Classifications

• • 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
• • 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
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/145—Ultrafiltration
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • 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
  • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
• • 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
  • B01D2311/10—Temperature control

Definitions

• This invention relates generally to a protein fractionation process. More specifically, the invention is directed to novel combinations of technology that allow for the simultaneous addition of potentially harmful de ⁇ naturing solvents to protein solutions at controlled rates, the simultaneous efficient exchange of heat gener ⁇ ated by the addition of these solvents to liquid, the control of concentration of protein, solvent, pH and ionic strength in complex mixtures of proteins such as blood plasma- Also this methodology can be utilized to remove organic solvents from protein solutions circumventing the harsh denaturing effect of solvent removed by freeze drying, flash evaporation or lyophilization and eliminat ⁇ ing need for centrifugation in certain phases of the Cohn cold ethanol process, specifically Cohn methods 6 and 9. The method is ideally adapted to a continuous cold ethanol process for protein or plasma fractionation.
• the Cohn process is currently practiced as batch process. Cooled plasma is treated with reagents i very large vessels. That is, after pH adjustment of th plasma or subfraction thereof, the protein solution i cooled to a specific temperature at or below 0° C t decrease solvent denaturing of the protein and to achiev conditions for correct precipitation. Precipitation i achieved by adding the precipitant (usually ethanol) wit stirring, the quantity being predetermined to achieve final concentration appropriate to the separation of th desired protein fraction.
• the precipitant usually ethanol
• Watt U. S. Patent No. 3,764,009
• W has attempte to control temperature and alcohol addition by introducin a method for the fractionation of protein solutions, par ticularly plasma, in which spatially projected convergen jets are combined with the protein precipitant to form mixture in which plasma protein is instaneously precipi tated and from which the protein is subsequently sepa rated.
• the degree that the five variables are con trolled determines the yield, purity, and extent of dena turation of the resultant protein fractions.
• Denaturatio of protein can occur from a combination of any or all o the following - turbulence and foaming, high alcoho concentrations, and heat generated by the alcohol addition or failure to keep temperature of the solution constant or, in the last step, when alcohol is removed from the protein.
• concentration of protein ' which is only initially controlled at the start of the process, these variables are all essentially uncon ⁇ trolled until near the final stages of each step.
• the present invention incorporates a number of principles for the precise control at any time of the six major variables of the Cohn method including (1) protein concentration, (2) rate of alcohol addition, (3) tempera ⁇ ture control by a unique heat exchange system, (4) control of pH and (5) conductivity, and (6) controlled agglomera ⁇ tion of the precipitating protein.
• This allows for the first time control of the significant variables of the Cohn method. Time required for completion of the complete fractionation cycle is reduced from 7 to 3 days, yields and purity are increased, and degree of denaturation is significantly reduced.
• the invention is a controlle rapid protein fractionation method which comprises th following steps.
• a cold protein-containing solution, suc as plasma, is circulated through a confined space define by one side of a porous membrane, such as a hollow fiber
• the temperature of the protein solution is controlled b circulating a liquid protein precipitant solution i contact with the opposite side of the membrane.
• the precipitant solution is caused to penetrate the mem brane and cause the precipitation of fine particles o plasma protein along the first side of the membrane.
• the the precipitated protein particles are separated. Th system may be backflushed with buffers to adjust p levels, Na+ concentration, and ionic strength.
• FIGURE 1 is a schematic representation of on form of system incorporating the present invention
• FIGURES 2a and 2b are schematic representation of one form of fractionation cell showing one mode o operation in FIGURE 2a and another mode of operation i FIGURE 2b;
• FIGURE 3 shows graphically the rate and concen tration of alcohol in plasma, the temperature of ingoin and outgoing plasma and the temperature of ingoing an outgoing alcohol, as measured in the practice of Exampl 1;
• FIGURE 4 shows graphically the rates of" alcoho addition and concentration in plasma, temperature o ingoing and outgoing plasma before and after alcoho addition and the temperature of the ingoing and outgoin heat exchanging alcohol measured in the practice o Example 2;
• FIGURE 5 shows graphically the condition measured in the practice of Example 3.
• the sys- tern includes an insulated plasma vessel 10, an insulated alcohol vessel 11, and a buffer vessel 12, each equipped with a stirrer 13, heat exchange means 14 and temperature indicating means 15.
• tank 12 is provided with conductivity sensor 16 and pH sensor 17.
• a pair of alcohol and plasma mixing and heat exchange fractionation vessels 18 are provided, arranged in parallel.
• each vessel or cell 18 includes a plurality of porous membrane fibers 19, all communicating at one end with an entry ⁇ chamber 20 and communicating at the other end with a discharge chamber 21.
• Each vessel is provided with an inlet port 22 and discharge port 23, each provided with a temperature sensor 15. All of the fibers 19 extend through and are surrounded by a central chamber 24 having an inlet port 25 and discharge port 26, each also having a temperature sensor 15.
• FIGURE 2a shows the cell in one mode of operation with ethanol (indicated by shading) passing through fibers 19 and plasma passing through the space surrounding the fibers .
• FIGURE 2b shows the oppo- site mode 2 in which the plasma passes through the fibers and the alcohol, again indicated by shading, passes through the surrounding space.
• Plasma vessel 10 is connected to the mixing cells 18 by means of pipe or tube forming line 27 extending from vessel 10 to the entry ports 22 of the mixing cells.
• Line 27 includes a controlled metering pump 28 to circulate the plasma.
• a temperature sensor 15, pH sensor 17, alcohol concentration sensor 29, protein sensor 30, flow gauge 31, pressure control 33, and pressure gauge 34 are all provided to detect and control conditions of liquid flowing through the line.
• Three-way valves 32 in the line enable the flow to be directed as desired.
• the discharge ports 22 of the mixing cells 18 are connected back to plasma vessel 10 through pipes o tubes forming line 35, these lines having temperature sen sors 15, pH sensors 17, alcohol sensors 29, flow gauge 31, pressure valves 36, and pressure gauge 34 to permi detection and control of flow conditions.
• Alcohol vesse 11 is connected through line 37 and pump 28 to the entr port 25 to the mixing cell 18 and through line 38 to th discharge port 26 of that cell.
• Line 37 includes flo gauge 31, pressure valve 36, and pressure control 33
• Buffer vessel 12 is similarly connected through lines 3 and 40 to the other of the mixing cells.
• the process involves preferably the use o hollow fibers of either Teflon, divinyl acrylonitrile polysulfone or cellulose acetate constitution.
• Alternat systems can include the utilization of spiral wound tubu lar membranes, plate and frame systems utilizing membrane of similar composition and pore size.
• the preferred hollow fiber membranes are manu factured as single, coherent structures that can tolerat pressures on either side of the active membrane surfac without rupture or damage to a support structure.
• Hollo fibers are made by "spinning" a polymeric solution whic upon setting or solidifying forms the anisotropic membran in a tubular configuration.
• These hollow fibers consis of a very thin smooth membrane surface on the inside o lumen of the fiber with a controlled pore density an molecular rejection coefficient, and a rough spongy mem brane structure on the outside of the fiber with a loose or more open pore configuration.
• This membrane structure lends itself to pressur izing both the inside and the outside of the hollow fiber
• Operating the hollow fiber module in an ultrafiltratio mode is accomplished by pressurizing the inside of th hollow fiber.
• Operating the hollow fiber module in "backflush” mode is accomplished by pressurizing the out side or shell side of the hollow fiber.
• the capability o backflushing hollow fibers is important to the use o hollow fibers for the addition of cold ethanol in the Coh process - Q .
• ethanol is a preferred protein precipi ⁇ tant
• solvents and solutions may be used. These include ethanol, butanol, acetone, mem ⁇ bers of ' the glycol series, " polyethylene glycol, dioxane, etc., neutral salts such as phosphate, sulfates, etc., or a mixture of reactants such as alchohol and salts, and finally block copolymer of ethylene oxide and polyisopro- pylene.
• the invention is applicable to the fraction- ation of protein solutions from whatever source, animal (human, bovine, porcine, etc.), bacterial, or plant.
• Plasma at between 4° C and -7° C is pumped through the lumen of the hollow fibers.
• This temperature can be sensitively and accurately controlled by pumping 50 per cent to 80 per cent ethanol at temperatures between -20° C to -10° C on the outside of the hollow fibers.
• the cold ethanol is added to the recirculating plasma inside the fibers simply by increasing the flow rate and adjust ⁇ ing the back pressure on the outside of the hollow fibers sufficient to cause the alcohol to penetrate the pores of the hollow fiber.
• local heat buildup is minimal due to the velocity (2-10 ft/sec) of the plasma through the hollow fiber and the thin (monomolecular) layer of alcohol continuously contacting the plasma at the membrane sur ⁇ face.
• This high surface area contact created by the hollow fiber membranes causes very small particles to pre ⁇ cipitate promoting sharp fractions, high yield, and mini ⁇ mal contact time for complete precipitation.
• This critical temperature control of plasma at the alcohol plasma interface is achieved by adjustments of the following easily controlled factors.
• the temperature and flow rate of the alcohol act in concert to effect efficient and instantaneous heat exchange and act syner- gistically with the flow rate and temperature of the plasma. Therefore, heat is effectively dissipated, local ⁇ ized concentrations of alcohol are minimized, and the plasma protein concentration is kept at concentrations favoring optimal formation of optimally sized protei precipitates.
• buffers can be carried out i similar * "backflushing" manner allowing for rapid an smooth equilibration to various pH levels, Na+ concentra tion, and ionic strength.
• the addition of cold alcohol and buffer solu tions can be accurately controlled by the pressure differ ential between the inside (lumen) of the hollow fibers an outside (shell) of the hollow ibers.
• the high velocit of the plasma flowing through the lumen of the fiber promotes the most efficient contact of alcohol with plasm and acts as an agglomerator to create fine particles o precipitating plasma while minimizing any heat of reactio caused by the mixing of plasma and cold alcohol.
• hollo fibers to add protein precipitants such as cold ethanol t plasma is the elimination of the need for open batc tanks, thereby reducing the risk of pyrogenic lots o plasma proteins.
• This process can be carried out i closed tanks since the pH, temperature, and alcohol addi tion are controlled within the hollow fiber module Special mixing devices and alcohol addition devices in th tanks are also eliminated since mixing and alcohol addi tion is carried out in the hollow fiber. Since the tem perature, particle size, and pH can be finely controlle by the use of hollow fibers, the need for very expensiv low shear pumps is eliminated and more moderately price low shear modified centrifugal pumps can be employed.
• the alcohol may be added to the plasma by reversing the compartments, i.e., alcohol flow- ing through the inside of the hollow fiber with plasma circulating on the outside. See FIGURE 2.
• FIGURE 2a the alcohol is circulated inside the hollow fiber.
• FIGURE 2b the alcohol is circulated outside the hollow fiber.
• FIGURE 2 Processing Data - Cohn Run #12 - Mode 1, FIGURE 2 -Cryo-Precipitated Plasma Sep -*-aration of Fraction I
• FIGURE 1 The pilot plant used for these examples i illustrated in FIGURE 1.
• the alcohol and plasma compart ments in cells 18 are shown in FIGURES 2a and 2b. Th mixing of alcohol and plasma and the heat exchange occur also in this interface.
• Starting cryo-precipitated plasm (1.6 liters, 12.2 mS at 24° C, containing 93 grams tota protein: 54.4 grams albumin and 13.5 grams IgG) is coole to +1° C without permitting ice to form.
• T obtain a final ethanol concentration in the plasma of per cent
• 178 ml of 80 percent ethanol were added to th plasma by increasing the rate of the ethanol flow until 2 psi of back pressure is observed.
• the addition of alcohol was completed in 70 minutes
• the plasma ethanol concentration wen from 0 per cent to 8 per cent
• the plasma temperatur dropped from 0° C to -2.5° C
• the ethanol temperatur climbed from -11° C to -8.5° C.
• the rate and concentra tion of alcohol in plasma, the temperature of ingoing plasma (prior to mixing with alcohol), the temperature of outgoing plasma (after alcohol addition), and the tem ⁇ perature ' of ingoing and outgoing alcohol are illustrated in FIGURE 3.
• Precipitate I was removed by centrifugation for 15 minutes, 27000 x G. Precipitate I was resuspended with ice to 420 ml. This precipitate is mainly fibrinogen. It contains 1.9 grams total protein (0.6 gram albumin and 0.4 gram IgG) . Supernatant I contains 87 grams total protein (51 grams albumin, 12.1 grams IgG) in a volume of 1550 ml. The conductivity of Supernatant I is 8.7 mS at 24° C. The protein cooling tank and the hollow fiber outside are cleaned with distilled, water and rinsed with 20 per cent ethanol.
• Supernatant I is placed into the plasma cooling tank and maintained at -2.5 to -3° C.
• the pH is adjusted to 6.8 ⁇ 0.05 by the addition of 3.1 ml of sodium acetate buffer.
• the supernatant is brought from 8 per cent ethan ⁇ ol to 20 percent ethanol by the addition of 310 ml of 80 per cent ethanol using the same flow rate and pressure conditions as outlined in the "Separation of Fraction I".
• FIGURE 3 shows that during this 30 minute addition, the supernatant I temperature is lowered to -6° C as the ethanol temperature climbs from -13° C to -6° C.
• Precipitate II + III is removed by centrifuga ⁇ tion for 15 minutes, -6° C, 27000 x G.
• Precipitate II + III is resuspended with ice to a volume of 1.16 liters. It contains 19.7 grams of total protein (1.9 grams albumin and 10.5 grams IgG).
• Supernatant II + III contains 60.5 grams total protein (43.7 grams albumin and 1.4 grams IgG) in a volume of 1.7 liters. Its conductivity is 4.8 mS at 24° C.
• the protein cooling tank and hollow fiber outside are rinsed with distilled water and 40 per cent ethanol. The 80 per cent ethanol is allowed to drop to -15° C in preparation for the separation of Fraction IV.. Separation of Fraction IV-
• Supernatant II + III is placed back into th plasma cooling tank and maintained at -6° C.
• the pH i adjusted' to 5.4 ⁇ 0.1 with the addition of 13 ml of sodiu acetate buffer.
• the supernatant is diluted to 18 per cen ethanol with the addition of 190 grams of distille crushed ice which is allowed to mix until the ice i dissolved. This step takes 25 minutes.
• Precipitate IV is removed by centrifugation fo 15 minutes, -6° C, 27000 x G. It is resuspended with ic to a final volume of 830 ml.
• Precipitate IV- contains 6. grams of total protein (1.6 grams albumin and 0.9 gra IgG).
• Supernatant IV.. contains 50.4 grams of total pro tein (39.4 grams albumin and 0.2 gram IgG) in a volume o 1.8 liters. Its conductivity is 5.3 mS at 24° C.
• the protein cooling tank is cleaned with dis tilled water.
• the 40 per cent ethanol is evacuated fro the outside of the hollow fiber. Separation of Fraction IV.
• Supernatant IV.. is placed into the cooling tan and maintained at -6° C.
• the pH is adjusted to 5.8 ⁇ 0.0 by the addition of 20 ml of 1.0 M NaHCO,.
• the supernatan is brought from 18 per cent ethanol to 40 per cent ethano by the addition of 998 ml of 80 per cent ethanol.
• Th same initial flow rates are used as in separation of Frac tion I, but during the addition the ethanol flow is in creased until 25 psi of back pressure is observed.
• FIGUR 3 shows that during this 80 minute addition, the plasm temperature is maintained between -6° C and -9° C as th ethanol temperature is kept below -7° C.
• Precipitate IV. is removed by centrifugation fo 15 minutes, -6° C, 27000 x G and is resuspended in ice t obtain a final volume of 730 ml. It contains 6.6 grams o total protein (1.6 grams albumin and 0.9 gram IgG) Supernatant IV. contains 40.2 grams of total protein (35. grams albumin and 0.0 gram IgG) in a volume of 2.6 liters. Its conductivity is 2.5 mS at 24° C.
• Supernatant IV is placed into the cooling tank and maintained between -7° C and -11° C.
• the pH is ad ⁇ justed to 4.810.05 by the addition of 38 ml of sodium acetate buffer.
• the plasma becomes milky white. This step takes 60-70 minutes and the plasma temperature is below -7° C throughout.
• Precipitate V is removed by centrifugation for 15 minutes, at -6° C, 27000 x G and is resuspended to a volume of 3.0 liters with ice.
• This precipitate is mainly albumin. It contains 37.5 grams of total protein by Biuret determination (37.7 grams albumin by immunochemical assay and 0.0 gram IgG).
• Supernatant V contains 2.6 grams total protein (0.1 gram albumin and 0.0 gram IgG) in a volume of 2.18 liters. Its conductivity is 3.0 mS at 24° C.
• Stabilized human plasma was prepared by the method outlined in U.S. Patents No. 3,998,946 and No. 4,136,094 (Condie). Stabilization of plasma results in the removal of fibrinogen, the clotting factors, the complement system, the kininogens, the lipoproteins, and the proteolytic enzyme plasmin plasminogen. Since it is free of fibrinogen, separation of Fraction I is not re ⁇ quired.
• the protein is recircu ⁇ lated through the outside of the hollow fiber and is referred to as Mode #1 (FIGURE 2a).
• Mode #2 When the addition of alcohol is accomplished with the protein on the inside of the hollow fiber, it is referred to as Mode #2 (FIGURE
• the protein is recircu lated through the outside of the hollow fiber (Mode #1) a a rate of 2500 ml/min.
• the cold ethano is recirculated through the inside of the fibers at a rat of 300 ml/min.
• the protein is recircu lated through the inside of the hollow fiber at a rate o 400 ml/min.
• the back pressure due only t the plasma is 12 psi.
• a screw clamp is applied to th ethanol out line until the total back pressure reaches 2 psi. This addition takes 70 minutes.
• the ethanol tem perature is kept below -5° C and the protein temperature is kept below -3° C throughout the addition.
• Precipitate II + III was removed by centrifuga ⁇ tion for 15 minutes, at -5 " ° C, 27000 x G. Precipitate II + III was resuspended with ice to 1270 ml. This precipi ⁇ tate is mainly IgG. It contains 132. grams total protein (3.4 grams albumin and 8.4 grams IgG). Supernatant II + III contains 89.1 grams total protein (75.9 grams albumin, 2.5 grams IgG) in a volume of 2160 ml. The conductivity of Supernatant II + III is 5.0 mS at 24° C. The protein cooling tank and the hollow fiber outside are cleaned with distilled water and rinsed with 40 per cent ethanol. Separation of Fraction IV.
• Supernatant II + III is placed back into the cooling tank and maintained at -3.5° C.
• the pH is ad ⁇ justed to 5.4 ⁇ 0.1 with the addition of 12 ml of sodium acetate buffer.
• the supernatant is diluted to 18 per cent ethanol with the addition of 240 grams of distilled crushed ice which is allowed to mix until the ice is di ⁇ - solved. This step takes 25 minutes.
• the addition of ice causes the plasma temperature to drop to -8° C (see FIGURE 4-IV.. ).
• Precipate IV.. is removed by centrifugation for 15 minutes, -6° C, 27000 x G. It is resuspended with ice to a final volume of 960 ml.
• Precipitate VI.. contains 5.1 grams of total protein (2.0 grams albumin and 1.4 grams IgG).
• Supernatant IV. contains 86.3 grams of total pro ⁇ tein (77.1 grams albumin and 1.0 gram IgG) in a volume of 2.34 liters. Its conductivity is 5.4 mS at 24° C.
• the protein cooling tank is cleaned with dis ⁇ tilled water.
• the 40 per cent ethanol is evacuated from the outside of the hollow fiber. Separation of Fraction IV.
• Supernatant IV.. is placed into the cooling tank and maintained at -6° C.
• the pH is adjusted to 5.8 ⁇ 0.05 by the addition of 17 ml of 1.0 M NaHCO .
• the supernatant is brought from 18 per cent ethanol to 40 per cent ethanol by the addition of 1294 ml of 80 per cent ethanol.
• Mode #2 When Mode #2 is used, the conditions are t same as in Fraction II + III Mode #1 except that the ba pressure is allowed to reach 25 psi.
• the ethanol temper ture is kept below -8° C and the protein temperature kept below -5° C throughout the addition.
• the additi was adjusted to completion in 250 minutes.
• Precipitate IV. is removed by centrifugation f 15 minutes, -6° C, 27000 x G and is resuspended in ice obtain a final volume of 1100 ml. It contains 19.9 gra of total protein (13.0 grams albumin and 0.9 grams IgG Supernatant IV. contains 64.6 grams of total protein (56 grams albumin and 0.0 gram IgG) in a volume of 3. liters. Its conductivity is 2.4 S at 24° C.
• the protein cooling tank is cleaned with di tilled water and is separated from the hollow fiber.
• T hollow fiber and ethanol cooling tank can be cleaned this time. Separation of Fraction V
• Table III summarizes the IgG and albumin yields of five runs of cryopoor plasma and four runs of sta- bilized human plasma.
• the albumin yields averaged 69.5 ⁇ 3_1 per cent for the stabilized human plasma runs and 69.8 ⁇ 1.8 per cent for cryo-precipitated human plasma runs.
• the albumin isolated during these runs contained less than 2 per cent contamination by other plasma proteins.
• the IgG Precipitate II + III contained appre ⁇ ciable concentrations of albumin with the yields of IgG being 76.6 ⁇ 7.5 per cent for stabilized human plasma and 73.9 ⁇ 4.3 per cent for cryo-precipitated human plasma. Reprocessing Precipitate II + III in this system following Cohn method 6 gave an essentially pure IgG with an overall yield based on the IgG present in starting plasma of 64.7 per cent.
• This fine control of temperature is achieved and controlled by: (a) the alcohol temperature and alcohol flow rate through the hollow fiber, and (b) plasma temperature and plasma flow rate through the hollow fiber.
• the alcohol serves as the major source of heat exchange and this control is enhanced by the design and construction of the hollow fiber syst when utilized according to this invention.
• Time " Required t- ⁇ Fractionate Plasma According to Cohn Method 9 Another significant feature of the process the reduction in time required to fractionate human plas completely to albumin. As can be seen in FIGURE 3, t time required for each step is short with a total elaps time being 10-1/2 hours, albumin yield averages 70 p cent with less than 2 per cent non-albumin contaminants.
• Example 2 When stabilized plasma is used . (Example 2 a FIGURE 4), the total time is reduced to 8 hours and r sults in an essentially pure albumin with 70 per ce yields.
• Example 3 When stabilized plasma is used . (Example 2 a FIGURE 4), the total time is reduced to 8 hours and r sults in an essentially pure albumin with 70 per ce yields.
• Diafiltration Diafiltration may be used to replace centrifug tion and solvent removal. The following is an example its use to process albumin eliminating centrifugation a solvent removal by other denaturing methods. After t addition of ethanol to 40 per cent, diafiltration may used to concentrate the protein and to remove the ethanol The process is identical to Examples 1 or 2 until aft the addition of ethanol to a plasma concentration of 4 per cent.
• FIGURE 5 The essential features of plasma temperatur plasma volume, protein concentration, and plasma ethan concentration are presented in FIGURE 5.
• the plasma containing 40 per cent ethanol i concentrated from 3.45 liters to 1.88 liters. This pr cess was adjusted for completion in 250 minutes.
• T protein temperature is kept below -3° C.
• the protei concentration of the plasma is increased from 26 g/ml 48 mg/ml.
• the plasma is pumped through the inside of t hollow fiber at a rate of 3000 ml/min which gives a ba pressure reading of 25 psi. Filtrate removal proceeds an average rate of 390 ml/hr at the temperature described.
• the concentrated plasma is now centrifuged for 15 minutes, at -6° C, 27000 x G.
• composition of Pre ⁇ cipitate IV. and Supernatant IV ⁇ is the same as described for Examples 1 and 2.
• the " volume of Supernatant IV., how- ever, has been reduced to 1.70 liters at a protein concen ⁇ tration of 42.5 mg/ml.
• Supernatant IV is placed into the cooling tank and kept at -6° C. Instead of reducing the pH to 4.8 fol ⁇ lowed by centrifugation, the plasma is simply brought to 0 per cent ethanol by diafiltration with ice and cold water.
• the flow rate of plasma will be 3000 ml/min.
• the filtrate flow rate averages 900 ml/hr during the ice addition which is accomplished in 120 minutes.
• the ethanol concentration in the plasma is reduced to 10 per cent, it is advantageous to allow the plasma temperature to increase above 0° C.
• the concentra ⁇ tion rate of the plasma is highly temperature dependent and the rate of filtrate out increases from 900 ml/hr to 5800 ml/hr as the plasma temperature increases to +14° C.
• the reduction of the ethanol concentration from 10 per cent to 0 per cent is accomplished by adding 3.5 liters of cold distilled water to the plasma over a two hour period. Since the rate of concentration is greater than the rate of water addition, the plasma volume decreases to a final volume of 1.6 liters and a final protein concentration of 45.5 mg/ml.
• the resulting albumin is free of alcohol and IgG, is in a concentrated state, and was never allowed precipitate.
• separation of albumin by di filtration is illustrated, the same method may be used remove and separate alcohol and other solvents from pr tein fractions from any step of the overall system.

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• 1980
  • 1980-04-29 GR GR61811A patent/GR68187B/el unknown
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