US 3657116 A
A process for separating blood components, wherein an auxiliary substance is added to blood plasma or serum to precipitate fractions thereof. The auxiliary substance-containing fractions are introduced into a chromatographic column filled with a porous solid, and are eluted from the column with an aqueous solvent. The eluant is collected into fractions containing the pure serum or plasma components, free from the auxiliary substance. The porous substance is glass, produced by heat treating, comminuting and leaching with acid and alkali the solidified product of melting together B2O3, SiO2 and RO, wherein R is an alkali metal, alkaline earth metal or heavy metal.
Description (OCR text may contain errors)
1151 3,657,116 1 Apr. 18,1972
United States Patent Haller 210/198 C ....2l0/l98 C 210/198 C Liberti 1/1970 Dalton et al..
 PROCESS FOR THE SEPARATION OF BLOOD COMPONENTS Primary Examiner-Charles N. Hart  Filed:
ABSTRACT A process for separating blood components, wherein an aux-  Appl. No.:
iliary substance is added to blood plasma or serum to precipitate fractions thereof. The auxiliary substance-containing fractions are introduced into a chromatographic column filled with a porous solid, and are eluted from the column with an aqueous solvent. The eluant is collected into fractions containing the pure serum or plasma components, free from the auxiliary substance. The porous substance is glass, produced by heat treating, comminuting and leaching with acid and alkali the solidified product of melting together B 0 SiO and R0, wherein R is an alkali metal, alkaline earth metal or heavy metal.
3 Claims, 1 Drawing Figure  References Cited UNITED STATES PATENTS 3,114,692 12/1963 MacDonnell.......................
wmm OmZq MILLILITERS MINUTES PROTEIN ETHANOL PATENTEDAPR J 8 m2 Tmm nmzq MILLILITERS I MINUTES PROTEIN BACKGROUND OFTl-IE INVENTION This invention relates to a method for the separation of blood components, and more particularly the separation of plasma or serum components by way of s'teric chromatogap y Blood fractionation is a well-established and important part of clinical practice. In therapeutics, for example, fractions rather than whole blood are advantageous or even mandatory for certain treatments. Large amounts of the beneficial fraction may be introduced into a patient without overloading his system with other, potentially harmful, components. Many fractions are tolerated even if the stem from donors of a different blood type or even from animals, if the other components which are type or species specific are removed. Also, theioptimum conditions for preservation differ for the various substances in the blood; by fractionation each substance can be preserved and stored under its ideal conditions. Finally, it is more economic to fractionate blood and to administer its components to various patients, each receiving the fraction which is beneficial to his particular pathological condition.
The first step in blood fractionation is usually the removal of the corpuscular elements, erythrocytes, platle'ts, etc. These components sediment from the collected blood by gravity, or are removed by centrifugation. The supernatant clear liquid is called plasma and contains a large number of physiologically active substances. Serum, which is similar to plasma except that certain clotting-substances, notably fibrinogen, are missing, is obtained when the blood is collected without the addition of an anticoagulant. Coagulation sets in after collection; the coagulate is removed with the corpuscular components, leaving the clear supernatant, serum.
Both plasma and serum contain many components (mostly proteins) and various schemes for fractionati'ng them have been suggested and practiced in the past. Theone most widely used is fractional precipitation, in which the composition of the solvent for the plasma components is manipulated in such a way as to cause insolubiliz'ation of certain proteins or groups of proteins while the others stay in solution. For example, the pH, ionic concentrations, or temperature of the solvent may be varied. Or, precipitation-causing chemicals such as salts or solvents less polar than water, like alcohol or acetone may be added to the solvent. Disadvantages of the process are its complexity, the denaturing of certain proteins such as immunoglobulins due to the effect of the chemicals, and the need to remove the added chemicals.
The chemicals generally are removed by dialysis, or, if the chemical is volatile as in the case of ethanol, by freeze drying. Dialysis is 'very time consuming, particularly at the low temperatures which are required to prevent denaturation of the proteins. Dialysis furthermore requires a'membrane that is ab solutely inert against the involved chemicals. Freeze drying on the other hand requires complex equipment, close attention over a period of many hours, and has been'found to denature certain proteins, possibly because of the build-up of high salt concentrations during the evaporation process. It further requires the expensive maintenance of 7 low temperatures, especially when alcohol has been added to the plasma or serum.
My copeinding' application discloses a purely physical,
- molecular-sorting process for isolating desired protein fractions, such asimmu'nologic'ally active globulins from blood plasma or serum. The plasma or serum may be whole or p'refractionated by the conventional processes. Because these globulins are highly sensitive to chemical denaturation, my prior application addressed itself particularly, but not exclusively, to the isolation'of the globulins.
SUMMARY OF THE INVENTION The present invention provides a fractionation process for blood proteins, wherein the proteins or groups of proteins are first prefractionate d by conventional precipitation processes and the added precipitants introduced during these fractionation process arerernoved by way of steric chromatography on a suitable chromatographic substrate.
The described process is particularly useful in, but not limited to, fractionating proteins which are less sensitive then globulin to chemical denatu'ration, as for instance albumin.
Further objects and details of this invention will become apparent from v the following examples which are to be interpretedas illustrative and not in a limiting sense.
BRIEF DESCRIPTION OF THE DRAWING The single FIGURE of the drawing is a graph of protein and alcohol concentration in the effluent from a porous glass column, operated according to this invention.
Preparation of Porous Glass My prior application contains a general description of the process for preparing the chromatographic material used in this invention.
EXAMPLE I An alkali borosilicateglass is produced by mixing in a ball mill analytical grade sodium carbonate, boric acid and ground quartz in a proportion equivalent to a Na 0 to B 0 to SiO, weight ratio of 6.9 to 25.7 to 67.4, respectively. The mixture is fused in an electric furnace at l,200 C. until the major amount of H 0 and C0, is expelled. The temperature is then elevated to 1,450 C. and maintained while stirring for 5 hours. The melt is chilled by pouring into cold water. The resulting glass is then heated in an electric muffle furnace for 2 hours at 564 C.
The heat-treated glass is crushed into small pieces in a steel mortar and fractionated by screening between stainless steel screens of 50-100 mesh size (U.S.S. ASTM).
The glass has a silica-poor phase which is removed by contacting the particles with 3N HCl at 509 C. for a period of 6 hours. This contact also serves to remove any iron contamination picked up from the mortar. The acid solution is decanted and the fractionated glass particles subjected to a second acid leaching treatment for a period of 18 hours. The ratio of original glass powder to acid is maintained at essentially 50 grams to 400 milliliters. After the acid leach treatment, the discrete particles of glass are washed with water until the supernatant liquid is neutral and free of visible colloidal silica. In addition to water-soluble sodium borate, the silica-poor phase contains some silica which is precipitated in colloidal form during the leaching process. Most of this silica remains in the pores of the silica-rich particles.
The colloidal silicais removed from the pores of the silica rich particles'by contacting the particles with a 0.5 N NaOl-l solution at 25C. for 2 hours. Thereafter, the glass is washed with water until neutral, stirred with cold 3 N HCl for 2 hours and extracted with cold water for 20 hours. The essentially colloidal silica-free particles are then washed with boiling water for 4.5 hours in an extractor and vacuum dried at 100 C. for 24 hours. v
Subsequently, mercury intrusion pore size and pore volume are determined and calculated according to the method described in A5TM Bulletin (February 1959) by N. M. Winslow and J. J. Shappiro. The average pore diameter is A. and the pore volume is 0.5 cc/gram.
Fractional Precipitation of Blood EXAMPLE ll Blood containing an anticoagulant is obtained in the usual way, and its corpuscular components are removed by centrifugation. The resulting plasma is frozen and stored at 20 to 18 C. until further processing.
The frozen plasma is thawed by immersion in a water bath of 37 C. It has a pH of approximately 6.8. While progressively lowering the temperature of the mixture to 2.5 to C, cold 95% ethanol is added in a slow stream until the mixture contains 8% alcohol. Simultaneously a predetermined amount of 0.1 M sodium bicarbonate solution is slowly added to result in a pH of 7.2. The precipitate formed in this operation is removed in a Sharples centrifuge.
To the supernatant fluid resulting from above operation, sufiicient amounts of cold 95 per cent ethanol and 10 M acetic acid are added in slow streams to result in an ethanol concentration of 21 per cent and a pH of 6.8 while the temperature of the mixture is lowered to 6 C. The precipitate formed in this operation is removed in a Sharples centrifuge.
Water and sufficient 10 M acetic acid are now added to above supernatant until the mixture has an ethanol concentration of 19 per cent and a pH of 5.2 while maintaining its temperature at 6 C. The formed precipitate is removed by centrifugation.
To the supernatant of the last operation now enough 95 per cent alcohol is added to adjust the mixture to an ethanol concentration of 40 per cent while lowering its temperature to 7 C. The precipitated protein is allowed to settle for 24 hours and is recovered by centrifugation. The resulting paste consists typically of 28.5 per cent albumin, 28.5 per cent ethanol and 43 per cent water.
Steric Separation EXAMPLE III To the protein precipitate recovered from the last operation of Example ll one adds sufficient water to dissolve the precipitate while simultaneously adjusting the pH to 7.5 with 1.0 M sodium hydroxide. Typically it will require 140 cc water for 100 cc paste, resulting in a solution containing 12 per cent protein and 12 per cent ethanol.
The glass particles prepared according to Example I are now vibrated into a chromatographic column of cm length and 1.8 cm diameter, having a volume of 250 cc.
Water is then pumped at a rate of 25 cc/minute through the packed column. At 8 A minutes intervals, 40 ml samples of the redissolved protein paste are injected into the head of the column. The effluent of the column is collected.
The figure shows a diagram of time respectively effluent volume versus concentration of protein and alcohol in the fractions. The curves give the protein and alcohol concentrations from the effluent of the first injection only. The protein (left peak) emerges before the alcohol (right peak). Arrow A indicates the period of introduction of the first 40 ml portion. Arrow B indicates the introduction of the next following portion 8 minutes later. From each cycle, fractions CDE and FGH are pooled and pools from subsequent cycles are combined. The 1 meter long and 1.8 cm diameter column yields in 24 hours 5,000 cc of the CDE-pool with a concentration of 10.1 per cent protein and 5,000 cc of the FGH-pool with 5.2 per cent protein. Both pools do not have any detectable amounts of alcohol. The pools are now adjusted with water, sodium chloride, caprilic acid and sodium hydroxide to a protein concentration, isotonicity, pH and buffer capacity which is required for the particular clinical application. After filtration and heating for 10 hours to 60 C. the solutions are ready for use.
What is claimed is:
1. In a process for the fractionation of plasma or serum wherein plasma or serum is separated into fractions by way of fractional precipitation, induced by precipitants, and wherein the precipitants are subsequently removed, the improvement WhlCh comprlses that the precipitant-containing plasma or serum fraction is introduced into a chromatographic column filled with a porous glass, is eluted from the chromatographic column with an aequeous solvent and from the eluant emerging from the column such portions are selected which contain plasma or serum components but are free from said precipitant, said porous glass having been produced by heat treating, comminuting and leaching with acid and alkali, the solidified product of melting together B 0 SiO; and R0, wherein R is an alkali metal, alkaline earth metal or heavy metal.
2. The process according to claim 1 wherein said precipitant is ethanol.
3. The process according to claim 1 wherein said precipitant is ammonium sulfate.