R-der-Controllera
for AU Process Vessels Are in This Centrally Located Conuol Room
A Staff-Indur’-g Collaborative Report Assmiate Editor in collaboration w i t h b. fi.
HINES, R: M. MCGHEE, AND K. A. SHUK1r;n Commercial Soloents G r p . , Term Haute, I d .
I .
T HAS bean estimated (6)by the armed forces that they need one pmt of plaama per year for each man in the service. Civilian defense authoritiee say that for adequate emergency preparedneas each metropolitan area should stockpile one pint for each person living there. When these estimatee are compared with a normal civilian use of about 4,000,wOpints of blood per year, it can be seen that needs are far from being met from donor sources. In addition to the very limited supply of blood and blood plaama, there are several other serious drawbacks to their use: Processing muat be rapid; expensive and extensive facilities are reqnired; plasma may contain a virus which will c a u e homologous serum jaundice in persons receiving the p l m . (The virus has an incubation time of from Bo to 160 days, and during this time i t cannot be detected in donors, donated blood, or proceased plasma. Blood being processed for its plaama is normally worked up in batcbes conmating of pinta from a number of donors; one “bad” pint and the entire batch is contaminated.
In an effort to minimize crospcontamination, some blood prow Mors have reduced the batch size from about 200 pints to about 25. Ultraviolet irradiation is also used. However, possibility of spreading the diseaee still exists.) While work on new blood sterilizing techniques continues, i t is not surprising that there hae been extensive work on effective substitutes. (Prop erly speaking, the materials are “substitutes” only in that they may be used in place of plaama. Correct terminology, 88 a p proved by National Research Council, b “plasma volume expander.”) The search haa centered around macmmolecular and colloidal mabrinls. Such materials have been shown to act like nstural blood colloids (83)which maintain correct blood volume. Milk infusions were popular during the last 20 years of the nineteenth century (11,84), but thereweremanyfsilures. T h e h a t conaiab eutly successful attempta to une a colloid were those by Hogan (19)in 1915, who used a gelatin-aaline solution, but simultsnwus work by Hnrwitz (14)in 88n FranCieco and Bayliss (S)in London
April 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
the following year with g u m arabic establihed it as the preferred material for the next 20 years. Gum arabic was used extensively by the British in France during the later stages of World War I ; production of sterile gum arabic near the front reached 75 liters per day a t one point during 1917. During the early days of World War 11, German workers (22) at Bayer Co. developed polyvinylpyrrolidone (PVP), a synthetic, water-soluble polymer for use as a plasma expander. P V P has been used in G e m a n y (Kollidon and Periston), Italy (Subtosan), and England (Plasmosan), and it is being produced in the United States by General Aniline & Film. While attention centered on PVP in Germany, Swedish workers Gronwall and Ingelman (7, 8 ) turned their attention t o dextran, a polysaccharide produced by the action on sucrose of enzymes liberated by the bacterium Leuconostoc mesenteroides. A patent was issued to them in 1948 (9). Two of $he four companies Producing dextran expanders in the United States are licensed under it (Commercial Solvents Corp. and Dextran Corp.).
693
smaller fragments. Clinical dextranisseparated from the hydrolyzate by fractional precipitation with organic solvents, generally methanol or ethanol. It is then dried and is ready for storage or bottling. Satisfactory Dextran Expander Requires High Proportion of 116 Bonds
T~~~of bond is of considerable importance in making a suitable dextran expander. For instance, in glycogen, a blood sugar and also a glucose polymer, linkages between units are ru-1,6 and 01-1,4; straight chains are linked 1,4, and branching occurs a t the (21)e dextran, just the is true: 1,6 ]inks are L‘straight~jand branching at the other positions (9, $0).
Several hundred strainsof L. mesenteroides have been checked for their dextran-producing ability. The percentages of 1,6 bonds in these dextrans varies from about 100 to about SO%, depending on the strain used and, t o a lesser extent, on process conditions. Sucrose Is Necessary to Produce Dextran A significant, point here is t h a t while 01-1,4glucose bonds are attacked rapidly by body enzymes, 0(-1,6 bonds are attacked “Dextran” is a generic name for polymers of glucose in which much more slowly. Obviously, a substance which is degraded at least 60% of the glucosidic bonds are ~r-1,6. Some forms have rapidly to low molecular weight material (which would happen plagued the wine and sugar industries for years, clogging filters to substances having a high proportion of 0r-1,4 bonds) would and retarding crystallization. Pasteur is reported (16) t o have not be a suitable expander; i t would leave the blood stream too investigated dextran first in 1861. Its empirical formula was rapidly. determined in 1874 by Scheibler, who named the material “dexFrom a bottle of unpasteurized root beer that elicited notice Tieghem showed that enzymes liberated by ‘’ because of its uniquely viscous contents, Northern Regional mesenteroides acting on sucrose were responsible for its production. Research Laboratory (of the Department of Agriculture’s A selected bibliography on dextran has been prepared by Jeanes Bureau of Agricultural and Industrial Chemistry) isolated in (15). 1943 a strain of L. mesenteroides and placed it in ita culture Dextran is produced according to the following reaction: collection under the number NRRL B-512. Other workers a t NRRL established conditions for the preparation of native CHzOH dextran from this strain. Results on the fermentation CHzOH production and chemical characterization of the B-512 Enzymes from dextran were published in 1948 (2’7). After Commercial L. mesenteyoides+ Solvents Corp. workers investigated a number of dextran-producing strains of L. mesenteroides for possible use in preparing clinical dextran, they selected B-512 as the etratin t o use. It produces a dextran having about OH H 9501,0r-1,6bonds. B-512 has since proved t o be the strain adopted as standard by all U. S. clinical dextran producers. NRRL has also sent B-512 cultures t o Swedish, English, and South / African producers; whether they are using B-512 in their produc+ tionisnotknown, however. Much of the fundamental chemistry of dextran and the development of test methods suitable for the rigid control of the quality HO of the product were the result of a collaborative project among H OH H OH Northern Regional Research Laboratory, Division of Organic and Fibrous Materials and Division of Chemistry of the National CHzOH H Bureau of Standards, and Commercial Solvents Corp. These l/O\l efforts, under the supervision of the Committees on Surgery and m (n - m ) HzO ( C ~ H I O O S ) , - ~ Blood and Related Problems of the National Research Council, have aided materially in expediting the availability of clinical dextran.
[HJlJ/cH201 +
+
As far as is known, only the glucose part of sucrose appears in dextran. Part of the fructose is consumed by the organism, and a large part appears as a by-product. Dextran cannot be produced from glucose alone or from mixtures of glucose and fructose; sucrose is necessary. Dextran as produced by the bacterium is called “native dextran,” and it cannot be used directly as a plasma expander. Molecular weight of native dextran ranges from several million to several hundred million, whereas material for use as an expander (clinical dextran) must have an average molecular weight of about 75,000. Thus, native dextran is subjected t o partial acid hydrolysis t o split the high molecular weight material into
C14Techniques Give Material Balance on Injected Dextran
A group headed by Stavely of Commercial Solvents cooperated with workers headed by Scully a t Argonne National Laboratory in synthesizing carbon-14 labeled sucrose (2.9). This they did by allowing canna leaves in a hermetically sealed jar to photosynthesize sucrose in the presence of C1402. This CI4 sucrose was then extracted and clinical dextran prepared from it. Clinicians accounted for essentially all the radioactivity in excretions and expired carbon dioxide. Of the 20-odd materials which have been tried as plasma expanders (IO),about 10 have been brought to the attention of
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Vol. 45, No. 4
dextran a t a yearly rate equivalent t o 1,OOO,OOO units. B unit is 500 ml. (delivered) of a 6% solution of dextran in physiologic saline (0.901, sodium chloride). ,411 U. S. producers make bulk dextran which is bottled by firms specializing in preparing injection solutions. Other companies in the field are shown in Table I. A possible newcomer to the field is Vitamins, Inc., Chicago manufacturer of vitamin concentrates (6).
TABLE I. DEXTRAX PRODUCTIOK
PREOlPlTATlOH TANKS LABORATORY
Producing Companies Commercial Solvents Corp., Terre Haute Ind. R. K. Laros cb Bethlehem F’A J. T. Baker Chemical Co., Philliusbure. N. J. Dextran Corp.’ Yonkers, N.’Y. Total As of April 1953.
Unn I ELEVATOR
CLINICAL SOLUTION TANKS
0
0
INSTRUMENT
STERILIZER LABORATORY
I 1 lcoool HYDROLYZERS
FERMENTATION
RECOVERY
STAIRS
1
3
WASH ROOM
ELEVATOR
I
I
rDO 1
SUPPLES
I
Yearly Production Ratea, Units of 6% Solution
1,000,000 600,000 360,000
600,000 2,560,000
Commercial Solvents, although licensed under the Swedish patent, has modified the process slightly. For instance, the expensive vacuum tray drying technique has been supplanted by the much speedier spray drying. Process conditions have been altered a t several points, also. R. K. Laros, which also spray dries its product, uses a fermentation process somewhat different from that of other American producers. I n the Laros process, L. mesenteroides is cultured in a sucrose solution containing nitrogen, essential mineral, and vitamin source materials. Laros reports that after the necessary culturing time, this medium containing cells, enzymes, and metabolic by-products is pumped to a large, open tank where further synthesis of dextran takes place under nonsterile conditions, The balance of Laros’ process is similar t o that of othei American producers. -411 dextran currently being produced is going to the armed forces. CSC has met its present military requirements and is announcing the product for civilian use on April 1. After that time, dextran preparations should begin to appear on the civilian market, provided they have complied with FDA regulations. Expandex, CSC clinical dextran, has already complied with these rpgulations.
i DEIONIZERS
CSC Produces Clinical Dextran i n Its New Plant at Terre Haute, Ind.
Figure 1. Floor Plan of Dextran Building
the Xational Research Council. Of these, KRC has recommended dextran and P-20 gelatin for use by the armed forces. PVP has been recommended by NRC to the Department of Defense and the Federal Civil Defense ildministration for stockpiling for use only in the case of a national emergency. Modified gelatins, such as oxypolygelatin and a modified fluid gelatin made by Knox, are under intensive study but as yet have not been approved by KRC. Dextran was released for general medical use in Sweden in June 1947, and Pharmacia, Ltd., became the world’s first commercial producer. Next on the scene was Dextran, Ltd., a subsidiary of the British firm East Anglia Chemical Co. (East Anglia also has a subsidiary in South Africa which is producing a dextran.) Dextran, Ltd., has since been bought by Glaxo Laboratories. Commercial Solvents Corp. was the first t o have its product approved in the United States. It is now producing bulk clinical
Commercial Solvents first became interested in dextran and its possible use as a plasma expander in April 1949. By September, dextran had been produced, hydrolyzed, and fractionated in the laboratory, and animal toxicity and pyrogen studies had been completed. A pilot plant was built and in operation by June 1951. During the next year, i t produced a 50,000-unit order for the armed forces. A certificate of necessity was granted for the full scale plant July 12, 1951 (rapid write-off of 80% of the 81,750,000 plant). In order to meet contract commitnients, it was necessary to schedule construction during the winter months of 1951-52. The entire structural steel frame was enclosed within a wood scaffolding which was covered rvith canvas tarpaulins. Portable, oilfired heaters were used to keep the temperature a t 40’ to 50’ F. inside the canvas enclosure. Completion date of the plant was upped about 2 months as a result. The three-story dextran plant is divided vertically in half into a fermentation section and a recovery section (Figure 1). This eases sanitation problems by isolating those sections handling clinical material. It also localizes recovery areas where large volumes of methanol are used, reducing health and safety problems.
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
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Air Containing C1402Was Circulated around Canna Leaves ( r i g h t ) Which Synthesized C14-Labeled Sucrose
A flow sheet for dextran production a t Terre Haute, Ind., is shown in Figure 2. First step in the process is preparation of the fermentation medium. The medium consists mainly of refined sucrose and water, but it also contains nitrogen and vitamin sources plus small amounts of essential minerals. Raw materials are received by truck and stored in a building separate from, but adjacent to, the dextran plant. This avoids bringing bags and other extraneous materials into processing areas, reducing contamination. Also located in the raw materials storage building is the 1500gallon medium slurry tank. (Materials of construction for this and other process equipment are discussed later.) The raw materials are weighed on nearby platform scales and introduced directly into the slurry tank. Sufficient water is added to bring the total volume to 1060 gallons. The tank is heated (15-poundper-square-inch steam in the jacket); the contents are agitated; and temperature is raised to GO" C. to facilitate solution of the solids. The medium is then pumped a t a rate of 10 gallons per minute to the second floor of the dextran building for sterilization. A two-section, plate-type heat exchanger (2OE) is used to sterilize the medium solution prior t o its being charged to the fermentors. The medium is sterilized in the first section and cooled t o fermentation temperature (25 C.) in the second. Hot water under pressure and a t 150" C. is circulated a t approximately 100 gallons per minute on one side of the plate. The fermentation medium circulates on the other side and is heated from 60" to 142' C. in this section. In the cooling section, the medium is cooled to 25" C. using 15" C. cooling water. Ratio of cooling mater t o medium solution is 3 gallons to 1. After sterilization, the 1060 gallons of medium continues directly to the third floor, part of it going to one of four 1300gallon fermentors (18E) and part to one of four 206-gallon seed tanks (18E).
As in most fermentations, inoculation of the batch is a stepwise process, beginning with a small sample from a stock culture. CSC uses a multiple-stage increase in inoculum size prior to inoculation of the seed tank. All stages are grown in duplicate to ensure a t least one being available should an accident occur. Sterile Air Bloww Final Inoculum Stage into Seed Tanks
Adjacent to the fermentor and seed tank room is the bacteriological laboratory (Figure 1). Cultures are stored and transferred in this lab. Leading off the main room are two small rooms: a culture room, where beginning stages are stored during their proper growing times; and a transfer room, where transfers are made from one stage to the next. Both these small rooms have ultraviolet lights in the ceiling to help keep air-borne contaminants to a minimum. All rooms are air conditioned, and 0.5' C . the temperature is controlled a t 25" Transfers are made by hand in the transfer room, but the seed tanks cannot be inoculated in this manner. Danger of contamination is too great in pouring the relatively large volume of the final stage. The final stage, therefore, is cultured in a 10-gallon stainless steel vessel called a bazooka (Figure 3 ) . After remaining in a constant temperature bath for the proper culturing period, the bazooka is connected to the seed tank by flexible stainless steel lines. The lines are purged with steam, and the contents are blown with sterile air into the seed tank. Contents of the seed tank are later blown directly to the opposite fermentor to inoculate the main batch. Temperature of the fermentor is maintained a t 25" =k 0.5" C. by cooling water in the vessel jackets. CSC uses 15' C. well water for all cooling operations in this plant. Water a t such a tempcrature actually would overcool, so CSC uses a combination of
IN D U S T R I A I A N Q 8 N GIs E 8 R I N 0
Axmil 1953
water and steam to maintaI0 the proper temperature. While thi~ is m e w h a t mom d y than using a highm temperatwe water, closer t a m p m t u r e w n h l is posaible without a great deal of &o&. The amount of steam is automatioally controlled
to give the-
C
---M I S T R Y
697
L
temperature.
Eecb fermentor is equipped with a turbine agitator. It may be umd continuously or intermittently to reduce temperature gradients w i t h the fermentor and to aid in temperature wntrol. Four fermentors are dropped every 24 hours. Ib the I ~ ~ e ~ pmceeda, t a t i ~pH drop and Viscosity riw. Fermentation ~amplesare t8k.m every 2 hours, and wben the p H brm dropped to approximst~ly4.5, fermentation is judged to be between 400 and wmplete. The Visaosity a t this point &ea 700 os. when the fermentation is complete, the native dextran solution is pumped by p a r pump (916)a t a rate of 42 d o n s per minute to one of two a4oppauon precipitation veesdn (116) located on the m ~ e r side y of the building. Here the solution is agitated, the pH adjnStea, and a quantity of methanol approximately equal to the f m t e d medium volume is added. The t e m w is wntrolled within 10 During tbia operation, essentially all the native dextran is precipitated. Lmge quautitiw of impurities, such BS unreacted sucrose, hctoea, higher alcohols, organic acids, nihgenous material$ and proteins remain in the supernatant alwhol solution. The precipitated dextran is rrllowed to eettle, and the supernatant liquors are pumped from the tank to a 4OWl-gallon run-downtank. Material from the run-down tank is continuously pumped to's methanol recovery ares. Here the methanol is redistilled to CSC's methanol product specifications and then returned to
c.
4
)I
Fi-3.
'i
Bazooka
All water added to the dextran after ita steriliaat~onmust be pyrogen-free water. A vapor compreesion still (6E) constructed of stsinless steel is used to produce the 400 gallons of pyrogen free water required &hour. Water softened with d u m 5eoIite is sent into the tube side of the evaporator tube bundle where it is vaporized. T h e vapor pwes though an entrainment separator smd into a rotary mpreaeor. Compressor suction p r e e w e is approximately 3 pounds
Raw Materials Are Slurried in Tank in Nearby Building k m i d mntrmilution in d-4 by ksepip.barn,
ProCalPs-ll.
-.,
outafdn
per square inch gage, and discharge preeaure is 6 pounds per square inch gage. The wmpreaeed steam is discharged into the shell side of the evaporator tube bundle where i t condenaee, giving offita heat to evaporate more feed water. Steam is umd only in the initial warm up to elevate the evaporator temperature to the boiling point. Thereafter, only a mall, regulated supply of steam is required to mske u p for radiation lasses. Only 16 B.lu. equivalent of electrical energy is used to evaporate 1 pound of water. The pymgen-free water produced is equivalent to doubledis WIed water, with a totsl solids content of less than 1.5 p.p.m. I1 is stored in an insulated and heated, 4 O W l - g d l ~stainless steeL tank located outaide the building. The precipitated native dextran is redissolved in 60"to 70" C. pyrogen-free water, and the precipitation process is repeated to remove contaminants occluded in the 6rnt precipitation. After removal of the supernatant liquors, which are also pumped to the --down tank for methand recovery, the native dextrsn is once more dissolved in 133' to '70' C. pyrogen-free water. The solution is pumped (196) a t a rate of 30 gallons per minute to one of three lOOO-gallon, glazslined hydrolysis vessels ( I @ ) located on the Boor Mow. Here the d e x h n content is adjusted by adding pyrogen-fma water. Befor+ hydrolysis is started, a combination of vacuum p d heat is used to remove the d g traces of methanol. Hydrochloric acid is added, and steam is applied to the jacket of the vessel to maintain an internal hydrolysis temperature of about 100° to 105O C.
Hydmlysis End Point Is Predicted from Viscosity Data Course of the hydrolysis is followed by viSWEitY meBBure menta. During hydrolysis a t leaet aiP readings are made until the viscosity is about 10 ca A t this point, readings are taken every 5 &utea and plotted on spaoilogarithmic paw. Fmm these data the exact end point can be predicted. The rate of hydrolysis is retarded by cooling the solution in a manner nuch
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 45, No. 4
( L e f t ) Plates Are Removed from Continuous Sterilizer and Scrubbed by Hand. ( R i g h t ) Following Sterilization of Medium, Acid Solution Is Circulated through Sterilizer to Clean It that when the solution has been finally cooled and neutralized its viscosity is less than 5.0 cs. The solution is then cooled and neutralized with sodium hydroxide. Roughly 35 pounds of diatomaceous earth is added to the solution in the hydrolysis tank, and the solution is pumped to a horizontal plate filter press (16E) located on the first floor. After filtering t o polish the solution, it is pumped up to one of two 1800-gallon fractionation vessels ( 1 1 E )on the third floor. Fractionation tanks are scale mounted (7E). Process piping, hot water piping to the jackets, and power conduits t o the agitators have flexible connectors so that weights accurate within 1 pound can be made on total net weights of 18,000 pounds. Dial scales are used with the dial mounted so that the operator who is adding the dextran solution or methanol to the tanks can see the dial. Stainless steel pivots are used throughout to eliminate the possibility of erroneous weights or sluggish scale action resulting from rusted pivots. Fractional Precipitation Separates Clinical Dextran from Unwanted Material
The solution after hydrolysis contains dextran molecules varying in molecular weight from a few hundred to about 1,000,000. During fractional precipitation, dextran molecules correctly sized for clinical use (molecular lveight from 25,000 to 200,000) are separated from the high and low molecular weight fractions. Temperature is controlled within 1 C. dhring fractionation. The filtered solution is weighed in the fractionation tank, and a calculated weight of methanol is added. The solution is agitated during and after methanol addition. A material that contains mostly high molecular weight dextran is precipitated. Agitation is stopped, and this material is allowed to settle and is withdrawn. Agitation is resumed and an additional amount of methanol is weighed into the vessel. During this operation, material having a molecular weight approximately in the clinical range is precipitated. This material is dropped to the second floor from the fractionation vessel into one of two redissolving tanks directly below.
Mostly low niolecular xeight dextran remains in the supernatant alcohol solution. The supernatant is pumped to the methanol run-down tank for subsequent methanol recovery. Sufficient pyrogen-free water is added to the redissolving tank to loxer the viscosity of the dextran to a point suitable for easy pumping The solution is then pumped back to the third floor to one of two smaller (750 gallons) fractionation vessels ( I I E )also on weigh scales. Because fractionation procedures are imperfect, repeated fractionations are necessary to produce material having the narrow molecular weight range required for clinical dextran. As in the first fractionation, high molecular weight material is withdrawn first. The next material withdrawn consists of dextran which has a molecular weight in the desired range. T h r remaining supernatant containing the light fractions is sent to the methanol recovery run-down tank. The clinical dextran is dropped from the final fractionation tank to one of two smaller (300 gallons) redissolving tanks (11E) directly below on the second floor. Enough water is added to reduce viscosity to a point where the solution will flow easily through the deionizing column. One of two mixed-bed deionizing units (10E) is used to dcionize the clinical solution just prior to its concentration and subsequent spray drying. Each unit has a capacity of 1F,500 grains, but as safe, standard operating procedure these units are regenerated after approximately 8000 grains have been rcmoved. Khile one unit is deionizing, the other is regenerated. Fifty per cent aqueous caustic soda and 66" BB. C.P. sulfuric acid are used for regeneration. Rinsing to remove the acid and caustic is done with pyrogen-free distilled water. Piping and valves on the unit are Type 304 stainless steel, and the resin chamber is neoprene-lined. Quality of the effluent from the columns is checked continuously by a conductivity cell mounted in the column outlet line. A specific resistance greater than 25,000 ohms is maintained. From the deionizing column, the effluent is pumped directly into a tube evaporator. The evaporator is a single-pass tube type having 60 square feet of heat transfer surface. Evaporation is started as soon as the tubes are filled; evaporation and deionization then proceed simultaneously. After all
April 1953
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
the material from the deiontinF, column haa reached the wap orator, Bvaporatioa continues ytil a wncentratiou suitable for ' s p y d r y h g , h s a been reached. M a t e d Circulated in the evap0&0r 8t.a rate of 36,000 POUndE WI' bur. ' The clinical dextran solution ia pwwi through a polishing filter ( l 6 E ) with about 0.5% of filter aid. It ia then pumped to one or m e r d of four 1 W l o n spray ~ F Y Wfeed tanks ( I I E ) , depending on the batch size.
.
699
product collection, and packaging room. concentrated the top of the 7-foot diameter drying chamber by a ter disk S P i n n e r l F w 4) rotating a t 21,000 r.p.m. The dextran h in conk& with the for about 5 seconds and never reaches a temperature hot over m0 Qutlet air temperature is 300" F.
:a,=
Yields uniformly eolubla Roduct
J
Final Btsp in the pmoees in spray dryiiog. The concentrated clinical wlution in dried to a moisture content of about 1%. The &ud is a white powder having an average particle &e of 40 micmnS. A primary conaideration in the drying opantion ia obtaining sterile air. Tno thoneand cubic feet of air per minute ia canpressed to a presnue of 15 inohea of water h a c8ntrXugal blower @E). The sir pMles througa imphmnent 6Itm (SE)coated with 8 VkCOEh &CkW Which m O V 0 S 8ppWXh8tdY 75% Of the sir-borne particles (se determined by the d e method for tenting sir filters, American Soaiety of Heating and Ventilating
b-
4
I08 Of lWUT IU 8 SO8 ff(IYSPEIOB)(YLO.TO
Wo*DIW
wawr
. .
.
The dextran powdv in collected in a &ea of four cyclone separatom mounted in parallel. Air containing dextran not col-
... ..
Figure 4.
.
.
s p n y Dryer 4tambm
The air then pmses through electrostatic precipiMors @E), the plates of which sse oil-coated to f a d i t a t e dust removal. Electrostatic precipitation is followed by filtration through a glass fiber filter (#E) approximately 20 square feet in cross-section arm. The glass fiber used is composed of glass filamenta having a diameter of 1.5 microns. The .air h then bested to 500" F. by direct heat from natural gas Barnes. Testa conducted by Brookhaven National Laboratory show that only O.CuX% of the spores and other microorganisms (diametem of 0.2 to 0.5 micron) in the air get by this treatment. Tbanpray dryer (+LE)h located outside the building adjacent Engineers).
'
..
Dextran from the cyclones is dropped into a central hopper
barrier in the wall, a rubber-gdeted snapring lid, and a polyethylene liner. Dextran is extremely hygroscopic, and these extra preoautiona are necessary to prevent water absorption as well as to prevent contminatiou. Fiber drums contsinidg huk clinical dextran are transferred to the pharmaceutical warehouse and held until the material haa been analyzed and approved for shipment. If the material pfmen all tests, it is approved for bottling and shipped to the packaging plant. Packaging is done on a contract basis. It includes dinsolving the bulk material in pyrogen-free water, adjusting'the dextran concentration to a 6% w./v. (6 grams per 100 ml.) solution, adding nu5cient sodium cbloride to make a 0.9% solution (staodard physiologic d i n e for injections), filtering, and bottling. Each plasma-type bottle is filled with enough solution to ensure delivery of 500 ml. Air h withdrawn, and the bottle h aealed, capped, sterilized; and packaged with the neOeasary injection apparatus. Each lot ia sampled, and the bottles are stored until the appropriate analytical testa have been made. In addition to meeting armed forces specifications, shown in Table 11, the material must also pass two additional CSC tests: color
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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Bazookas Are Autoclaved Prior to Use for Culturing Final State Note wrappings on pipes to keep them sterile
must be less than L4PHA50 and solvent (methanol) concentration must not be more than 20 mg. per 100 ml. Sanitary Construction Is Used Throughout
The dextran building is a structural steel frame building with brick exterior walls and glazed tile interior walls. All steel beams are concrete encased, both for fire protection and to provide a smooth, easily cleaned surface. All concrete surfaces are painted with a plastic-base enamel ( I E ) . Floors in the recovery side of the building are red quarry tile cemented in place with Lumnite cement. Floors in the fermentation and raw material sections of the plant are concrete, treated with a silicofluoride floor hardener. Offices, laboratories, and instrument control room have asphalt tile floors. The steel-sashed windows are glazed with hammered-surface blue glass to minimize glare inside the building and to decrease t h e air-conditioning- load. Sash in the air-conditioned sections is fixed; projected sash is used in the nonair-conditioned sections. Steel pipe is used for steam lines and cast-iron pipe for all drains except those in the laboratory, which are lead. Type 304 stainless steel pipe is used for prrogen-free distilled water. Galvanized pipe is used for hot water and steel pipe is used for condensate returns, service water, Bprinklers, and gas. Lighting circuit conduit is embedded in the walls, and power circuit conduit is exposed. Galvanized conduit with fittings conforming with National Electric Code Class I, Group D, are used throughout. I n general, all equipment that comes in direct contact with dextran during any stage in its processing is Type 304 stainless steel. Type 304 meets standards set by the three associations (International Association of Milk Sanitarians, U. S. Public Health Service, and Dairy Industry Committee) known as the 3A. Since the process must be sanitary, tanks, pumps, and pipe
conform to 3A standards (28, 19). In order to conserve stainless steel, thin-walled sanitary tubing was used in place of Schedule 40 or standard pipe. Exceptions to the use of light-gage stainless steel are the fermentors, nhich are ASlME code stainless pressure vessels to allow for steam sterilizing pressures, and the glass-lined hydrdyzers, which handle low pH corrosive material. 911 process vessels are equipped with temperature rccorder-controllers. All recording instruments are located in a sectionalixed control room. Recorder-controllers ( S E ) in the fermentation section have a divided scale. The 0 " t o 50" C. section, two thirds of the scale range, is graduated every degree, and the 50" to 150" C. section is graduated every five degrees. Accurate control is required in the 0' to 50' C. range, but it is only necessary
TABLE 11. ARMEDFORCES DEXTRAN SPECIFICATIONS" Property Dextran content, grams/100 ml. Sodium chloride, gram/100 ml. ?%osity, cp. at 250 C. Xitrogen (rnax.), mg./100 mi. Heavy metals max as P b ) , mg./100 mi. Ash (max. resi6ue o;;ignition), ing./100 mi. Weight average Molecular weightb The high fraction (?-lo%) shall not exceed 200.000; low fraction (5-10%) shall not be less t h a n 25 000 Intrinsic viscosiLy b, deciliter/gram Color
Value for 6 % Solution
5.7-6.3 0.85-0.95
5 .O-7.0 2.5-3.5 1.0
0.5 0.056
75,000 4z 25,000
0 . 2 3 zt 0 . 0 5 Colorless t o light yellow
Buffering capacity (rnax.), ml. of 0.100 NNaOH/100 ml. of dextran solution 3.0 Vacuum in bottle (min.), inches of Hg 1.5 Toxicity Negative Pyrogenicity Negative Antigenicity Xegative Sterility Negative a Armed Forces Medical Procurement Agency, Specification 1-161-890 ( M a y 21, 1952). b Determined by light scattering.
April 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
t o obtain and record temperatures sufficiently high (121 ' C.) for sterilization during cleaning portions of the cycle. A split scale arrangement permits this to be done on one instrument for each vessel. Units in the recovery section are standard recorder-controllers (12E). The fermentors are fitted with platinum resistance thermometers instead of the usual thermocouples. Resistance thermometers provide greater accuracy at temperatures in the neighborhood of.25" C., which is the temperature used for most operations. In addition to the recording instruments located in the control room, there are pressure gages on each of the services (steam, water, distilled water, air, gas). Level indicators show the quantities of pyrogen-free water and methanol in the storage tanks. Level alarms ( I S E ) are connected t o an annunciator on the control room panel board. This rings an alarm bell and lights a red light specifically indicating the tank where low or high liquid conditions exist. There are no instruments that use mercury in the entire plant. Even laboratory thermometers are of the alcohol type. There is, therefore, no possibility of mercury pickup in the product.
101
Sanitary pumps which can easily be disassembled for cleaning are used for all dextran in process. For clear liquids where a high liquid head is required, gear pumps (81E)are used. These have either constant- or variable-speed drive, depending on the application. They are used for flow rates up t o 50 gallons per minute a t discharge pressures up to 70 pounds per square inch gage. Solutions containing suspended solids, such as filter aids, are pumped with helical rotor pumps (16E). These pumps have Ameripol case liners; other parts are of stainless steel. Quantities handled vary from 5 t o 30 gallons per minute at discharge pressures of 80 t o 100 pounds per square inch gage. For general pumping services not demanding high pressure, sanitary centrifugal pumps (19E) are used. Only one size was purchased in order t o minimize the inventory of spare parts required for normal maintenance. Lightweight (18 gage), welded stainless steel tubing is used throughout. I n the recovery section, tubing is demountable for easy cleaning. The ends of the tubing are expanded into sanitary bevel seat unions and ferrules having standard Dairy-Acme threads. In the fermentation section of the plant, tubing is welded in place. Saunders type diaphragm valves ( 9 E ) are used throughout for materials in process. I n the fermentation section these valves are connected t o all-welded stainless steel tubing. In the recovery section, they are connected t o sanitary union type fittings. Material of construction is electropolished Type 304 stainless steel; diaphragm is Buno S. Valve stems are fitted with telltale indicators showing the valve position; limit stops prevent crushing the diaphragms. Valves and piping conform t o 3A standards (18, 19). These standards call for a No. 4 mill finish polished with 180 grit. Lines and valves must be free from dead spots, crevices, sharp corners, and shoulders. Air Conditioning Keeps Methanol Concentration below 50 P.P.M.
(Top) After Precipitation of Native Dextran, It Is Dissolved in Pyrogen-Free Water Prior to Hydrolysis
( L e f t ) Vapor Compression Still Supplies 500 Gallons of Pyrogen-Free Water Each Hour
Handling large quantities (10,000 gallons per day) of methanol in the recovery side of the building presents a toxicity hazard. Maximum concentration of methanol vapors allowable ( 1 ) for continuous exposure is 200 p.p.m. The air-conditioning system is designed t o remove methanol vapors and t o keep the concentration below this limit. There is a complete air change in the building every 3 minutes. Fresh air s u p plied to the system varies from 20 t o 100% of thr circulating air, and the ratio of fresh air to recirculated air is controlled by inside and outside thermostats. Exhaust outlets are a t floor level to remove heavier-than-air methanol vapors. Methanol is removed from the recirculated air by water spray nozzles located immediately ahead of the airconditioning cooling coils. When the plant was first put into operation, methanol content of the air was measured a t several different spots on each floor during periods
.
702
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 45, No. 4
search personnel were withdrawn and the operating group took over. In order to ensure full coordination of all groups, a stewing committee composed of representatives of research, production development and quality control, engineering, and production departments was formed. The success of this type of start-up is indicated b y the fact that in 26 days from start-up the plant xqras a t full capacity production of acceptable material. b
Steam and Hypochlorite Ensure Sanitary Conditions
Viscosity Samples
Talcen from Hydrolyzers Every Two Hours
Hydrolyzers are glass-lined to prevent corrosion resulting from low pH used i n hydrolysis
Since the plant was designed for 24-hour operation, 7 days per weeh, there is no over-all shutdown or start-up of equipment. However, as soon as a given section of the plant has completed a batch cycle, definite steps are taken to prepare i t for the next batch. Fermentation Equipment. As soon as the fermcntation cycle has been completed, all lines and vessrls are thoroughly rinsed lvith water, care being taken to flush all valves, bleeders, traps, and pumps. sterilizing steam at 15 pounds per square inch is then introduced and maintained in the system until the unit is ready for another batch. Common lines which 13-ill not have as long a sterilizing time as others are sterilized a t 30 pounds per square inch. The 15-pound pressure was selected as the minimum necessary for sterilization and the maximum permissible for satisfactory diaphragm life (average diaphragm life is 3 months). Recovery Equipment. On completion of a batch a t each stage (native dextran precipitation, fractionation, evaporation), all lines are flushed a i t h water and then removed and thoroughly cleaned by internal brushing. Simple washing in place cannot be counted on to remove the viscous dextran; actual scrubbing with a brush is necessary. These lines are then rinsed in pyrogen-free distilled water and placed on racks to dry. Large equipment, such as tanks and columns, are rinsed with hot mater and either filled with a sterilizing solution of hypochlorite (100 p.p.m.
A continuous tester is now in operation a t all times sampling - - the air a t points where the highest methanol concentrations were observed. The test units consist of gas absorption bottles through which measured quantities of air are drawn continuously for 24 hours. Methanol concentration in the water is then determined, and results are calculated in parts per million of methanol in the air. High concentrations of methanol resulting from spills have been reduced to 100 p.p.m. in less than 5 minutes. Under normal conditions, methanol concentration in the air is less than 50 p.p.m. Before the plant was put into operation, all technically trained men and group leaders in analytical control to be assigned to the plant were given an intensive training course in the pilot plant and the analytical section of the research department. At the start-up, key personnel from the foregoing groups of the research department were assigned to the plant on a temporary basis. These individuals assisted in on-thejob training of operating personnel. As soon as the engineering group had checked out the equipment, calibration and operation of the equipment was done by the research group to determine whether the equipment Fractionation Is Carried Out in These Scale-Mounted Tanks w a s t e c h n i c a l l y sound. When t h e Here the operator adds methanol while watching soale dial at left. Note static grounding couneetion on methanol pipe and tank plant was operating smoothly, re-
of a t least 1 week.
, April 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
703
chlorine) or atmospheric steam, depending on type and location of equipment. Since the main process equipment is under instrument control for temperature and flow of liquids is either metered or weighed, there are only the normal operating techniques of setting lines, starting and stopping agitation, etc. The process lends itself t o analytical control, and most end points of reaction are determined in the laboratory and relayed to operations. The most critical operations are those of “coasting” the hydrolyaers t o the proper final viscosity and separation of the interfaces during fractionation. Timing is the most important operating technique. Any holdup in the recov;ry process will delay the dropping of a fermenter and a subsequent change in the product going t o the recovery steps. It will also affect the age of the seed step in the fermentation. This close scheduling is maintained by active supervision on the part of the shift superintendent. Preventive maintenance is stressed in order to maintain the close operating schedule. Minor emergency repairs are usually made by the operating staff; however, all crafte from the central maintenance pool are on 24-hour call. Scheduled maintenance is handled by the various crafts from the central maintenance pool. Analytical Control Is a “Must” if Specifications Are to Be Met
There are no unusual sampling devices or techniques involved in this process. Most samples are approximately 250 ml. Fermentation samples are taken with aseptic precautions in sterile flasks. I n the recovery section, all samples are taken with stainless steel beakers which are rinsed with pyrogen-free water prior t o introduction into the vessel. All sample bottles and flasks are prepared by the laboratory t o ensure clean, uncontaminated containers. Fermentation samples are taken every 2 hours; pH, viscosity, and purity of culture are determined. All p H determinations, either in the plant or the laboratory, are made on Beckman p H meters, and viscosity is determined b y Ostwald-Fenske pipet. The main analytical control load is in the recovery section and final product specifications. Before any batch can proceed t o the next step, it must be cleared through the laboratory since the subsequent steps are based on dextran concentration. At each step of the process, p H is checked by the laboratory even though the p H was determined in the plant.
Dextran Building under Construction To complete construction on sahedule, structural ateel frame was enclosed with tarpaulins, and oil-fired heaters kept interior temperature at about 50” F. during winter
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Vol. 45, No. 4
( L e f t ) All Process Pipes in Recovery Section Are Demountable for Easy Cleaning Here operator hooks up a line to the hydrolyzer for the next batch
( R i g h t ) Pipes Are Cleaned in This Tank A brush is necessary (to left of operator’s hand) t o remove viscous dextran completely
The initial precipitation is followed by pH, viscosity, per cent dextran (weight per cent by anthrone), and specific gravity measurements. Adjustment of volume of the prehydrolyxate is made using these data. Solvent concentration is also determined a t this point. At the fractionation stage, careful control is necessary on solvent concentration. Specific gravity and dextran content (weight per cent by optical rotation) are determined to maintain the proper dextran-solvent ratio. Dextran content is again determined prior to deionization and vacuum evaporation. Concentration is carried out to a definite dextran coritent (weight per cent by rotation). After the material has been spray dried, a complete analysis is made of the bulk material prior to bottling. Since more than one batch is used to produce a bottle lot, additional tests are made on the finished, bottled material. Table I11 shows service requirements. Approximately 90% of the analytical control section and 100% of the operating personnel are on a shift basis. Four separate shifts are required for full time operation; work week for each shift is 40 hours. Operating a t rated capacity, this unit employs 106 people. There me 14 technically trained men directly connected with this operation; nine are supervisory and five are concerned with
TABLE 111. SERVICE REQUIREMENTS Utility
a
N o t including methanol recovery.
Amount
analytical control. Administration and service personnel are supplied by over-all plant management. Total Enzymatic Synthesis Is Possible Newcomer
One new process looming on the horizon is a total enzymaticone (86). In this proposed synthesis, L. mesenterozdes (B-512) is cultured in a manner conducive to yielding a liquor rich in extracellular dextran-producing enzyme. By proper control of reaction conditions between sucrose and enzyme, it is possible to synthesize a dextran predominantly of molecular weight suitable for clinical use. No hydrolysis is required, and only a limited amount of fractionation is necessary to “size” the material. Pilot plant work on this process is currently under way a t Northern Regional’ Research Laboratory. Some work has also been done on substituting other hydrolysis methods for acid hydrolysis : enzymatic (86), ultrasonic (6),and thermal (27). While CSC has no plans a t present for changing its clinicall dextran production, it is looking into the possibilities for nativr dextran. Native dextran acts as a thickener and has been used or suggested for use in oil well drilling compounds, plasticizers, paper coatings, textile printing. and foods. These usey, however, a t the moment appear to be of rather small volume, say a total of a few million pounds per year. One medical use for clinical dextran that shows promise is the treatment of various syndromes which involve edema such as toxemia of pregnancy and nephrosis. Edema is a condition in which excess water is present in intercellular tissue space. Plasma expanders act as oncotic diuretic agents. They cause the excess fluid to return to the circulatory system from which it can be excreted by the kidneys. Biggest potential use for clinical dextran, however, is in routine treatment during injury or surgery. Here it is possible to think
INDUSTRIAL AND ENGINEERING CHEMISTRY
April 1953
in terms of millions of units per year. Until recently dextran was restricted to stockpiling and t o rather rigidly controlled clinical tests b y the armed forces; it was released by the Surgeon General in February for general army use, wherever possible, in place of blood plasma. Successful armed forces use will pave the way for unrestricted civilian use. This does not mean there will be any lessening of the need for donated blood. Where oxygen carrying capacity is needed (high loss of red cells), only blood will suffice. It does mean, however, t h a t the limited blood supply can be used more efficiently. It means blood and blood fractions (such as gamma globulin) can be put t o use where specifically called for and that “routine” plasma volume expansion can be left t o the synthetic products.
705
J . M i l k and Food Technol., 10,277-81 (1947). J . Milk Technod., 9,12-21 and 152-5 (1946). Lohmar, Rolland, J . Am. Chem. Soc., 74, 4974 (1952). Meyer, K. H., Advances in Enzymol., 3, 109 (1943). Scully, N. J., Stavely, H. E., Skok, J., Stanley, A. R., Dale, J. K., Craig, J. T., Hodge, E. B., Chorney, W., Watanabe, R., and Baldwin, R., Science, 116, 87 (1952). (23) Starling, E. H., J. Physiol. ( L o n d o n ) , 19,312 (1896). (24) Thomas, T. G., N . Y . M e d . J., 27, 449 (1878). (25) Tsuchiya, H. M., Jeanes, A., Brucker, H. M., and Wilham, C. A., J . Baeteriol., 64,513 (1952). (26) Tsuchiya, H. M., Hellman, N. N., and Koepsell, H. J., J. Am. Chem. Soc., 75,758 (1953). (27) Wolff, I. A , , Watson, P. R., Sloan, J. W., and Rist, C. E., IND.ENG.CHEM.,45, 755 (1953).
(18) (19) (20) (21) (22)
Processing E q u i p m e n t References
American Standards Association, Standard Z 37.14 (1944). Barker, S. A., Bourne, E. J., Bruce, G. T., and Stacey, M., Chemistry & Industry, 1952,p. 1156. Bayliss, W, M., Proc. Roy. Soc. (London), B89, 380 (1916). Brewer, D. B., Proc. Rou. Soc. Med., 44, 561 (1951). Chem. Eng. News,29,650 (1951). Ibid., 31, 735 (1953). Gronwall, A. J. T., and Ingelman, B. G. A , , Acta Physiol. Scand., 7,97 (1944). Ibicl., 9, 1 (1945). Gronwall, A. J . T., and Ingelman, B. G. A. (to Aktiebolaget Pharmacia, Stockholm, Sweden), U. S. Patent 2,437,518 (March 9, 1948). Gropper, A. L., Raisz, L. G., and Amspacher, W. H., Intern. Abs. Surg., Surg., Gynecol., Obstet., 95,521 (1952). Guthrie. C. C.. and Pike, F.H.. Am. J. Phvsiol., 18, 14 (1907). (12) Hecht, G., and Weese, H., M u n c h . med. Wochschr., 90, 11 (1943). (13) Hogan, J. J., J . Am. M e d . Assoc., 64, 721 (1951). (14) Hurwits, S. H., Ibid., 68,699 (1917). (15) Jeanes, Allene, “Dextran-A Selected Bibliography,” Northern Regional Research Laboratory, Peoria, Ill., 1952. (16) Jeanes. A,. and Wilham, C. A,, J . Am. Chem. Soc., 72, 2655 (1950). (17) Jeanes, A,, Wilham, C. A., and Miers, J. C., J . Bid. Chem., 176, 603 (1948). I
,
’
O p t i c a l R o t a t i o n Measu r e m e n t s Are M a d e to C h e c k Per C e n t Dextran in Process S o l u t i o n s Control is a “must” if correctly sized dextran is to be produced
(1E) Amercoat Corp., South Gate, Calif., Amercoat N 4 : No. 13 prime, No. 13 seal, and two coats of No. 33. (2E) American Air Filter Go., Louisville, Ky. (3E) Bailey Meter Co., Cleveland, Ohio, Model WS 35, Class QAP. (4E) Bowen Engineering, Inc., North Branch, N. J., No. 2 spray
dryer.
(5E) Cleaver-Brooks Co., Milwaukee, Wis., Model DVC-30E dis-
tillation unit.
(6E) Continental Can Co., Chicago, Ill. (7E) Fairbanks, Morse & Co., Chicago. HI., ZO,OOO-pound, overhead
suspension, pipe lever scales. (8E) General Blower Co., Morton Grove, lll., standard turboblower. (9E) Hills-McCanna Co., Chicago, Ill. (10E) Illinois Water Treatment Co., Rookford, Ill., Model MB 570. (11E) Metal Glass Products Co., Belding, Mich. (12E) Minneapolis-Honeywell Regulator Go., Minneapolis, Minn., Brown ElectroniK Model 152 P 13W-8611. (13E) Moore Products Co., Philadelphia, Pa., Model 25 C 415. (14E) Pfaudler Co., The, Rochester, N. Y., Model Type R. (15E) Robbins & Meyers, Inc., Springfield, Ohio, Moyno F-6 and F-4. (16E) (17E) (18E) (19E) (20E) (21E)
Sparkler Manufacturing Co., Model 18-5-15.
Ibid., Model 33-XS-21.
Superior Welding Corp., Decatur, Ill. Tri-Clover Machine Co., Kenosha, Wis., Model EJ. Walker-Wallace, Inc., Buffalo, N. Y., Model HERE. Waukesha Foundry Co., Waukesha, Wis., Model 25BB.
