LANTS 4NTIBIOTICS DEVELOPMENT
A
W. B. FORTUNE, S. L. McCORMICK, H. W. RHODEHAMEL, JR., AND J. J. STEFANIAK Eli Lilly and Company, Indianapolis, Indiana resins and plant scale chromatography are leading to the development of relatively new unit processes and equipment which are designed and developed in the purification section of the pilot plant. Among other examples, the development of a unique process for drying antibiotics illustrates the typical handling of a problem in the pilot plant.
T h e fermentation of penicillin and streptomycin, as well as new antibiotics, invoIves many variables, such as rate of aeration and agitation, components of the media, types of antifoam agents, temperature control, sterilization of media, sterility control during the operation, and size of inoculum. The proper control of such variables can best be achieved througk effective utilization of a well equipped pilot plant designed specifically for antibiotic research and development. The fermentation unit described ranges in capacity from 500-ml. shake-flasks through banks of 30-liter jar fermentors to coated and uncoated 1600-gallon fermentors. The purification of penicillin and streptomycin and the isolation and purification of new antibiotics present everchanging problems of varying complexity involving modifications of present unit operations as new types of equipment and materials become available. The introduction of the ion exchange
Figure 1 .
T
HE urgent necessity for the production of penicillin in quantity for the use of the armed forces,as soon as laboratory cxsperimentation showed surface culture to be feasible, was responsible for by-passing usual pilot plant work on this new antibiotic when it first came on the pharmaceutical scene. From the small, laboratory Petri dish holding perhaps 25 ml. of medium, the process was translated directly to large scale operation in 2- or 3-quart bottles, usually with thousands of bottles of penicillin media being “harvested” per day. Contamination in one bottle in a large incubator often meant the loss of a whole day’s
Thirty-Liter Jar Fermentor Unit
191
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INDUSTRIAL AND ENGINEERING CHEMISTRY
production, and sometimes even a week’s production, because of rapid spreading of such contamination. As soon as operation on a plant scale had been initiated and a few grams of penicillin were being produced per day, scientific thought turned toward newer and more efficient methods of growing the mold and purifying the final product A great deal of work at the Northern Regional Research Laboratories, under Robert D. Coghill and Andrew J. Noyer (9, I O ) was directed toward the production of penicillin in submerged culture. One of the first results of this intensive work was the discovery of the value of corn steep liquor and lactose in the fermentation process. A great deal of research effort was spent on the search for new strains capable of producing higher potency. Soon after the advent of the new strain of Penicillium designated NRRL 1251 B 25, and the discovery that it could be grown in submerged culture, Eli Lilly and Company determined to build a submerged culture production plant. During the construction of this production unit, crude pilot plant work was carried out in a single 2000-gallon fermentor in an attempt to ascertain roughly the conditions requisite to growth of the mold in the 8000gallon tanks in the plant under construction. Upon completion of this plant, further experimental Fork was carried out in the production tanks themselves. At about this time, a project was set up in the Organic Research Divisionof the Lilly Research Laboratories, as a part of the wartime cooperative work carried on by various industrial and universitr groups, for the purpose of studying the biosynthesis of new
Vol. 42, No. I
penicillins. The results of this work have been published in a series of eight papers by Behrens et al. (1-6, 8, 12). I n order to grow the mold in the presence of the precursors being tested, it was necessary to set up a small pilot plant consisting of two 50gallon stainless steel fermentors and the necessary purification equipment for processing according to the then used methods. This pilot plant was used throughout the study of the above problem. The difficulty of doing pilot plant work in production tanks and with production purification equipment soon became apparent with the result that, when plans were drawn up for the expansion of the original plant into a new building, a completely equipped pilot plant was designed to be included in one section of the production unit. FERMEUTATION EQUIPMENT
The fermentation equipment is divided into three general size groupings: shake-flasks, 30-liter jar fermentors, and tank fermentors. The shake-flask equipment consists of a Gump rotary shaker with a platform modified to hold 21 2-liter flasks and 168 1-quart square fruit jars. The 2-liter flasks are used to grow inocula for the 30-liter jar fermentors. Several designs of vessels were tried for use on the rotary shaker. Erlenmeyer flasks (500 ml.) were modified by blowing a series of projections inward to serve ae baffles during the shaking period. It was hoped that this would
January 1950
c
ri
INDUSTRIAL AND E N G INEERING CHEMISTRY
produce more efficient aeration by breaking up the swirling motion of the medium. However, results indicated very little difference between these and the square 1-quart fruit jars used as controls. Other types of baffles were tried with no appreciable improvement. The radius of rotation used on this shaker is approximately 2.75 inches. For most fermentations, the shaker is operated at a speed of 240 r.p.m. The 30-liter jar fermentor unit, shown in Figure 1,represents a composite of the best features of several similar units. Modifications were found to be desirable through actual use. The water bath and general over-all design are very similar taa unit prepared by the Commercial Solvents Corporation. Modifications incorporated in our units include a different type of variable speed drive and several improvements in the head as used on the jar. The unit is shown schematically in Figure 1. The physical unit is built throughout of stainless steel on a channel iron framework. A indicates one of the water baths in which the jar fermentor unit is operated. Each of the two water baths has a capacity for eight jar fermentors. Each bath operates as an individual unit entirely separate from the other. The temperature is controlled automatically by means of a Brown temperature recording controller, shown a t H . Thermocouples placed in the water bath and in the circulating water line indicate continuously the temperature of the water in the bath. A thermostat, B, and a Calrod heating unit, C, control the increase of temperature in the water bath BS required by the controller. Each water bath has a pump, D,which continuously circulates the water in the bath. Cooling of the bath is controlled by the addition of chilled water through an automatic valve also controlled by the Brown recording instrument. The agitators in the jar fermentors are driven by a V-belt from a two-step pulley, F. Power for each half of the unit is transmitted from a 1.5-hp. electric motor through a Reeves.variable speed transmission, E, to a single drive shaft. Individual agitators are connected to this main drive shaft through the two-step pulley and a helical gear arrangement, C. An air line coming from the 25-pound production supply enters a surge tank, J , to reduce fluctuations in flow; the rate of flow is indicated with the flowmeter, I . From the fermentor, air passes to an exhaust header, K , which opens to the roof of the building. The entire unit is designed to require a minimum of operating personnel. The individual 30-liter glass jar fermentor is shown in Figure 3. This fermentor is described in full detail on page 188 of this issue (11).
a
Twenty complete jar units are available for use in this equipment, thus permitting four units to be free a t all times for modifications or preparation of new experiments. Another water bath is available for eight jar fermentor units, but without mechanical stirring equipment. This unit has been found to be of real use in fermentation experiments on aeration alone, and for growing inocula. The tank fermentation unit consists of three 85-gallon seed tanks, three 350-gallon “bump” tanks, and three 1600-gallon fermentors. In each size gsoup, two of the tanks are coated with a polymerized coating; the third, of mild steel, is uncoated. One other unit consists of two 50-gallon stainless steel fermentors with removable heads. The large units are illustrated in Figures 4 and 5. The header systems on these units are so interconnected that transfers of media or seed may be made from one unit to another under sterile conditions. Separate antifoam containers are available for each tank, or any or all of the tanks may draw antifoam from the production antifoam system. Agitation may be controlled in each tank by means of various size pulleys. The agitator blades are of the cutting type, cutting through the medium and throwing the medium against the walls of the tank.
Figure 3.
193
Thirty-Liter Glass Jar Fermentor
Aeration is accomplished by forcing the sterile air through sparger tubes at the base of the tanks. Various size holes in the sparger tubes control the dispersion of the air. Air exhausts from the fermentors through a fixed orifice; hence, the rate of flow of air into the tanks is controlled by the pressure maintained on the tank. After medium preparation, sterilization is accomplished in the tanks by injecting 30 pound steam through the steam sparger ring, meanwhile agitating the medium. After fermentation in the 1600-gallon tanks, the broth may be harvested into holding tanks in the purification pilot plant or may be harvested directly to the production purification plant through special header systems. Automatic safety devices to be effective in case of accidental shutdowns in air or water supply are incorporated into the tank equipment. VARIABLES IN FERMENTATION OF PENICILLI\ AND STREPTOMYCIN
The variables encountered in the submerged fermentation of penicillin and streptomycin were found to be multitudinous. A proper balance of these variables was deemed necessary to obtain good yields-for example, high aeration and rapid agitation rates give rise to excessive antifoam usage which may be detrimental to the fermentation. Some of these variables will be considered. Aeration and Agitation. A number of experiments were made to determine optimal aeration and agitation rates for submerged penicillin fermentation in jar fermentors. Fourteen liters of medium for the penicillin fermentation were sterilized for 30 minutes a t 20 pounds per square inch gage steam pressure in an autoclave. The media were cooled in the water bath with the maintenance of a positive air pressure inside the jar to avoid contamination. The jar fermentors were inoculated with a 40- to 48- hour vegetative culture and incubated at stipulated temperatures. Table I summarizes the data on these experiments. The 450-r.p.m. agitation with 0.5 or 1.0 volume per volume of air is optimal for maximal yields. Higher rates of agitation add
INDUSTRIAL AND ENGINEERING CHEMISTRY
194
TABLE I. EFFECT OF AEn.kTIos
AND
AGITATIOX RATESO N ,JAR FERXENTORs
TABLE
11.
AERATION AND -&GITA4'I'1OS RATESW I T H
Streptomyces Griseus
PENICILLIN PRODUCTION I N 30-LITER Experiment
Agitation. Jar
1 2
1
2
1 2 1 2 1
2
Incubation Temp., C. 26
R.P.11 450 450
700 700 550 550 450 450 450 450
Medium, 70 Lactose, 3 5 Cerelose, 0 . 5 Corn steep qolids, 2 0 ZnSOa, 0 005 CaC03, 0 1 Piecursor, 0 03
icration, Vol /Val. 1 0 1.0
0.5
0.5 0.5 0.5 0.5 0.5
1 0 I .o
Max. Yield, Cnits/Cc 780 832 535 525 1102 1001 I269 998 1212 1159
Antifoam, % Vegifat YOctadecanol 3
Age,,
Hour. 102 122 92 122 91 120
Euperi-
inent 1
102
122 92 111
t
200 300
6
7 8 ii
10
II
to the foaming problem which accounts for the lower aeration rates with the greater agitation. The air sparging problem was a difficult one in that the viscosity or consistency of submerged cultures changes n-ith age of fermentation. Small air bubbles, which give good air-liquid surface contact, are easily obtained early in the incubation period. The increase in the viscosity of the culture promotes coalescence which results in air "blubbing" from the surface of the culture. Pressures necessary to maintain open holes in a sparger tube are usually not available, and approximately 50% of the holes in a sparger tube in the 30-liter jar fermentors tend to plug. A pressure greater than the pressure drop through the sparger is necessary to prevent plugging. The number of sparger holes, hole size, and sparger tube diameter all influence the pressure drop. The conditions and procedures for the streptomycin fermentation are similar to those of the penicillin fermentation. A comprehensive study of the optimal stirring and aeration rates has
Figure 4.
Iricuhatiori Temp., " C .
26
eo0
?
5
Inoculum, % Vegetative, 7
Agitation, R.P.M. 200 200 200 200
1
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300 400 400 400 400 400 400 400 400 450 450 450 450 600
GO0
Aeration, Vol./Vol 0 5 0 5
1 0
0
1 5
1 5 1 5 1 5
110
0 5 0 5
443 403 323 613 GOO 585
1 0
1 0 1 5 1 5
2 0 2 0
1.0
Medium, % Cerelose, 1,s Soybean oil meal, 1.5 Corn steep solids, 0 . 5 NaC1, 0 . 2 5 CaC03, 0 . 1
215 0 323 230 0 0
1 0
1 0 1 0 2 0 2 0 i~ n.
Alau. Yield, Units/Cc.
304
300 463 500 500 525 .
n
60
Antifoam, 70 Pogifat POctadecanol R
Age a t Max., Hours 82 72 117 I02 82 82 72 82 112 92
92 71 82 72 72 82
1i2 Inoculum, G7c Vegetative, i
been completed. The data in Table TI show that the optimal stirring rate is 400 r.p.m. with aeration a t 1.0 to 1.5 volume pc'i volume of air per minute. Aeration rates are governed by the agitation rate, since a t low stirring speed more air can be used because the foaming properties of the media do not interfere. However, yields do not follow the increase in aeration rate as one might expect. A number of types of stirrers have been mounted on the shafts of the jar fermentors and tested. The blade type of stirrer with a pitch of 45" was found to give the best stirring. Reversal
Sixteen Hundred-Gallon Fermentors
January
..
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
of the stirrgr, which reversed the vertical flow of the culture in the jar, did not influence the yield of antibiotic. Ingredients of the Medium. Ingredients gf the medium were significant in so far as the source was concerned. Different suppliers prepared raw materials in a variety of ways. Corn steep water from four different suppliers might give four different yields of penicillin when all other variables were eliminated. Solids content might vary a8 much as 30% in various lots from the same supplier. Reducing sugars often varied 100% between suppliers. The content of certain amino acids varied greatly between products labeled the same. Table 111 indicates variations seen between corn steeps from various suppliers. A series of tests with different types of carbohydrates was conducted. Considerable variation in carbohydrate material under the heading of glucose sirup was found. The constituents of these sirups varied widely with the type of sirup and the manufacturer. Table IV gives the sirups tested and information concerning the yield of streptomycin obtained with these carbohydrate sources. The data indicate that Puritose and cerelose give higher yields than the other sirups tested. Antifoam Agents. A study of antifoam agents was initiated early. The initial phase of such a study was the determination of thelimiting toxicity of the antifoam agent; this study was carried out in shake-flasks. When such results indicated that the toxicity was below certain limits set up in our laboratories, the study passed into the second phase wherein the knockdown and holddown qualities of the antifoam agents were studied. T o date, more than 35 such antifoam agents have been studied, and the end result has been that only six havepassed the required standards. Of these, only two or three have been economically feasible foruse. Although certain of these antifoam agents apparently increased the yield of total penicillin, the increase was misleading as the antifoam agent apparently contained a precursor capable of forming other penicillins which were undesirable? Temperature Control. The determination and control of the temperature of the fermentation has always presented a problem. Both the energy put into the fermentation process through vigorous agitation and the heat produced by the fermentation must be controlled and/or dissipated within very close limits for efficient results. Large tanks may either be jacketed or contain cooling coils. Jars must be operated in a constant temperature water bath. Shake-flasks must be handled in some type of incubator. Much work is required in the determination of the optimal fermentation temperature for maximum yields. An important factor invoIved in the pilot plant jar fermentation has been that of the insulating effect of glass. This factor must be considered during two phases of the fermentation varia-
TABLE111. EVALUATION OF CORNSTEEPSIN RELATION TO
PENICILLIN PRODUCTION
Experiment
Supplier
J a r No.
Max. Yield, Units/Cc.
Age at Max., Hours
Figure 5.
Incubation Temp., C.
26
D
A B
2 1
2 1 2 1
C
1 2
E
1
Medium, % Lactose 2.5 Cerelosd o 5 Corn stiep'solids, 2 .O
ZnSOc 0 005 C a d 0.1 PrecurAor, 0.05
660 498 525 1375 1330 1152 968 1017 738 Antifoam, % Vegifat YOctadecanol3
119 119 112 120 102 120 120 112 102 Inoculum, % Vegetative, 7
Eighty-Five-Gallon Seed Tanks
ble study: first, during sterilization, as will be discussed in the next section; and secondly, during the actual fermentation in the constant temperature bath. The 30-liter glass jars have a wall thickness of approximately 0.25 t o 0.375 inch and, hence, have an appreciable insulating effect. This effect sets up a temperature differential between the water bath and the medium within the jars; the temperature of the bath must be maintained approximately 2 O C. lower than the desired temperature within the fermentation jar. At the present time experiments are being carried out involving the use of a metal jar in place of glass, thus eliminating the heat transfer variable. Sterilization. The problem of proper and complete sterilization represents another variable occurring both in the fermentation medium and in the air supplied to the fermentation. Where
TABLE IV. INFLUENCE OF CARBOHYDRATES SOURCES ON STREPTOMYCIN PRODUCTION I N 30-LITER JAR FERMENTORS Exgeriment 1
2 1 1 1 2 2 2 2 2 2
195
Carbohydrate Cerelose Puritose
3
Special high purity
4
Corn sirup 8430
5
Cere 1ose
6
High purity
7
Malt sirup
Incubation Temp., a C .
26
Medium, % Carbohydrate (dryweight) 2 . 0 Soybean oil meal, 2 . 0 Corn steep solids, 0.5 NaCl 0.26 c a c d s , 0.1
Jar No. 1 2 1 2 1 2 1 2 1 2 1 2 1 2
Max. Yield, Units/Cc.
Age a t Max., Hours
351 495 420 540 330 375 300 300 700 538 340 540 200 109
82 86 86 86 52 86 86 80 82 90 82 90 90 82
Antifoam, % Vegifat YOctadecanol3
Inoculum, % Vegetative, 7
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 42, No. 1
Figure 6. Part of Purification Pilot Plant Equipment a completely liquid medium is utilized, only the insulation properties of the glass jars are effective as variables, but when a medium composed of both liquids and solids, such as soybean oil meal, is utilized, a second insulating quality, that of the solid, enters the picture. Thermocouples placed in the jars indicate that, although the temperature of the autoclave reaches 115’ to 120 C. within a few minutes after autoclaving begins, the temperature of the liquid-solid medium in the bottom of the jar does not exceed 100’ C. until at least 30 minutes later and usually does not exceed 110 C. during a sterilization time of 30 to 60 minutes. Although the utilization of metal jars in place of the glass will reduce the glass insulation variable to a minimum, no practical stirring device has been developed to minimize the insulating effect of the solids in the medium during sterilization. The sterility of the jar is maintained after inoculation by a positive air pressure of 1 to 2 pounds per square inch inside the fermentor. A valve from the outlet line of the fermentor connected to the air exhaust header through a mercury manometer permits accurate pressure regulation. O
FERMESTATIOS OF NEW ANTIBIOTICS
The preliminary selection of new antibiotics is carried out in the microbiological research department under the direction of J. hf. McGuire. After determining that the microbiological spectrum of a new isolate appears interesting to the research personnel, further work is discussed with the development group and plans are made to continue the investigation of the isolate. The research group reports the details of the shake-flask work that they have completed and the methods of testing for potency. The culture is then released to the pilot plant for preliminary fermentation studies. Experimentation is carried out simultaneously in shake-flasks, and in 30-liter jar fermentors, in an attempt to produce yields high enough to supply starting materials for the purification pilot plant work. I n the shake-flask studies, various types of media are utilized to determine the type which will give the highest production of the desired antibiotic. All of the variables mentioned underpenicillin and streptomycin are studied in a preliminary way with
respect to the new antibiotic. Data secured from the shake-flask experiments are translated to the 30-liter fermentors and verified on a larger scale. At this point, yields in the 30-liter fermentors become of prihary importance for further purification work. Simultaneously with the above, work is initiated on testing procedures which can be utilized both for fermentation and purification. Such tests are developed in isolated laboratories which have been set up specifically for new antibiotics work in order to eliminate possible cross-contamination with penicillin and s t r e p tomycin testing. Preliniinary testing is usually carried out using a broth or agar dilution test. It cannot be emphasized too strongly that neither fermentation nor purification work can be carried very far until a suitable test, a t least semiquantitative, has been developed. Some standard must be adopted to determine the relative potencies obtained. Usually, by the time the test has been developed for daily use, 30-liter fermentor work has progressed far enough to warrant production in 85-gallon fermentors. Shake-flask and 30-liter jar fermentor data are translated t o the tanks, and the broth from the tanks is turned over to the purification group. Work continues until a decision is reached on the value of the new antibiotic, and it is either put into routine production or is dropped. Generally, the data obtained in the 30-liter jar fermentors can be translated directly to production scale. PURIFICATION
The purification of streptomycin and penicillin and the isolation and purification of new antibiotics present ever-changing problems that may best be solved in a purification pilot plant. The design of new types of equipment and the discovery of new materials and methods require constant development work in a pilot plant in order to adapt these changes to commercial, competitive’production. Functions. The functions of the purification pilot plant are twofold: the development and control of new processes and methods for the purification of commercially-produced antibiotics; and the isolation and purification of new antibiotics produced in the fermentation pilot plant. The development of new processes and methods of production
lanuary 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
197
Figure 7 . More Purification Pilot Plant Equipment of penicillin and streptomycin represents a continuing challenge to remain competitive in the field. Beginning with the yellowcolored, low potency, amorphous sodium or calcium penicillin, the development group has improved the methods of purification of penicillin until the present time when penicillin-GI crystalline, potassium, is packaged as a white, theoretically pure, 100% crystalline powder produced at a cost many times lower than that of the original impure compound. The discovery in these laboratories of insoluble, slow acting procaine penicillin ('7, I S ) , requiring but a single injection per day in either oil or water menstruum, represents a phase o f t h e atudy of several hundred water-insoluble compounds of penicillin produced in the development laboratories and pilot plant. The need for solvents, used in the isolation and purification processes of penicillin and streptomycin, which do not present serious industrial health hazards has been the basis for a continuing pilot plant problem. The isolation and purification of new antibiotics continuously present new problems to the pilot plant staff. The harvesting of a few liters, probably of relatively low potency, of a new antibioticcontaining broth by the fermentation pilot plant sets the stage for intensive, preliminary, laboratory scale work. The personnel assigned to the study must determine the general chemical and physical make-up of the new compound. An acidic compound will be attacked one way, a basic compound another. Existing unit processes are studied with respect to the new compound, and preliminary isolation work begins. Concurrent with concentration and purification of the compound, studies must be initiated on toxicity of the concentrate. Full cooperation must exist between the purification and fermentation personnel in order to have additional material available a t times when i t is needed. As soon as sufficient data have been accumulated to indicate potentialities of a new compound, the fermentation group is instructed to grow larger tanks, and the purification problem proceeds to semiplant scale. Fermentors of 1600-gallon volume must be processed, and a highly purified compound must be prepared in quantities sufficiently large to initiate extensive animal and clinical investigation.
Only after the completion of the above studies and the approval of the clinical testing group can plant production be considered. At this time, the pilot plant engineering personnel make the necessary translations of data and equipment to commercial production scale. Equipment. The physical layout of the Lilly purification pilot plant consists of three laboratories for bench scale work and a large major equipment installation. For efficiency of work and proximity to the source of problems, all of the pilot plant physical layout is located as a unit in one section of the production plant. The three laboratories are equipped as research laboratories. In operation, one laboratory has become predominantly organic in the character of work done; the second has developed chiefly into a chromatographic installation; and the third contains solvent extraction and hydrogenation equipment. Each is staffed with highly trained personnel. The major equipment installation is shown in part in Figures 6 and 7 . Three 1600-gallon broth collection tanks do not appear in the figures. Two 36-inch Sperry filter presses with coated plates and frames appear in the background of Figure 6. Thirteen agitated tanks are available for purification work. Likewise, a 24inch coated Sperry filter press may be seen. The operator a t the right of Figure 6 is carrying out an experiment on a Sharples airdriven supercentrifuge. A Sharples centrifuge and a DeLaval centrifuge are available for use as needed. Figure 7 shows a 100gallon and a 25-gallon still on the platform to the left. A steamvacuum table is available for concentration work in 22-liter flasks and smaller equipment. Two Devine drying ovens are seen at the right of Figure 7. I n so far as possible, all of the tanks and processing equipment in the pilot plant are of stainless steel construction. The header systems, also of stainless steel, are so devised that any tank may be connected to any other tank, either directly, through the presses, or through the centrifuges. The entire pilot plant has one very important characteristic often missing in pilot plants today-hamely, space to expand, with a design permitting the temporary installation of many new pieces of equipment, The unit processes available are complete with the required
198
INDUSTRIAL AND ENGINEERING CHEMISTRY
equipment, Adsorption-elution processes may be investigated in the broth collection tanks where the broth may be acidified, or alkalinized, and the adsorbent added with agitation. Various sizes of Sperry presses can be connected to these tanks and the adsorbent material collected. The antibiotic material may be eluted from washing-type presses by recirculating the eluting solvent from one of the tanks, through the presses, and back into the same or another collection tank, or by batch methods. The solvent extraction process has many possibilities. The antibiotic-rich solution from one tank, chilled by circulating cold water in the jacket, if necessary, may be mixed with chilled solvent, from a second tank, in a third tank if desired. From the mixing tank the two-phase system may be blown or pumped to the Sharples or DeLaval centrifuges for separation. Or, the antibiotic-rich solution in one tank and the solvent in a second tank may be sent through header systems to a Podbielniak countercurrent centrifugal extractor and the antibiotic-rich solvent colLected in a third tank. The unit process, chromatography, is readily available in several size ranges. Glass column installations of 2- and 4inch size are available for small scale work, including the necessary header systems and pumps. Stainless steel columns (8- , 16- , and 22inch) are available with all the necessary accessory equipment for large scale work. Paper chromatographic equipment is available as an adjunct to large scale work in one of the laboratories. An automatic recording Podbielniak fractionating column is installed with all accessory equipment for work with multiphase solvent systems encountered in the purification processes or for control of solvent recovery. ADAPTATION OF DATA TO I’I(ACTICAL SCALE
An example of the application of data from pilot plant experimental work to a practical scale may be cited in the development of the vacuum-drum drying process for antibiotics. In the early purification of penicillin, it was necessary to dry the yellow fmal product from a water solution of the sodium salt a t a concentration of approximately 100,000 units per ml. Previous experience indicated that drying from the frozen state in vacuo was a possible solution to the drying problems in the finishing of penicillin. However, there were several distinct disadvantages to the method. Shell-freezing in small bottles was a time-consuming process. Furthermore, during the drying process, a short-time loss of vacuum allowed the frozen cake to melt and boil out of the bottles. Once drying was completed, the product was difficult to remove from the bottles, and the losses due to the material sticking to the sides of the bottles were unduly high. Various other methods of drying aqueous solutions were investigated extensively, and it was finally determined that the method involving vacuum drum drying seemed the most practical. A small dryer of the double drum type was available, and work was initiated in the pilot plant to adapt this equipment to the drying of the penicillin solution. Several difficulties were immediately encountered: I. Method of feed 2. Method of collection of output 3. Maintenance of sterility during operatiori 4. Entrainment losses during the process 5. Control of vacuum Boiling occurred during the feeding operation because of the air dissolved in the solution and the relatively high vacuum maintained within the dryer. It was necessary to devise and build an apparatus, which would fit within the dryer, to provide for deaeration of the solution entering the dryer without disturbing the pattern on the drum itself. This apparatus was fabricated of glass and, with minor modification, i t is still used today. The output of the dryer was scraped from the drum, after drying, by a knife and dropped down into thb base of the dryer. A method for continuous removal of the dried powder from the dryer, without disturbing the vacuum, was seriously needed. This problem was solved by building a funnel through the base of the machine. An external valve with a special adapter permits the
Vol. 42, No. 1
connection of 22-liter glass flasks to the funnel. These flasks are held in place against a rubber base on the adapter by the vacuum within the dryer. Loss of fine powder caused by the product dropping into the flask is prevented by building a vacuum by-pass outside the dryer, with an opening over distilled water in another 22-liter flask. Dust collects in the water in the second flask and can be salvaged in the next run. Maintenance of sterility of the final product was accomplished by presterilizing the machine before the drying operation was begun and by releasing the vacuum in the machine with air, which had been passed through special Seitz filters, at the end of a run. Presterilization was accomplished by the use of 3 pounds per square inch gage of steam pressure for a t least 8 hours prior to the beginning of a drying operation. As soon as the steam for the sterilization cycle was turned off, vacuum was immediately applied to the machine throughout the cooling stage prior to beginning the actual drying operation. Entrainment loss during the drying operation was very serious during the early stages of development but was circumvented by the installation of circular baffles immediately below the vacuum exhaust stack. The baffles were so designed as to throw the entrained material out away from the opening of the stack itself and allow it to drop back into the base funnel or into the shell framework of the machine where it could be later recovered. Control of vacuum was a very important problem, as too high a vacuum caused the liquid feeding into the machine to freeze; freezing was undesirable, and too low a vacuum did not permit proper and efficient drying. Since high pressure steam ejectors were used, this problem was quite complicated. I t was finally solved by the use of a needle valve to permit a slow leakage of air into the dryer shell. The air entering the shell is first filtered through a Berkefeld sterilizing filter. Careful control of the needle valve permits control of the vacuum within very close limits. After the completion of the pilot plant work on the laboratory model of the drum dryer, sufficient data had been collected t o permit the engineering personnel to design a machine with a much larger drum embodying all of the modifications described. .4lthough the example of the adaptation of a pilot plant problem to a practical scale, as described, was originally carried out in the purification of penicillin, later developments in the purification process leading to the direct crystallization of penicillin from solution have been reason for the abandonment of the use of this equipment for the production of penicillin. However, further modifications of the original design have permitted the later application of this method of drying to other products. Although the original investment is quite high, a pilot plant specifically designed for development work in the field of antibiotics is a vital necessity if a commercial organization is to maintain its position in the competitive field of antibiotic production. The proper utilization of such an installation will produce dividends in a short period of time. BIBLIOGRAPHY
(1) Behrens, 0. K., Corse, J., Edwards, J. P., et al., J . Biol. Chem., 175, 793 (1948).
(2) (3)
Behrens, 0. K., Corse, J., Huff, D. E., etal.,Ibid., 175,771 (1948). Behrens, 0. K., Corse, J., Jones, R. G., et al., Ibid., 175, 785
(1948). (4) Ibid,751.
( 5 ) Behrens, 0. K., and Kingkade, M . J., Ibid.,
176,
1947
(1948). (8) (7)
Corse, J., Jones, R. G., et al., J . Am. Chem. Soc., 70, 2837 (1948). Herrell, W. E., Nichols, D. R., and Heilman, F. R., Proc. Stag
Meetings, Mayo Clinic, 22, 587 (1947). ( 8 ) Jones, R. G., Soper, Q. F., et al., J . Am. Chem. Soc., 70, 2843 (1948).
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~
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RECEIVEDOctober 4, 1949. Presented before the Division of dgrioulture and Food Chemistry at the 115th Meeting of the AMBRICAN CHExfIcAL SOCIETY,San Francisco, Calif., 1949.
