PRODUCTION OF THERAPEUTIC FRACTIONS OF
HUMAN BLOOD SERUM
REMOVAL OF SALTS WITH ION-EXCHANGE RESINS*
ALLEN F. REID, P H . D . , AND FRANCES JONES, B.S.
From Baylor University, Graduate Research Institute, Dallas, Texas
INTRODUCTION AND SURVEY
The separation of blood serum protein fractions in greater than.milligram
quantities depends on the relative solubility of the proteins in different mediums.
Since protein molecules are large colloidal particles, their solubility is dependent
upon the concentration of the ions of the solution. In their separation, in order
to avoid denaturation of the proteins, it is necessary to keep the pH of the solution comparatively close to neutral. Three general methods may be employed
to fractionate the serum proteins by precipitation from their solutions:
1. The decrease of the ionic strength of the solution by removal of salts or by
dilution,
2. The increase of the ionic strength of the solution by addition of salts, and
3. The decrease of the dielectric constant of the solution by dilution with a
water-miscible organic solvent.
An excellent review of the considerations pertaining to these methods is given
by Anson and Edsall, 1 so that some aspects are only briefly set forth here.
The separation of plasma proteins by removal of salts from their solutions has
until recently been done by dialysis. This method was not found easily adaptable to quantity production, but was quite valuable for preparation of small
research fractions. The lowering of the ionic strength sufficiently for the precipitation of proteins by the dilution of a solution, on the other hand, resulted in such
a low protein concentration that no very effective precipitation was possible
except upon adjustment of the pH to the isoelectric point of the protein. Even
with this process the separated fractions were found to be complex mixtures of
many components.1 However, with the advent of ion-exchange resins, which
have the properties of sorbing both anions and cations, another and practical
approach was opened to the problem of decreasing the ionic strength of protein
solutions for fractionation; and this is the main subject of this paper.
The separation of plasma proteins by the addition of salts ("salting out") is a
conventional analytic method, at least as far as gross fractions are concerned.
In the precipitated proteins large quantities of salts are carried down, most of
which should be removed to give satisfactory therapeutic products. Until
recently, as in precipitation by salt removal, the only feasible method was
dialysis, which was impractical for large-scale production. The use of ionexchange resins for salt removal in such a process must be considered.
Large-scale separation of plasma proteins has been carried out with precipi
* Received for publication, August 20, 1948.
10
BLOOD SERUM FRACTIONS
•
11
tation technics involving the use of ethanol as a diluent. 1 ' 4 Because of the
denaturing effect on proteins by ethanol solutions, such fractionation has been
carried out at considerably reduced temperatures to minimize denaturation.
Because of the cost of the close engineering control of the low temperature
ethanol fractionation procedure, preparation of the human plasma fractions for
clinical use has remained in a few large commercial production centers. The
importance of a method which could be used by smaller organizations, such as
large blood banks and medical centers, is at once evident since it would afford
local utilization of a supply of raw material otherwise difficult to tap. It is
apparent that if ion-exchange resins could be successfully used as outlined above
for quantity production of plasma protein fractions, these products would be more
generally available to the medical profession as a whole at costs not beyond the
reach of the average patient.
METHODS AND APPLICATION
Cation-exchange resins normally have active phenolic, carboxyl or sulfonic
groups which are saturated with hydrogen ions or other cations.2 These cations
can be exchanged for one another, as, for example, when the hydrogen form of a
resin is placed in a solution containing excess sodium ions, some of the hydrogen
ions in the resin are replaced by sodium ions and an equal number of sodium
ions in the solution are replaced by hydrogen ions:
+
+
(1) HR + Na ?=± NaR + H
Anion exchange resins normally have active amine groups,7 which are bases for
anion exchange in a similar manner:
(2) ROH + CI ^ RC1 + OH
Thus, removal of NaCl from a solution can be accomplished by passing the
solution alternately through beds of suitable cation- and anion-exchange resins.
A practical property of the ion-exchange resins, which are available in granular
form, is their application to "multiplate" sorption or exchange. Thus, if it is
desired to remove an ion, A, quantitatively, a long column, packed with resin
free of the ion but able to sorb it, may be set up and the solution to be treated
introduced at the top. In the top layer the resin exchanges some of its sorbed
ions for A ions, the system approaching the steady state in which the rate of
sorption of A would be equal to the rate of desorption. The A-depleted solution
flows down to the next layer and is further depleted. This continues until, if
the column is long enough, the concentration of A in the solution is negligible.
After sorption to its working capacity, a resin may be "regenerated" by treating with a solution which will substitute other ions for the sorbed species. Theoretically, with a stable resin, the cycle of sorption-regeneration could continue
indefinitely; however, there is a practical limit.
By using ion-exchange resins to remove salts from plasma, protein fractions
were selectively precipitated out at the lower than normal concentrations at
which they were insoluble. Control of the pH to avoid denaturation was accomplished by using small quantities of resin in the alternate cation and anion
12
REID AND JONES
sorption steps. In a well buffered solution, such as plasma, the pH swing upon
substituting hydrogen ions to the solution for the cations sorbed by the resin is
less pronounced than in poorly buffered solutions. A similar buffer function
controls the pH swing upon substituting hydroxyl ions for the anions sorbed.
For extreme cases requiring very precise pH control, a mixture of anion and
cation exchange resins may be used; however, sorption effectiveness of the resins
is decreased since the exchange capacity of the resins for hydrogen and hydroxyl
ions is dependent on their concentrations in the solution.
In the utilization of ion-exchange resins for removing salts from blood serum or
plasma, cation- and anion-exchange resins were employed alternately keeping the
FLOW
DIAGRAM OF ALBUMIN
PRODUCTION
CENTRIFUGATION
4 i
PASTEURIZATION
K>
ADO
SODIUM
CAPRYLATE
DENATURED
HEMOGLOBIN
VACUUM
DRYING
CONC.
CAPRYLATE
REMOVAL
PYROGEN
REMOVAL
BACTERIAL
FILTER
FIG.
1
pH between 6 and 8. As the salt concentration continued to go down, globulins
precipitated out until the solution was essentially salt-free. At this time only
euglobulins, albumin, hemoglobin and other impurities were left in the solution.
Then, upon bringing the pH down to 5, the euglobulins precipitated. A solution
of albumin containing the hemoglobin which was in the original plasma remained.
Figure 1 is a schematic representation of the production of albumin from serum or
plasma.
The precipitated globulins were fractionated by dissolving in suitable aqueous
salt solutions and reprecipitating by ion-exchange removal of the salts and/or dilution. Desired degrees of refinement were obtained by repeated fractionation
using modifications of the procedure. This method of separation has shown
itself to be adaptable to varied systems allowing considerable latitude in meeting
individual requirements.
BLOOD SERUM FRACTIONS
13
A typical laboratory separation is outlined in the following first stage operation:
Portions of a cation-exchange resin and an anion-exchange resin were shaken repeatedly
with distilled water. On 15 ml. of the cation-exchange resin, 250 ml. of normal citrated
human plasma was poured. T h e mixture was stirred for two minutes, making sure t h a t
the p H did not drop below 6. The plasma, from which some of the metallic ions had been
removed, was then decanted onto 15 ml. of the anion-exchange resin. After stirring for
five minutes, making sure t h a t the p H did not rise above S, the plasma was decanted onto
a fresh 15 ml. portion of cation-exchange resin. This cation-anion exchange procedure
was repeated three times, after which considerable amounts of globulin had come out of
colloidal suspension. This was then centrifuged off and the resin t r e a t m e n t and globulin
removal continued for three more centrifugations. As the salt concentration became lower
the contact time of the resins with the solution became greater, up to about 20 minutes
at the end. Measurement of t h e sodium concentration then showed a concentration of
less than 0.1 millimole per liter. T h e s u p e r n a t a n t solution was then brought to a p H of 5
and the precipitated euglobulins centrifuged off. The separated globulins were pooled
and leached with successive 25 ml. portions of phosphate buffered (pH 7.4) saline solutions.
The ionic strengths of the solutions were progressively 0.001, 0.01, 0.03, 0.07, 0.09, 0.12,
0.14, 0.1S. In the first-stage separations:
a globulin predominated in the solution of 0.001 ionic strength,
/3 globulin predominated in t h e solution of 0.03 ionic strength, and
7 globulin predominated in the solution of 0.09 ionic strength.
Fibrinogen was recovered practically pure in the solution of 0.14.
In the above solutions boundary concentrations of the other globulins were found; and
in the other solutions mixtures of the proteins contiguous in solubility were present.
Sodium caprylate was added to .05 molarity of albumin solution and the pH
adjusted to 7.4. Upon reduction of the pH to 5.0 the hemoglobin was denatured
and was quantitatively removed by centrifugation. The caprylate also stabilized
the albumin so that it could be pasteurized. After removal of the sodium caprylate similarly to the removal of the other salts, pyrogenic contaminants, if
present, were quantitatively removed by passage of the solution through successive columns of a stable cation-exchange resin. Vacuum desiccation from the
frozen state of the albumin solution safely carried the albumin to any concentration desired.6 Passage of the albumin solution through a bacterial filter removed any particulate contaminants.
The alternative method of salting out the protein fractions and subsequently,
removing the salts by ion-exchange from solutions of the precipitated proteins
did not appear as practical for large-scale production, because it was found that
greater quantities of salts came down with the proteins than were in the original
serum or plasma. Furthermore, with the same precision of control, the fractions
precipitated by salting out were less discrete, at least when using the criterion of
electrophoretic mobility.1 The merit of the salting out method, as in all fractionation methods is dependent on the denaturation of the various proteins.
In an attempt to evaluate the relative amount of denaturation of proteins
fractionated by the various processes, pilot runs were made on the steps necessary
for the gross separation of the fractions containing titratible antibodies. Specimens containing the Rh antibodies were carried through the cold-alcohol fractionation procedure detailed by Cohn et aL,4 as "Method 6," but the final antibody
14
REID AND JONES
yield* was disappointingly small. Accordingly, since the technics for this fractionation have probably not attained the high degree of refinement in our laboratories as is used commercially, a program was worked out which would minimize
handling technics and evaluate almost directly the denaturation effects of the
conditions of fractionation. Each serum was adjusted to the salt concentrations,
ethanol mole fraction, pH and temperature as specified in "Method 6" for the
precipitation of the crude and globulin fractions. The precipitate was redissolved and the antibody titer compared with that of the original serum. Two
orders of Rh antibodies (saline agglutinins and cryptagglutinoids5) were used as
tracers in these experimental runs and in all cases the antibody activity after
this single precipitation was between 25 per cent and 40 per cent of the original.
Similarly, precipitation of the globulins from aliquots of the same serums was
accomplished by salting out in aqueous solution with sodium sulfate. The
precipitate was redissolved and the antibody activity compared to the original.
In the salting out process precipitation was carried out both at room temperature
and with a one-hour incubation period at 37 C. No appreciable loss in titer was
noted in the room-temperature runs. However, in the incubated runs some
antibodies were lost, indicating that the method inherently denatures some of
the less stable proteins.
In the simple salt-removal process, using the same serums, no appreciable loss
of antibody activity was evident even after repeated solution and precipitation
at room temperature to obtain a refined product.
Since urea has an appreciable denaturing effect on plasma proteins, 3 an additional advantage of the process using ion-exchange resins as described is the
quantitative removal of urea.
In the treatment of serum or protein solutions with certain anion-exchange
resins, some of the resin may be dissolved, causing undesirable reactions upon
intravenous injection. Small amounts of this contaminant can be quantitatively
removed upon subsequent treatment with a cation-exchange resin.8 However,
of the anion-exchange resins with suitable sorption and regeneration properties,
the relatively insoluble ones should be chosen for this purpose.
Similarly, as indicated in the description of the process, bacterial pyrogens can
be quantitatively removed on ion-exchange resins. In tests made of the
efficiency of this removal, a pyrogenic solution,f which gave a strong reaction
when diluted by a factor of 100 and a weak reaction when diluted by a factor of
10,000, after being passed through 107 cm. column of mixed anion- and cationexchange resins and then through an 84 cm. column of cation-exchange resins,
gave no detectible pyrogenic response. In the normal processing of the protein
solutions by the method described, the solutions are subjected to this treatment
* Rh antibody assay work reported in this paper was done by Dr. Sol Haberman and
Ruth Guy of the Department of Immunology, Baylor University Hospital.
t The pyrogens were kindly supplied to us for this purpose by Dr. M. V. Valdee of the
National Institute of Health, having been prepared from Aerobacter cloacae by the process
described by Welch et al." Pyrogenic assays were furnished by Dr. E. E. Muirhead of
Baylor University Hospital.
BLOOD SERUM FRACTIONS
15
at least twice, thereby probably reducing the original bacterial pyrogens by a
factor of more than 100,000,000. A positive indication of the safety of the
products is that in no instance was any pyrogenic reaction noted upon use of
products from this process in animal experiments or in clinical application.
Further demonstration is given in the reported use of quantities of ion-exchange
resins in the direct circulatory path of living animals with no detectable pyrogenic
reactions or untoward effects.8
SUMMARY
Large-scale production of human blood plasma protein fractions for therapeutic
use has been limited until the present to the use of cold-alcohol precipitation procedures. It has been found that, by the utilization of ion-exchange materials for
salt removal, fractions suitable for clinical use can be obtained. Deterioration
in the normally occurring properties of the proteins, which may occur under more
artificial conditions, apparently does not occur to an appreciable extent when
these proteins are precipitated at conservative values of pH by salt removal with
jon-exchange resins.
Acknowledgments.
T h e authors wish t o acknowledge the assistance of Margaret Robbins
and Finis Robbins on this project. They wish t o express their appreciation t o D r . J . M .
Hill and D r . Sol H a b e r m a n for their stimulating discussions and suggestions.
REFERENCES
1. ANSON, M . L., AND EDSALL, J . T . , E d i t o r s : Advances in Protein Chemistry. Vol. 3.
New Y o r k : Academic Press, Inc., p p . 383-479, 1947.
2. BAUMAN, W. C , AND EICHLORN, J . : Fundamental properties of a s y n t h e t i c cation exchange resin. J . Am. Chem. S o c , 69: 2830-2836, 1947.
3. BOYBR, P . D . , BALLOU, G. A., AND LUCK, J . M . : Combination of f a t t y acids and related
compounds with serum albumin; stabilization against urea and guanidine denaturation. J . Biol. Chem., 162:199-208,1946.
4. C O H N , E . J., STRONG, L. E . , H U G H E S , W. L . , J R . , M U L F O R D , D . J . , A S H W O R T H , J . , M E L I N ,
5.
6.
7.
8.
M., AND TAYLOR, H . L . : Preparation and properties of serum a n d plasma proteins;
system for separation into fractions of protein and lipoprotein components of biological
tissues and fluids. J . Am. Chem. S o c , 68: 459-475, 1946.
H I L L , J . M., HABERMAN, SOL, AND J O N E S , F . : Hemolytic R h immune globulins; evidence
for a possible third order of antibodies incapable of agglutination or blocking. Blood,
Special Issue 2 : 80-100, 1948.
H I L L , J . M . (Dallas, Texas), AND P F E I F F E R , D . C : New a n d economical desiccating
process particularly suitable for preparation of concentrated plasma or serum for
intravenous use; adtevac process. Ann. I n t . Med. 14: 201-214, 1940.
K U N I N , R., AND M E Y E R S , R . J . : Anion exchange equilibria in anion exchange resin.
J. Am. Chem. S o c , 69: 2874-2878, 1947.
M U I R H E A D , E . E . , AND R E I D , A. F . : Resin artificial kidney. J . L a b . and Clin. Med.,
33: 841-844, 1948.
9. W E L C H , H . , CALVARY, H . O., M C C L O S K Y , W. T . , AND P R I C E , C. W . : M e t h o d of p r e p a r a -
tion and test for bacterial pyrogen.
J . Am. P h a r m . A. (Scient. E d . ) , 32: 65-69, 1943.