Process for the production of cell stimulating agents

The invention concerns a process for obtaining cell respiratory stimulating active agents having a molecular weight in the range of about 300 to 8000 Dalton, by defibrination of calves blood, hemolyzing the solution obtained thereby, separating the proteins and substances having a molecular weight of over about 8000 Dalton comprised in the hemolyzed solution by a membrane separation procedure, concentrating the resulting protein- and high molecular weight-freed solution under reduced pressure at a temperature not exceeding 40.degree. C. to a density in the range of 1.10 to 1.15 g/ml at 20.degree. C. and partially separating the inorganic salts from the resulting concentrated solution, wherein the membrane separation procedure employed is a continuous multi-step ultrafiltration procedure employing membranes with a molecular weight exclusion limit of about over 8000 Dalton and the partial separation of the inorganic salts is carried out by electrodialysis with the aid of membranes with a membrane permeability up to a molecular weight of about 300 Dalton.

BACKGROUND OF THE INVENTION 
This invention relates to a process for the production of cell stimulating 
agents. More particularly, the invention relates to a process for 
obtaining a solution of cell respiration stimulating active agents from 
calves blood. 
It is known from German Pat. No. 10 76 888 that one can obtain cell 
respiration stimulating active agents from bovine blood. In the process 
there described, one for example defibrinates fresh calve's blood, 
subjects the solution obtained to hemolyses, separates off the higher 
molecular weight components from the hemolysed solution obtained, such as 
in particular proteins, carries out a dialysis procedure and directly 
obtains a solution of active agent suitable for therapeutic uses by 
carrying out a subsequent mild concentration to a dry substance weight of 
30 to 60 mg/ml. The dialysis required in this procedure can be carried out 
with any well known and conventional dialysis material to achieve the 
objects therein stated, whereby the use of cellophane tubes are indicated 
to be particularly suitable. 
Although the above procedure does lead to a product with the desired 
therapeutic activity, it however possesses the disadvantage that the 
dialysis required is comparatively time-consuming and difficult to 
perform. Also, the product obtained is not the same uniform product since 
the upper molecular weight limit and accordingly its composition are 
subject to variations. Furthermore the yield of product obtained leaves 
much to be desired. 
The process of the German Pat. No. 10 76 888 furthermore also leads to a 
product with a relatively high content of inorganic salts, in particular 
sodium chloride and potassium chloride. Such salts can not be separated 
off by the procedure therein described. The salt content of active agent 
solutions obtained can for example make up about 25 to 80% by weight of 
the solids content, so that corresponding injectable solutions or high 
concentration topical forms are several times hypertonic. As a result of 
this hypertonicity, intramuscular administration of such active agent 
solutions is on the one hand accompanied by pain and on the other hand by 
cell damage, which is disadvantageous to the desired cell regeneration 
effect. 
The undesirable excessively high salt content of cell respiratory 
stimulating agents obtained by the procedure of the German Pat. No. 10 76 
888 could in principle be lowered by several different methods, so that an 
isotonic or only slightly hypertonic solution of active agent is thereby 
obtained, namely for example by dialysis, ultrafiltration, ion-exchange or 
gel-filtration. All of these procedures are either not at all useable or 
are only useable with very unsatisfactory results. In accordance with 
German Pat. No. 25 12 936, the problem of desalting or partial desalting 
of the cell respiration stimulating active agent solution under 
discussion, should be solved by employing a very special gel-filtration 
procedure, namely by bringing the strongly salt-containing solution of the 
cell respiratory stimulating active agents into contact with a filtration 
layer situated on a perforated centrifuge drum and consisting of a gel 
with a high degree of cross-linking at a proportion of solution to gel 
volume of about 1:2.5 to 1.4.5. In this German Patent there are also at 
the same time described in detail the disadvantages of the other known 
procedures which are in principle possible for obtaining a partial 
desalting. All of these possible process procedures, including the process 
described in German Pat. No. 25 12 936, however, possess the disadvantage 
that their separation efficiency is very low and the apparatus required is 
very large, since as a rule these procedures have to be carried out at 
high dilutions. Furthermore, they are accompanied by higher or lower 
losses of active agent, since additional to the desired separation off of 
the inorganic salts, an adsorption of the cell respiration stimulating 
active agents on the separating medium also takes place. The essential 
regeneration of the gels and ion-exchange resin which is thereby necessary 
also requires a great amount of time and enables only a discontinuous 
working procedure with continuous change of the separating conditions. 
The process for obtaining cell respiration stimulating active agents known 
from German Pat. No. 10 76 888, as a result of above, possesses the most 
differing disadvantages, and these could also not be solved by the 
additional considerations of the process described in German Pat. No. 25 
12 936 for separating the excessively high content of disadvantageous 
inorganic salts. 
It is therefore an object of the present invention to provide a new process 
for obtaining cell respiration stimulating active agents of the nature 
under discussion, which does not possess the disadvantages of the known 
working procedures and which can in particular be carried out simply and 
continuously, which is controllable in clean-cut fashion so that a product 
is obtained with a consistent upper--and to an extent also 
lower--molecular weight limit and further which leads directly to a 
solution of the cell respiration stimulating active agents by the 
provision of a controlled partial desalting procedure. 
SUMMARY OF THE INVENTION 
The process in accordance with the invention for obtaining a solution of 
cell respiration stimulating active agents from calves blood, comprises 
the step of defibrinating fresh calves blood, hemolysing the defibrinated 
calves blood, subjecting the hemolysed calves blood to a membrane 
ultrafiltration procedure with the aid of an ultrafitration membrane 
having a molecular weight exclusion limit of about over 8000 Dalton, 
collecting the ultrafiltrate solution comprising respiration stimulating 
active agents having a molecular weight of below about 8000 Dalton, and 
subjecting the collected ultrafiltrate solution to an electrodialysis 
procedure with the aid of electrodialysis membranes having a permeability 
up to a molecular weight of about 300 Dalton, whereby inorganic salts and 
other ionic lower molecular weight substances are partially separated out 
from the solution of cell respiration stimulating active agents. 
The process in accordance with the invention leads to a therapeutic product 
in which the cell respiration stimulating activity is essentially the same 
as the products obtained in accordance with German Pat. No. 10 76 888, but 
provides through the employment of the combination of ultrafiltration and 
electrodialysis a significant improvement in a number of respects. The 
ultrafiltration provides a uniform product with a clean-cut separation of 
the proteins and other high molecular weight substances, with the 
simultaneous advantage of an improved economy regarding time required, 
space required and yield. The fact that one can employ the solution 
obtained by the ultrafiltration after a certain reduction in volume 
followed by electrodialysis, possesses the advantage that on the one hand 
one can employ a relatively concentrated solution, for example a solution 
with a solids content of 15 to 30% by weight, and that one can thereby 
work in a practically stationary equilibrium which sets in after an 
initial phase on the ion-exchange membranes. By changing the parameters of 
current density, temperature and time, it is furthermose possible to 
control the desalting procedure in determined directions and to so 
optimise that only a minimum of organic substance is lost. The degree of 
the particular desalting is most easily provided by a continuous 
measurement of the conductivity or also by a continuous determination of 
the osmolarity.

DETAILED DESCRIPTION OF THE INVENTION 
The known or inventive measures taken in the present process are otherwise 
carried out as follows: 
The calves blood required as starting material is, immediately after having 
been taken, defibrinated by intensive stirring, if desired with cooling, 
and then filtering off the fibrin thus formed. The step is normally 
carried out directly in the particular abbatoirs. 
After completion of the defibrination, the blood, if desired after mixture 
with a conservation agent, is immediately frozen and stored in frozen 
condition until required for further use. 
In order to liberate the cell respiration stimulating active agents, it is 
necessary to subject the defibrinated blood or blood frozen for storage to 
a hemolysis, namely a break down of the red blood cells. For this purpose 
the cell membranes of the blood particles must be ruptured in some or 
other fashion. 
Such rupturing can be achieved by both chemical and mechanical methods. For 
chemical hemolysis, one treats the defibrinated blood for example with 
ferments or bacteria, which attack and rupture the cell membranes. The 
mechanical hemolyses can take place either by addition of agents which 
lead to an elevation of the osmotic pressure within the cells, for example 
by the addition of water or organic solvents such as ethanol, diethyl 
ether or acetone, whereby the cell membranes then burst, or hemolysis can 
be obtained by the socalled ice-hemolysis, in which the blood is slowly 
cooled in such a fashion that larger ice crystals can develop which 
penetrate the cell membranes. After the hemolysis, practically all red 
blood cells are ruptured and the substances contained therein are set 
free. 
The hemolysis is conveniently also carried out in the presence of a 
conservation agent. 
The blood obtained hereby is then ready for carrying out the first step in 
accordance with the invention of the present process, namely, 
ultrafiltration employing membranes having a molecular weight cut-off 
limit of over about 8000 Dalton, and preferably over about 5000 Dalton. 
The membranes employed in the ultrafiltration can consist of the most 
different materials, whereby membranes of cellulose triacetate or 
hydrophylic polyolefine are preferred. 
Membranes of the above nature with a molecular weight cut-off limit of 8000 
Dalton are characterised by their permeability, which is 100% separation 
of dextran with a molecular weight of 20,000, a nominal 85% separation of 
dextran with a molecular weight of 10,000, a nominal 50% separation of 
polyethyleneglycol with a molecular weight of 6000 and a nominal 10% 
separation of lactose with a molecular weight of 342. 
Membranes with a molecular weight cut-off limit of over about 5000 Dalton 
are in respect of their permeability characterised by a 100% separation of 
dextran with a molecular weight of 10000, a nominal 97% separation of 
polyethyleneglycol with a molecular weight of 6000 and a nominal 30% 
separation of polyethyleneglycol with a molecular weight of 1000. 
The solution obtained by ultrafiltration of calves blood after 
defibrination and hemolysing with employment of the membranes of the 
nature described, provides a finished product with an upper molecular 
weight limit of about 8000 Dalton or of about 5000 Dalton. 
For carrying out a continuous multiple-step ultrafiltration, an 
ultrafiltration apparatus consisting of four units is advantageously 
employed, as is described in greater detail in the Example in conjunction 
with FIG. 1. Such an apparatus comprises ultrafiltration modules with type 
designation B 1, as is described in more detail in the brochure BPL 3/73 
2M of the firm Paterson Candy International Ltd., Reverse Osmosis 
Division, Whitchurch, Great Britain and which is also described in German 
Patent Application No. 20 65 812. This special apparatus comprises 
membranes with a molecular weight cut-off limit of over about 8000 Dalton. 
It can however equally well be fitted with membranes with another 
molecular weight cut-off limit, for example a molecular weight cut-off 
limit of over about 5000 Dalton. 
The ultrafiltration apparatus is operated with the use of pressures of for 
example 8 to 10 bar and cooling of the solution to be ultrafiltered to a 
temperature of for example 18.degree. to 22.degree. C. It is 
advantageously supplied with a solution of calves blood which has been 
conserved with a conventional conservation agent, for example an alcoholic 
solution of 4-hydroxy-benzoic acid methyl ester or 4-hydroxy-benzoic acid 
propyl ester, whereby an ethanolic solution of a combination of these two 
conservation agents is preferred. 
Between the first step and the last step of the continuous multiple step 
ultrafiltration as diluent for the concentrated and then again to be 
ultrafiltered blood solution advantageously such an amount of a 
conservation agent solution is added that the drained off amount of 
ultrafiltrate is compensated by maintaining an about constant level of 
blood solution to be ultrafiltered again in the next step. Advantageously 
all steps but the last step of the continuous multiple step 
ultrafiltration are conducted while maintaining a near constant level of 
blood in the solution to be ultrafiltered. Only in the last step 
preferably no dilution of the blood solution coming from the last but one 
step is made so that here the blood load is totally dialysed under 
reduction of volume. 
The waste blood resulting eventually from the ultrafiltration is discarded. 
The ultrafiltrate obtained from the ultrafiltration, freed of proteins and 
higher molecular weight substances is subjected to a further step of 
volume reduction under reduced pressure at a temperature of not more than 
40.degree. C. The solution is reduced in volume to a density in the range 
of 1.10 to 1.15 g/ml at 20.degree. C., for example by distillation under 
reduced pressure at a temperature of for example 30 to 35.degree. C. The 
purpose of this concentration is firstly to remove the organic solvents 
which were optionally employed for hemolysis or for the organic solvent 
required for the specific conservation agent with simultaneous removal of 
the conservation agent, and secondly a concentration of the ultrafiltered 
solution to a dry content, namely for example a dry content of 180 to 230 
mg/ml, suitable for carrying out the subsequent electrodialysis step of 
the invention. The precipitated conservation agent resulting from this 
concentration is best separated off by filtration, whereby residual 
conservation agent, if any, in the solution can be precipitated by 
suitable adjustment of the pH-value of the solution, for example, by 
addition of small amounts of concentrated hydrochloric acid up to a 
pH-value of 5, and then filtered off. The distillation required for this 
concentration is conveniently carried out in a suitable vacuum evaporator. 
The concentrate obtained by the above concentration is then subjected to 
the second of the process steps of the invention, namely, the step of 
electrodialysis with the aid of a cell stack of alternatively arranged 
cation - and anion-exchange membranes with a membrane permeability up to a 
molecular weight of about 300 Dalton and preferably up to a molecular 
weight of about 400 Dalton, for partially separating off the inorganic 
salts therein contained and for forming an isotonic to slightly hypertonic 
solution having an osmolarity in the range of 250 to 550 mOsmol or a 
conductivity in the range of 40 to 75 mS/cm or a density at 20.degree. C. 
in the range of 1.05 to 1.08 g/ml. 
The membranes employed for this purpose are characterised by a permeability 
for inorganic or organic ions having a molecular weight up to about 300 or 
preferably up to about 400, whilst these membranes are closed to 
oligopeptides and larger molecules. 
With the employment of such membranes, a lower molecular weight 
permeability limit of about 300 Dalton or preferably of about 400 Dalton 
is provided for the active agents. 
The electrodialysis can be carried out under conditions which are 
conventional for separating inorganic salts from corresponding aqueous 
concentrates, so that the step of the invention here may for example work 
with a current density range of 5 to 70 mA/cm.sup.2 free membrane 
cross-section, preferably 20 to 50 mA/cm.sup.2 free membrane 
cross-section, a direct current voltage of 0.2 to 2 V per membrane pair, 
preferably 0.5 to 1 V per membrane pair, and a temperature range of 
5.degree. to 30.degree. C. 
The above step of electrodialysis can be carried out by employing 
conventional electrodialysis apparatus of a cell stack of alternately 
arranged cation- and anion-exchange membranes with the specifically 
desired permeability. In the example, an electrodialysis apparatus is 
employed which is in accordance with FIG. 2, which is later described in 
more detail. This is an apparatus of type designation BEL 2 from Berghof 
GmbH, Tubingen, Germany. The membranes of this apparatus reflect a 
membrane permeability up to a molecular weight of about 400 Dalton. A 
detailed description of the cell stack of this electrodialysis apparatus 
is shown in published German Patent Application No. 29 46 284. 
The aqueous solution with a content of cell respiration stimulating active 
agents obtained in accordance with the process of the invention may, 
dependent on the intended form of application, either be employed directly 
for therapeutic uses, or if desired diluted further with pyrogen-free 
water or where required further concentrated. The corresponding 
pharmaceutical form may thus for example be solutions, aerosols, pastes, 
creams or gels. The cell respiration stimulating activity is, as already 
mentioned, essentially the same as for the substances obtained by German 
Pat. No. 10 76 888. Accordingly such medical forms are especially used for 
the promotion and control of healing processes. The dosage of active agent 
employed for this purpose is dependent on the mode administration as well 
as the nature and the seriousness of the condition being treated. Types of 
wounds which may be treated with the cell respiration stimulating active 
agents obtained by the present process are for example burns, ulcers or 
decubitus. For this purpose, the active agent can be administered 
intravenously, intraarterially, intramuscularly, or in the case of open 
wounds, also topically. In an injection therapy, such as in the treatment 
of ulcers in human subjects, an intravenous or intraarterial daily dose of 
200 to 800 mg of active agent is for example required, and thereafter a 
follow-up treatment by intravenous or intramuscular administration of 
daily doses of 80 to 200 mg active agent. 
Concerning this and other possible uses of such an active agent, reference 
is made in general to the brochure "Solcoseryl reaktiviert den gestorten 
Energiestoffwechsel der Zelle, regeneriert das Gewebe" of Solco Basel AG, 
Birsfelden, Switzerland, with the Publication no. SS-CH/d-6.81. 
An important requirement for the successful employment of the process of 
the invention is that the biological activity of the calves blood extract 
is not influenced, and this particularly in the ultrafiltration and more 
particularly in the electrodialysis. In accordance therewith the original 
biological activity must be fully maintained. According to the process of 
the invention, a blood extract should be obtained which corresponds at 
least to the biological activity of product obtained by the German Pat. 
No. 10 76 888 or which is even higher. 
The danger of a possible loss in biological activity during the 
ultrafiltration to be performed in accordance with the invention may be 
regarded as small, since in this step of the process only proteins and 
higher molecular weight substances are separated off and the major 
proportion of the biological activity determined by corresponding 
experiments should reside in the lower molecular weight range of the cell 
respiration stimulating active agents recoverable by the present process. 
It can also be taken that the ultrafiltration will not lead to any 
additional influence on the blood extract than the simple dialysis to be 
performed according to the process of German Pat. No. 10 76 888. A 
comparative test on the possible influence on the biological activity by 
the ultrafiltration of the invention is thus not necessary. 
Similarly, the concentration of the solution following the ultrafiltration 
is also carried out under conditions which can not be associated with any 
influence on the biological activity of this blood extract. Thus, with 
this step also, comparative tests are not necessary. 
On the other hand, the separation of inorganic salts comprised in the 
active agent concentrate by electrodialysis could lead to a negative 
influence on the biological activity of the blood extract. Here, 
relatively high amounts of salts must be separated off, and the molecular 
weights of the salts to be separated are comparatively close to the lower 
molecular weight limit of the cell respiration stimulating active agents 
to be obtained in accordance with the invention. It is therefore necessary 
to determine with the aid of suitable experiments whether the biological 
activity of the active agent solution obtained by electrodialysis, as 
compared to the starting material concentrate, has been influenced. Also, 
it is of course to be determined how the physical characteristics are 
influenced by this electrodialysis as compared to the starting material 
concentrate and which important substances additional to the inorganic 
salts are removed. 
The comparative experiments related to the particular level of biological 
activity are performed by three different methods, namely by 
(a) Testing of the growth-stimulating activity on fibroblasts after 
damaging by carbonate withdrawal (Providing of the fibroblast activity), 
(b) Testing of the O.sub.2 -metabolism increasing activity according to 
Warburg (Providing the Warburg activity) and 
(c) Testing the wound healing promoting activity with standardized, dorsal 
burn wounds on rats (Providing the wound healing time against 
physiological saline) 
The first two in vitro tests reflected that the full biological activity is 
retained under all test conditions and even improved in part, since the 
cell damaging salts are no longer present. The wound healing experiments 
provided the expected results that the partially desalted blood extracts 
obtained by the present process are better tolerated. 
In all of these comparisons, the activity of the non-desalted starting 
concentrate and the partially desalted extract are of course carried out 
under normalised conditions, that is with employment of solutions with the 
same content of organic active agent but not with the same dry content. 
For this purpose, the solutions to be compared are always diluted to a 
determined content of total nitrogen, for example to a nitrogen content of 
1 mg/ml of injection solution, and this total nitrogen content is taken as 
representative of the available content of organic material in the 
particular solutions. Of course, one proceeds in precisely the same 
fashion also for solutions to be employed for therapeutic purposes, 
wherein each charge is additionally tested in the Warburg test and in the 
fibroblasts-test. 
The changes in dry weight, conductivity, osmolarity and ash content are 
obtained with the aid of comparative tests. The changes between the 
starting concentrate and that obtained by separating off the low molecular 
weight substances, namely mainly the anionic ions and other low molecular 
weight products, are obtained by comparison of the chemical analyses for 
characteristic substances and parameters. 
The results obtained in comparative experiments carried out with the aid of 
two different starting concentrates and eight partially desalted extracts 
obtained in accordance with the invention can be seen in Table I of the 
Example. They support that additional to the inorganic ions lower carbonic 
acids and amino acids are partially removed, the biological activity 
however being fully retained. 
For the purpose of further supporting that the electrodialysis carried out 
in accordance with the invention does not lead to a change or only leads 
to an insignificant change in the composition of the blood extract as 
related to its organic components, high pressure liquid chromatographic 
experiments were carried out. Here, the chromatograms obtained were each 
time identical for the starting concentrate and the partially desalted 
extracts. 
The process of the invention is further illustrated by the following with 
the aid of an Example. 
EXAMPLE 
Ultrafiltration 
An ultrafiltration apparatus of the nature mentioned in the introduction is 
employed (B1 from Paterson Candy International Ltd.). This four step 
ultrafiltration apparatus and its functioning can be seen schematically 
from FIG. 1. 
Each step of the total four step apparatus (steps 1 to 4) is equipped with 
two ultrafiltration tube modules (U.sub.1 to U.sub.4) each having a 1.7 
m.sup.2 filter surface. Their membranes consist of cellulose acetate with 
the permeability characteristics already mentioned, namely a molecular 
weight permeability limit of over about 8000 Dalton. The pressure transfer 
pumps P.sub.1 to P.sub.4 are rotary piston pumps, which possess a capacity 
of about 1000 l/h at a pressure of 10 bars. The supply pumps P.sub.5 to 
P.sub.7 and the drain pumps P.sub.8 to P.sub.10 are dosage pumps, which 
can be freely adjusted within a range of 2 to 20 l/h. The total number of 
four tanks T.sub.1 to T.sub.4 each contain about 300 l. The added 
solutions are maintained at a temperature of 18.degree. C. by coolers 
K.sub.1 to K.sub.4 cooled with cold water. PI in each case refers to 
pressure indicator devices, whereas FI refers in each case to flow 
indicator devices. The other abbreviations referred to therein have the 
following meanings: 
B=defibrinated and hemolyzed blood solution 
KL=Conservation agent solution 
UF=ultrafiltrate 
AB=waste blood 
To start the apparatus one supplies each of the tanks T.sub.1 (step 1), 
T.sub.2 (step 2) and T.sub.3 (step 3) with each 250 l of defibrinated and 
hemolyzed calves blood B, which contains as conservation agent solution 15 
volume percent of a 2% (weight/volume) alcoholic solution of a 9:1 mixture 
(weight/weight) of 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic 
acid propyl ester. Then the pressure transfer pumps P.sub.1 to P.sub.3 are 
set in operation, whereby the ultrafiltrate UF begins to run out and the 
content of the tanks T.sub.1 to T.sub.3 reduces to the same degree. As 
soon as the content of the tanks T.sub.1 to T.sub.3 is about 150 l, the 
supply pumps P.sub.5 to P.sub.7 and the drain pumps P.sub.8 to P.sub.10 
are set in operation. 10 l/h of conservation agent containing defibrinated 
and hemolyzed calves blood of the nature already described above are 
supplied over supply pump P.sub.5. At the same time, 10 l/h of a 
conservation agent solution KL of 85 volume percent water and 15 volume 
percent of a 1% (weight/volume) alcoholic solution of a 9:1 mixture 
(weight/weight) of 4-hydroxybenzoic acid methyl ester and 4-hydroxy 
benzoic acid propyl ester is supplied to each of the tanks T.sub.2 and 
T.sub.3 over supply pumps P.sub.6 and P.sub.7. This leads to dilution of 
ultrafiltered loads. The drain pumps P.sub.8 to P.sub.10 are adjusted so 
that the level in the tanks T.sub.1 to T.sub.3 remains constant at 150 l. 
In accordance therewith the tanks T.sub.2 and T.sub.3 are each supplied 
with such an amount of dilution agent KL that the amount of ultrafiltrate 
UF flowing from the individual ultrafiltration tube module U.sub.2 and 
U.sub.3 is compensated by maintaining a constant level of blood load. The 
same level of blood load is also maintained in tank T.sub.1. Only in tank 
T.sub.4 the level of blood load is lowered while carrying out the 
continuous multiple-step ultrafiltration by the amount of ultrafiltrate 
coming from ultrafiltration tube module U.sub.4, because here no dilution 
agent is added in order to keep the level of blood load at about the same 
level as in the previous steps. 
As soon as about 120 l of solution is available in tank T.sub.4 (step 4) 
from the step 3, the pressure transfer pump P.sub.4 is switched in. The 
waste blood passing out of the overflow of this tank T.sub.4 is discarded. 
After about 36 hours, an equilibrium has set in in all of the four steps. 
The ultrafiltrate UF flowing from the ultrafiltration tube modules U.sub.1 
to U.sub.4 is thus representative of the present process, so that it may 
be collected for further use (subsequent concentration). The ultrafiltrate 
UF flow is about 4.7 l/h with step 1, about 11 l/h with step 2, about 10.4 
l/h with step 3, and about 1.8 l/h with step 4. The amounts of 
ultrafiltrate UF transferred are accordingly 5.3 l/h for the drain pump 
P.sub.8, 4.3 l/h for the drain pump P.sub.9 and 3.9 l/h for the drain pump 
P.sub.10, whilst the overflow of waste blood AB from the tank T.sub.4 
(step 4) is 2.1 l/h. 
CONCENTRATION 
The ultrafiltrate UF obtained by the above four stage ultrafiltration is 
then concentrated to a density of 1.130 mg/ml at 20.degree. C. by 
distillation under reduced pressure of 30 mbar and a temperature of 
35.degree. C. and is hereby freed of the alcohol comprised in the 
conservation agent. With this concentration, most of the conservation 
agent also precipitates out, which is filtered off. The filtrate obtained 
is treated in small portions with concentrated hydrochloric acid until a 
pH value of 5 is reached, whereby the remaining amounts of conservation 
agent precipitates out. This precipitate is once again filtered off. 
The material obtained by the above ultrafiltration and concentration is 
combined and serves as the starting concentrate with the designation 
733.10 for the following step of electrodialysis in accordance with the 
invention. 
Repetition of Ultrafiltration and Concentration 
The above two process steps are repeated in every detail at a later point 
in time employing calves blood of a different origin, whereby one obtains 
a further starting concentrate with the designation 350.03 for the 
following step of electrodialysis. 
Electrodialysis 
The starting concentrates no. 733.10 and no. 350.03 obtained in the above 
fashion are once divided into two parts and the other time divided into 
six parts and are each added separately to the electrodialyses apparatus 
shown in FIG. 2. In this manner, one obtains the electrodialysates no. 12 
and no. 13 with employment of the starting concentrate no. 733.10, and the 
electrodialysates No. 14, no. 15, no. 16, no. 17, no. 18 and no. 19 with 
employment of the starting concentrate 350.03. 
An electrodialysis apparatus of the type mentioned in the introduction is 
employed (BEL 2 from Berghof GmbH). This apparatus and its function can be 
seen schematically from FIG. 2. 
The central section of this apparatus is a cell stack which consist of 
seven pairs, each of an anion exchange membrane a and a cation exchange 
membrane k (Type CMV and AMV) and comprising on each side an additional 
membrane k as last membrane. (In the schematic drawing only three ion 
exchange membrane pairs are shown for simplicity reasons). The membranes 
employed have a permeability up to a molecular weight of about 400. 
The following three liquid circuits are connected to the cell stack: 
The solution to be desalted circulates from tank T.sub.1 over a pump 
P.sub.1 and a cooler K.sub.1, which analogous to the literature is 
designated as diluate D. 
An aqueous solution which takes up and enriches the salts, circulates from 
tank T.sub.2 over a pump P.sub.2 and a cooler K.sub.2, and this solution 
is designated as concentrate K. 
An aqueous solution of 2% sodium sulphate (weight/volume) circulates from 
tank T.sub.3 over a pump P.sub.3, and this solution is designated as 
electrode rinse solution EL. 
The heat generated by friction is conducted away by the coolers K.sub.1 and 
K.sub.2 which maintain the solutions at 25.degree. C. 
A conductivity measuring cell LF is built into the diluate circuit D after 
the cooler K.sub.1. The flow of concentrated down ultrafiltrate UK is 
regulated over a two-point regulator R. 
A portion of the concentrated down salt solution KA is continuously carried 
away on the concentrate side and is replaced by distilled water W over the 
pump P.sub.5. Additionally, a certain water transport takes place in the 
cell stack from the diluate side to the concentrate side. 
A portion of the diluate D after measurement of the conductivity in the 
conductivity measuring cell is continuously withdrawn and after passing 
over a sterile filter SF is stored as finished product PR in tank T.sub.4 
at 0.degree. C. until further working up into the corresponding finished 
pharmaceutical form. 
To initiate operation of the electrodialysis apparatus, the 2 l-containing 
tank T.sub.1 is added with 1.2 l of the concentrated ultrafiltrate UK 
produced in accordance with the procedure described above. 1 l of 
distilled water is added to the tank T.sub.2 of the same size and 750 ml 
of a 2% NaSO.sub.4 solution is added to the 1 l tank T.sub.3. After the 
cell packet and all circuits are filled and freed of air, the three pumps 
P.sub.1 to P.sub.3 are set in operation, which each transport about 90 
l/h. A direct current is applied to the two electrode plates (+ and -), 
whereby the voltage is so chosen that a current of about 20 mA/cm.sup.2 
flows on the free membrane cross-section. Thus, a total current of about 
75 mA flows in the cell stack employed having a free membrane 
cross-section of 37 cm.sup.2. The starting voltage of about 16 V is 
adjusted downwardly within 1 hour to a stationary value of about 5.5 V for 
the cell stack consisting of seven membrane pairs. 
The conductivity has lowered to a value of between 45 and 50 mS/cm after 10 
hours. The regulator R now regulates the supply of further concentrated 
ultrafiltrate UK over a dosage pump P.sub.4 in such a fashion that the 
conductivity of the diluate remains between 45 and 50 mS/cm, in average at 
about 100 ml/h. 
The dosage pump P.sub.5 is set at 200 ml/h, and the outflow of the salt 
concentrate KA from the tank T.sub.2 is about 220 ml/h. 
The results obtained with the above dialysis several times repeated 
employing the two different starting concentrates no. 733.10 and no. 
350.03 and formation of the different electrodialysates no. 12 and no. 13 
as well as no. 14, no. 15, no. 16, no. 17, no. 18 and no. 19 are provided 
in the following Table I together with the applied conditions of 
temperature, current density and direct voltage as well as with the 
different analysis data for the starting concentrates and the recovered 
electrodialysates and the comparative data on the specific biological 
activity. 
The concentrated diluate obtained by the above electrodialysis is 
advantageously diluted with sterile water--for example by five times--in 
order to form an isotonic to slightly hypertonic active agent solution 
having an osmolarity in the range of from 1 to 250 to 550 mOsmol. 
The test results show that by employment of the process of the invention a 
cell respiration stimulating active agent solution can be prepared from 
calves blood in a particularly elegant and economical fashion, which at 
least has the same or in part an even better biological activity than the 
corresponding active agent solution obtained by the known procedures. 
__________________________________________________________________________ 
Dimension 
Starting- 
Electro- 
Electro- 
Starting- 
Electro- 
concentrate 
dialysate 
dialysate 
concentrate 
dialysate 
No. 733.10 
No. 12 
No. 13 No. 305.03 
No. 14 
__________________________________________________________________________ 
Solids content mg/ml 
212,1 103,0 
108,7 213,11 
109,7 
Density (20.degree. C.) g/ml 
1,129 1,057 
1,060 1,129 1,060 
Conductivity mS/cm 149 56 68 145 56 
Osmolarity mOsm 1053** 
404*** 
497*** 1052** 
414*** 
Ash content % solids content 
66,25 33,90 
41,80 75,57 34,55 
Total Nitrogen % solids content 
2,55 4,83 4,57 2,73 5,59 
Amino Nitrogen % solids content 
0,65 1,23 1,25 0,64 1,38 
Chloride % solids content 
35,88 15,74 
19,31 37,17 12,28 
Sodium % solids content 
27,19 17,30 
19,49 24,79 18,03 
Potassium % solids content 
4,34 1,59 2,14 3,78 1,20 
Glucose % solids content 
10,91 22,30 
20,52 3,87 8,65 
Urea % solids content 
2,15 3,59 3,43 2,43 4,76 
Total Phosphorus % solids content 
0,78 1,52 1,39 0,77 1,75 
Lactate % solids content 
9,72 17,58 
17,71 9,78 20,62 
Acetic Acid % solids content 
1,83 3,45 3,18 2,37 4,48 
Fibroblast-Activity Growth stimulation 
1,6* ** 
1,5* *** 
1,5* *** 
2,0* ** 
1,1* *** 
compared to 
Standard-Culture* 
Warburg-Activity % compared to 
-2** +10*** 
+14*** -6** + 5*** 
Standard 
Wound healing time against 
9,9** 13,7/10,9*** 
15,7** 
physiological Saline % Shortening 
Temperature .degree.C. 25 25 25 
Current density mA/cm.sup.3 
50 50 50 
Direct current per membrane V 
0,7 1,0 1,0 
__________________________________________________________________________ 
Dimension 
Electro- 
Electro- 
Electro- 
Electro- 
Electro- 
dialysate 
dialysate 
dialysate 
dialysate 
dialysate 
No. 15 
No. 16 
No. 17 No. 18 
No. 19 
__________________________________________________________________________ 
Solids content mg/ml 
106,37 
107,58 
111,37 129,62 
101,28 
Density (20.degree. C.) g/ml 
1,057 1,058 
1,060 1,073 1,052 
Conductivity mS/cm 50 55 56 40(6.degree. C.) 
46 
Osmolarity mOsm 378*** 
410*** 
428*** 510*** 
355*** 
Ash content % SC 32,48 36,11 
36,72 43,90 28,10 
Total Nitrogen % SC 
6,26 5,61 5,87 5,13 6,16 
Amino Nitrogen % SC 
1,56 1,46 1,45 1,28 1,64 
Chloride % SC 9,20 11,89 
12,69 16,30 7,30 
Sodium % SC 16,99 18,23 
18,36 20,40 17,30 
Potassium % SC 1,07 1,10 1,12 1,53 0,92 
Glucose % SC 9,81 9,06 9,09 7,95 10,28 
Urea % SC 5,39 4,56 5,11 4,34 5,04 
Total Phosphorus % SC 
1,91 1,75 1,67 1,56 1,94 
Lactate % SC 22,72 21,29 
21,63 18,32 22,60 
Acetic Acid % SC 4,58 4,56 4,62 4,01 4,68 
Fibroblast growth 1,2* *** 
1,3* *** 
0,7* *** 
1,8* *** 
2,0* *** 
stimulation compared to 
Standard-Culture* 
Warburg-Activity % compared 
+6*** -7*** 
+30*** +9*** +30*** 
to Standard 
Wound healing time against 9,0*** 
physiological Saline % Shortening 
Temperature .degree.C. 
25 25 25 6 25 
Current density mA/cm.sup.2 
50 22 50 50 20 
Direct current per membrane V 
1,2 0,8 1,2 1,3 0,4 
__________________________________________________________________________ 
SC = Solid Content 
*All values over 1.0 are effective; quantitative lower graduations are no 
longer possible 
**These data are based on an ultrafiltrate diluted by five times with 
sterile water 
***These data are based on an electrodialysate diluted by five times with 
sterile water.