Figure 1. Plasma Fractionation Laboratory, Armour and Company, Fort Worth, Tex.
Fractiona J. B. LESH, IC. SCEIUETZ, AND J. D. PORSCHE .4rmotir arid Compuny, Chirago, I l l .
S V I E W of the steadily
]Procedures have been described for the large scale make availahle for civilian use increasing experience in fractionation of na tive water-soluble proteins by precipitathe various plasma fractions. tion with alcohol at low temperatures. The application of These operations constitute a the medical a,pplication of fractions of human plasma, these methods to the fractionation of human blood plasma part of the peacetime blood the large scale fractionation in a plant designed specifically for t.his purpose is discussed donor program for the Red here with emphasis on the processing equipment involved. Cross which is planned to of plasma has become a rout,ine chemical operat>ion. The handling of large volumes of fluids at controlled low provide, for emergency uses, bIethods for t.he separation of temperatures and the precautions necessary in the prepawhole blood, plasma, and proteins from such protein ration of parenteral prodncts therefrom are described in plasma fract,ions on a nationmixtures were developed early detail. Such procedures are applicable to processing of wide scale. inany pharmaceutical products, particularly those inSince the plant was dein Jl-orld War I1 from techtended for parenteral use. niques based on research by signed specifically for prepaE. J. Cohn and associates ration of parenteral a t Harvard Medical School. products by the low hemperature alcohol fractionation procedures, special precautions Details of these methods have been wpoyted ( 2 , 3, 5 ) and their application to large scale operations has been described were taken to ensure cleanliness in all operations and t o es( 1 ) . It is the purpose here to discuss the mechanical aspects of clude contamination from the outside. The building shown such large scale operations with particular reference to a plant in Figure 1 %vas constructed in such a way as to exclude designed specifically for this purpose. dust and dirt, a t every point possible. Floors, ceilings, and walls were finished in moistureproof materials with DESCRIPTION O F PL smooth surfaces to facilitate cleaning. The entire building is inaint'ained under positive pressure vith the aid of suitable air T~,as constructed under xavy to fratIn 1913 a conditioning equipment. hll incoming and all recirculated air tionate the plasma obtained by t,lle Red Cross from is passed t'hrough oiled, spun glass filters, then oyer activated 8 ~ donors o ~ per This plant was located charcoal, and finally through a self-cleaning, oiled precipitron at Fort Worth, T ~ ~ in ,order , to comp13, lvith the o f the unit t o remove the last traces of dust. L!.ir supplied to sterile Red crossthat blood available in the southffest area of the rooms receives a second precipitrou treatment, followed by exUnited States be utilized. qrith the ternlination of intensive posure to ultraviolet irradiation. blood collection at the end of the war, operations a t this plant Processing equipment is so constructed that the plasnia and were temporarily discontinued. Recently, however, some of the all reagents used are allowed to come in contact only with glass, outdated surplus dried plasma prepared for the armed services rubber, or stainless steel, with the object of avoiding excessive is being fractionated under Red Cross contract to recover and
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metallic contamination. All tanks employed are glass-lined and equipped with steel jackets to carry refrigerant.
1377
the plan is the fact that the distilled water is always uncomfortably hot. Technicians, however, soon become accustomed to the situation and no serious difficulties have been encountered.
REAGENTS
_I
With regard to reagents, perhaps the greatest single source of difficulty was a result of the presence of pyrogenic material in the reagents. Pyrogens are generally considered to be products of bacterial metabolism capable of producing a rise in body temperature following intravenous injection. Consequently, i t is absolutely essential to avoid any possibility of introducing pyrogens. The early difficulties were greatly simplified once it was discovered by Strong (6) that most pyrogenic materials are adsorbed by albumin, apparently quantitatively. Since, at one stage in the process, albumin is diluted to a concentration of approximately 0.85%, it is easy to see that a 30-fold concentration of albumin and consequently of pyrogens is effected when the albumin is finally put up in 25% solution ready to use. Once this discovery was made, i t became a routine matter to test all reagents at concentrations 30 times the maximum concentration at which they were used. Materials showing contamination at those elevated levels are refined or rejected, I n order to eliminate one of the sources of pyrogens, all alcohol taken into the plant or recovered after processing is distilled in a conventional pot-type still with a specially constructed entrainment trap made of stainless steel and filled with Berl saddles. The trap was designed to eliminate any possibility of entrainment in the vapors. A single distillation is sufficient since 53% is the maximum concentration of recovered alcohol required. Distillate from a glass-lined condenser flows through stainless steel pipe lines to glass-lined storage tanks. The production of the necessary quantities of pyrogen-free water presented a somewhat more difficult problem owing to the fact that water exposed to air becomes pyrogenic in a relatively short time, presumably because of the growth of microorganisms. This problem was solved by the storage of pyrogen-free water obtained from a conventional-type still at temperatures approximately 80 O C. in steam-heated, glass-lined tanks. Distribution of the pyrogen-free water throughout the building is accomplished by gravity flow through stainless steel pipe lines. The water in these lines is kept hot by the simple device of loosely fastening a 0.25-inch steam line to the distilled water line and wrapping the bundle with suitable insulation. This scheme results in a continuous supply of pyrogen-free water. The only drawback to
Figure 2.
TEMPERATURE CONTROL
Close control of temperatures is of primary importance in all processing rooms where protein solutions are handled. The entire room shown in Figure 2 is maintained at 23' * 1' F. by constant recirculation of air through a wet-type refrigeration unit. As an additional precaution, all of the stationary tanks are jacketed and refrigerated with brine a t 20" to 22" F. The refrigerant serves t o remove the heat of dilution of ethyl alcohol and the heat of neutralization produced as a result of buffer addition, thereby preventing any appreciable rise in temperature during reagent addition. Each of the precipitating tanks is equipped with both an indicating and a recording thermometer in view of the extreme importance of temperature control, and in all cases the smallest possible temperature differentials are utilized in the many refrigerating steps requiring precise control. FRACTIONATION OF PLASMA
Inasmuch as absolute sterility is not essential to successful plasma fractionation, continuous separation of plasma from the red cells was selected for convenience. The process is accomplished satisfactorily with standard types of continuous centrifugals, the yield and color of the plasma being at least as satisfactory as that obtained by cup centrifugation. It was found desirable, however, to use several small units rather than one large unit to permit separation of small amounts of blood without appreciable loss. No difficulties have been encountered as a consequence of interaction between blood types. Plasma is collected in tared, portable, glass-lined tanks capable of withstanding 25-pound internal pressure. It is then weighed on a floor-level platform scale and transferred to one of the processing tanks by means of air pressure. Stainless steel sanitary tubing, fittings, and valves are employed in making the necessary connections. Tubing is cut into lengths short enough to permit thorough cleaning and steam sterilization. It is of interest to point out at this juncture the remarkable ability of protein solutions to pick up traces of metals. It was found that utilization of stainless sanitary tubing which had been sweated into the necessary fittings with solder caused a barely perceptible, though significant, increase in the lead and tin content of some of the protein fractions. It was, therefore, desirable to utilize rolled fittings in order to avoid even slight exposure of the protein solutions to the heavy metals. Some of the synthetic rubbers contain appreciable quantities of s u b s t a n c e s soluble in ethyl alcohol even at the low concentrations employed in this work. The so-called pure gum rubber tubing was found satisfactory, however, following treatment with hot alkali to remove pyrogenic material. The transfer technique referred to, utilizing air under pressure in place of a pump, proved to be very satisfactory. I n some instances evacuation of the receiver proved t o be more convenient. When air under pressure is employed, i t is desirable t o compress the air with a water-sealed rotary pump; to reduce the moisture content of the compressed air by passing i t through a condenser Main Proceseing Room Held at 23" F.
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Vol. 42, No. 7
port,able glass-lined tanks. Jets are located as close to the propeller blades as possible to ensure rapid dilution of ethyl alcohol and buffer solutions. All reagent,s are weighed in the portable glass-lined tanks and int,roduced into the protein solutions by means of air pressure, a,s previously described. Flowmeters are employed in order to permit accurate control of the rate of reagent addition. Fifty-three per cent alcohol is prepared in 1000-gallon scale tanks in the follomTing manner: Redistilled alcohol is drawn from the basement storage tanks by vacuum, and pyrogen-free water flows by gravity from t'lic. second floor dist'illed water t,anks. Weight and specific gyavity determinations permit precise control of concentration. The temperature of the warm mixture is brought tmo23" F. uiitler vigorous agitation, t,he heat being removed by means of refrigel,a n t circulated through the jacket. Smaller, jacketed tanks w e used for the preparation of the necessary buffer solutions.
INFUSION TUBE
Figure 3.
DRAIN TUBE
.ifter addition of sufficient 53.370 alcohol to the plasma with agitation, so that the resulting over-all concentration is 8qc, the precipitate, fraction I, primarily fibrinogen, is removed in specially constructed refrigerated centrifuges of the continuou.: t>ype. Without refrigeration a considerable temperaturc risci occurs during operation of these machines. It,was found possible, however, to eliminate this by lining t,he shells with a coil of small bore t,ubing through m-hich a refrigerant is passed. The authors' experience has been tha.t eit,her direct expansion Freon 11or brine cooling is sat,isfactory, although somewhat more uniform results are obtained by the latter method if vigorous circulation of brine at, a temperature 20" F. below the desired tcmperat,urrl is maintained. This technique permits t,he collection of protein precipitates in stainless steel bowls without appreciable rise in temperature in either continuous or batch operations.
Cap Plate Assembly for Infusion 0.5 inch = 1 foot
maintained a t -70" F.; and to equip each outlet with a porous, oiled, stone filter and a reducing valve with a pressure gage. 'This combination permits a supply of clean air with flexibility of pressure control. By this means it is possible to avoid the vigorous agitation of the protein solutions and the stuffing box contamination so common when pumps are employed to effect liquid transfers. The most satisfactory valves, even for the largest tanks, proved to be short pieces of heavy-walled, gun1 rubber tubing provided with a V-clamp. This arrangement permits quick action in closing or opening the outlet and simplifies both maintenance and cleaning. Agitation of large volumes of liquid is effected by means of rather large, slow-moving, screw-type propellers mounted a t an angle so as to produce a slow but vigorous roll without foaming. Two speeds were found sufficient to provide satisfactory control of agitation as the volume of the tank contents is varied. Suspension of the large tanks from a mezzanine proved to be very satisfactory in view of the convenience of having both the top and the bottom of the tanks readily accessible. In the early work on laboratory scale the addition of a reagent, particularly alcohol, was accomplished by dialysis in order to eliminate exposure of the proteins to local high concentrations of water-miscible solvents. This procedure was impractical on a large scale and it was found that satisfactory results could be achieved by injection of solvent through capillary jets with efficient agitation. Figure 3 illustrates the type of capillary jet employed for the introduction of reagents into solutions of the plasma proteins contained in the large tanks. This device, of welded stainless steel construction, is clamped onto the bottom flange of the tank while empty. Rubber tubing is used to effect connection with the previously mentioned sanitary tubing of the
Figure 4. Refrigerated Centrifugals for Separation of Precipitated Proteins
A typical arrangement for the collection of protein precipitatcls is shown in Figure 4. In the background can be seen the bottom of a large, suspended precipitating tank. These tanks are connected by means of sanitary stainless steel tubing to the inlets of the centrifuges, the insoluble protein being collected in the bowl and the supernatant being allowed to flow into glass-lined, portable tanks. A demountable piping arrangement is available
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completion of the drying and after closure of the main valve. Four-stage steam ejectors are found, on the whole, to be satisfactory for the purpose of maintaining the low pressure required, provided adequate supply of steam and water is maintained. Both McLeod gages and recording Pirani gages are employed for measuring the pressures within the system. It was found relatively easy to maintain a pressure of 200 to 300 I* without resorting to use of a refrigerated condenser in the system. One point which should be emphasized in connection with vacuum installations of this type is the occasional occurrence of surges in the low pressure system. These surges have been observed t o carry dry protein powder into the vacuum system and to carry solids from the condenser water into the cans. In order to circumvent this exchange of solids, a special filter was designed which does not impede the flow of vapors but does prevent the loss of protein and the contamination of protein with dust from the system. It consists simply of a stainless steel wire frame reaching close to the bottom of the can with a layer of Canton flannel in the form of a sock fastened securely over it, as shown in Figure 6. Another difficulty which may occur at intervals in this type of system is the flash back resulting from sudden changes
Figure 5.
*
Equipment for Drying Plasma Fractions from the Frozen State
to permit use of from one to eight of these centrifuges on any single batch. The supernatants from centrifugation are weighed, and transferred to a second large tank for precipitation of the next fraction. These operations are continued until removal of all of the four fractions has been accomplished. The h a 1 supernatant is allowed to flow by gravity into a basement tank which serves as a reservoir for the alcohol recovery still. Careful records are maintained of the weights of solutions and precipitates. Nitrogen determinations are run on plasma pools and all supernatants. In this way it is possible to set up a double accounting system on the basis of weight and on the basis of protein. By rigidly following and enforcing these procedures, losses are kept near the zero mark. Removal of the precipitates from centrifuge bowls has never been a very satisfactory procedure. The use of a combination of a stainless steel shovel and stainless steel spatula results in reasonably complete removal of the precipitates, but is still a time-consuming task. The removal of alcohol from the protein precipitates is effected by drying from the frozen state. The paste-for example, albumin-is suspended in sufficient distilled water to reduce alcohol concentration to below 20%. One and one-half gallon quantities of the resulting suspension are placed in chilled stainless steel containers having a capacity of approximately 8 gallons. The containers are rotated in horizontal position in a freezing bath a t -70" F. This procedure results in the formation of a frozen shell on the interior surface of the can. After insertion of the necessary filter, the can is quickly connected to suitable high vacuum equipment, and sublimation is allowed to proceed while the protein suspension is maintained in the frozen state. Heat required for sublimation is provided by utilizing the drying room as an air bath. Temperatures of 130' F. are attainable and may be desirable during the terminal stages of drying. A panoramic view of the drying equipment is shown in Figure 5. It will be noted that pairs of the cans are connected to the main header by means of a 4-inch vacuum valve. Each pair is provided u-ith a small valve through which air is admitted after
I
30''
M
Figure 6.
I6 GA. S.S. VACUUM
SHELL
fl
Vacuum Safety Valve Assembly and Drying Container 0.25 inch = 1 foot
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Figure 7.
Container for Storage of Sterile Liquids i n Bulk
in n-ater or steam pressure. This phenomenon may result in the flooding of one or more of the cans with condenser water. I n order to guard against this, a flap valve tyas designed utilizing a circular diaphragm lying on a perforated stainless steel plate. The diaphragm is of such weight that it can readily be lifted by vapors leaving the can but also pressed firmly over the holes by any flow of gas or fluid in the opposite direction. It was found convenient to bolt these flap valves permanently onto the outlets from the vacuum header. Theoretically and practically the drying system described is not the most efficient conceivable. Its principal deficiency lies in the difficulty of transferring heat from the air to the frozen shell after drying has begun. Obviously the shell is in intimate contact with the stainless steel after its formation. However, owing to this very fact, drying occurs preferentially a t the interface between the stainless steel and the frozen protein suspension. This results in the formation of vapors a t the interface and consequent separation of the protein from the stainless steel. Thus, soon after drying begins, the shell is separated from the source of heat by alcohol-water vapors a t extremely low pressure and is consequently well insulated. In spite of this fact, there is sufficient radiant heat from the stainless steel to permit drying in 36 to 48 hours. PACKAGING PRODUCTS OF PLASMA FRACTIONATION
The primary products of plasma fractionation-albumin and the r-globulins-are generally packaged as sterile solutions in M concentrated form as possible. In the case of albumin aqueous solutions containing 25 grams of protein per 100 ml. are prepared. Gamma globulins in solution are much more viscous than albumin so that concentrations of 16.5% are as high as can be conveniently handled. With either product the equipment employed in reconstitution, clarification, and sterile filtration is of standard design. Special containers for receiving and storage of sterile filtrates in bulk were designed to provide adequate means for sampling without danger of contamination. The receptacle employed for sterilized protein solution is a stainless steel, cylindrical container with a removable, flanged top fitted with a gasket and a total of seven outlets, as shown in Figure 7 . One of these is connected to an air filter, two more
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serve as main outlets, and the remainder are connected to sterile sample containers. The main advantage of this arrangement is that it permits removal of a sterile sample for testing without exposure of the bulk of the solution to possible contamination attendant upon the introduction of sampling devices. It has the additional advantages of being capable of sterilization as a unit and also withstanding the necessary air pressure to permit bottling. The tracks on the periphery of the container are designed to fit on wheels arranged in such a way that the container could be immersed in a tank of water of any desired temperature should it be necessary to change the temperature of the sterile solution. Agitation, of course, can be effected by simply rotating the container. Sterile solutions are stored in such containers pending completion of control tests for sterility, toxicity, purity, and stability. Final packaging is carried out under rigorously aseptic conditions to avoid contamination, since neither albumin nor globulin solutions contain preservative. The albumin solution, however, does contain a stabilizer to permit pasteurization in the final container at a temperature of 60" C. for 10 hours. This treatment has been found effective in inactivating viruses and most vegetative forms of bacteria, but does not kill spores. The globulin solutions also contain a stabilizer, but will not withstand pasteurization temperatures. Control tests of the final packages are made in accord with regulations of the Division of Biologics Control of the National Institutes of Health regarding such products. DISCUSSION
The same types of equipment are used in the preparation of other fractions of plasma. These include fibrinogen and antihemophilic globulins from fraction I, thrombin from fraction 111-2, isoagglutinins from fraction 111-1. Fibrin products such as film, foam, and plastics may require additional types of drying and sterilizing equipment. The methods of preparation are essentially those described in the laboratory scale (4). Other fractions of plasma, the application of which is still limited to clinical investigation, may be prepared in similar equipment whenever demand for them is sufficient to warrant large scale operation. These include a t the present time prothrombin, plasmin, hypertensinogen, phosphatases, and ironbinding globulins. The success of all such fractionations in producing preparations of satisfactory purity, stability, and potency depends largely upon careful control of all variables, particularly temperature, solvent concentrations, and pH, regardless of the scale of operation. SUMMARY
Procedures have been described for the large scale fractionation of native water-soluble proteins. The fractionation methods used are based on the simultaneous control of pH, ionic strength, protein concentration, temperature, and ethyl alcohol concentration. LITERATURE CITED
Callaham, J. R., Chem. Eng., 53,101 (June 1946). (2) Cohn, E. J., Luetscher, J. A., Jr., Onoley, J. L., Armstrong, S. H., Jr., and Davis, B. S., J. Am. Chem. Soc., 62, 3396 (1940). (3) Cohn, E. J., Strong, L. E., Hughes, TV. L., Jr., Mulford, D. J., Ashworth, J. N., Melin, M., and Taylor, H. L., Ibid., 68, 459 (1)
(1946). (4) (5)
Ferry, J. D., and Morrison, P. R., Ibid., 69, 400 (1947). Oncley, J. L., Melin, M., Richert, D. A., Cameron, J. W., and
(6)
Strong, L. E., private communication.
Gross, P. M., Jr., Ibid., 71, 541 (1949).
RECEIVED July 7, 1948. Presented before the Division of Industrial and Engineering Chemistry at the 114th Meeting of the AMIBRICAN CHEMICAL SOCIETY,Washington, D. C.
