CA1102694A - Process of preparing a serum protein composition for intravenous application - Google Patents

Abstract

TITLE
PROCESS OF PREPARING A SERUM PROTEIN
COMPOSITION FOR INTRAVENOUS APPLICATION

INVENTOR
Markus RADOWITZ
ABSTRACT
A process of preparing a serum protein composition for intravenous application or administration, in which process, starting from a human blood protein solution, a stablized, universally applicable preparation or composition is prepared which contains dissolved in an aqueous isotonic solution, such proteins characterized by performing the following steps:
(a) fractionating the human blood plasma and removing coagulation factors;
(b) recovering, during the said fractionation step, precipitates and supernatants at each differend fractionation step;
(c) mixing and resolving of precipitates of the different fractionation steps, in which blood proteins, especially albumin and globulins, are preserved in a native form, said precipitates mixed and resolved in a chemically and physiologically adapted solvent, said solvent eventually containing also supernatants of the fractionation steps;
(d) adjusting the mixture to a content of 11 - 60% albumin and - 89% globulin, (e) stabilizing, purifying and drawing off the preparation.

Description

6~4 Process of preparing a serum protein composition for intravenous application The present invention relates to a process o preparing a serum protein composition for intravenous application or administration, in which process, starting from a human blood protein solution, a stab~ized, universally applicable preparation or composition is prepared which contains dissol-ved in an aqueous isotonic solution, such proteins in which the ratio of globulins and albumins corresponds substantially to that of the native blood serum.

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; Serum protein preparations or compositions represent valuable ~
, . .
~ blood derivatives. The albumin contained in such compositions . .
functlons as a transporting protein. These compositions ; contain further functional proteins, such as transferrin, eruloplasmin, ~ -antikripsyn and particularl~ the immune globulins. Of importance ara the antibodies which are present in the ~ -globulin. Due to the passive immunization caused by these substances, they are suitable for prophylactic :
therapy, and they strengthen the body weakened by loss of blood.
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A serum protein composition which should be suitable to be intravenously administer d to a human patient has to comply with two strict requkements:

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1 1) The composition must form a storable product;
¦~ 2) infection carriers (vixuses or bac-terla) should be ! neutràlized reliably. This applies particularly to the carrier of virus hepatltis.

Serum protein compositions are normally provided in the form of an aqueous solution of the stable proteins ` ~; having an electrolyte content that is toler~ble to the ~ody. The total protein content is normally 5 percent by weight ~i.e. 5,000 milli-I grams per 100 milliliters)~
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The l~terature shows to ~e known a process for the preparation of a serum protein composition suitable for - intravenous application or administration, which composition is obtained directly from human blood in a number of process steps (W. Stephan r "Hepatitis-Free and Stable Human Serum for Intravenous Therapy"; XXIVth Sc. Meeting ;~ of the Blood Research Institute, Vox Sanguin, V. 20, (p. 422 - 457));

The conventional process i5 performed in the followin~

steps:

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(a~ Recovery of a natural serum from donor blood upon removal of the solid constituents ~blood corPuscles, blood platelets) and recovery of a serum having a protei~ concentrat~on of about 7.5 % and a ~H of from 7.2 to 7.8.

3269~

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(b) For the removal of the lipoproteins which can be easily denaturalized~ 2.0 g of Aerosil 2491~380 (Aerosil: registered trademark of the firm Degussa, for a colloidal silicic acid) are added per 100 ml of serumO The suspension is agltated for 4 hours at 45C, cooled, and the sediment is removed ~y centrifuging. The lipoproteins are therebv ~ quantitatively bonded to the Aerosil so as to be : removed from the blood serum.
: i (c~ FilterinYof the solutlon through a microfilter for ~;~ the removal of any suspended particles still present ~- in the so~ution~ :

(d) For sterilization, the solution is mixed with 0.3 g B-propiolactone per 100 ml of solution at a temperature of 5C (pH 8.0~ and irradiated with ultraviolet light.
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The thus obtained serum has the following composition ~: (based upon S,000 milligrams contained in 100 milliliters):

4, SubstanQe ontent Function Albumin 3,060 On~tic activity; transport of nutritive materials, vitam-ins, hormones, medicaments.
IgG 820 Ant1body to viruses and bacterla, IgA 185 Antibodies protecting the mucous membrane.
IgM 75 Anti~odles to bacteria and toxines.
~; Prealbumin 17 Tyroxine bond.
. ~
5~ Oæ1-antitrypsin162 d42-macroglobulin 141 Inhi~itors to pr~teolytic enzymes.
~aptoglobulin ~10 ~onding and transport of free haemoglobulin.
Haemopexin 75 Bondlng and transport of free haemin.
Transferrin 195 Transport of Fe+~
Coeruloplasmin1~ Transport of Cu~ as peroxidase.
Cholines~erase3.0 Elml Splitting of succinylcholin.
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;;,; , . ~ ~he above Table also reveals the functions of the variOus constltuents of the serum protein composition.

he composition prepared in accordance with the conventional process is commercially available. This conposition mav be :' ' ;:, applied without complication.
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However, it is considered to be disadvantageous with respect to the preparation and application tha-t the composition is very costly because of the high-quality starting material - i.e. human blood - and cannot be made available to a sufficient extent. Add to this that the contents of the particularly highly active antibodies IgG, IgA, IgM is relatively low in correspondence with the presence of these substances in the human blood~

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Accordingly, it is the object of the present invention to provide a process for the preparation of a serum protein composition for intravenous administration, in which the starting substances are less expensive and rare. This prccess should allow to use for the preparation of valuable serum protein compositions such starting substances which have no, or inadequately only, been processed or recovered heretofore. It is a further object of the invention specifically to increase the content of active antibodies or other proteins of the serum without thereby increasing the cost of preparation to any substantial degree.

In the above mentioned process, this object is sol~ed hy usin~ as the starting material, fractions of human blood plasma which are recovered from human serum with the use l~OZ694 of one or more fractionating steps ~erving to enrich (concentrate) or ~solate, respect1vely~ individual ones of the blood protein ~onstituents with these latter constituen~s being substantially preserved in their native form;
and ~haracterized by the fact that the fractions of the starting material are proviaed with a composition o-E
albumins and globulins equivalent to that of the blood serum, by adjustment (standardization) of the mixture, stabilization, purification ana concentration or dilution, respectively. Especially, this i~ performed by the steps ~ - d of claim 1.
The generalized wording o the claim for patent proteation ~tates that, although it is started with human blood as the starting material, the preparation of serum protein composition in accordance with the present process is no longer performed directly on the basis of the native serum, but that other intermediate ractionation products are employed which result from the fractionation of blooa~ In this connection, it is an objec~ of the present invention to employ well~known process products which h~ve not been utilized heretofore, to useuncomplicated process steps, and to increase the recovery of the proportions of useful products present in the hum~n blood.

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In particular, the process according to the invention makes use of the fact that the ~lood fractionation according to COHN yields certain fractions which could heretoore not, or incompletely only, be further processed.

~, The process according to COHN is based upon the -~
fractionation of the blood plasma with the use of e~hanol as a precipitant under precisely determined conditions of temperature, of the pH, of the ion densities and of the ethanol content. The COHN process is described, for lnstanc~, in the following pu~lications:

~OHN, E.J. et al.: "JOAmer.Chem.Soc." 72 (1950), p~ 465 - 474 COHN, E.J. et al.: "~.Amer.Chem.Soc." 68 ~1g46), p. ~59 - 475 U.S. Patents 2,390,074 and 2~469,193.
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The enclosed flow diagram shows the various Process steps~
In thls diagram, the expression "~ of ethanol" always refers to percents by volume, measured at 25C. The startin~
plasma is obtained from donor blood. 500 milliliters o~
human blood are each collected in 50 milliliters of a 4 % solution of sodium Citrate. Initially, t~e solid constituents of the blood are separated from the plasma;

the plasma of a plurality of donors is psoled.

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i In step a accor~ing to the flow diagram, the crude ~fibrionogen is removed from the plasma by addin~ th~eto '. cold 53.3 ~ ethanol up to a concentration o:E 8 ~.
The temperature is maintained at from -2t5 to -3,0VC.
The precipltate P 1 - fibrinogen - is removea ~y centrifuglng. The supernatant S 1 is further processed.

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Step b again comprises the addition of 53.3 '~ ethanol to the supernatant liquid S 1 until a concentration of from 18 to 25 ~ of ethanol in the liquid reachad. ~he temperature is kept at -5~C, and the pH is adjusted to .
5.8. The precipitate P 2 is precipitated, which is designated as fraction II~III or as y-globulin fraction~
This precip1tate includes the immune globulins and other physiologically si~rificant protein5. The precipitate P 2 is removed by centrifuging at -5C. The supernatant liquid S ~ is further processed.
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Step c involves the further treatment of the supernatant S 2. The ethanol concentration is set to 40 ~. Similarly as ln ~tep b, the temperature and the pH value are set to -7 C and to 5.8, respectLvely. By centrifuging, the precipitate P 3, also termed fraction IV, is o~tained.
The latter contains the ~-globulins and B-~lobulins.

' :i The supernat,ant liquid - Supernatant S 3 - is thereaf ter ~' further processed. To this end, the conditions of temperature ;~
~ 7C, pH = 408)and ethanol concentration = 40 % are '~ malntained. In this wayl precipitate P 4 is precipitated, namely to the so-called crude al~umin, which is purified an steriliæed in a manner known per se. The supernatant : liquid - Supernatant S 4 - is disposed of.
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The ~-globulin fraction II~III is further processed hy the following steps: ~
-.
..

Pr~cipitate P 2 is suspended in a citrate phosphate ~uffer a~ a pH of from 7.0 to 7.4. The temperature is held at 15C. 3 % of polyethylene glycol (PEG) 4,000 or 2.5 ~ of PEG 6,000 are added.

The PEG used in practice comprises a mixture of non-voIatile polyethylene glycols which are solu~le both in water and in organic liquids and which have a molecular weight on the order o from 4,000 to 20,000. A mixture of polyethylene glycols of this type having an average weight of 4,000 :is termed PEG 4,000.

Upon thorough agitation and after a reaction period of from 1/2 40 4 hours, the mixture is centrifuged, whereby ~z~
I

Supernatant S 5 and fraction III-1 (precipitate P 5) are ~ , , .
formed. Supernatant S 5 is again treated under modified conditions ~pH 4,6; PEG 4,000 up to about 5.5 %;
temperature = i5C) and fractionated. Fraction III-2 (precipitate P 6) and Supernatant S 6 are formed, the latter containing primarily the immune globulin IgG The globulins of fractions III-1 and III-2 are enriched or concentrated to substantial degree with the immune globulins IgG, IgA and IgM. Further, ~ antitripsin, ~2- haptoglobulin, coeruloplasmin, transferrin and haemopexin are present in concentrated form.

In general, fractions III-1 and III~2 are not further process a. These fractions are available in large quantities, because it is in the first line the a~bumin that is recovered from the blood, while the ~-globulins play a secondary role only.

As the starting products for the process according to the invention, mainly fraction IV as well as fractions III-1 and III-2 of the above-described fractionation are employed ~precipitates P 3, P5, P 6).

These fractions are mixed in predetermined rations bv adjusting the ratio to from 50 to 70 % of albumin of from 50 to 30 % of globulins, respectively, based on the total protein. X-t is possible in th~s way to select in the above-~entioned ratio the same values as are present in the native serum proteins, too.

Elowever, hy correspondingly increasing the additio:ns of the globulin portion or of the proportions of fractions III-1 and III-2, it is also possible to specifically increase the proportion of globulins in the form o IgM
~nd IgA.

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In a preferred mode of operation, it is contemplated that fractions IV, III-1 and III-2 ~sediments) are suspended in a normal saline solution or in a suitable bu~fer solution, respectively, i.n the following quantities:

Fraction IV fxom 30 to 80 percent ~)~ weight Fraction III-1 fxom 0 to 70 percent by weight Fraction III-2 from 0 to 70 percent by weight.

based upon a total protein content corresponding to 100 % each.

Likewise, it is possible to recover the serum protein compound from fractions III-1, III-2 or IV only~ and to prepare these fractions from separate fractions in a z~

stable, dissolved form as the.preparation or composition, for the concentration of specific plasma proteins~
Similarly, desired ratios of components may be produced - upon separately preparing the flnal products o~tained from fractions III~ 2 and from fraction IV.

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~he substanc~s mixed in a fi~ed ratio and suspended in a suitable buffer or saline solution are subjected to a pre-purification. In such pre-puriication, the lipid factors of the ~- and ~-glo~ul~n series are remaved by adsorption to suitable adsor~ents (Aerosil, bentonite or other sillcic acid containing complex compounds).

Thereupon, th~ product is purified by filtering and dialyzed, and the solution is concentrated. Normally, a seperate sterilizing step is not necessary. However~
such sterilizat~on may be performed in the well-known manner. For example, two methods are used for eliminating viruses:

(a~ Pasteurizing by heating to 60C for 1~ hours.
Thi.s method suffers from the drawback that numerou~
serum proteins are denaturaliæed under the above conditions. ~, - 13 - .

(b) Combination of B propiol~cthonetreatment and ultrav}olet irradiation~method according to Lo Grippo; Fed. Proceedings l5, page 518, 1959).
~his method allows to sterilize sera and plasmas under mlld conditions; the storage stability is not improved thereby, however.

In general, in accordance with the present process the mixture of the starting components, after having been aaiusted or standardized to a specific protein content and to a specific ratio of the proteins, is subjec~ed to a similar process as is conventional for the serum ~W. Stephan, loc. cit;). Surprlsin~ly~ it is in this .:
way possible to prepare a serum protein composition which is superior to the conventional composition in ;
lts e~ectiveness or activity, due to its increased content of immune globulines Ig~ or IgA, respectivelya Of course, it ~s feasible to remove from the individual fractions as such the constltuents tending to beCome denaturalized and thereafter to mix, concentrate an~ I
stabilize the purified fractions. Also, it is feasible to use as the startin~ material a ~ractionation product from a ~ractionation with rivanol. To its end, fractions/

1, 6~ -I II, III and IV according to STEINB~CH are suitable, I which fractions are described in Vox Sanguin.(V. 23, pages 92 to 106)~

Preferably, operating with adsorbents is effec-ted in a concentration of from 200 to 500 milligrams of silicic `~
acid per gram of total protein and at a temperature of up to 50C. The substances are intimately mixed, whereupon the adsorbent loaded with the lipoProteins is separated. `;

As experiments have shown, the most favorable results are obtained at temperatures of between 20 and 50C or at p~ values of between 6.5 and 8, respectivelv.

The followings examples serve to explain the process in greater detail. :~

Example 1 To 300 liters of normal saline solutian (aqua destillata containing 1.7 percent by weight of NaCl, adjusted to ~
pH of 4.6 by using 0j2 n of HCL) - termed solvent in the following -, 10 kp of COHN fraction IV showing the folloNing analysis of the protein constituents, are .

Zfi~4L r ~1 5 added and suspended therein:

55 % of albumin 10 % of ~1-glo~ulin 8 % of ~2-glo~ulLn 15 ~ of B-glo~ulin 12 ~ of ~ globulin Fraction IV contains about 50 g of solids CProtein. salts) and 50 % of residual moisture (N20; residual ethanol).

Further, S kp (kiloponds) of COHN fraction III-1 (subfraction) are added, w~ich fraction shows the following analysis of the solid constituents ~percent by weight):

15 % of albumin

2 % of ~1-globulins 13 % of ~2-globulins 17 ~ of B-globullns 53 % of ~ globullns, and 5 kp of COHN subfraction III-~ are suspended there~n, whlch subfraction has the follow1ng composition:

2~

8 ~ of al~umin ~ % Of ~.1-globulins 9 % Qf ~2-globulins 25 % of ~--globulins 55 % of ~-globulins.

The ration between solids and residual moisture is substantially identical in all of these fractions.

The 20 kp pf "moist" fractions are suspended and s~irred ~n the so~ent. ~ereby, the p~ is maintained at a constant value of 4~6. Soluble substances present in the fractions a~e sissolved or suspended ln the solvent. Coexisting, insoluble components cause turbidity. The insoluble substances comprise a portion of 12 percent by weight, based on the total content of solid substah~es. The .
lnsoluble substances involve particularly denaturalized globulins, including lipoproteins wh~ch are not required in the further course of the process. These latter substances are therefore removed in a centrifuging s-tep.

The supernatant li~uid is slightly turbid and of yellowish color.

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. By adding 0.2 n of NaOH, the pH is adjusted to 7.4. Pure colloidal silicic acid is added to the solution untiL a concentration of 5 percent by weight is reachecl, and this silicic acid is admixed by stirring. The :Liquid is heated to 45C (about 1C per minute), whi.le ]ceeping the p~l constant. When the final temperature of 45C is reached, the liquid i5 agitated for 4 hours. Duriny this process step, the storage instable proteins are bonded to the silicic acid to form a precipitate which is removed by centrifuging.
The supernatent liquid from the centrifuying step is subjected to a clarifying filtration over active carbon filters (~,anufactured by the firrm Seitz (AKS-Filter)). The thus obtained filtrate is a clear liquid of arnbe~ color.
The liquid is concentrated to about 25~ of the initial vol~ne by means of a dialysis concentration method (Millipore~ cassette syste, pore size of the membrane or diaphragm 10,000 Daltons). Following this step~ the solution has a total protein concentration of from about 3.4 to 4.5 percent by weight.
An analysis shows that substantially all of the blood constituents having a moleeular weight of less than 10,000 have been removed from the concentrate by the dialysis concentration step. Tbese constituents include particularly those substances which are undesirable in a protein e.g. the oligo pep-tides showing a vaso-active effectiveness.
Following this, the concentrate is su~jected to a dialysis with three times the volume of a 9 percent weight NaCl solution for the final removal of the above-ment:ioned "impurities" and for the adjustment of the electrolyte. The ` - 17 .: . .

dialysis is performed in the same devices and with the same diaphragms as have been employed in the concentration step.
This processing is followed by another concentration step at the termination of which step a total concentration of the protein of from 6 to :l0~ is present.
Then, the phy~iological conditions of the isotonic pararneters accordlrig to the human blood are standardized by means of a NaCl solution, and the total protein concentration is set to 5 percent by weight.
The analysis of the protein recovered according to ~
example 1 shows the following:values: ~ ;
(based on 100 milliliters and a Protein - content of 5,000 milligrams) Albumin 2,925 "
IgG ~ 1,500 IgA 3 ao IgM ~ 285 "
The thus standardized;solution is additionally filtered for clarification through a germicidal filter (EKS
~20 II; Seitz,:Kreuznach). The sterlle filtration is performed with the aid of membrane filters.
In its finally bottlel ~tate, the composit.ion is now ready for intravenous administratlon.
~XAMPLE 2 ~ ~ In the salne manner as in example 1, the following ,~
: materials are dissolved in 300 liters o~ solvent:
7,5 kp of COHN fraction IV
4 kp of COHN fraction III-l and 8,5 kp of COHN fraction III-2 The composition of these fractions is identical to that of exarnple 1. The fractions are mixed by stirring with . - ..

the solvent, and the pEI is maintained at 4.6.
60 grams of Aerosil 2491/380 per liter of solution are added to the latter, and the pH i.s brought to 7.6. The IlliXtUre i5 agitated for 4 hours at 47C and thereafter coolecl to 18C and subjected to alluvial filtratiorl. q~his filtration step is perfor~ed in an upright alluvial ~r precoat filter, type CHF-S of the firm Schenk, E'ilterbau, Schwab. Gmund. As tXe filtering aid, ~yflo~ Super Cel is employed in a concentration of 8 percent by weight, based upon the filter volwne. Likewise, kieselguhr Celite~ 545 in a concentration of 5% may ~e used as the filtering aid.
EXAMPL~ 3 Following the purification, the various solutions are combined and subjected to a dialysis concentration method equivalent to that of example 1. However, the solutions, if desired, may be processed into the final concen-trate in the rnanner explained in example 1, whereupon these solutions may be selectively used individually or cornbined i.n any desired manner.

lQ kp of fraction III-l and 10 kp of fraction III-2 are di.ssolved in 200 liters of a phosphate-citrate buffer solution (0,066 moles of~phosphate-citrate), within a period of about 3 hours. The pN is set to 7~50, and after a further dilution to 300 liters by using the above-mentioned , ~, ~'` buffer solution and with the addition of 100 grams/liter of Aerosil/bentonite ~2:1), the solution is stirred or agitated for 4 hours at 45C. After cooling to 10C, the suspension is bright filtered on an alluvial or precoat filter precoated with 1,0 kg of kieselguhr-celite~ 545 per scluare meter of filter area with a filter performance of 50 liters/square meter hour. I~is is followed by purification filtration (using a Seitz-AKS4 filter) and by dialysis concentration a~, explained in examples 1 to 3. The fincll concentration of the solution ready for use is set to 5~ of protein. This provides the following average composition:
(Protein 5,000 milligrams) albumin about 1,800 to 2,000 rmilligranls globulins 3,000 to 3,200 milligrams IgG about 1,450 rnilligrams IgA " 750 "
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IgM " 500 ÆXAMPLE 5 Instead of a fractionated precipitation, the COEIN
fractions I to III and IV-l may be precipitated in a single step at a concent~ration of 40~ of ethanol and a pH of 5.8.
Fraction IV-l means a sub~raction of fraction IV (eompare e.g. U.S. Patent 2,710,293). The precipitate is isolated.
20 kp of the precipitate are suspended in 300 liters of solvent according to example 1 and further processed in the manner described in example 1.

It is known to effect a fractionated precipitation of the h~nan plasma with Rivanol (2-ethoxy-6, 9 diarnino-acidinil~acetate) (STEINBUCH; Vox Sanguin. 23, p~ 92 to 106). In accordance with the processes described in the latter publication, precipitates II, III and IV are obtained. These precipitates may be processed to yield the serum protein cornposition according to the invention, too.
10kp of precipitates II, III and IV each, a total of 30 kp, are suspended in 300 litres of the solvent accordiny to exarnple 1 and processed in accordance with the process explained in example 1.

Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process of preparing a serum protein composition for intravenous application or administration, in which process, starting from a human blood protein solution, a stablized, universally applicable preparation or composition is prepared which contains dissolved in an aqueous isotonic solution, such proteins characterized by performing the following steps:
(a) fractionating the human blood plasma and removing coagulation factors;
(b) recovering, during the said fractionation step, precipitates and supernatants at each different fractionation step;
(c) mixing and resolving of precipitates of the different fractionation steps, in which blood proteins, especially albumin and globulins, are preserved in a native form, said precipitates mixed and resolved in a chemically and physiologically adapted solvent, said solvent eventually containing also supernatants of the fractionation steps;
(d) adjusting the mixture to a content of 11 - 60% albumin and - 89% globulin, related to the entire protein content;
(e) stablizing, purifying and drawing off the preparation.

2. The process according to claim 1, characterized by the following process steps:
(a) Suspending the COHN fractions (sediments) IV, III-1 and III-2 in a suitable normal saline solution or a buffer solution, in the following proportions:
fraction IV 30 to 80 percent by weight fraction III-1 0 to 70 percent by weight fraction III-2 0 to 70 percent by weight each based on the total protein content =
100%, wherein the suspended solids are adjusted to a ratio of from 50 to 70% of albumin and from 50 to 30% of globulins, albumin and from 50 to 30% of globulins, (b) precipitating the components of the mixture which tend to become denaturalized, particularly lipoproteins, by adsorbing them to adsorbents of a specific affinity, and subsequently removing the concentrated of loaded adsorbents;
(c) concentrating and stabilizing the thus prepared solution.

3. The process according to claim 2, characterized by the following proportions of the COHN fractions:
fraction IV 40 to 60 percent by weight fraction III-1 20 to 30 percent by weight fraction III-2 20 to 30 percent by weight

4. The process as claimed in claim 2 or 3, characterized in that the separate fractions are individually separated from the components tending to become denaturalized, whereupon the purified substances are mixed, concentrated and stabilized.

5. The process according to claim 1, characterized by using as the starting substances, fractionation products of the fractions with Rivanol (fractions II, III and/or IV
according to STEINBUCH).

6. The process according to claim 1, characterized by using as said adsorbents, colloidal silicic acids in a concentration of from 200 to 500 milligrams of silicic acid per gram of total protein, which adsorbents are intimately admixed at temperatures of up to 50°C and subsequently separated.

7. The process according to claim 6, characterized by carrying out the process at temperatures of between 20 to 50°C.

8. The process as claimed in claim 6 or 7, characterized by carrying out the process at a pH in a range of from 6.5 to 8.