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Description  |
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FIELD OF THE INVENTION
This invention relates to the separation of complex protein mixtures and
more particularly to the fractionation and partial resolution of such
mixtures by combinations of electrodialysis and at least one of the
following steps: forced-flow electrophoresis, electrodecantation and
alcohol precipitation.
BACKGROUND OF THE INVENTION
Biological fluids usually contain a mixture of several proteins, and one of
the major achievements of modern biochemistry is to have devised methods
for their separation. Best example if blood plasma or serum, where methods
are available for identification and separation of at least 25 major
protein components (Schultze and Heremans: Molecular Biology of Human
Proteins, Elsevier, 1966). Other examples of naturally occurring complex
proteins mixtures is milk or whey, urine, spinal fluid, egg white, etc.
For the purpose of the present disclosure, it is helpful to define the
following protein nomenclature, the classification being based on their
solubility in a variety of solvents: (1) albumin is the major protein
component of plasma, serum, and egg white, and is characterized by being
soluble both, in half-saturated ammonium sulfate and in distilled water;
(2) globulins are those proteins of plasma or other biological fluids
which precipitate in half-saturated ammonium sulfate; (3) euglobulins are
those globulins which are not only precipitated in half-saturated ammonium
sulfate, but also in deionized water, as they apparently need some salts
to be soluble. Obviously, this classification is arbitrary, though widely
used in protein chemistry, as the solubility of all proteins depends also
on the pH of the solution, temperature, and other solutes present, such as
alcohol; (4) euglobulin-like materials; the term "euglobulin-like" is used
herein for those proteins which precipitate in deionized aqueous solutions
only in presence of various amounts of alcohol. These proteins are not
true euglobulins, being soluble in deionized water in absence of alcohol,
yet they possess some of the characters of the euglobulins, being
solubilized by even low concentrations of salts.
It will further help to define, for the purposes of this invention, the
following electrical membrane processes:
1. Electrodialysis is primarily used for the desalting of aqueous
solutions. Uusally, this is accomplished by means of ion-selective
membranes, said membranes allowing preferential passage of either
positively or negatively charged ions, as described in a variety of
patents including U.S. Pat. Nos. 2,694,680, 2,848,402, 2,860,091,
2,777,811. The usefulness of this technique for the separation of proteins
has not previously been recognized. Ion-selective membranes can also be
substituted by essentially electrically neutral membranes, with inclusion
of polyelectrolytes into certain compartments, these polyelectrolytes
becoming polarized along the membranes under the influence of an
electrical field, thereby conveying to the neutral membranes an element of
ion-selectivity as taught in U.S. Pat. Nos. 3,677,923 and 3,725,235. In
other electrical membrane processes, discussed in the following two
sections, some electrodialysis is unavoidably superimposed to other
effects sought, being a direct result of the passage of electrical
current. For the purpose of the present invention, the term
electrodialysis will be reserved to these electrical processes, the
primary purpose of which is desalting, preferentially accomplished either
with ion-selective membranes or with the use of polyelectrolytes.
2. Electrodecantation is an electrical process for concentration and
separation of a variety of colloids including proteins as taught in U.S.
Pat. Nos. 2,057,156, 2,292,608, 2,762,770, 2,800,448, 2,801,962. These
teach devices which contain a multitude of essentially
electrically-neutral membranes in a parallel array, the colloids or
proteins accumulating under the influence of the electrical current or
fields in the immediate neighborhood of said membranes and are decantable
along said membranes as a result of density gradients. An analogous method
is sometimes referred to as electrophoresis-convenction (in U.S. Pat. No.
2,758,966), where usually only a single pair of electrically neutral
membranes is employed for the purpose of creating electrodecantation in
the protein solution. These techniques have been widely used for protein
fractionation, principally for preparation of gamma globulins, these
proteins of plasma being isoelectric and not decanting. This technique has
not been taught for the preparation of serum albumin, the most mobile of
plasma proteins (in terms of electrophoresis). Among the objectives of the
present disclosure is to teach utilization of such techniques for
fractionation for the preparation of serum albumin.
3. Forced-flow electrophoresis includes devices similar to those for
electro-decantation, which also utilize a parallel array of electrically
neutral membranes, but the partitions are located between adjacent pairs
of membranes. Such partitions permit better control of flow patterns
within the apparatus, and also act as diffusion barriers. Two such
electrophoresis devices are described in U.S. Pat. Nos. 2,878,178 and
3,079,318, and the technique also is described as "forced-flow
electrophoresis" by M. Bier; "Electrophoresis", Academic Press, 1959, page
295.
The processes of electrodecantation and forced-flow electrophoresis are
similar in principle and results and for purposes of convenience will be
often referred to herein as electrofield separations. As set forth below
they may be used interchangeably.
The most important protein products of commerce are those obtained from
human or animal plasma or serum. Two such proteins, serum albumin and
gamma globulins, either from human or animal origin will be used as the
principal examples but the scope of this invention can also be applied to
other biological fluids or other proteins without modification. The
present commercial methods of obtaining these fractions are based on
alcohol fractionation, a process developed by Cohn et al and described in
U.S. Pat. Nos. 2,390,074, 2,770,6l6. This technique is essentially based
on sequential precipitation of various protein fractions by alcohol, under
controlled conditions of temperature, alcohol content, pH, and salt
content as summarized by C. A. Janeway, Adv. in Internal Med. 3, 295,
1949. This technique requires a large installation, the yield of certain
fractions is low, it requires prolonged exposure of proteins to high
alcohol content, which has a denaturing effect on some protein fractions.
The technique is also limited to production of certain fractions of plasma
only; other protein fractions are not recoverable in sufficient states of
purity.
THE INVENTION
The present invention relates to the fractionation and partial resolution
of protein mixtures, principally mixtures such as but not exclusively
plasma, serum, or their derivative fractions, using one or more of the
electrical processes above defined, either alone or in combination with
each other, and in combination with alcohol fractionation. The
fractionation scheme of this invention permits far greater flexibility in
terms of fractions obtainable as the electrical processes can replace some
or all of the fractionation steps in conventional alcohol fractionation,
resulting in substantial savings of money, time, installation costs, and
provide increased yield of products. More specifically, the invention
includes:
1. the process of fractionation of proteins including plasma or plasma
fractions, comprising causing the precipitation of an euglobulin fraction
by means of electrodialytic desalting under controlled conditions of
temperature, pH, and conductivity and recovering the dialysate.
2. the process of fractionation of proteins including plasma or plasma
fractions, comprising precipitation of an euglobulin-like fraction by
means of electrodialytic desalting under controlled conditions of
temperature, pH, conductivity, and alcohol content.
3. process of fractionation of proteins including plasma or plasma
fractions, comprising preparation of an euglobulin-like precipitate by
electrodialytic desalting under controlled conditions of temperature, pH,
conductivity, and alcohol content, followed by selective dissolution of
albumin-enriched fraction by re-adjustment of temperature, pH,
conductivity, or alcohol content.
4. process of plasma fractionation, comprising the steps including a first
precipitation by alcohol, a second step of electrodialytic desalting of
the supernatant of said first precipitation, said second step causing
precipitation of an euglobulin-like fraction, an elective third step
comprising selective dissolution of an albumin rich fraction from said
euglobulin-like precipitate, and a last step of alcohol precipitation of
an albumin-rich fraction from the combined supernatants of electrodialytic
desalting step or, alternatively from the combined supernatants of the
second step and the elective third step of fractionation.
5. process of improving fractionation of protein mixtures by
electrodecantation or forced-flow electrophoresis, comprising the
reduction of their salt content through prior electrolytic desalting, said
desalting causing also precipitation of euglobulins or euglobulin-like
materials.
6. process of improving fractionation of protein mixtures by
electrodecantation or forced-flow electrophoresis, comprising the
reduction of their salt content through prior desalting, and subsequent
addition of a buffering salt, said buffering salt being an ampholyte such
as glycine, said desalting causing also precipitation of euglobulins.
7. process of plasma fractionation, comprising a first precipitation by
alcohol, a second step of electrodialytic desalting of the supernatant of
said first precipitation, said second step causing precipitation of an
euglobulin-like fraction from said euglobulin-like precipitate, and a last
step comprising the selective concentration of an albumin-rich fraction by
means of electrodecantation of forced-flow from the supernatant of the
desalting step or alternatively the combined supernatants from the
desalting and the elective third step of fractionation.
8. products of manufacture suitable for use as plasma expander, and
comprising at least 90% of albumin, obtained by above processes, in
particular by processes 4 or 7.
These and other aspects of the invention will become clear from the
following detailed description.
DETAILED DESCRIPTION
A. Electrodialysis is widely used for desalting of aqueous solutions. In
the field of proteins it has received usage in desalting of milk, whey
(U.S. Pat. Nos. 3,433,726; 3,447,930; 3,595,766; 3,757,005; 3,754,650),
but these patents have no relations to present invention, as they are only
concerned with reducing the salt content of whey, rather than with the
incorporation of the desalting process into a complex scheme of
fractionation.
It is also well known that desalting causes precipitation of euglobulins.
The U.S. Pat. Nos. 2,669,559; 2,761,809; 2,761,811; 3,234,199 and
3,429,867 disclose the application of desalting by means of ion exchange
resin beds, for purposes of plasma fractionation, but this process has too
limited flexibility in terms of products obtainable to be of significant
practical value. In addition, ion exchange columns are difficult to
maintain in suitable state of cleanliness and sterility necessary for
protein fractionation.
It is the essence of this invention that it was discovered that
electrodialytic desalting can be a valuable tool in an overall scheme of
plasma fractionation, if used in conjunction with other techniques,
because the results of combining various techniques together increases the
usefulness of each in a previously unsuspected manner. Specifically:
Addition of various amounts of alcohol to desalted proteins causes
additional precipitation of unstable or euglobulin-like proteins. The
process is not quite identical to alcohol fractionation of proteins,
because the quality and composition of the precipitated fraction if
inordinately sensitive to temperature, pH, and smallest quantities of
electrolyte, characteristic of all euglobulin fractionations. The value of
this discovery is particularly significant in as much as the current
scheme of alcohol fractionation separates, in the first step, a so-called
Cohn fraction II and III, the precipitate comprising most of the gamma
globulins. The supernatant contains approximately 20% of alcohol already,
and if this is now desalted, one obtains a precipitation of
euglobulin-like fraction which contains most of the remaining globulins of
plasma (called alpha and beta globulins), which are undesirable in the
preparation of serum albumin. Moreover, because of the danger of hepatitis
infection, it is highly desirable to prepare the gamma globulins in the
time-honored manner by alcohol fractionation, the resulting product being
non-infectious. Thus the use of electrodialytic desalting fits into the
present scheme of fractionation by providing a gamma globulin product
prepared by presently acceptable methods, and additionally offering a
shortcut in albumin preparation. Hepatitis is not a problem in albumin
preparations, as they can be pasteurized by heat treatment as taught in
U.S. Pat. Nos. 2,705,230; 2,958,628.
In addition, because of the inordinate sensitivity to temperature, pH and
ionic strength of the composition of euglobulin fraction, this fraction
can easily be used to provide a variety of subfractions, yielding products
useful for the preparation of other plasma fractions.
The euglobulin-like fraction obtained at 20% alcohol content also contains
a significant amount of albumin which can be recovered by selective
dissolution, as shown in the examples. Again from such a source a further
variety of fractions can be obtained. It is not necessary to first
separate the euglobulin-like fraction from its supernatant to effectuate
selective dissolution, but the pH, temperature, conductivity, or alcohol
content of the desalted protein mixture can be adjusted in a multitude of
ways, again to be explained in the examples, showing that a new and
versatile tool of fractionation is obtained when combining the
before-mentioned factors of alcohol content, pH, conductivity and
temperature.
It is also not necessary that the first step in the fractionation be an
alcohol precipitation step, say the Cohn fraction II and II separation.
This procedure only fits best in the present scheme of gamma globulin
preparation. But it is also possible to first desalt the plasma, separate
or not separate the euglobulins formed, and then add alcohol to the
desalted plasma, to bring about additional precipitation of
euglobulins-like fraction. This euglobulin-like fraction precipitates
already at 10% alcohol content, while the Cohn fractionation requires
twenty percent of alcohol content for its first step, thus significant
amounts of alcohol can be saved. This fractionation scheme is particularly
attractive if only albumin is desired, and not gamma globulins, as is the
case with many animal sera, where albumin is the most significant product
of commerce. It is also useful if separation of the so called
macro-globulins or IgM immunoglobulins is desired. These are presently
lost in the scheme of Cohn alcohol fractionation, but can easily be
recovered in the first euglobulin fraction, being insoluble even in
absence of alcohol.
Optimal precipitation of euglobulins or euglobulin-like proteins occurs at
their isoelectric points, which is the point of their least solubility. It
is characteristic of properly carried out electrolytic desalting
procedures that the final mixture automatically comes to the pH
corresponding to the average isoelectric point of proteins in the mixture,
as all free ions are removed and only proteins are retained. In the case
of plasma, this corresponds to a pH of 5.3 .+-. 0.2 pH units. Because of
protein-protein interaction, there is co-precipitation of several
proteins, but the composition of the precipitate can be altered and
modified by adjusting the pH to a range of pH values from pH 6 to 4.8,
thereby significantly altering the composition of the precipitate, and
permitting selective precipitation of certain proteins, including the
aforementioned macroglobulins.
Precipitation of euglobulins in plasma begins at a specific resistance of
above 1,000 ohms.cm, but increases progressively until maximum desalting.
For best fractionation of euglobulins or euglobulin-like fractions, a
resistance in excess of 50,000 ohms.cm is necessary, most of the
fractionations having been carried out in the range of between 50,000 and
200,000 ohms-cm.
Temperature plays a significant role in the precipitation of the
euglobulin-like fraction. Most of the fractionation is carried out in the
temperature range of below 5.degree.C, but subfractionation of the
euglobulin-like fraction can be carried out at temperatures from about
15.degree.C and lower.
Summarizing, then, the optimal fractionation of proteins by electrodialytic
desalting is obtained within the following narrow ranges of conditions:
temperature below 15.degree.C, pH 5.3 plus minus 0.2, resistance above
100,000 ohms-cm. The influence of these parameters will be made more
specific in examples of actual fractionation.
The equipment for carrying out electrodialytic fractionation is not of
critical design, and several commercial instruments can be utilized. Most
of the experiments reported here were carried out with instruments
obtained from the Ionics Corp., B Watertown, Mass. It is important to
properly select paired ion exchange membranes which will cause
proportionate removal of positively and negatively charged ions from
solution, thus avoiding excessive changes in pH values. This has been
obtained with the Ionics Corp. membranes. Other instruments, would no
doubt give equally good results, and in some of the work home-made
apparatus was used, similar to that described in U.S. Pat. Nos. 3,079,318
and 3,677,923. Desalting can also be carried out using the process
described in the just mentioned U.S. Pat. No. 3,677,923, thus avoiding the
necessity of using ion-exchange membranes.
The protein solution is continuously circulated through the electrodialysis
apparatus, refrigerated by means of heat exchangers, and a d.c. electric
field superimposed across the membranes to cause electrodialysis. The
electrolyte brine bathes the alternate sides of the membranes, and
gradually becomes more salt concentrated as it receives the salts from
plasma. This brine can be of any usually suitable composition. Its
composition or conductivity is not critical to the process.
In most fractionations using ion exchange membranes, a solution of about
0.5 gms/liter of sodium chloride was employed. Other electrolyte solutions
have been equally acceptable.
In experiments based on electrically neutral membranes, the "brine" was a
0.2% solution of polyacrylic acid, adjusted to pH 6 with sodium hydroxide.
For best temperature control, the brines are also cooled by cooling means
such as heat exchangers.
With a properly balanced system, the pH of the plasma gradually decreases
toward its average isoelectric point of pH 5.2 .+-. 0.2. Precipitation
begins at a specific resistance of about 1,000 ohms-cm, and a pH of about
5.6. If the starting product is not plasma, but an
alcohol-precipitation-derived fraction thereof, precipitation occurs when
a resistance of about 6,000 ohms-cm is reached, as some of the euglobulins
have already been eliminated. In either case, precipitation is most
complete at highest possible desalting, when the resistance is above
100,000 ohms-cm.
The protein should be circulated vigorously through the appropriate
chambers of the electrodialytic apparatus, in order to provide maximum
turbulence within the apparatus. This is well established in the art of
electrodialysis. A circulation pressure of 25 lbs/sq. in. was employed in
most experiments. This turbulence is also necessary to prevent deposition
of precipitating proteins within the apparatus, thus clogging of its
channels of flow.
In order to minimize the clogging problem, it is also possible to install a
continuous centrifuge in the flow circuit of the protein solution.
Precipitation of euglobulins is rapid, and their complete centrifugation is
obtained at relatively low speeds of centrifugation, 2,000 rpm being
sufficient. This expedient has certain advantages, as it enables
fractionation of the euglobulins as they are being formed, by collecting
and segregating the precipitates separately at the different pH or
resistance values. It is also advantageous to insert into the pathway of
protein circulation suitable monitoring instruments for automatic or
operator actuated monitoring for control of pH, resistance, and
temperature during the desalting process.
Should clogging of any part of the apparatus become apparent, as indicated
by a sudden increase of pressures, this can be easily remedied by adding a
suitable alkalinizing agent, such as sodium hydroxide solution in amounts
to raise the pH of the protein solution above pH 6. This causes rapid
dissolution of all precipitates. This declogging does not cause great
delay in the overall process of desalting. Electodialysis of sodium ions
is much more rapid than that of many other ions in the protein solution.
The overall time requirement for complete desalting is mainly limited by
these slower electrodialysing ions and not by the sodium ions. Thus it is
possible to completely desalt the protein mixture, then add sufficient
alkali to redissolve all the precipitates (which of course, decreases the
resistance), and then obtain a final product in a further, final pass
through the electrodialyzer, by which the added sodium hydroxide will be
removed. This avoids accumulation of the precipitate in the apparatus.
Most of the precipitation being sufficiently time-delayed, it occurs only
after exit from the dialyzer.
Another method to avoid precipitation and clogging within the apparatus is
by a periodic reversal of current polarity.
Because of the requirement of numerous recirculations of the plasma through
the apparatus before complete desalting is obtained, the process is
essentially a batch process. However, it can be rendered semi-continuous,
by a sequenced operation wherein an intermediary vessel receives a portion
of the total protein solution, this portion is then completely desalted by
repeated circulation through the electrodialyzer, and then replaced by a
new batch, as is well known in the art of process automation. By
sequencing the passage of the protein solution through successive dialysis
chambers the salt content in each successive chamber is reduced until the
final chamber where the salt content is at a minimum.
The power requirement for the electrodialysis is not critical. Most of the
experiments have been started with a current density of about 0.03
amps/cm.sup.2, necessitating less than 25 volts/cm. As the resistance of
the electrodialyzer progressively increases, due to increased resistance
of the protein solution, the voltage is gradually increased up to 100
volts/cm. Final current density is low, usually less than about 0.03
amps/cm.sup.2. The main limitation to the power is that is causes heating
of the solution. Control of the total power input is based upon monitoring
the temperature of the effluent streams. The temperature can be maintained
below any desired value, consonant with the stability and sanitary
management of the protein solutions, i.e., it can be maintained throughout
the experiment at below about 5.degree. or 10.degree.C.
Sanitation is of utmost importance in protein fractionation. The complete
electrodialyzer apparatus, all connections, tubing, pumps, etc., are
sanitized in situ by conventional procedures, such as rinsing with dilute
sodium hydroxide, hydrochloric acid, hypochlorite or other suitable
agents. Rinsing with sodium hydroxide is preferred as it is also an
effective means of removing precipitated proteins. B. Both, forced-flow
electrophoresis and electrodecantation have been used for fractionation of
plasma proteins as taught in U.S. Pat. Nos. 2,801,962; 2,878,178;
3,079,318. The usual objective has been the isolation of gamma globulin.
The reason for this focusing on gamma globulin is that these methods are
easily and directly applicable because gamma globulin is isoelectric or
near isoelectric over a relatively broad pH range around neutrality. The
above two methods essentially differentiate only between isoelectric and
mobile components. In both methods, the mobile components are brought to
electrodecant, and the supernatant is composed mainly of isoelectric or
near isoelectric components, which (by definition of the term
"isoelectric" -- having equal positive and negative charge, i.e. having
zero net charge) are not affected by the applied electric field. The
decanting fraction contains most of the albumin, which is the
electrophoretically most mobile major component of plasma. Albumin so
fractionated however is heavily contaminated by globulins of intermediate
mobility, broadly referred to as alpha and beta globulins.
These methods of forced-flow electrophoresis or electrodecantation have not
yet found application for commercial production of gamma globulins or any
other plasma fractions. The reasons for it are numerous, and include:
1. Gamma globulin is not in short supply. More albumin is required than
gamma globulins.
2. The Cohn alcohol fractionation method yields a product free of
infectious hepatitis agents. It is not yet certain whether other methods,
such as the above electrical processes, would consistently yield an
equally safe product. As a result, many legal specifications require the
alcohol fractionation process. In view of the abundance of this product,
there are few incentives to change the process.
Equally important are, however, some purely technical shortcomings of these
two electrical processes, which are overcome by the present invention:
1. Plasma contains a number of relatively unstable proteins, which
precipitate readily, either as a result of low inherent solubility or
because of denaturation. As a result, when plasma is used, longevity of
the multi-membrane assemblies, used in these two electrical processes, is
limited because membrane fouling occurs as a result of precipitation. As
the assembly of these multi-membrane apparatus is an important cost
element, this renders the processes expensive. By practice of this
invention this problem is completely eliminated by either of the two
treatments discussed in previous sections: (a) alcohol prefractionation
resulting in the so-called Cohn fraction II + III supernatant, or (b) the
electrodialytic desalting. Either of these two initial fractionation steps
eliminate the unstable proteins, and no traces of membrane fouling is
observed.
2. Though the membranes employed in these two processes of electrophoresis
and electrodecantation are essentially electrically neutral, their
character is altered as a result of protein polarization along the
membranes caused by the electrical field, as observed and explained in
U.S. Pat. No. 3,677,923. An element of electrodialysis is thus
superimposed upon the fractionation process, and there is partial
desalting of the "isoelectric" fraction causing premature precipitation.
Euglobulins tend therefore to precipitate, and contribute to the
aforediscussed problem of membrane fouling. Obviously, this problem is
avoided in the present invention, as all euglobulins have been eliminated
in the electrodialytic desalting.
3. Plasma has a high salinity, corresponding approximately to 0.9% sodium
chloride. This severely limits the electrical field which can be applied
because the Joule heating caused by the electrical field is proportional
to the conductivity, i.e. salt content, of the processed fluid. As a
result, the processing rates are low (being again proportional to the
field applied), and, at best, marginal, from a commercial point of view.
Dilution has been advocated to remedy the high salt content in U.S. Pat.
No. 2,878,178, Example 3 but this is, at best, a palliative effect, and it
commensurately increases the volumes to be processed.
In the present invention, this problem is entirely eliminated. The effluent
of the electrodialytic desalting has no residual salts -- and thus
excessive heating as a result of the electrical field, is avoided. Heating
is deleterious, of course, because of purely sanitary considerations as
well as causing chemical degradation. Higher electrical fields can be
applied by the process of this invention thus resulting in faster
production rates, making the process economically more attractive.
4. Most of the salt content in plasma is actually sodium chloride, which
has no buffering action at the pH range where protein fractionation is
carried out. Thus, the pH of processed fluid is poorly controlled with
resulting uncertainty regarding the actual sharpness of the fractionation,
as the electrophoretic mobility of proteins are strongly pH dependent. The
addition of suitable buffers to untreated plasma can ameliorate the
situation, but is also adds to the overall conductivity of the solution,
which, as outlined above, is highly undesirable. Prior desalting of the
liquid being processed permits the suitable addition of any number of
buffers, such as phosphate, tris (hydroxymethyl) aminomethane, glycine,
and others, which exert their maximum buffering action in the desired pH
range, while maintaining the conductivity of the medium at an order of
magnitude lower than that of untreated plasma. For this purpose,
particularly suited and preferred are amphoteric buffer salts, for example
glycine, which while stabilizing the pH, do not contribute significantly
to the conductivity of the medium. Other amphoteric substances are the
various other amino acids, including alanine, or di or tri-peptides,
including glycylglycine and glycyl-glycyl-glycine. Such products are
readily available in commerce, and provide a sutiable range of isoelectric
points.
5. Prior investigators have been unable to use techniques such as
forced-flow electrophoresis of electrodecantation for the production of
any other plasma fractions except gamma globulin. Albumin, in particular,
was not possible to prepare in sufficient purity for use as a plasma
expander, by any of the previous inventigators. This has been remedied in
the present invention, as a result of:
a. elimination of the precipitate of the Cohn II and III fractionation
steps;
b. the precipitation of euglobulins or euglobulin-like materials by
electrodialytic desalting;
c. by the improved conditions prevailing during the fractionation as a
result of the lower salt content and introduction of appropriate buffer
into the processed fluid, as explained under 3 and 4 above; and
d. finally, by permitting the use of special conditions during the
fractionation itself.
These special conditions (d) merit more detailed discussion:
In either electro-decantation or forced-flow electrophoresis, the influent
stream is divided into two fractions. The most mobile components are
segregated into the decanted fraction, at the bottom of the
membrane-defined compartments. These include the desired albumin fraction.
The less mobile or isoelectric components, are segregated to the top of
the membrane-defined compartments. The relative distribution of components
in the two effluents, which will be referred to, for brevity's sake,
hereinafter as the top and bottom effluents, is a function of many
factors, including the applied field, conductivity, temperature, relative
concentrations and the mobility of each component of the mixture.
As a rule, at constant top flow, the slower the bottom flow the higher its
total protein content. It has now been discovered that paralleling this
increased concentration of protein, there is also an increased purity of
the albumin fraction, recovered in the bottom effluent. For optimum
protein concentration, it is necessary to maintain the bottom effluent at
a concentration between 15-25% total protein content. This is preferably
achieved by maintaining the top to bottom flow rates in a ratio of between
8:1 and 15:1, depending on the concentration of the starting supply.
Forced-flow electrophoresis and electrodecantation, according to this
invention, have been performed using the components of equipment as
described in U.S. Pat. No. 3,079,318. Three different modes of operation
have been successfully used. These are schematically illustrated in the
figures which are schematic presentations of the side views of the
membranes and filters used in this type of apparatus. The figures do not
show the spacers maintaining the components in their proper place, which
may include the inlet and outlet means. The solid lines represent
membranes which are of the type generally used in passive dialysis, i.e.
electrically neutral membranes, such as regenerated cellulose sold under
the trade name "Visking" by Union Carbide. The broken line represents
filters. These can be of many different types, including filter paper (for
instance Whatman No. 54), microporous filters as sold by the Millipore or
Gelma Corp., or certain type of battery separator elements as utilized by
the Mallory Corp.
The essential difference between filters and membranes, above described, is
that filters are permeable to proteins and permit gross liquid flow
through them. Membranes, on the other hand retain proteins and do not
allow gross liquid flow through them, but only slow
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