg of a defined mediator requires a culture solution volume of about 1,000 l with respect to a 10% recovery after purification. As regards the number of cells to be used, it can be calculated that in view of the molecular efficiency of the cells, about 10.sup.14 leukocytes are necessary for obtaining a quantity of about 100 nmol proteins. This corresponds to about 1 mg of a mediator with the molecular weight of 10,000 dalton. This means that for the isolation of mediators in physical amounts about 50 kg of leukocytes are necessary for the culture. For reasons of availability, leukocytes or man, cow, horse, pig, sheep, dog, cat, rabbit, rat, mouse or guinea pig are preferred. The process described in the German unexamined patent publication DE-OS No. 30 09 126 is especially suitable for the preparation of large amounts of leukocytes; see also J. H. Wissler et al., Hoppe-Seylers Z. F. Physiol. Chemie. 361 (1980), p. 351 to 352.

Apart from leukocyte cultures, the mitogens of the invention can also be obtained from inflamed tissue sites. There, they are formed by the accumulation of leukocytes in the course of inflammatory processes induced by tissue injuries. The inflamed tissue can be obtained in the usual manner and used for the preparation of the mitogens. Inflamed tissues are homogenized in buffer solution and soluble constituents or exudates are separated from insoluble structural components by means of centrifugation.

Preferably, inflamed, infarcted heart mucle tissue is used which was formed by ligation of 24 hours of the left anterior descendent branch of the left coronary artery by a transfemoral catheter technique. The leukocyte-containing inflamed heart muscle site is separated at 0.degree. to 4.degree. C. from the remaining non-infracted tissue.

As shown above, the preparation and isolation of the mitogens of the invention requires the processing of a very large culture solution volume. Therefore, at the beginning of the purification process effective reduction of the solution volume to be processed is necessary. In addition to the small amounts of the proteins produced, the culture solution contains the mixture of the components of the medium. Preferably, in the first step of the purification process a separation of the formed proteins from the medium components with a concomitant reduction of the large volume of aqueous solution is achieved. This can be effected by selective salting-out precipitation of the proteins from the supernatant culture solution, for instance by adding a sulfate or a phosphate. In the following, the salting-out precipitation of proteins is exemplified by adding ammonium sulfate to the culture solution.

By saturation of the supernatant culture solution with ammonium sulfate, a major portion of the proteins formed is precipitated together with serum albumin present as medium component. The proteins precipitated are recovered e.g. by centrifugation. They are then separated into the individual components of the mixture as described below. Thereby, some mitogens are obtained. On the other hand, some other mitogens are salt-soluble and remain in the supernatant solution of the salting-out precipitation process. This supernatant also contains all soluble components of the medium. It is concentrated and the proteins obtained are processed in the manner described below.

If the protein-containing supernatant culture solution is saturated with ammonium sulfate, a major portion of proteins is precipitated. In this way, a protein mixture is obtained consisting of numerous different proteins. Their separation into the individual protein components is obviously laborious. Therefore, in a preferred embodiment of the inventive process the protein mixture of the supernatant culture solution is already separated into several fractions by the salting-out precipitation step. The separation into several crude protein fractions is possible, since groups of individual proteins precipitate at different ammonium sulfate concentrations. Preferably, in the process of the invention, ammonium sulfate is therefore added stepwise to the culture solution up to a specific degree of saturation. Each fraction contains a group of proteins, the solubility product of which corresponds to the range of salt saturation. Hence, in the process according to the invention a crude separation into groups of proteins can be achieved in this first step by suitable choice of the saturation limits.

For instance, the supernatant culture solution is first brought to a 35% saturation with ammonium sulfate. The protein precipitate obtained is separated off. The 35% saturation of the supernatant solution is then increased to 45% by further addition of ammonium sulfate. A protein precipitate is again formed which is separated off. Thereafter, the 45% salt-saturated supernatant solution is brought to a 90% ammonium sulfate saturation. The protein precipitate formed is again separated off. The supernatant solution of this precipitate is concentrated e.g. by dehydration dialysis or ultrafiltration.

The salting-out precipitation of proteins is preferably carried out at a temperature of about 0.degree. to 10.degree. C., especially of about 0.degree. to 4.degree. C. The subsequent purification steps are performed under the same conditions. The solutions used for the purification have a pH value of between 5 and 9, in particular between 6 and 8. In order to achieve a constant pH-value of the solution, a strong buffer, for instance 0.1 mol/l of phosphate buffer is preferably added prior to the salting-out precipitation. To maintain the redox potential of the proteins, cysteine is preferably added in an amount of 0.001 mol/l to all solutions throughout the process. The protein purification does not require sterile conditions.

After dissolution in a protein-compatible medium, the proteins obtained by salting-out precipitation can be directly subjected to purification and separation in the manner described below. The 90% salt-saturated supernatant of the last precipitation step is concentrated. For instance, by dehydration dialysis or ultrafiltration, all compounds having molecular weight higher than about 300 to 500 daltons are obtained as a retentate fraction. They can also be further processed for purification of salt-soluble chemorecruitins.

The protein fractions obtained in the step described above contain the mitogens of the invention in admixture with numerous foreign proteins, e.g. other secreted proteins, in part serum albumins and in part CON. These foreign proteins form the major part of the constituents of this mixture. The mitogens must be further purified by a sequence of further purification steps. Foreign proteins must be removed to avoid interference with the molecular-biological specifity of mitogens. In addition, mitogens themselves form a class of protein compounds which must be separated into individual, specifically acting structures.

In general, purification processes for proteins and other natural substances comprise sequences of combined separation techniques. Subtle differences in molecular size, charge, form, structure stability and nature of the molecular surfaces between the desired natural substance and the accompanying inactive foreign materials are used in such purification steps for their separation. Accordingly, a large number of combinations of various modifications of preparation techniques can be devised for the purification of a protein. The nature and the conditions of the preparation steps used, but also their sequential combination, are of paramount significance for operational properties, technical practicability, possibility of optional automatization and for the economical performance of a purification process and also for the yield and molecular quality of a natural product investigated. Particular attention has to be given to the optimum form of separation steps and on their ingenious combination into a purification sequence within the framework of structural and functional stability and other molecular parameters of the substance under investigation. This implies that the use of identical or similar separation principles (molecular sieve filtration, dialysis, ion exchange adsorption, etc.)--however in a different combination--can be of specific and paramount importance for the practice and economical performance of the purification process as well as for the yield and quality of the product obtained. In some cases, the use or omission of a single technique (.e.g. hydroxyapaptite chromatography, zone precipitation chromatography, etc.) at a certain point in the purification sequence or within a partial sequence, is of decisive significance for the yield and quality of the desired natural product as well as for the practice and economical performance of the purification process. These general relationships and basic principles inherent to the purification processes of natural products are clearly illustrated e.g. by some well known facts. Thus, within an economically and technically operable process for the purification of a natural product, initial dialysis, ultrafiltration or lyophilization steps are not recommended prior to reduction of original volumes of crude starting extracts by a factor of at least 500 to 1000 through other techniques.

For the purification of the individual protein fractions, a plurality of purification steps so far known in biochemistry can be used. Examples of such purification steps are: Preparative and analytical molecular sieve chromatography, anion and cation exchange chromatography and batch adsorption techniques, chromatography on hydroxyapatite, zone precipitation chromatography and recycling or cascade molecular sieve filtration.

It is possible to remove a considerable amount of accompanying foreign proteins from mitogens by only one performance of these purification methods. However, proteins contained in the fractions tend to adhere together very strongly. Therefore, for example, in spite of different molecular weights of proteins, using molecular sieve filtration, no complete (ideal) separation of protein polyelectrolytes according to their exact molecular weight is obtained immediately. Hence it is necessary to perform at least two of the mentioned separation processes in sequence. A particularly preferred embodiment of the process in accordance with the invention uses three of the mentioned purification steps in sequence for the purification of mitogen activity from the protein fractions.

All combinations of the mentioned separation steps constitute objects of the invention. It is evident, that certain sequences of separation steps are of less advantage than other combinations. Thus, for example, it is imperative to perform a preparative molecular sieve filtration before an analytical molcular sieve filtration: In reverse order of performance, difficulties in handling, economic efficiency and yield are obvious.

Molecular sieve filtration achieves separation of proteins according to their molecular weights. Since the bulk of the foreign proteins have molecular weights different from those of mitogens they can be separated off in this manner. A hydrophilic water-swelling molecular sieve as matrix is used for separation of the proteins by molecular weight. Examples of suitable molecular sieve matrices are dextrans cross-linked with epichlorohydrin (Sephadex), agaroses cross-linked with acrylamides (Ultrogels), and three-dimensionally cross-linked acrylamides (Biogels). The exclusion limits of the matrices used are higher than the separation limits.

If several separation steps are used, the molecular sieve filtration is preferably carried out as one of the first separation steps. Depending on the length-to-diameter ratio of the column used and the particle diameter of the gel matrix, molecular sieve filtration is termed "preparative" or "analytical". A molecular sieve filtration is "preparative" when the chromatography is performed on columns with a length-to-diameter ratio of up to 10:1 and a charge of the column of up to 1/3 of its capacity in terms of the total separation volume of the matrix. "Analytical" molecular sieve filtration means a length-to-diameter ratio larger than 10:1, and preferably about 50:1, and a maximum charge of the column of up to 3% of its capacity.

In preparative molecular sieve chromatography, gel matrices with the largest possible particle size are used for maximum flow-through rates of mostly viscous protein solutions applied at reasonably low pressures. In analytical molecular sieve filtration the particle size ranges of the gel matrix are selected as small as possible, to obtain a maximum number of theoretical plates, a flow rate of the mobile phase in the range of 2 to 4 cm/h combined with a pressure which is limited to technical and safety aspects. These parameters are dependent on the structure of the gel matrix and may vary from gel to gel.

If several preparative molecular sieve filtrations are performed in sequence, graduated separation limits can be selected. This can be followed by an analytical molecular sieve filtration with correspondingly graduated separation limits. The exclusion limit of the gel used must in all cases be higher than about 10,000 daltons to allow a volume distribution of mitogens between the stationary gel matrix phase and the mobile aqueous buffer phase.

The "exclusion limit" is a hydrodynamic parameter of a dissolved particle, which corresponds to the pore size of the gel matrix. Particles with a greater hydrodynamic parameter cannot penetrate the gel matrix (volume distribution coefficient K.sub.D =0). The "separation limit" refers to a hydrodynamic parameter which has been chosen for the separation of dissolved particles from others and which has a value of between the volume distribution coefficient K.sub.D =0 and K.sub.D =1.

For molecular sieve filtration, the proteins are applied to the molecular sieve after dissolution in a protein-compatible liquid. A special example of a suitable solvent is 0.003 mol/l sodium-potassium phosphate solution containing 0.3 mol/l NaCl and 0.001 mol/l cysteine and having a pH of 7.4. After filtration, the mitogen-containing fractions are concentrated in the manner described below and optionally subjected to a further purification step.

Examples of suitable anion exchangers are dextran matrices cross-linked with epichlorohydrin (Sephadex) or cellulose matrices carrying functional groups with anion exchanger capacity. These exchangers can be regenerated for repeated further use. It is preferable to use a weak anion exchanger in the Cl.sup.- form such as DEAE-Sephadex A-50, pre-swollen and equilibrated in a buffer. Swelling and equilibration is preferably carried out at a pH of 8 to 10. A special example of such a buffer solution is 0.01 mol/l tris-HCl containing 0.04 mol/l NaCl and 0.001 mol/l cysteine and having a pH value of 8.0.

The anion exchanger is added to the protein fraction in an amount sufficient for complete adsorption of the mitogens and of the other positively adsorbing accompanying proteins. Two volume parts of swollen anion exchanger per volume of concentrated protein solution are normally sufficient. The reaction can be carried out either as chromatographic process or as an easy and fast batch adsorption technique. In the latter case, the supernatant liquid containing negatively adsorbed proteins is separated from the anion exchanger which is charged with the positively adsorbed mitogens or other proteins, e.g. by filtration in a chromatographic column, by decantation or centrifugation. The charged anion exchanger is freed from adhering negatively adsorbing compounds by washing with water or a salt solution having a maximum ionic strength equivalent to 0.04 mol/l NaCl, preferably at a pH of 8 to 10.

The maximum preferred temperature is about 15.degree. C. A special example of salt solution suitable for the washing-out process is the said HCl buffer of pH 8.0.

The anion exchanger on which mitogens and other proteins are adsorbed and which is freed from the negatively adsorbed compounds is eluted with a protein-compatible aqueous salt solution having an ionic strength higher than 0.04 mol/l NaCl and a pH of between 4.0 and 10.0. A salt solution of high ionic strength and a pH of between 5.0 and 7.0 is preferably used. A special example of such a salt solution is a 2.0 mol/l NaCl solution buffered to a pH of 6.5 with 0.01 mol/l piperazine-HCl and containing 0.001 mol/l cysteine.

If the anion exchange reaction is carried out as a chromatographic process, elution of the mitogens and other positively adsorbed proteins can also be done by a linear NaCl concentration gradient.

Examples of cation exchange matrices suitable for the purification of the protein fraction are dextrans crosslinked with epichlorohydrin (Sephadex) or cellulose matrices carrying functional groups with cation exchange capacity. These can be readily regenerated after use and employed again. It is preferable to use a weakly acidic cation exchanger such as CM-Sephadex C-50 having Na.sup.+ as mobile counter-ion, and to perform the exchange reaction at a pH between 4 and 6. To facilitate the charge process and to approach more ideal equilibria conditions prior to treatment with the cation exchanger the protein fractions should be diluted with a protein-compatible salt solution having a maximum ionic strength equivalent to 0.04 mol/l NaCl. This salt solution can be used at the same time to adjust the pH. A special example of a salt solution for this purpose is a 0.001 mol/l potassium phosphate-acetate buffer containing 0.04 mol/l NaCl and 0.001 mol/l cysteine and having a pH of 4 to 6. This cation-exchange reaction may be performed as a chromatographic process, or technically easier, as a batch process.

The swollen cation exchanger is added to the protein fraction in a quantity sufficient to adsorb it. As a rule, about 2 volume parts of swollen ion exchanger per volume part of protein solution is sufficient for this purpose. The supernatant is then separated from the cation exchanger charged with proteins, for example by decantation or centrifugation. The charged cation exchanger is free from adhereing, negative adsorbed compounds by washing with water or a salt solution, having a maximum ionic strength equivalent to 0.04 mol/l NaCl. Preferably a pH of about 4 to 6 and a maximum temperature of about 15.degree. C. is used. A special example of a salt solution suitable for the washing out process is the mentioned potassium phosphate-acetate buffer having a pH of 5.0.

The washed protein-charged cation exchanger is now eluted with a protein-compatible aqueous salt solution. A salt solution of high ionic strength with a pH of about 4 to 10 is preferably used for this purpose. Special examples of such salt solutions are aqueous 0.5 mol/l potassium phosphate with a pH of 6.5 to 7.5 or a 2 to 5 mol/l NaCl with the same pH.

For chromatography on hydroxyapatite, salts, e.g. ammonium sulfate and especially phosphates, possibly present from preceding steps are removed from the protein solution, preferably by dialysis or ultrafiltration at membranes with an exclusion limit of 500 daltons prior to the application of the proteins to hydroxyapatite. Apart from viscosity increase by accompanying salts, however, only the phosphate concentration of the protein solution is critical for the chromatography on hydroxyapatite. The mitogens are eluted by a potassium phosphate concentration gradient which is preferably linear. The mitogens containing fractions are collected and then concentrated in the manner described below.

The use of hydroxyapatite is of essential significance for the structure-conserving isolation of pure mitogens. However, in general, for technical and economic reasons, considerable difficulties arise from chromatography of larger volumes of protein solutions on hydroxyapatite columns. On the one hand, larger protein amounts contribute to the strong tendency of hydroxyapatite to clog, thus becoming unusable as stationary matrix in chromatography. On the other hand, hydroxyapatite is very expensive. Its use on larger scales is not economical. For these reasons, in the process of the invention, the separation of a large part of the accompanying foreign proteins by appropriate biotechnical purification steps from the mitogen-containing protein fractions is preferred for considerably reducing the volume of the protein solution prior to its chromatography on hydroxyapatite.

In the zone precipitation chromatography (cf. J. Porath, Nature, vol. 196 (1962); p. 47-48), residual protein contaminations in the mitogens are separated by salting-out fractionation of the proteins by means and along a salt concentration gradient. The basic principle of separation of proteins in zone precipitation chromatography are different, structure-related, reversible solubility characteristics of proteins. They belong to the most sensitve molecular separation criteria and are often used for demonstration of molecular homogeneity of a protein. Two variants of this technique for development of the chromatogram are known: Fractional precipitation zone chromatography and fractional elution zone chromatography. Both types of techniques may have selective advantages in specific cases as described for fractional precipitation and fractional elution methods in protein separation. Temperature and pH, column characteristics can all be varied within relatively wide limits.

The temperature for zone precipitation chromatography can be between 0.degree. and 40.degree. C. Preferably, a temperature range from about 0.degree. to 10.degree. C. is used, especially from about 4.degree. to 6.degree. C. The pH can be between 4 and 10; preferably, a pH range of 6 to 8 is used, especially a pH of about 7. The length-to-diameter ratio of the column used should be greater than about 10:1. A ratio of 30 to 100:1 and especially of about 50:1 is preferred. All protein-compatible salts having salting-out properties for proteins are suitable. Examples of such salts are sodium-potassium phosphate, ammonium sulfate, and sodium sulfate. Ammonium sulfate is preferred.

The salt concentration gradient can have any desired shape provided that salting-out criteria of proteins achieve protein separation. Linear concentration gradients are preferred, especailly an ascendent linear concentration gradient from 25 to 100% ammonium sulfate saturation. The maximum column charge is about 5% and preferably about 1% of total column volume.

The recycling or cascade molecular sieve filtration can be performed under the conditions described above for the analytical molecular sieve filtration. The same molecular sieves and the same column conditions can be used. Sephadex G 50 as stationary matrix is preferred in a column of a length-to-diameter ratio of at least about 50:1 and a maximum charge of about 3% of the column volume. The solvents used in the analytical molecular sieve filtration are also preferred as solvents for the elution in this method.

In recycling molecular sieve filtration, the distribution equilibria are disturbed continuously and the eluate is recycled onto the same column with fixed separation limits. In this way, the separation length of the migrating protein distribution bands are differentially extended. Alternatively, in cascade molecular sieve filtration, distribution equilibria are disturbed by continuous transfer of the eluate into a new second column with the same or similar, defined parameters at fixed separation limits.

Between the above-described purification steps, and if necessary at any stage for special purposes, protein solutions can be separated and freed from unwanted salts and water as well as concomitantly concentrated. The concentration (separation of a major portion of aqueous salt solution of the protein) can be achieved in different ways. Dehydration dialysis or ultrafiltration against protein-compatible liquid, preferably a sodium potassium phosphate buffer, are such methods. Dehydration dialysis is carried out preferably against polyethylene glycol (molecular weight 20,000 daltons) at membranes with exclusion limits of preferably 500 daltons. Ultrafiltration is preferably achieved at membranes with an exclusion limit of about 500 daltons. Small amounts of protein precipitates formed are removed by intermediary centrifugation to result in a clear protein solution. A desalting molecular sieve filtration on matrices with appropriate separation and exclusion limits can as well be used for this purpose, e.g. on Sephadex G 10, G 15 or G 20 as matrices. Furthermore, by selecting an appropriate mobile phase in the usual way, a usual molecular sieve filtration step can also be used concomitantly for this purpose.

To prevent sulfhydryl group oxidation, about 0.001 mol/l of cysteine is preferably added to protein solutions throughout.

In the molecular sieve filtration purification steps about 0.4 mol/l ammonium sulfate is preferably added to the protein solution. In contrast to higher concentrations of this salt, at this concentration ammonium sulfate exerts a strong salting-in effect on proteins. Thus, proteins are better kept in solution during the molecular sieve filtration. Moreover, ammonium sulfate prevents growth of microorganisms and inhibits certain enzymes. Hence, it contributes to stabilization of the mitogen structure which is important when chromatography is performed at higher temperature (above about 20.degree. C.) and under nonsterile conditions.

Mitogens which can be salted out are preferably completely precipitated alone or together with accompanying proteins by adding ammonium sulfate up to a concentration of about 3.25 to 3.7 mol/l (80 to 90% saturation). For this purpose 630 g/l ammonium sulfate are added (about 90% saturation). The pH value is preferably kept between 4 and 9 and the temperature up to 40.degree. C., preferably between 0.degree. and 8.degree. C. The mitogen-containing protein precipitate is separated from the protein-free supernatant solution by filtration, decantation or centrifugation. Unless otherwise staed, centrifugation is preferably carried out at least at 10,000.times.g for a minimum of 45 min, and preferably for 1 h, in a one-step process. Or it can be carried out in two stages, at lower forces in the first stage for removal of the bulk of precipitated proteins; and then, for the supernatant of the first stage containing residual fine protein particles at higher forces, e.g. 20,000 to 50,000.times.g, by flow-through centrifugation.

The temperature and pH conditions during performance of the purification steps are not particularly critical. If the native conformation of the protein is to be preserved, an optimum temperature range is about 0.degree. to 8.degree. C., and preferably about 0.degree. to 4.degree. C. Moreover, the separation and purification steps must be carried out under essentially physiological pH and salt conditions. An essential advantage of the process of the invention consists in that these conditions are for the first time easy to adhere to.

The mitogens obtained can be stored in a buffered physiological saline, e.g. in 0.0015 mol/l sodium-potassium phosphate solution containing 0.15 mol/l (0.9 w/v%) NaCl, 0.001 mol/l cysteine and having a pH of 7.4. After usual sterilization by filtration (pore diameter 0.2 .mu.m), the protein preparation remains native and biologically active at room temperature for at least 200 h or frozen at -25.degree. C. for at least 5 years. This stability of the protein can be considered, among others, to be one of the criteria of molecular homogeneity. Mitogen solutions are safely stored at temperatures of between -20.degree. and +50.degree. C. in the presence of 2.0 to 3.6 mol/l ammonium sulfate (50 to 90% saturation). At this high osmotic pressure mitogen solutions are protected against infection and degradation by microorganisms and bacterial growth. For their physiological, therapeutical and any other use, the mitogens are again freed from salts by dialysis or ultrafiltration against an appropriate saline as described above.

The invention will now be given in detail by examples describing the isolation of the mitogen protein preparation starting from leukocytes of porcine blood. However, the invention is not restricted to this embodiment. Leukocytes and inflamed tissues of other mammalians can be used too.

The production of mitogens in a culture solution of a mixed population of leukocytes and the separation of monocytoblastogen (MBG), granulocyto-blastogen (GBG), monocytohistiomitogen (MHM) and lymphocyto-lymphomitogen (LLM) from the other components of the culture supernatant are described. All process steps are carried out at 0.degree. to 8.degree. C. in the presence of 0.001 mol/l cysteine, unless otherwise specified. The centrifugation is carried out in the manner described, either as a one or two step procedure (as flowthrough centrifugation).

50 kg (about 10.sup.14) leukocytes are isolated as mixed cell population of physiological composition from 10,000 l of porcine blood and cultured in 20 batches of 2.5 kg (about 5.times.10.sup.12 cells) under sterile conditions. The medium indicated in table V is used as culture solution. 50 l of culture medium are used per batch. Culturing is performed in glass vessels (Duran 50 or Pyrex glass). Initially, the cell density is about 10.sup.8 cells/ml. The culture is maintained at 37.degree. C. in an atmosphere of 1 v/v % CO.sub.2 over 40 hours. During this period, the cell suspension is slowly stirred (to r.p.m.) and flooded with sterile, water-washed and heat-decontaminated air bubbles (<1 mm). The heat-decontamination of air is performed at about 500.degree. C. by flowing through a silica tube. In addition to the partial oxygen pressure, the pH value (7.1) and the D-glucose level are measured and maintained constant. During culturing, the cells are induced to mitosis by the polyvalent mitogen content (CON) of the culture medium. The number, differential and morphological viability (dye exclusion test) of the cells are continuously determined by usual methods of hematology and cell culture techniques. The functional viability of cells is measured by their motility and their ability to respond to chemokinetic and chemotactic proteins. Mitoses are determined by chromosome count. The morphological viability of the cells after their biotechnical culturing is 95%. The entire loss in cells (mainly granulocytes) during culturing is at most 20% which is normal for primary cell cultures.

The culture is terminated by separating the cells from the supernatant solution by centrifugation for 10 minutes at 400.times.g and 10.degree. C. The cells are washed twice in a salt solution containing 0.15 mol/l NaCl, 0.0015 mol/l sodium potassium phosphate and having the pH-value 7.1. They can be used for another purpose.

The culture supernatant solution is then centrifuged again for 1 hour at 10,000.times.g and at 4.degree. C. to remove suspended particles. The resultant clear supernatant culture solution which has a total volume of 1000 liters and contains about 1,400 g protein as well as other macromolecules and salts is directly subjected to salting-out fractionation with ammonium sulfate (A2). Unless otherwise stated, all further steps are carried out at 0.degree.-4.degree. C.

0.5 mol/l sodium-potassium phosphate buffer solution with a pH value of 6.7 is added to the supernatant culture solution (A1) up to a final concentration of 0.1 mol/l. Furthermore, solid L-cysteine is added up to a concentration of 0.001 mol/l.

This buffered supernatant culture solution is then adjusted to 35% saturation of ammonium sulfate by addition of 199 g of ammonium sulfate/l solution. During the addition, the pH-value of the protein solution is continuously controlled and maintained at 6.7 by the addition of 2 n ammonia. Part of the proteins is precipitated from the solution. The protein precipitate formed is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hour at 10,000.times.g. The precipitated crude protein fraction I is obtained as ammonium sulfate-containing protein sludge which contains about 100 g protein. This crude protein concentrate fraction I may separately be processed for its constituents according to the procedure described below for the crude protein concentrate fraction III.

Then the 35% salt-saturated supernatant culture solution is adjusted to 45% saturation of ammonium sulfate by adding 60 g of ammonium sulfate/l solution. The pH value of the protein solution is continuously controlled and maintained constant at 6.7 by 2 n ammonia. Another portion of proteins is precipitated from the solution. The protein precipitate is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hours at 10,000.times.g. The precipitated crude protein concentrate fraction II is obtained as ammonium sulfate-containing protein sludge, the protein content of which is about 60 g. This crude protein concentrate fraction II may be processed separately for its constituents, according to the procedure described below for the crude protein concentrate fraction III.

The 45% salt-saturated supernatant culture solution is then adjusted to 90% saturation of ammonium sulfate by adding 323 g of ammonium sulfate/l of solution. The pH-value of the protein solution is again continuously controlled and maintained constant at 6.7 by 2 n ammonia. Another portion of the proteins is precipitated from the solution. The protein precipitate is separated from the supernatant containing salt-soluble proteins by centrifugation for 1 hour at 10,000.times.g. The precipitated crude protein concentrate fraction III is obtained as ammonium sulfate-containing protein sludge the protein content of which is approximately 1,080 g. This fraction also contains the bulk of the serum albumin as component of the culture medium. This crude protein concentrate fraction III is processed for the contained mitogens MBG, GBG, MHM and LLM according to the procedure described below. The 90% salt saturated supernatant fraction IV of the crude fraction III contains 160 g of salt-solution proteins and other macro-molecules (>500 daltons). It may also be processed for its constituents.

The crude protein concentrate fraction III obtained above (A2) is dissolved in a minimum volume of buffer solution B (0.01 mol/l of tris-HCl solution containing 0.04 mol/l NaCl and 0.001 mol/l cysteine and having a pH value of 8.0). The resultant slightly turbid solution (20 l) is clarified by centrifugation and then freed of salts by dialysis at a membrane with the exclusion limit of 500 dalton against buffer solution B until no sulfate ions are detectable. The clear solution obtained is then applied to a column of a swollen regenerated anion exchanger (Cl.sup.- as mobile exchangeable ion). It has a dextran matrix cross-linked with epichlorohydrin (DEAE-Sephadex A 50) which is equilibrated in the above-mentioned buffer system B.

The column has four times the volume of the protein solution and a length-to-diameter ratio of 10:1. The gel column is then washed with the above-mentioned adsorption buffer solution B until the extinction of the filtrate at 280 nm is .ltoreq.1.0.

For elution of the mitogens and the adsorbed proteins, the charged ion exchanger gel is eluted with a NaCl-concentration gradient during 2 days. The gradient is linearly ascending from 0.04 to 2.0 mol/l NaCl, whereas the pH value, the tris/HCl and the cysteine concentrations are maintained constant. The same shape of gradient is then used for lowering the pH from 8 to 6.5 for further elution of the compounds. It is made up by 0.01 mol/l piperacine-HCl-buffer containing 2.0 mol/l NaCl and 0.001 mol/l cysteine and having the pH 6.5.

The mitogen-containing fractions are collected separately (MBG, GBG, MHM and LLM are separated in this step). They are, therefore, separately processed in further purification steps described below (A.3.2-A.3.6)

After concentration of the proteins in the fractions (A.3.1) by salting-out precipitation with ammonium sulfate, the protein precipitate containing either MBG,GBG,MHM or LLM is dissolved in a minimum volume of buffer solution C (0.003 mol/l sodium-potassium phosphate containing 0.3 mol/l NaCl and 0.001 mol/l cysteine and having a pH value of 7.4). After removal of a small amount of insoluble compounds by centrifugation, the solution is applied to a column of a molecular sieve matrix of agarose cross-linked with acrylamide (Ultrogel AcA 34, particle size 60 to 160 .mu.m) for preparative molecular sieve filtration. The column has 10 times the volume of the protein solution and a length-to-diameter ratio of 20:1. The column is then eluted with an upward flow (3 cm/h) of the mentioned buffer solution C. For MBG, the fraction with the separation limits of 20,000 and 30,000 dalton, for GBG, the fraction with separation limits of 70,000 to 100,000, for MHM, the fraction with separation limits of 10,000 to 16,000 and for LLM, the fraction with the separation limits of 14,000 and 20,000 dalton are collected. For the concentration of the proteins, the fractions are lyophilized, and ultrafiltered at a membrane with the exclusion limit of 500 daltons or are adjusted to an ammonium sulfate concentration of 3.7 mol/l. In this case, the protein precipitates are separated from the supernatant by centrifugation and further processed as described below (A.3.3).

The resultant MBG, GBG, MHM or LLM-containing protein precipitates (A 3.2) are dissolved in 1.5 volume parts of buffer solution D (0.01 mol/l sodium-potassium phosphate, 0.04 mol/l NaCl, 0.001 mol/l cysteine, pH 6.0). The solutions are centrifuged at 10,000.times.g for 1 hour for removal of a small amount of insoluble material.

The clear solution is dialyzed against the buffer solution D at a membrane with the exclusion limit of 500 dalton until no sulfate ions are detectable. The clear solution obtained is then applied to a column of swollen, regenerated cation exchanger, based on a dextran matrix cross-linked with epichlorohydrin (CM-Sephadex C 50). The exchanger is equilibrated in the above-mentioned buffer system D (Na.sup.+ as mobile exchangeable ion).

The column has four times the volume of the protein solution and a length-to-diameter ratio of 10:1. The gel column is then washed with the above-mentioned adsorption buffer solution D, until the extinction of the filtrate at 280 nm is .ltoreq.1.0. MBG, MHM and LLM are eluted in this step.

For elution of GBG and the adsorbed proteins, the charged ion exchange gel is eluted with an NaCl-concentration gradient during 2 days. The gradient is linearly ascending from 0.04 to 2.0 mol/l NaCl whereas the pH-value and the phosphate and cysteine concentrations are maintained constant. For further elution, the same shape of gradient is then used for increasing the phosphate concentration from 0.01 to 0.5 mol/l at a pH of 8.0, whereas the NaCl (2 mol/l) and cysteine concentrations are kept constant.

The MBG, GBG, MHM or LLM-containing fractions are collected and concentrated in the usual manner and further processed as described below (A.3.4).

The mitogen-containing protein precipitates (A.3.3) are dissolved in a minimum volume of 0.0001 mol/l sodium-potassium phosphate buffer solution E containing 0.001 mol/l cysteine and having a pH of 7.20. The solutions are then desalted with this buffer by molecular sieve filtration, ultrafiltration or dialysis (exclusion limit 500 dalton), until no sulfate is detectable in the dialysis buffer. Thereafter, a small portion of insoluble material is removed by centrifugation at 10,000.times.g for 1 hour.

The clear MBG, GBG, MHM or LLM-containing protein solutions obtained are separately applied to a column of hydroxyapatite. The length-to-diameter ratio of the column is 10:1 and it has four times the volume of the protein volume to be applied. The column has been equilibrated with the mentioned buffer E used in an amount five times the column volume (flow 3 cm/h).

The negatively adsorbed proteins are washed out with the buffer solution E used for equilibrating the column. The elution of the MBG, GBG, MHM or LLM-containing fractions is carried out with a phosphate concentration gradient for 4 days. The gradient is linearly ascending from 0.0001 mol/l to 0.5 mol/l sodium-potassium phosphate having a constant pH value of 7.4 and constant cysteine concentration. MBG is eluted at an average phosphate concentration of about 0.003 mol/l, GBG at about 0.1 mol/l, MHM at about 0.006 mol/l and LLM at about 0.01 mol/l. The elution gradient is measured and controlled by means of conductivity. The mitogen-containing fractions are concentrated in the usual manner and further processed as described below (A.3.5).

The mitogen-containing fractions (A.3.4.) are dissolved in 0.1 mol/l sodium-potassium phosphate solution F containing 0.1 mol/l NaCl, 0.001 mol/l cysteine and 1 mol/l ammonium sulfate and having a pH value of 7.4. The resultant solution is applied at a temperature of 4.degree. C. to a column of swollen molecular sieve matrix of dextran cross-linked with epichlorhydrin (Sephadex G-25). In the matrix, an ascendent, linear ammonium sulfate concentration gradient is established with the mobile buffer phase from 1.0 to 4.0 mol/l ammonium sulfate (25 to 100% saturation). The slope of the gradient is +2% of the ammonium sulfate saturation/cm of column height (0.08 mol/l (NH.sub.4).sub.2 SO.sub.4 /cm). The range of the gradient extends over approximately half the length of the column.

The length-to-diameter ratio of the column is 50:1, the column volume is 100 times higher than the protein solution volume to be applied. The flow rate is 2 cm/h.

The elution is carried out with the above-mentioned sodium-potassium phosphate solution F containing 1 mol/l of ammonium sulfate. The mitogen-containing fractions which are eluted at 72% (MBG), 52% (GBG), 65% (MHM) and 61% (LLM) ammonium sulfate saturation, respectively, are collected. The proteins are concentrated in the usual manner and further processed as described below (A.3.6.).

The mitogen-containing fractions (A.3.5.) are dissolved in buffer C (0.003 mol/l sodium-potassium phosphate containing 0.3 mol/l NaCl and 0.001 mol/l casteine and having a pH value of 7.4). Removal of a small portion of insoluble substances is achieved by centrifugation for 30 minutes at 48,000.times.g.

The resultant clear solution is then subjected to analytical recycling molecular sieve chromatography. For this purpose, the solution is applied at a temperature of 4.degree. C. to a column of Ultrogel AcA 44 having a particle size of 60 to 140 .mu.m. The column has 50 times the volume of the protein solution and a length-to-diameter ratio of 50:1. The elution is carried out with the mentioned buffer C. The eluates are recycled three times at separation limits of either 30,000 dalton (MBG), 100,000 dalton (GBG), 20,000 dalton (MHM) or 24,000 dalton (LLM). After usual protein concentration, approximately 6 mg of MBG, 8 mg of GBG, 6 mg of MHM and 5 mg of LLM are obtained. The mitogens have a molecular homogeneity of >95%, as indicated by conventional methods.

In the following flow sheet the above-described process for preparing the mitogens of the invention is schematically represented.

3.5 kg (about 7.times.10.sup.12) monocytes obtained from porcine blood are cultured under the conditions described in example A. During culture, the polyvalent mitogen (CON) in the medium induces the mitosis of the cells.

The MBG and MHM secreted to the culture solution are isolated according to the preocedure described in example A. They are thereby obtained in a highly purified state. The yields obtained are comparable to those of example A.

The preparation and isolation of mitogens from inflamed tissue are described. 500 g of infarcted, inflamed canine heart muscle tissue are used. The heart muscle tissue is ground at 0.degree.-4.degree. C. 0.05 mol/l sodium potassium phosphate buffer solution containing 0.001 mol/l cystein and having a pH of 6.8 is added in a quantity three times the amount of the tissue. The resultant suspension is homogenized in a homogenizer (ultraturax). Thereafter, the supernatant containing the soluble compounds of the inflamed tissue is separated from the insoluble constituents by centriguation at 10,000.times.g and 4.degree. C. The resultant supernatant solution is then centrifuged for 3 hours at 100,000.times.g. The clear supernatant solution obtained is siphoned off from the flotating lipid layer.

The mitogen-containing clear supernatant protein solution is then subjected to fractional salting-out precipitation with ammonium sulfate according to example A. The resultant protein fraction III is processed as described in example A. From the 500 g of tissue, mitogens are obtained in a yield of approximately 0.02 mg of MBG, 0.04 mg of GBG, about 0.02 mg of MHM and about 0.03 mg of LLM.

Leukocytes are prepared from blood according to example A. A homogenate of 500 g of leukocytes is prepared as shown in example C for muscle tissue. The isolation of the mitogens contained in the leukocytes is performed according to example A. The leukocytes cultured without stimulation contain only relatively small (about 1%) amounts of monocyte- and lymphocyte-mitogens. The yields are approximately 5 .mu.g of GBG, 1 .mu.g of LLM, about 1 .mu.g of MBG and also about 1 .mu.g of MHM.