CA1043703A - Fractionation of proteins by electrical means - Google Patents
Fractionation of proteins by electrical meansInfo
- Publication number
- CA1043703A CA1043703A CA225,941A CA225941A CA1043703A CA 1043703 A CA1043703 A CA 1043703A CA 225941 A CA225941 A CA 225941A CA 1043703 A CA1043703 A CA 1043703A
- Authority
- CA
- Canada
- Prior art keywords
- euglobulin
- albumin
- fraction
- plasma
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
- B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
- B01D61/44—Ion-selective electrodialysis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2642—Aggregation, sedimentation, flocculation, precipitation or coagulation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/124—Water desalination
Abstract
ABSTRACT
A process for the fractionation of liquid solutions of protein mixtures which includes the steps of subjecting such solutions to electrodialysis in the pH range 4.8 to 6, until desalting of the solution commences, said electrodialysis being conducted at temperature below 15°C: and continuing said electrodialysis until the specific resistance of the mixture exceeds 1000 ohm-cm whereby a fraction of said protein mixture precipitates; and recovering the dialysate. Another aspect of the invention is an albumin concentrate for use in the preparation of plasma extenders, having at least 90% albumin, free from salts, euglobulins and euglobulin-like materials.
A process for the fractionation of liquid solutions of protein mixtures which includes the steps of subjecting such solutions to electrodialysis in the pH range 4.8 to 6, until desalting of the solution commences, said electrodialysis being conducted at temperature below 15°C: and continuing said electrodialysis until the specific resistance of the mixture exceeds 1000 ohm-cm whereby a fraction of said protein mixture precipitates; and recovering the dialysate. Another aspect of the invention is an albumin concentrate for use in the preparation of plasma extenders, having at least 90% albumin, free from salts, euglobulins and euglobulin-like materials.
Description
:
~37 [)3 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.
~ACKGRO~ID OF THE INVENTION
BiologiCal fluids usually contain a mixture of several 10 proteins, and one of the major achievements of modern bio-chemistry is to have devised methods for their separation.
Best example is blood plasma or serum, where methods are available for identification ancl separation of at least 25 major protein -components (Schultze and Heremans Molecular 15 Biology of Human Proteins, Elsevier, 1966). Other examples of naturally occurring complex proteins ~ixtures is milk or ~:
whey, urine, spinal fluid, egg white, etc.
For the purpose of the present disclosure, it is ~.
helpful to dçfine the following protein nomenclatuxe, 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 bv being ~ soluble hoth, .in half-saturated ammonium sulfate and in distilled water; (2) globulins are those proteins of plasm.a or other biological fiuids which precipitate in half-saturated ammonium sulfate; (3) euglobulins are those globulins which ~2--,, 7 01~
are not only precipitated in half-saturated ammonium sulfate, but al50 in deionized water, as they apprently need some salts to be soluble. Obviously, this classification is arbitrary, though widely used in protein chemistry, a~ 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 t'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 watex in absence of alcohol, yet they possess some of the characters of the euglobulins, being olubilized by even low concentrations of salts.
It will further help to define, for the purposes of this invention, the following e:lectrical membrane processes:
(1) Electrodialysis is primarily used for the desalting of aqueous solutions Usually, this is accomplished by means of ion-sele¢tive membranes, said membranes allowing preferential passage of either positively or negative1y charged ions, as described in a variety of U.S. patents including 2,694,680, 2,848,402,
~37 [)3 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.
~ACKGRO~ID OF THE INVENTION
BiologiCal fluids usually contain a mixture of several 10 proteins, and one of the major achievements of modern bio-chemistry is to have devised methods for their separation.
Best example is blood plasma or serum, where methods are available for identification ancl separation of at least 25 major protein -components (Schultze and Heremans Molecular 15 Biology of Human Proteins, Elsevier, 1966). Other examples of naturally occurring complex proteins ~ixtures is milk or ~:
whey, urine, spinal fluid, egg white, etc.
For the purpose of the present disclosure, it is ~.
helpful to dçfine the following protein nomenclatuxe, 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 bv being ~ soluble hoth, .in half-saturated ammonium sulfate and in distilled water; (2) globulins are those proteins of plasm.a or other biological fiuids which precipitate in half-saturated ammonium sulfate; (3) euglobulins are those globulins which ~2--,, 7 01~
are not only precipitated in half-saturated ammonium sulfate, but al50 in deionized water, as they apprently need some salts to be soluble. Obviously, this classification is arbitrary, though widely used in protein chemistry, a~ 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 t'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 watex in absence of alcohol, yet they possess some of the characters of the euglobulins, being olubilized by even low concentrations of salts.
It will further help to define, for the purposes of this invention, the following e:lectrical membrane processes:
(1) Electrodialysis is primarily used for the desalting of aqueous solutions Usually, this is accomplished by means of ion-sele¢tive membranes, said membranes allowing preferential passage of either positively or negative1y charged ions, as described in a variety of U.S. patents including 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 reco~nized. Ion-selective membranes can also be substituted by essentially electrically neutral membranes, with inclusion of polyelectrolytes into certain compartments, these polyelectrolytes becoming polarized alon~ the membranes under the influence of an electrical ~ie}d, thereby conveying to the neutral ~f'(~3 membranes an element of ion-selectivity as taught in : U.S. Patents 3,677,923 ~nd 3,725,235. In other electrical membrane processes, discussed in the following two sections, some electrodialysis is unavoidably superimposed to other effects sou~ht, being a direct result of the passage of electrical current. For the purpose of the present invantion, the term electrodialysis will be reserved to these electrical processes, the primary purpo~e of whi~h . 10 is desalting, preferentially accomplished either with ion-selective membranes or with the use of polyelectrolytes.
(2) Electrodecantation is an electrical process or -`, concentration and separation of a variety of colloids including proteins as taught in U.S. patents 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 electrisal current or fields in ~ the immediate neighborhood of said membranes and are i decantable along said membranes as a result of density gradients. An analogous method is sometimes referred i to as electrophoresis-convenction (in U.S. patent 2,758,966), where usually only a single pair of ! electrically neutral membranes is employed for $he purpose of creating electrodecantation in the protein solution. These techniques have been widely used for , ........ . . .
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6;!t3 protein fractionation, principally for preparation of gamma globulins, these proteins of plasma being iso~lectric 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.
(2) Electrodecantation is an electrical process or -`, concentration and separation of a variety of colloids including proteins as taught in U.S. patents 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 electrisal current or fields in ~ the immediate neighborhood of said membranes and are i decantable along said membranes as a result of density gradients. An analogous method is sometimes referred i to as electrophoresis-convenction (in U.S. patent 2,758,966), where usually only a single pair of ! electrically neutral membranes is employed for $he purpose of creating electrodecantation in the protein solution. These techniques have been widely used for , ........ . . .
~, . . . .. .
if - ~
6;!t3 protein fractionation, principally for preparation of gamma globulins, these proteins of plasma being iso~lectric 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 membrcmes. 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. patents 2,878,178 and 3,079,318, and the technique also is described as "forced-flow electrophorasis" by M. Bier, 2D "~lectrophoresis", ~cademic Press, 1959, paae 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 ~5 be used interchangeably.
~he most important protein products o~ commerce are those obtained from human or anima] plasma or serum. Two such proteins, serum albumin and gamma ~lobulins, either _5_ , .
3'~6~3 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 modi-fication. 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. patents 2,390,074, 2,770,616. 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 requi.res 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 fra ~ions are not recoverable in sufficient : :
states of purity.
THE INVENTION
According to the invention there is provided a process for the fractionation of liquid solutions of protein mixtures selected from the group consisting of plasma, serum and fractions derived therefrom which includes the steps of a) subjecting such solutions to desalting electrodialysis in the pH range 4.8 to 6; b~ conducting said electrodialysis at temperatures below 15 C; c) continuing said electrodialysis until the specific resistance of the resultant mixture exceeds , 1000 ohm-cm and a fraction of said protein mixture precipitates;
,, d) separating the precipitate from the supernatant soluble protein solution; and e) recovering said supernatant soluble protein solution.
~ - 6 -.," ~
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 conven-tional .
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., .
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alcohol fractionation, rssulting in substantial savings of money, time, installation costs, and provide increased yield of pxoducts, More specifically, the invention includes: .
5` ~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 temperatllre-, pH, and conductivitv and recovering the dialysate. :
~2) the process of ractionation of proteins including plasma or plasma fractions, comprising pre~cipitation I of an euglobulin-like fraction by means of electro-¦ dialytic 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 an albumin-enriched~fraction I by re-adjustment of temperature, pII, conductivity, ,1 or alcohol content,
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 ~5 be used interchangeably.
~he most important protein products o~ commerce are those obtained from human or anima] plasma or serum. Two such proteins, serum albumin and gamma ~lobulins, either _5_ , .
3'~6~3 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 modi-fication. 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. patents 2,390,074, 2,770,616. 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 requi.res 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 fra ~ions are not recoverable in sufficient : :
states of purity.
THE INVENTION
According to the invention there is provided a process for the fractionation of liquid solutions of protein mixtures selected from the group consisting of plasma, serum and fractions derived therefrom which includes the steps of a) subjecting such solutions to desalting electrodialysis in the pH range 4.8 to 6; b~ conducting said electrodialysis at temperatures below 15 C; c) continuing said electrodialysis until the specific resistance of the resultant mixture exceeds , 1000 ohm-cm and a fraction of said protein mixture precipitates;
,, d) separating the precipitate from the supernatant soluble protein solution; and e) recovering said supernatant soluble protein solution.
~ - 6 -.," ~
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 conven-tional .
` 1 0 ~ ~
., .
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alcohol fractionation, rssulting in substantial savings of money, time, installation costs, and provide increased yield of pxoducts, More specifically, the invention includes: .
5` ~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 temperatllre-, pH, and conductivitv and recovering the dialysate. :
~2) the process of ractionation of proteins including plasma or plasma fractions, comprising pre~cipitation I of an euglobulin-like fraction by means of electro-¦ dialytic 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 an albumin-enriched~fraction I by re-adjustment of temperature, pII, conductivity, ,1 or alcohol content,
(4) process of plasma fractionation, comprising the steps includinq a first precipitation by alcohol, a second step of electrodialytic desalting of the supernatant of said ~irst precipitation, said second step causing precipitation of an euqlobulin-like ~ 7 ~(~4;37(:3~
fraetion, an eleetive third step comprising seleetive dissolution of an albumin rich fraction from said euglobulin-like precipitate, and a last step of al-cohol precipitation of an albumin-rich Eraetion from the eombined supernatants of electrodialytic desalting step or, alternativel~ from the eombined supernatants of the second step and the eleetive third step of fraetionation.
fraetion, an eleetive third step comprising seleetive dissolution of an albumin rich fraction from said euglobulin-like precipitate, and a last step of al-cohol precipitation of an albumin-rich Eraetion from the eombined supernatants of electrodialytic desalting step or, alternativel~ from the eombined supernatants of the second step and the eleetive third step of fraetionation.
(5) process of improving fractionation of protein mixtures by electrodeeantation or foreed-flow electro-phoresis, eomprising the reduction of their salt eontent through prior electrolvtic desaltin~, said desalting causin~ also precipitation of eu~lobulins or euglobulin-like materials,
(6) proeess of improving fractionation of pr~tein mixtures by eleetrodecantation or foreed~flow electro-phoresis, comprising the reduetion of their salt eontent through prior desalting, and suhsequent addi-tion of a buffering salt, said buffering salt being an ampholyte such as ~ eine, said desaltin~ eausing also preeipitation of euglobulins.
(7) process of plasma fractionation, comprisiny a first - precipitation by aleohol, a second step o eleetro-dialytic desalting of the supernatant of said first preeipitation, said seeond step eausing preeipitation ~, of an euglobulin-like fraction from said euglo~ulin-like preeipitate, and a last step comnrising the selective eoneentration of an albumin-rich fraction . .
7~
by means of electrodecantation or forced-flow from the supernatant of the desalting step or alternatively - `
the combined supernatants from the desalting and the elective third step of fractionation.
7~
by means of electrodecantation or 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 9Q~ 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 descxiption.
DETAILED DE~SCRIPTION
A - Electrodialysis is widely used for desalting of aqueous solutions. In the field of p~oteins it has received usage in desalting of milk, whey (U.S. patents 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.5. patents 2,669,559; 2,761,809 2,761,811; 3,234,199 and 3,429,867 disclose ~he application of desalting by means of ion exchan~e resin beds, for purposes of plasma fractionation, but this process has too limited flexibility in terms of products ohtainable to be of signi-ficant practical value In addition, ion exchange columns are difficult to maintain in suitable state of cleanliness and sterility necessary for protein fractionation.
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It is the essence of this invention that it was discovered that electrodi~lytic desalting can be a valuable tool in an overall scheme of plasma fractionation, if used -~
in conjunction with other techniques, because the results of co~bining various techniques together increases the 'usefulness of each in a previously unsuspec~ed 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 is inordinately sensitive to temperature, pH, and smallest quantities of electrolyte, characteristic of all euglobulin fractionations.
The value of this discovery i5 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~ o~
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 ' 3!703 fractionation by providing a gamma globulin product prepared - by pre~ently acceptable methods, and additionally offering a shortcut in albumin preparation. Hepatitis is not a problem in albumin preparations, as they can be pasteurized S by heat treatment as taught in U.S. patent 2,705,230; 2,958,628.
In addition, because of the inordinate sensitivity to temperature, p~ and ionic strength of the composition of euglobulin fraction, this fraction can easily be used to provide a variety of subfractions, yielding products useful I 10 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 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, conducti~ity, 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 fractiona-tion is obtained when combining the before mentioned factors of alcohol content, pEI, 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 ~ separation. This procedure only fits best in the present scheme of gamma ~lobulin preparation.
But it is also possible to first desalt the plasma, separate or not separate the euglobulins formed, and then add alcohol ( , .
3~3 to the des~lted plasma, to bring about additionalprecipitation 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 fir~t step, thus significant amounts of alcohol can be ~avod. ~his fractionation scheme is particularly attractive if only albumin is desired, and not gamma giobulins, 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 IaM immuno-globulins 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 ~hat the final mixture automatically comes to the pH cor-responding 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 + O.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 o the precipitate, and permitting selective precipitation .. . . . .. . .
~3~3 of certain proteins, including the aforementioned macro-globulins.
Precipitation of euglobulins in plasma begins at a specific re~istance of above 1,000 ohms.cm, but increases progressively until maximum desalting. For best fractiona-tion of euglobulins or euglobulin-like fractions, a re-sistance in excess of 50,000 ohms.cm is necessary, most of ~he fractionations having been carried out in the range of be~ween 50,000 and 200,000 ohms-cm.
Temperature plays a significant role in the pre-cipitation of the euglobulin-like fraction. Mo5t of the fractionation is carried out in the temperature range of below 5C, but subfractionation of the euglobulin-like fraction can be carried out at temperatures from about 15C and lower.
Summarizing, then, the optimal fractionation of ¦ proteins by electrodialytic desalting is obtained within the following narrow ranges of conditions: temperature below 15C, 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 desi~n, and several com-mercial instruments can be utilized. Most of the experimen~s 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 p~l valu~s. This has been obtained with the IonicB Corp~ mQ~brQ~s, 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 ~.S. patents 3,079,318 and 3,677,923.
Desalting can also be carried out using the process described in the just rnentioned U.S. patent 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 qradually becomes more salt concentrated as it receives the salts from plasma. This brine can be of any usuall,y 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, `~
i the brines are also cooled by cooling means such as heat exchangers, ~ ?ith a properly balanced system, the pll of the plasma gradually decreases toward its average isoelectric point of -:
A~ .`! :
.
, . .
.
3 7 ~3 pH 5,~ + 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 a~out 6,000 ohms-cm is reached, as some of the euglobulins have aIready been eliminated. In either case, precipitation is most complete at highest possible desalting, when the resistance is abov~ 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 proteîns 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 o~tained at relatively low speeds o centrifugation, 2,000 rpm being sufficient. This Pxpedient has certain advantages, as it enables fractionation of the euglobulins as they ara 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 de-salting proces~
... .
, 37q~3 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 dl~olution of all precipitates. This declogging does not cause great delay in the overall process of desalting.
~lectrodialysis of sodium ions is much more rapid than that of many other ions in the protein solution. The overall time requ~rement for complete desalting is mainly limited by these ~lower 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 ohtain a final product in a further, final pass through the electrodialyzer, by which the added sodium hy-droxide will be removed. This avoids accumulation of the precipitate in the apparatus, Most of the precipitation being ~ufficiently 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 requiremen of numerous recirculations of the plasma through the apparatus before complete desalting is obtained, the process i5 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 ,, .
,, 37~)3 de alted hy repeated circulation through the electrodialyzer, and then replaced by a n~w batch, as is well known in the art of process automation. By sequencing the passaae of the protein solution through successive dialysis chambers the salt content in each successive chamber is reduced u~til 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/cm2, necessitating less than 25 volts/cm, As the resistance of the electro-dialyzer progressively increases, due to increased re-sistance of the protein solution, the voltage is qradually increased up to 100 volts/cm Final current density is low, usually less than about 0.03 amps/cm2. The main limitation to the power is that it causes heating of the solution Control of the total power input is based upon monitorin~
the temperature of the effluent streams. The ~emperature 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 or 10C.
Sanitation is of utmost importance in protein frac-tionation The complete electrodialyzer apparatus, alI
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. ~insing with sodium hydroxide is preferred as it is also an effective means of removing precipitated proteins.
3'~$
;
B - Both, forced-flow electrophoresis and electro-decantation have been used for fractionation of plasma proteins as taught in U.S. patents 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 becau~e gamma globulin is isoelectric or near isoelectric over a rela ively broad p~ 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 iso-electric 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 oE 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 me~hod yields a product free of infectious hepatitis agents. It is not yet cextain whether other methods, such as the above electrical ?
_18-..
~3~03 processe~, would consistently yield an equally safe product.
A~ 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, longevitv of the multi-membrane assemblies, used in these two electrical processes, is ~ limited because membrane foulin~ occur.s as a result OL
rZ precipitation. As the assembly o these multi-membrane apparatus is an important cost element, this renders the processes expensive. By practice o~ this invention this problem is completelv eliminated by either of the two treatments discussed in previous sections: (a) alcohol pre-fractionation resultin~ in the so-called Cohn fraction II ~ III
:' ?O 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 b~ the electrical field, as observecl and explained in U.S, patent, 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 approxi-mately 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 pro-portional 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. Patent 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 i6 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 con-siderations as well as causing chemical degradation. tligher 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 p~l .
~ 37~;)3 range where protein fractionation i5 carried out. Thus, the pH of processed fluid is poorly controlled with resulting uncertainty regarding the actual sharpness of the fractionation, as the elactrophoretic mobility of proteins are ~trongly S pH dependent. The addition of suitable buffer3 to untreated pla~ma can ameliorate ~he situation, but it also adds to the overall conductivity of the solu~ion~ 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) amino-methane, glyclne, and others, which exert their maximum buffering action in the desired pH range, while maintaining tha 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 p~, do not contribute slgni~icantly to the conductivity of the medium. Other amphoteric substances are the various other amino acids, including alanine, or di or tri-peptides, including glycyl-glycine and glycyl-glycyl-glycine Such products are readily available in commerce, and provide a suitable range of i~o-electric points.
5. Prior investigators have been unable to use techniques such as forced-flow electrophoresis or electro-decantation for the production of any other plasma fractions exceyt gamma globulin. Albumin, in particular, was not possible to prepare in sufficient purity for u e as a ~ plasma expander, by any of the previous investigators.
j This has been remedied in the present invention, as a , , .. . . . . . .
37~
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 desaltlng;
(c) by the improved conditions prevailing during the fractionation as a result of the lower salt content and introduction of appropriate buffer into the proces~ed 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 ~etailed discussion:
In either electro~decantation or forced-flow electro-phoresis, the influent ~tream 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 fractionD The less mobile or isoelectric com~onents, are segregated to the top of the 2~ membrane-de~ined compartments. The relative distribution of components in the two effluent~, which will be referred to, for brevity's sake, hereinafter as the top and bottom effl~ents, 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 o~ the albumin '' , , , . , . .: . . : .,.;, .
37~3 fraction, r~covered in kh~ bottom effluent. For optimum protein concentration, it i8 nece sary to maintain the bottom effluent at a concentration between 15-25% total protein content. This iB preferably ~chieved by maintaining the top to bottom flow rates in a ratio of between 8:1 and 15:1, depending on the concentration of the s~tarting supply.
Forced-flow electrophoresis and electrodecantation, a~cording to this invention, have been performed u~ing the components of ~quipment as des~ribed in U.S. Patent 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 figure5 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 ~A~ the trade ~ "Visking" by Union Carbide. The broken line represents filters These can be of many different types, including filter paper ~for in~tance Whatman No. 54), micro!
porous filters a~ 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 ultrafiltration. The 3~7IJ3 arrows in the ~igures indicate the direction of the flow of the liquids through the apparatus.
Figure 1 illustrates the electrodecantation mode, while figures 2 and 3 illustrate two modalities of forced-flow electrophoresis, differing in the location of the feed inlet. These two modalities difer little in their resultc and may be used interchangeably.
The fi~ures also illustrate the essential diference between ele~trodecantation and forced-flow electrophoresis.
In forced-flow electrophoresis the filters separate each electrophoretic compartment into two subco~partments.
The filters act, essentially as frictional boundaries between downward and upward flowing port:ions of liquid, and thus add substantially to the efficiency of the procedure. While, therefore, forced-flow electrophoresis is the preferred technique, essentially similar results are achieved by , electrodecantation.
3 ~ desirable element in the process, though not essential, is the separation of individual electrophoretic compartments from each other by channels for the flow of suitable electrolyte. The electrolyte primarily provides for maximum of internal cooling of the apparatus, and to i this purpose, the electrolyte is circulated through an external refrlaerating heat exchanger.
It is preferred that this electrolyte be a bufer, and that the ~ame considerations apply to it as discussed above with regards to the buffering of the protein solution bcing processed. Thus, phosphate, alycine, sodium octanoate, or other buffers can be utilized The electrical conductivity -2~-. :
i ~L043r7~3 of the buffer should be of the same order a~ the conductivity of the protein solution bein~ processed to assure a relatively uniform electric field throughout the apparatus, This buff~r also plays an additional important role in the fractionation 5 ` of alcohol-containing protein solutions. By diffusion through the membranes, a substantial part of the alcohol can be eliminated from the effluent protein solutions, where it is undesirable EXAMPLE I
This example demonstrates the difference between euglobulins and euglobulin-like materials, i.e. the difference between precipitation of desalted proteins in absence or presence of 10% alcohol. Two liters of bovine plasma were desalted in an electrodialysis cell consisting of two pairs of cationic and anionic membrane~, 9 x 10", until the specific electrical resi~tance of 200,000 ohms-cm, at pH 5.2 was reached. A voluminous precipitate was obtained, which was centrifuged at 0C. Analysis of the supernatant showed .
the presen~e of 84% albumin, with still about 4~ of gamma globulins. The addition at 0C of 10~ alcohol by volume to the sup~rnatant resulted in a second precipitation, which-again was centrifuged ~ff at the same temperature, The resulting supernatant then analyzed 95~ albumin, and less than 0.5% gamma ~lobulin.
EXAMPLE II
This example demonstrates the recovery through desalting of an albumin-enriched fraction from the so-called Cohn II ~ III
supernatant fraction, obtained by precipitation of plasma with 18% alcohol. It also demonstrates the advantage of selective ; ~ rediQ~olution of part of the albumin, precipitated with the euglobulin-like fractlon, and the effects of electrical re-si~tance and pH on the purity of fractions thus ob~ained.
~wo liters of the Cohn II ~ III supernatant were desalted as in experiment I, and separated into 100 ml. aliquots.
~ach aliquot wa~ proces~ed separately, all liquids being kept at 0C throughout, even during centrifugation.
After thorough desalting, the resistance of the protein solution was 240,000 ohms-cm, pH 5.2. In some samples the resistance was decreased by the addition of concentrated sodiwm chloride, which had but negligible ef~ect on pH. In two samples the pH was raised by the ; addition of sodium hydroxide. This also resulted in a significant lowering of resistance. After these adjustments, all aliquot samples were centrifuged, decanted and the pre-' cipitatc washed in half of the original volume of either i~ distilled water, or an alcohol solution of indicated co`n-- centration. The precipitate was thoroughl~ resuspended in this wash, and the resulting suspension was again centrifuged.
The supernatant is the 'recoverable albumin', while the residue is the precipitateO Data in Table I show the resulting al~umin content in all fractions: `
TABLE I
It can be readily seen that the purity of the recovered albumin is strongly influenced bv the alcohol content of the suspension medium. ~hile precipitates ma~ still have rela-tively hiah albumin content, the loss of albumin, considering the small wei~ht of the precipitate is less than 8% of total albumin~ the recovery being in excess of ~0~ in all instances.
'' :
7~;i;
TABLE I
Effect o:E pH, re~ifitance and alcohol content on purity of proeein fractions, $ample # Spec, resistance pH Supernatant ~Nash Recovery Precipita~e ~.ohmæ-cm) Alb. % % alcohol ~0 alb, % alb, 240,000 5.2 95, S 15 93.1 70,5 2 240,000 5,2 95,3 10 95,9 62.5 3 240,000 S,2 95,8 5 91.0 35.0 4 240,000 S, 2 97,0 0 85,2 37, 100,000 5.3 94,6 0 81,4 ~ 35,4 6 45,000 5.3 96,0 0 83,5 33,4 7 21,000 5,3 92, 8 0 79, S 27, S
8 10,000 5,3 92.1 0 71,2 2,5
plasma expander, and comprising at least 9Q~ 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 descxiption.
DETAILED DE~SCRIPTION
A - Electrodialysis is widely used for desalting of aqueous solutions. In the field of p~oteins it has received usage in desalting of milk, whey (U.S. patents 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.5. patents 2,669,559; 2,761,809 2,761,811; 3,234,199 and 3,429,867 disclose ~he application of desalting by means of ion exchan~e resin beds, for purposes of plasma fractionation, but this process has too limited flexibility in terms of products ohtainable to be of signi-ficant practical value In addition, ion exchange columns are difficult to maintain in suitable state of cleanliness and sterility necessary for protein fractionation.
_g_ .
, 3'7~
It is the essence of this invention that it was discovered that electrodi~lytic desalting can be a valuable tool in an overall scheme of plasma fractionation, if used -~
in conjunction with other techniques, because the results of co~bining various techniques together increases the 'usefulness of each in a previously unsuspec~ed 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 is inordinately sensitive to temperature, pH, and smallest quantities of electrolyte, characteristic of all euglobulin fractionations.
The value of this discovery i5 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~ o~
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 ' 3!703 fractionation by providing a gamma globulin product prepared - by pre~ently acceptable methods, and additionally offering a shortcut in albumin preparation. Hepatitis is not a problem in albumin preparations, as they can be pasteurized S by heat treatment as taught in U.S. patent 2,705,230; 2,958,628.
In addition, because of the inordinate sensitivity to temperature, p~ and ionic strength of the composition of euglobulin fraction, this fraction can easily be used to provide a variety of subfractions, yielding products useful I 10 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 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, conducti~ity, 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 fractiona-tion is obtained when combining the before mentioned factors of alcohol content, pEI, 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 ~ separation. This procedure only fits best in the present scheme of gamma ~lobulin preparation.
But it is also possible to first desalt the plasma, separate or not separate the euglobulins formed, and then add alcohol ( , .
3~3 to the des~lted plasma, to bring about additionalprecipitation 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 fir~t step, thus significant amounts of alcohol can be ~avod. ~his fractionation scheme is particularly attractive if only albumin is desired, and not gamma giobulins, 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 IaM immuno-globulins 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 ~hat the final mixture automatically comes to the pH cor-responding 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 + O.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 o the precipitate, and permitting selective precipitation .. . . . .. . .
~3~3 of certain proteins, including the aforementioned macro-globulins.
Precipitation of euglobulins in plasma begins at a specific re~istance of above 1,000 ohms.cm, but increases progressively until maximum desalting. For best fractiona-tion of euglobulins or euglobulin-like fractions, a re-sistance in excess of 50,000 ohms.cm is necessary, most of ~he fractionations having been carried out in the range of be~ween 50,000 and 200,000 ohms-cm.
Temperature plays a significant role in the pre-cipitation of the euglobulin-like fraction. Mo5t of the fractionation is carried out in the temperature range of below 5C, but subfractionation of the euglobulin-like fraction can be carried out at temperatures from about 15C and lower.
Summarizing, then, the optimal fractionation of ¦ proteins by electrodialytic desalting is obtained within the following narrow ranges of conditions: temperature below 15C, 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 desi~n, and several com-mercial instruments can be utilized. Most of the experimen~s 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 p~l valu~s. This has been obtained with the IonicB Corp~ mQ~brQ~s, 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 ~.S. patents 3,079,318 and 3,677,923.
Desalting can also be carried out using the process described in the just rnentioned U.S. patent 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 qradually becomes more salt concentrated as it receives the salts from plasma. This brine can be of any usuall,y 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, `~
i the brines are also cooled by cooling means such as heat exchangers, ~ ?ith a properly balanced system, the pll of the plasma gradually decreases toward its average isoelectric point of -:
A~ .`! :
.
, . .
.
3 7 ~3 pH 5,~ + 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 a~out 6,000 ohms-cm is reached, as some of the euglobulins have aIready been eliminated. In either case, precipitation is most complete at highest possible desalting, when the resistance is abov~ 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 proteîns 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 o~tained at relatively low speeds o centrifugation, 2,000 rpm being sufficient. This Pxpedient has certain advantages, as it enables fractionation of the euglobulins as they ara 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 de-salting proces~
... .
, 37q~3 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 dl~olution of all precipitates. This declogging does not cause great delay in the overall process of desalting.
~lectrodialysis of sodium ions is much more rapid than that of many other ions in the protein solution. The overall time requ~rement for complete desalting is mainly limited by these ~lower 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 ohtain a final product in a further, final pass through the electrodialyzer, by which the added sodium hy-droxide will be removed. This avoids accumulation of the precipitate in the apparatus, Most of the precipitation being ~ufficiently 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 requiremen of numerous recirculations of the plasma through the apparatus before complete desalting is obtained, the process i5 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 ,, .
,, 37~)3 de alted hy repeated circulation through the electrodialyzer, and then replaced by a n~w batch, as is well known in the art of process automation. By sequencing the passaae of the protein solution through successive dialysis chambers the salt content in each successive chamber is reduced u~til 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/cm2, necessitating less than 25 volts/cm, As the resistance of the electro-dialyzer progressively increases, due to increased re-sistance of the protein solution, the voltage is qradually increased up to 100 volts/cm Final current density is low, usually less than about 0.03 amps/cm2. The main limitation to the power is that it causes heating of the solution Control of the total power input is based upon monitorin~
the temperature of the effluent streams. The ~emperature 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 or 10C.
Sanitation is of utmost importance in protein frac-tionation The complete electrodialyzer apparatus, alI
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. ~insing with sodium hydroxide is preferred as it is also an effective means of removing precipitated proteins.
3'~$
;
B - Both, forced-flow electrophoresis and electro-decantation have been used for fractionation of plasma proteins as taught in U.S. patents 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 becau~e gamma globulin is isoelectric or near isoelectric over a rela ively broad p~ 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 iso-electric 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 oE 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 me~hod yields a product free of infectious hepatitis agents. It is not yet cextain whether other methods, such as the above electrical ?
_18-..
~3~03 processe~, would consistently yield an equally safe product.
A~ 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, longevitv of the multi-membrane assemblies, used in these two electrical processes, is ~ limited because membrane foulin~ occur.s as a result OL
rZ precipitation. As the assembly o these multi-membrane apparatus is an important cost element, this renders the processes expensive. By practice o~ this invention this problem is completelv eliminated by either of the two treatments discussed in previous sections: (a) alcohol pre-fractionation resultin~ in the so-called Cohn fraction II ~ III
:' ?O 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 b~ the electrical field, as observecl and explained in U.S, patent, 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 approxi-mately 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 pro-portional 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. Patent 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 i6 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 con-siderations as well as causing chemical degradation. tligher 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 p~l .
~ 37~;)3 range where protein fractionation i5 carried out. Thus, the pH of processed fluid is poorly controlled with resulting uncertainty regarding the actual sharpness of the fractionation, as the elactrophoretic mobility of proteins are ~trongly S pH dependent. The addition of suitable buffer3 to untreated pla~ma can ameliorate ~he situation, but it also adds to the overall conductivity of the solu~ion~ 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) amino-methane, glyclne, and others, which exert their maximum buffering action in the desired pH range, while maintaining tha 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 p~, do not contribute slgni~icantly to the conductivity of the medium. Other amphoteric substances are the various other amino acids, including alanine, or di or tri-peptides, including glycyl-glycine and glycyl-glycyl-glycine Such products are readily available in commerce, and provide a suitable range of i~o-electric points.
5. Prior investigators have been unable to use techniques such as forced-flow electrophoresis or electro-decantation for the production of any other plasma fractions exceyt gamma globulin. Albumin, in particular, was not possible to prepare in sufficient purity for u e as a ~ plasma expander, by any of the previous investigators.
j This has been remedied in the present invention, as a , , .. . . . . . .
37~
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 desaltlng;
(c) by the improved conditions prevailing during the fractionation as a result of the lower salt content and introduction of appropriate buffer into the proces~ed 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 ~etailed discussion:
In either electro~decantation or forced-flow electro-phoresis, the influent ~tream 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 fractionD The less mobile or isoelectric com~onents, are segregated to the top of the 2~ membrane-de~ined compartments. The relative distribution of components in the two effluent~, which will be referred to, for brevity's sake, hereinafter as the top and bottom effl~ents, 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 o~ the albumin '' , , , . , . .: . . : .,.;, .
37~3 fraction, r~covered in kh~ bottom effluent. For optimum protein concentration, it i8 nece sary to maintain the bottom effluent at a concentration between 15-25% total protein content. This iB preferably ~chieved by maintaining the top to bottom flow rates in a ratio of between 8:1 and 15:1, depending on the concentration of the s~tarting supply.
Forced-flow electrophoresis and electrodecantation, a~cording to this invention, have been performed u~ing the components of ~quipment as des~ribed in U.S. Patent 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 figure5 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 ~A~ the trade ~ "Visking" by Union Carbide. The broken line represents filters These can be of many different types, including filter paper ~for in~tance Whatman No. 54), micro!
porous filters a~ 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 ultrafiltration. The 3~7IJ3 arrows in the ~igures indicate the direction of the flow of the liquids through the apparatus.
Figure 1 illustrates the electrodecantation mode, while figures 2 and 3 illustrate two modalities of forced-flow electrophoresis, differing in the location of the feed inlet. These two modalities difer little in their resultc and may be used interchangeably.
The fi~ures also illustrate the essential diference between ele~trodecantation and forced-flow electrophoresis.
In forced-flow electrophoresis the filters separate each electrophoretic compartment into two subco~partments.
The filters act, essentially as frictional boundaries between downward and upward flowing port:ions of liquid, and thus add substantially to the efficiency of the procedure. While, therefore, forced-flow electrophoresis is the preferred technique, essentially similar results are achieved by , electrodecantation.
3 ~ desirable element in the process, though not essential, is the separation of individual electrophoretic compartments from each other by channels for the flow of suitable electrolyte. The electrolyte primarily provides for maximum of internal cooling of the apparatus, and to i this purpose, the electrolyte is circulated through an external refrlaerating heat exchanger.
It is preferred that this electrolyte be a bufer, and that the ~ame considerations apply to it as discussed above with regards to the buffering of the protein solution bcing processed. Thus, phosphate, alycine, sodium octanoate, or other buffers can be utilized The electrical conductivity -2~-. :
i ~L043r7~3 of the buffer should be of the same order a~ the conductivity of the protein solution bein~ processed to assure a relatively uniform electric field throughout the apparatus, This buff~r also plays an additional important role in the fractionation 5 ` of alcohol-containing protein solutions. By diffusion through the membranes, a substantial part of the alcohol can be eliminated from the effluent protein solutions, where it is undesirable EXAMPLE I
This example demonstrates the difference between euglobulins and euglobulin-like materials, i.e. the difference between precipitation of desalted proteins in absence or presence of 10% alcohol. Two liters of bovine plasma were desalted in an electrodialysis cell consisting of two pairs of cationic and anionic membrane~, 9 x 10", until the specific electrical resi~tance of 200,000 ohms-cm, at pH 5.2 was reached. A voluminous precipitate was obtained, which was centrifuged at 0C. Analysis of the supernatant showed .
the presen~e of 84% albumin, with still about 4~ of gamma globulins. The addition at 0C of 10~ alcohol by volume to the sup~rnatant resulted in a second precipitation, which-again was centrifuged ~ff at the same temperature, The resulting supernatant then analyzed 95~ albumin, and less than 0.5% gamma ~lobulin.
EXAMPLE II
This example demonstrates the recovery through desalting of an albumin-enriched fraction from the so-called Cohn II ~ III
supernatant fraction, obtained by precipitation of plasma with 18% alcohol. It also demonstrates the advantage of selective ; ~ rediQ~olution of part of the albumin, precipitated with the euglobulin-like fractlon, and the effects of electrical re-si~tance and pH on the purity of fractions thus ob~ained.
~wo liters of the Cohn II ~ III supernatant were desalted as in experiment I, and separated into 100 ml. aliquots.
~ach aliquot wa~ proces~ed separately, all liquids being kept at 0C throughout, even during centrifugation.
After thorough desalting, the resistance of the protein solution was 240,000 ohms-cm, pH 5.2. In some samples the resistance was decreased by the addition of concentrated sodiwm chloride, which had but negligible ef~ect on pH. In two samples the pH was raised by the ; addition of sodium hydroxide. This also resulted in a significant lowering of resistance. After these adjustments, all aliquot samples were centrifuged, decanted and the pre-' cipitatc washed in half of the original volume of either i~ distilled water, or an alcohol solution of indicated co`n-- centration. The precipitate was thoroughl~ resuspended in this wash, and the resulting suspension was again centrifuged.
The supernatant is the 'recoverable albumin', while the residue is the precipitateO Data in Table I show the resulting al~umin content in all fractions: `
TABLE I
It can be readily seen that the purity of the recovered albumin is strongly influenced bv the alcohol content of the suspension medium. ~hile precipitates ma~ still have rela-tively hiah albumin content, the loss of albumin, considering the small wei~ht of the precipitate is less than 8% of total albumin~ the recovery being in excess of ~0~ in all instances.
'' :
7~;i;
TABLE I
Effect o:E pH, re~ifitance and alcohol content on purity of proeein fractions, $ample # Spec, resistance pH Supernatant ~Nash Recovery Precipita~e ~.ohmæ-cm) Alb. % % alcohol ~0 alb, % alb, 240,000 5.2 95, S 15 93.1 70,5 2 240,000 5,2 95,3 10 95,9 62.5 3 240,000 S,2 95,8 5 91.0 35.0 4 240,000 S, 2 97,0 0 85,2 37, 100,000 5.3 94,6 0 81,4 ~ 35,4 6 45,000 5.3 96,0 0 83,5 33,4 7 21,000 5,3 92, 8 0 79, S 27, S
8 10,000 5,3 92.1 0 71,2 2,5
9 6,300 5,3 96,2 0 6~.4 ` 9,2 58,000 . 5,4 94.9 0 74.6 3,1 11 30,000 5.7 91,2 0 60,2 3,2 .
3~03 EXAMPLE III
This example shows the effect of the temperature during the separation of the euglobulin-like precipitate from the alcohol-containing plasma. The starting material was the same as in experiment II, i.e. thoroughly desalted Cohn II ~ III supernatant. This was divided into 100 ml.
aliguot~, and centrifuged at the indicated (Table II) temperatures. All precipitates were resuspended in an equal volume of 10% alcohol in distilled water, and centri-~uged a second time, This washing step was repeated a second time, giving a ~econd recovexable albumin fraction and the final precipitate. All steps were carried out with strict control of temperature, as indicated, the data is reported in Table II.
T~BLE II
Percent albumin at the indicated temperatures Sample (0C) (5C) (10C) (15C) 1st supernatant97.7 94.0 31.8 85.5 1st recovery 94.1 91.2 90.1 85.5 2nd recovery 89.2 82.3 78~1 78.0 Precipitate 17.5 10.2 5.5 5.0 EX~LE IV
This example~illustrates the application of the process of the invention for the preparation of an albumin concentrate, using the steps of desalting of an alcohol~containing plasma , protein fraction, separation of a euglobulin-like protein i( precipitate, recovery of partiall~ precipitated albumin by a washin~ process in 10% alcohol, and, finally, concentration, and further purification using forced-flow electrophoresis .~ .
2~
:,. . , ~ , ~, .. .
7~)~
of the combined supernatants from the first euglobu~in-like precipitation and recovered protein.
The starting material was 10 liters of so-called Cohn Fraction IV-l supernatant which is a later intex-S mediary step in the current scheme of alcohol fractionation.
The starting material was first desalted as in Example I, using a five membrane-pair cell assembly. The precipitated euglobulin-like fraction was centrifuged and washed twice with one liter aliquots of a lO~ solution of alcohol in distilled water. The supernatants of this albumin recovery step were combined with the supernatant from the first centrifugation. The combined supernatants were adjusted to pH 7.5 using sodium hydroxide and concentrated by forced ,~ flow electrophoresis, using an assembly of five cells of `~ 15 the type illustrated in figure 2, yielding a bottom con-centrate of albumin and a top effluent fractian. The results of the fractionation are recorded in Table III.
T~BLE III
The albumin concentrate had only 5% alcohol content, while the original feed, namely the Cohn fraction IV-l had 40~ alcohol content. ~hus, the forced-flow concentration resulted in significant decrease of alcohol content due to dialysis. Desalting does not alter alcohol content.
.
.~ .
.
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:~ ~ ~ '`~ ~
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EX~MPLE V
~hiq experiment illustrates the new methodology used to recover valuable fractions from presently rejected materials. The starting material were 2,000 gms of wet pr~cipitate from the so-called step IV-4 of the Cohn alcohol fractionation scheme. This material contains mainly alpha and beta globulins, but also contains between 40 and 50% of albumin, which is presently wasted. This precipitate was suspended in 10 liters of water, and thoroughl~ desalted according to Example I. Then 1 liter of alcohol was added, while keeping the solution at 0C. This resulted in formation of a copious precipitate. The supernatant was clarified by centrifugation, and was found to contain about 1.6% of albumin, at 90.5% ~urity. A total of 180 gms of alhumin were recovered from the 2,000 gms. of paster having a total content of albumin of about 320 gms. Thus, over 50~ of albumin normall~ wasted was recovered.
- - .. :.
3~03 EXAMPLE III
This example shows the effect of the temperature during the separation of the euglobulin-like precipitate from the alcohol-containing plasma. The starting material was the same as in experiment II, i.e. thoroughly desalted Cohn II ~ III supernatant. This was divided into 100 ml.
aliguot~, and centrifuged at the indicated (Table II) temperatures. All precipitates were resuspended in an equal volume of 10% alcohol in distilled water, and centri-~uged a second time, This washing step was repeated a second time, giving a ~econd recovexable albumin fraction and the final precipitate. All steps were carried out with strict control of temperature, as indicated, the data is reported in Table II.
T~BLE II
Percent albumin at the indicated temperatures Sample (0C) (5C) (10C) (15C) 1st supernatant97.7 94.0 31.8 85.5 1st recovery 94.1 91.2 90.1 85.5 2nd recovery 89.2 82.3 78~1 78.0 Precipitate 17.5 10.2 5.5 5.0 EX~LE IV
This example~illustrates the application of the process of the invention for the preparation of an albumin concentrate, using the steps of desalting of an alcohol~containing plasma , protein fraction, separation of a euglobulin-like protein i( precipitate, recovery of partiall~ precipitated albumin by a washin~ process in 10% alcohol, and, finally, concentration, and further purification using forced-flow electrophoresis .~ .
2~
:,. . , ~ , ~, .. .
7~)~
of the combined supernatants from the first euglobu~in-like precipitation and recovered protein.
The starting material was 10 liters of so-called Cohn Fraction IV-l supernatant which is a later intex-S mediary step in the current scheme of alcohol fractionation.
The starting material was first desalted as in Example I, using a five membrane-pair cell assembly. The precipitated euglobulin-like fraction was centrifuged and washed twice with one liter aliquots of a lO~ solution of alcohol in distilled water. The supernatants of this albumin recovery step were combined with the supernatant from the first centrifugation. The combined supernatants were adjusted to pH 7.5 using sodium hydroxide and concentrated by forced ,~ flow electrophoresis, using an assembly of five cells of `~ 15 the type illustrated in figure 2, yielding a bottom con-centrate of albumin and a top effluent fractian. The results of the fractionation are recorded in Table III.
T~BLE III
The albumin concentrate had only 5% alcohol content, while the original feed, namely the Cohn fraction IV-l had 40~ alcohol content. ~hus, the forced-flow concentration resulted in significant decrease of alcohol content due to dialysis. Desalting does not alter alcohol content.
.
.~ .
.
3~
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:~ ~ ~ '`~ ~
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EX~MPLE V
~hiq experiment illustrates the new methodology used to recover valuable fractions from presently rejected materials. The starting material were 2,000 gms of wet pr~cipitate from the so-called step IV-4 of the Cohn alcohol fractionation scheme. This material contains mainly alpha and beta globulins, but also contains between 40 and 50% of albumin, which is presently wasted. This precipitate was suspended in 10 liters of water, and thoroughl~ desalted according to Example I. Then 1 liter of alcohol was added, while keeping the solution at 0C. This resulted in formation of a copious precipitate. The supernatant was clarified by centrifugation, and was found to contain about 1.6% of albumin, at 90.5% ~urity. A total of 180 gms of alhumin were recovered from the 2,000 gms. of paster having a total content of albumin of about 320 gms. Thus, over 50~ of albumin normall~ wasted was recovered.
- - .. :.
Claims (20)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the fractionation of liquid solutions of protein mixtures selected from the group con-sisting of plasma, serum and fractions derived therefrom which includes the steps of a) subjecting such solutions to desalting electrodialysis in the pH range 4.8 to 6;
b) conducting said electrodialysis at temperatures below 15° C; c) continuing said electrodialysis until the specific resistance of the resultant mixture exceeds 1000 ohm-cm and a fraction of said protein mixture precipitates; d) separating the precipitate from the supernatant soluble protein solution; and e) recovering said supernatant soluble protein solution.
b) conducting said electrodialysis at temperatures below 15° C; c) continuing said electrodialysis until the specific resistance of the resultant mixture exceeds 1000 ohm-cm and a fraction of said protein mixture precipitates; d) separating the precipitate from the supernatant soluble protein solution; and e) recovering said supernatant soluble protein solution.
2. The process according to claim 1 wherein the specific resistance of the resultant mixture upon completion of the electrodialysis is in the range of 50,000 to 200,000 ohm-cm and the temperature is main-tained below about 5° C.
3. The process according to claim 2 wherein the desalting electrodialysis is conducted until the final pH 5.3 ? 0.2 is reached, this being the isoelectric point of euglobulin and/or euglobulin-like proteins.
4. The process according to claim 3 wherein the precipitated fraction separated at d) is the euglobulin or euglobulin-like proteins in plasma.
5. The process according to claim 1 wherein said plasma-derived protein mixture is an albumin-containing fraction obtained by the Cohn alcohol process of plasma fractionation, said fraction containing at least 20% alcohol content, whereby the euglobulin and euglobulin-like fraction caused to precipitate by the electrodialysis process and separated at d) includes mainly alpha and beta globulins, thus resulting in greater purity of albumin in the recovered soluble protein fraction.
6. The process according to claim 5 wherein recovered desalted supernatant soluble protein solution from e) is treated with alcohol to precipitate its proteins, consisting mainly of albumin.
7. The process according to any one of claims l, 2 or 3 wherein the electrodialyzed solution or suspension is adjusted to a 10 to 20% of alcohol content, this alcohol content in electrodialyzed plasma or plasma-derived solutions or suspensions causing optimal frac-tionation of euglobulin or euglobulin-like proteins from supernatant soluble proteins, and resulting in higher albumin purity in the supernatant fraction and separating and recovering said supernatant fraction.
8. The process according to any one of claims 5 or 6 wherein the recovered euglobulin or euglobulin-like material is partially redissolved in a 5 to 15%
alcohol-containing solution and the resulting supernatant protein solution is combined with the recovered albumin-containing soluble protein fraction.
alcohol-containing solution and the resulting supernatant protein solution is combined with the recovered albumin-containing soluble protein fraction.
9. The process according to claim l wherein the recovered supernatant soluble protein solution from e) is further treated by buffering at the pH range of optimum protein electrophoretic mobility and then treated by an electrofield separation step.
10. The process according to claim 9 wherein the electrofield separation step is an electrodecantation.
11. The process according to claim 9 wherein the electrofield separation step is a forced-flow electro-phoresis step.
12. The process according to any one of claims 1, 2 or 3 wherein the precipitated fraction separated at d) is redissolved by adding an alkali solution to raise the pH to about 6 and then again electrodialyzing the redissolved precipitated fraction.
13. The process according to claim 9 wherein recovered supernatant soluble protein solution from e) is amphoteric, and is treated with amphoteric buffers having high buffering capacity and contributing little to increas-ing the specific electrical conductivity of the solution.
14. The process according to claim 9 or 13 wherein said amphoteric buffer is selected from the group consisting of tris (hydroxymethyl) aminomethane, glycine, alanine, glycyl-glycine, diglycyl-glycine.
15. The process according to claim 9 wherein said buffered product of the dialysis step is divided during said electrofield separation process into top and bottom effluents as a result of said separation process and the flow rates of said top and bottom effluents are maintained at a ratio of 8:1 to 15:1.
16. A process according to claim 1 for the preparation of an albumin concentrate suitable for use as a plasma extender which comprises the steps of treat-ing plasma with alcohol to precipitate and eliminate the Cohn II and III fractions, subjecting the supernatant to electrodialysis at temperatures below about 5° C until all euglobulins and euglobulin-like fractions have been precipitated, separating and recovering the precipitated fractions and the supernatant soluble protein solution, mixing the precipitated fractions in a 5 to 15% alcohol-containing solution, separating the residue from the supernatant to yield an albumin-rich supernatant, com-bining said albumun-rich supernatant liquid with supernatant soluble protein solution, buffering said com-bined liquids at the pH of optimum protein electrophoretic mobility and introducing said buffered liquid into an electrofield separation apparatus having an influent and top and bottom effluents, supplying an electrical potential to said apparatus to separate protein fractions contained in said buffered liquid between the top and bottom efflu-ents and collecting the albumin concentrate from said bottom effluent.
17. An albumin concentrate substantially free from ionic salts, euglobulins and euglobulin-like materials, prepared according to the process of claim 16.
18. An albumin concentrate from plasma for use in the preparation of plasma extenders, consisting of at least 90% albumin, said concentrate being substantially free from ionic salts, euglobulins and euglobulin-like materials.
19. A process for the fractionation of aqueous protein solution mixtures from naturally occuring biological fluids which includes the steps of a) subjecting said solu-tion mixtures to deionizing electrodialysis at a pH range of ? 0.2 of the isoelectric point of said mixtures and at temperatures below 15° C; b) continuing said deionizing dialysis until substantially all ionizable salts are removed from said mixture as indicated by the specific resistance or said dialysate increasing to above 50,000 ohm-cm; c) separating the precipitated protein fraction which is insoluble in the resulting deionized supernatant soluble protein fraction solution.
20. The process according to claim 19 wherein said protein solution mixture is derived from the class of naturally occurring biological fluids in the group consisting of milk, whey, urine, spinal fluid, egg white, blood plasma, serum and mixtures thereof.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/467,115 US3972791A (en) | 1974-05-06 | 1974-05-06 | Fractionation of proteins by electrical means |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1043703A true CA1043703A (en) | 1978-12-05 |
Family
ID=23854426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA225,941A Expired CA1043703A (en) | 1974-05-06 | 1975-04-30 | Fractionation of proteins by electrical means |
Country Status (13)
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|---|---|
| US (1) | US3972791A (en) |
| JP (1) | JPS50153696A (en) |
| BE (1) | BE828752A (en) |
| BR (1) | BR7502739A (en) |
| CA (1) | CA1043703A (en) |
| CH (1) | CH622436A5 (en) |
| DE (1) | DE2519909A1 (en) |
| ES (1) | ES437483A1 (en) |
| FR (1) | FR2270267B1 (en) |
| GB (1) | GB1496726A (en) |
| IT (1) | IT1032822B (en) |
| NL (1) | NL7505304A (en) |
| ZA (1) | ZA752829B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4204929A (en) * | 1978-04-18 | 1980-05-27 | University Patents, Inc. | Isoelectric focusing method |
| JPS5569298A (en) * | 1978-11-17 | 1980-05-24 | Kureha Chem Ind Co Ltd | Electrodeposition method of protein |
| US4238307A (en) * | 1979-02-14 | 1980-12-09 | Research Products Rehovot Ltd. | Electrodialysis process for the separation of essential amino acids from derivatives thereof |
| US4238306A (en) * | 1979-02-14 | 1980-12-09 | Research Products Rehovot Ltd. | Electrodialysis process for the separation of non-essential amino acids from derivatives thereof |
| FR2492269A2 (en) * | 1980-01-10 | 1982-04-23 | Ionics | ELECTRODIALYSIS APPARATUS AND METHOD FOR FRACTIONING PROTEIN MIXTURES |
| US4276140A (en) * | 1980-01-10 | 1981-06-30 | Ionics Inc. | Electrodialysis apparatus and process for fractionating protein mixtures |
| US4322275A (en) * | 1980-01-10 | 1982-03-30 | Ionics Incorporated | Fractionation of protein mixtures |
| US4351710A (en) * | 1980-01-10 | 1982-09-28 | Ionics, Incorporated | Fractionation of protein mixtures |
| DE3219248A1 (en) * | 1982-05-21 | 1983-11-24 | Solco Basel AG, Birsfelden | METHOD FOR OBTAINING CELL-BREATHING ACTIVE INGREDIENTS FROM CALF BLOOD |
| WO1988007406A1 (en) * | 1987-04-03 | 1988-10-06 | Gradient Pty. Ltd. | Improvements relating to separation of charged molecules |
| GB2225339A (en) * | 1988-11-15 | 1990-05-30 | Aligena Ag | Separating electrically charged macromolecular compounds by forced-flow membrane electrophoresis |
| US4963236A (en) * | 1989-03-08 | 1990-10-16 | Ampholife Technologies | Apparatus and methods for isoelectric focusing |
| US5160594A (en) * | 1989-03-08 | 1992-11-03 | Board Of Regents Of The University Of Texas System | Apparatus and methods for isoelectric focusing of amphoteric substances incorporating ion selective membranes in electrode chambers |
| US5650055A (en) * | 1993-04-07 | 1997-07-22 | Margolis; Joel | Electrophoresis separation method and apparatus using barrier separation and polarity reversing |
| AUPP790898A0 (en) * | 1998-12-23 | 1999-01-28 | Life Therapeutics Limited | Renal dialysis |
| CN103616429A (en) * | 2013-12-11 | 2014-03-05 | 郑秋月 | SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection method of true protein of dairy products and identification atlas database of method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3330749A (en) * | 1958-02-11 | 1967-07-11 | Takeda Chemical Industries Ltd | Process for treating amino acid solution |
| US3355375A (en) * | 1962-10-09 | 1967-11-28 | Swift & Co | Apparatus for electrophoretic fractionation of ampholytes |
| US3325389A (en) * | 1963-02-28 | 1967-06-13 | Ionics | Process for pretreatment of whey to increase demineralization rate |
| JPS4918226B1 (en) * | 1970-08-01 | 1974-05-08 | ||
| FR2131859B1 (en) * | 1971-03-30 | 1974-03-08 | Rhone Poulenc Sa |
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- 1974-05-06 US US05/467,115 patent/US3972791A/en not_active Expired - Lifetime
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- 1975-04-30 CA CA225,941A patent/CA1043703A/en not_active Expired
- 1975-05-01 ZA ZA00752829A patent/ZA752829B/en unknown
- 1975-05-05 GB GB18825/75A patent/GB1496726A/en not_active Expired
- 1975-05-05 DE DE19752519909 patent/DE2519909A1/en not_active Withdrawn
- 1975-05-06 CH CH582675A patent/CH622436A5/fr not_active IP Right Cessation
- 1975-05-06 JP JP50053312A patent/JPS50153696A/ja active Pending
- 1975-05-06 IT IT68154/75A patent/IT1032822B/en active
- 1975-05-06 ES ES437483A patent/ES437483A1/en not_active Expired
- 1975-05-06 BE BE156075A patent/BE828752A/en unknown
- 1975-05-06 BR BR3501/75A patent/BR7502739A/en unknown
- 1975-05-06 FR FR7514172A patent/FR2270267B1/fr not_active Expired
- 1975-05-06 NL NL7505304A patent/NL7505304A/en not_active Application Discontinuation
Also Published As
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| FR2270267B1 (en) | 1979-03-16 |
| JPS50153696A (en) | 1975-12-10 |
| NL7505304A (en) | 1975-11-10 |
| GB1496726A (en) | 1977-12-30 |
| DE2519909A1 (en) | 1975-11-20 |
| US3972791A (en) | 1976-08-03 |
| IT1032822B (en) | 1979-06-20 |
| ES437483A1 (en) | 1977-05-16 |
| CH622436A5 (en) | 1981-04-15 |
| BR7502739A (en) | 1976-03-16 |
| BE828752A (en) | 1975-09-01 |
| FR2270267A1 (en) | 1975-12-05 |
| ZA752829B (en) | 1976-04-28 |
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