Large-Scale Production of Protective Antigen of Bacillus anthracis inAnaerobic Cultures'

MILTON PUZISS, LEE C. MANNING2, JOE W. LYNCH, EUGENE BARCLAY, IRA ABELOW,AND GEORGE G. WRIGHT

U.S. Army Biological Laborator ies, Foert Detrick, Frederick, Maryland; Merck, Sharp and Dohme, West Point, Pennsylvaniia;and U.S. Army CBR Engineering Group, Fort Detlrick, Frederick, faryland

Received for publication 6 February 1963

ABSTRACT

PUZISS, MILTON (U.S. Army Biological Laboratories,Fort Detrick, Frederick, Md.), LEE C. MANNING, JOE W.LYNCH, EUGENE BARCLAY, IRA ABELOW, AND GEORGE G.WRIGHT. Large-scale production of protective antigen ofBacillus anthracis in anaerobic cultures. Appl. Microbiol.11:330-334. 1963.-A production-proving test was de-scribed for the preparation, by the anaerobic culturemethod, of large volumes of culture filtrate containingimmunologically potent protective antigen of Bacillusanthracis. The process consisted of the anaerobic cultureof a selected production strain in a chemically definedmedium. The culture was then clarified and sterilized byfiltration through sintered-glass filters. The sterile culturefiltrate was adsorbed onto a preformed aluminum hy-droxide gel, and the stabilized gel-antigen complex wasconcentrated. The final product had high immunizingpotency, as shown by both in vivo and in vitro assays, andwas well tolerated in man. Stability of the product toaccelerated aging was good, and storage at 4 C for 1 yearcaused only a minor loss in protective activity. Largevolumes of the highly antigenic gel-adsorbed protectiveantigen were readily produced by the method described.

The protective antigen elaborated by Bacillus anthracisduring aerobic growth has been shown to be effective inimmunizing animals and man against anthrax (Wright,Green, and Kanode, 1954; Schlingman et al., 1956; Brach-man et al., 1962). Subsequent research established thatprotective antigen was also elaborated in good yield underanaerobic conditions in chemically defined media (Wright,Puziss, and Neely, 1962). Adsorption of the culture filtrateantigen onto a preformed aluminum hydroxide gel, andpreservation and stabilization of the adsorbed product,was described by Puziss and Wright (1963). The stablefinal product had high antigenicity in experimental animalsand was well tolerated in man. These studies indicatedthat the anaerobic process could be readily adapted to

I In conducting the research reported herein, the investigatorsadhered to Principles of Laboratory Animal Care established bythe National Society for Medical Research.

2 Deceased, March 1962.

production of antigen on a practical scale. The objectiveof the work presented in this report was a study of therequirements for adaptation of the anaerobic culturemethod to provide a large-scale process for production ofanthrax protective antigen. ^

MATERIALS AND METHODS

This work was performed primarily at the facilities ofthe Merck, Sharp and Dohme Division of Merck & Co.,Inc., West Point, Pa. All animal immunization and chal-lenge studies were performed at Fort Detrick.

Cultures. Strain V770-NPl-R, a nonencapsulated, Inoni-proteolytic, and avirulent mutant of B. anthracis was usedas inoculum (Wright et al., 1962). Immunized animalswere challenged with the virulent Vollum strain of B.anthracis (Puziss and Wright, 1959).

Growth medium. The chemically defined 1095 mediumdescribed by Wright et al. (1962) was used; it was madeup as a 25-fold concentrated stock and diluted to required-volume in a fermentation tank. The chemicals were chemi-cally pure or reagent grade; all water was freshly distilledand obtained from the plant service line at 180 F. Com-position of the defined medium is presented in Table 1.

Production equipment. A glass-lined, jacketed, closedtank of 150-gal capacity (special type M; Pfaudler 1Ianu-facturing Co., Rochester, N.Y.) served for preparation ofmedium and growth of the culture. This vessel (Fig. 1)was equipped with a variable-speed impeller for agitationof the contents, and with the customary temperature andmotor controls. Cultures were filtered through a combina-tion of sintered-glass clarifier (Chem-Flow Corp., LittleFalls, N.J.) and sterilizer units, each 1 ft2. The clarifierfilter (5-,u porosity) was connected in series with two 1-,uporosity sterilizing filters. A flow rate of about 0.82 literper min was achieved; this required back-flushing of theclarifier filter with sterile water once per hr. A stand-by5-, porosity clarifier unit was used during the back-washlperiod. The back-flushed fluid was collected in a carboy,autoclaved, and discarded. The filtration arrangement isshown in Fig. 2.

Sterile culture filtrate was passed into a second, closed,glass-lined, jacketed tank (E.S. series; Pfaudler Manu-facturing Co., Rochester, N.Y.) for adsorption and holding

330

on Decem

ber 28, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

LARGE-SCALE PRODUCTION OF PROTECTIVE ANTIGEN3

at 4 C. This tank was also equipped with an impeller andthe customary refrigerant and motor controls. The gel-antigen complex was processed in an International centri-fuge (model 13L; International Equipment Co., Boston,MIass.).Aluminum hydroxide gel. The gel used as adsorbent and

adjuvant for the antigen was prepared by the method ofHansen (1953) in a glass-lined, steam-jacketed, open tankequipped with stirring paddles. A continuous-flow nozzle-discharge centrifuge (Sharples Corp., Philadelphia, Pa.)was used to remove and wash the gel. The final gel productwas tested for its antigen-adsorbing ability with a culturefiltrate of known immunizing activity. Standard gel ofproved antigen-adsorbing capacity, kindly supplied byInga Scheibel, Statens Seruminstitut, Copenhagen, Den-mark, was used as an adsorbent control (Puziss andWright, 1963). The gel product used in the present workresembled the standard Danish gel in its adsorptive capac-ity for antigen.

Assay methods. Culture filtrates were tested for in vitroantigen activity by the complement-fixation method ofMcGann, Stearman, and Wright (1961), and also by the

TABLE 1. Constituents requred for 300 liters of 1095 medium

Constituent* Concn

AB-G stock q/12 litersBiotin, crystalline .0.15Thiamine HCl .0.12Adenosine .0.30L-Alanine .2.70L-Tryptophan .3.12DL-Serine .6.24L-Arginine* HCI .................................. 6.24L-Proline .8.76Glycine .8.76DL-Methionine .9.00DL-Threonine (allo-free) .18.00DL-Valine ... 18.00DL-Aspartic acid .19.20DL-Isoleucine .19.20DL-Phenylalanine .20.40L-Histidine* HC .28.80DL-Leucine . 38.40L-Glutamic acid .50.40MnSO4*H20. 1.20MgSO4.7H20 . 3.00CaCl2*2H20 .4.44Glucose .300Guanine * HClt .................................... 2.76

* Included in 1095 medium were the solutions listed below. Cstock solution contained (g/12 liters): KH2PO4 , 204; and K2.HP04 , 261. Ferrous sulfate solution, which was heated to dis-solve and sterilized by filtration, contained: FeSO4-7H20, 0.87g/3 liters; and concentrated HCI, 4.5 ml/3 liters. Pyridoxal solu-tion, which was sterilized by filtration, contained 0.3 g/600 ml ofpyridoxal-HCl. Sodium bicarbonate solution, sterilized by auto-claving for 45 min at 121 C, contained 750 g/6 liters of NaHCO3 .

t Boiled to dissolve in 600 ml of water plus 9.0 ml of concen-trated HCI, added to AB stock, and brought to a final volume of12 liters.

agar gel diffusion method of Thorne and Belton (1957).In vivo-immunizing activity was assayed by immunizationand challenge of rabbits (Puziss and Wright, 1963). Allanimals received a single 0.5-ml subcutaneous immunizingdose of antigen diluted in saline., Then, 2 weeks later theanimals and unimmunized controls were challenged intra-cutaneously with 10,000 spores of the challenge strain ina 0.25-ml volume. This dose represented 500 to 1,000 LDIo.Residual glucose in the culture was determined by theanthrone method of Morris, as described by Neish (1952).The bacterial counts were estimated turbidimetrically in acolorimeter against a nephelometric standard suspensionof Salmonella typhosa obtained from the Division of Bio-logics Standards, National Institutes of Health, Bethesda,Md. Aluminum, aluminum oxide concentration, andformalin content in the final antigen product were deter-mined by the methods described in the ITSP. Sterility andsafety tests performed during production, as well as on thefinal product, were in accordance with Public HealthService Regulations (1961).

Process description of medium. AB-G and C stocks (12

FIG. 1. Steam-jacketed fermentation tank and controls, showingthe inoculating port (a), and the sampling tube (b).

VOL. 1 11 1963 331

on Decem

ber 28, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

32UZISS ET AL.

liters each) were added to 266.3 liters of distilled watercointainied in a 150-gal fermeintation taink. The taiik aiidits contents were sterilized by heatinig to 120 C anid cooliingimmediately to 37 C. After the temperature reached 100 C,pressure was equilibrated by allowing air to eniter througha sterile air filter; 6 liters of sterile sodium bicarboniate(12 U0) were thein added. Sterile ferrous sulfate and pyri-doxal hydrochloride solutionis, pooled in a 20-liter l'yrexglass bottle, were added to the contents of the tank, andthe tanik was inioculated with the seed culture. Final pHof the medium was about 7.9. A sterile tube, inisertedthrough the inocuilatinig valve, was used to introducenitrogen (sterilized by a filter) inito the tank to a pressureof 5 psi. The gas was initroduced close to the surface of themedium for 25 miii, anid displaced the air through the topvalve of the tank; the valve was then closed.The culture was incubated at 36 to 38 C under niitrogen

for approximately 27 hr, with agitation at a constant rateof 20 rev/miii. Samples for the various tests were removedthrough a sterile tube at selected intervals during growth.

Filtration and adsorption. At the terminatioin of incuba-tioni, the pH of the culture, which had dropped to pH 6.9,was raised to pH 8.0 with sterile 2 N sodium hydroxide andthe culture cooled to 18 to 20 C. A pressure of about 10psi of nitrogen was applied, and the culture was forcedthrough a glass valve in the bottom of the tank, throughthe clarifying anid steriliziing filters, and into the lholdingtank. Approximately 6 hr were required to filter the 300liters of culture.

Sterile aluminum hydroxide gel (12 liters) at a tempera-ture of 22 C was added to the filtrate. The mixture wasagitated for 30 sec at 6Q rev/min and then cooled to 4 C.Three times daily for 5 days, the suspension was agitatedfor 30 sec. An alternlate method, preferable when proper

FIG. 2. Chem-Flow filter assembly. The clarifier filters (a) are

attached to the sterilizer filters (b) arranged in series. Cualtitre fromthe fermnentor enters the assembly from the tube at right (c); sterilefiltrate leaves in the tutbe at left through an opening in the wall.

e(luipmneit is available, would be to agitate continuouslyat 15 to 20 rev/miil for 2 days (Puziss and Wright, 1963).Agitationi was disconitiiniied 20 hr prior to removal ofadsorbed anitigen fiom thie tanik to allow the material tosettle. Approximately :31 liters of the concentrated suispeni-sion of adsorbed anitigeni were removed through the bottomvalve. The supernatanit was discarded.

Final pr-ocessing. Tlhe adsorbed anitigen was distr ibutedaseptically into steiile 1 .5-liter l'yrex cenitrifuge bottles,eaclh containiilng several sterile glass beads. A centriflugalforce of 780 X g was applied for 10 min at 4 C, aiid thesupernatanit was removed aseptically by vacuum from eachbottle anid discarded. The gel sluirry remaining was str ailnedinto a sterile 45-liter Pyrex carboy through a sterile § 62mesh nylon straiinei. The pooled sluiry (approximately 10liters) was resuspended ini cold, sterile, physiological salinesolutioni to 28.5 liters. l'i'esei'vatives were added anid thecarboy was held at 4 C) utntil potency and safety tests werecompleted. The pieseivative conisisted of 750 ml of a1:1,000 solutioni of ieci'ystallized benizethonium chloride(Phemerol; Parke, Davis & Co., Detroit, Mich.) to a1:40,000 final concenitrationi. As a stabilizer, 750 ml of 1 %formalini were added to a final cotncentration of 0.0092 %formaldehyde. The finial voluime of product was 30 liters;this represented a telifold conicenitration from the original300 liters of culttuie filtrate. Determinations of sterilityby cultural tests, safety tests with mice and guiniea pigs,tests foi' antigenicity in i'abbits, and tests for aluminiumand formalin conteint were performed. Finally, vials wAerefilled with 20-ml (quanitities of the antigen. Approximately60,000 doses were obtainied from the 300-liter lot.

It ESULTS

In the early phases of the inivestigation, the fermenitationtank containing the cuilturle medium was inoculated with500 spores/ml of the productioni straini of B. anthracis, asdescribed previously (I'uziss and Wright, 1963). tUniderthese coniditions, the iniitiationi of growth was delayed,maximal turbidity anid uitilizationi of glucose were reduiced,and only small amouiits of pirotective antigen were detectedin culture filtrates.The difficulty was overcome by seeding the taiik with

an actively growinig vegetative iinoculum rather tlhain witlthe spore suspensioni. The cultture medium (10 liters) in acarboy was inoculated with 107 spores of strain V770-NP1-R, incubated statically in air for 26 hr at 37 C, anidadded to the main lot of medium. UTnder these coniditionis,good growth and maximwal accumulation of antigeni oc-curred in the 300-liter cuiltuire after incubation for approxi-mately 26 hr. At intervals during the incubation period,determinations of the pH, the bacterial count, the comple-ment-fixation titer, and the residtual glucose were carriedout; data compiled from several production experimentsare shown in Fig. 3. Glucose utilization was the mostsensitive anld useful indicator of the course of the fernmenita-tioin; earlier studies had showvn that maximal aiitigen

APPL. AlICROBIOL.33:2

on Decem

ber 28, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

LARGE-SCALE PRODUCTION OF PROTECTIVE ANTIGEN

accumulation coincided with the point at which glucosein the medium approached exhaustion (Puziss and Wright,1959). After 18 to 20 hr of growth, it was possible toextrapolate the glucose-utilization curve to estimate thetime at which antigen elaboration would be at a maximumand filtration of culture could be initiated. Antigenicityof five consecutive production lots as measured by in vivoand in vitro assays is presented in Table 2.

Stability to accelerated aging of the gel-adsorbed prod-uct at 37 C was studied. Antigen 9 was incubated at 37 Cand assayed in rabbits at weekly intervals to determinethe loss of immunogenic potency. The results indicatedthat the antigen had considerable stability to acceleratedaging; approximately 60 % of the test animals survivedchallenge after immunization with antigen heated at 37 Cfor 8 weeks (Fig. 4). Stability to accelerated aging of the' 1302

c 1000° 80a

.x 60

E 40

30

E0

u

omplement fixation titer

0

x

E"I

ag

14

10

8

6

Estimated bacterial countA

Glucose utilized

pH

19 20 21 22 23 24 25 26Fermentation time (hours)

7.47.37.27.1a7.0

6.9

E-Z co a

1000m 0

800 E -

700 - _0600 °

500.ot-AOO .S400,

s

0

0

cL

FIG. 3. Relationship of complement-fixation titer, bacterial count,glutcose utilization, and pH change in cutltures of Bacillus anthracis.

TABLE 2. Antigenicity obtained in six successive 300-liter cultures

Titers of culture filtrate Antigenicity of gel-adsorbed product*

Comple- Antigen dilutionLot no. ment Gel

fixation diffusion(501% end point 1:10 1:30 1:90

units/ml)

5 160 1:4 4/8; 6/86 160 1:4 6/87 160 1:4 4/8; 4/88 166 1:2 7/8; 7/8 5/8 5/89 160 1:2 7/8; 8/8 7/8 7/810 160 1:2 6/8

* The antigen product was diluted with saline as shown; asingle 0.5-ml immunizing injection was given. Results show thenumber of surviving rabbits over the total number challenged.Two control animals were used in each experiment, and nonesurvived.

product was equivalent to that of an experimental gel-adsorbed antigen prepared previously in the laboratory(Puziss and Wright, 1963). Several lots of antigen were

reassayed after storage for 1 year at 4 C. Results of theseassays indicated that antigenicity of the stored productdecreased to a slight but minor extent in some of the lots(Table 3).

Several lots of gel-adsorbed antigen have been used in a

continuing program of immunization of personnel occupa-

tionally exposed to anthrax, either in research laboratoriesor in industry. The antigen was well tolerated; mild andtransitory local reactions were elicited at the site of injec-tion in a very small percentage of those immunized. Fur-ther studies on the antigenicity of the product are inprogress.

DISCUSSION

The study achieved its objective, the development of a

laboratory method into a process suitable for large-scale

lOOr-

80

601-

40F

201-

5 6 7 8 9Weeks at 37C

FIG. 4. Stability of the gel-adsorbed product after heating at 37 C.

TABLE 3. Antigenicity of gel-adsorbed antigenafter storage at 4 C for 1 year

Survival ratio*

Lot no. Antigen dilution

1:10 1:30 1:90

8 5/6 3/69 5/6 4/6 3/610 8/8 - 6/8

* The antigen product was diluted with saline as shown; a sin-gle 0.5-ml immunizing injection was given. Results show thenumber of surviving rabbits over the total number challenged.Two control animals were used in each experiment, and nonesurvived.

VOL. 1 ly 1963 333

20r

on Decem

ber 28, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

334 PUZISS ET AL.

production of the protective antigen of B. anthracis. Theprocess described herein appears to be a reliable methodfor producing large volumes of the antigen in a form suit-able for immunizationi of man; it is adaptable to a furtherincrease in the scale of production should the need exist.Replacement of the spore inoculum with an actively grow-inig vegetative inoculum was the only change that wasrequired to adapt the laboratory procedure to use with300-liter lots of culture in tanks. Substitution of the vegeta-tive inoculum not only led to satisfactory growth andelaboration of antigen, but also reduced the incubationtime of the tank culture to approximately 26 hr. No ex-planation was established for failure of the tank-scalecultures to grow as satisfactorily as the laboratory-scalecultures when a spore inoculum was used.The preparations of antigen produced in the present

study appear to be at least equivalent to those preparedin the laboratory in all respects tested. Complement-fixation titers of the culture filtrates and the antigenicityof the adsorbed products in rabbits were generally similar(Puziss and Wright, 1963). Gel diffusion titers of the pres-ent filtrates were not significantly different from unpub-lished titers obtained with laboratory lots of filtrate; novalid comparison can be made with diffusion titers recordedby others because of the use of different antisera and theprobable influence of minor variations in technique. Thepresent preparations also resembled laboratory prepara-tions with respect to stability during storage at 4 C andduring accelerated aging at 37 C.The least satisfactory portion of the over-all process

was the centrifugation of the gel-adsorbed product. Thisprocedure is time-consuming and affords a potential sourceof contamination of the product. Newer concepts in theproduction method are under consideration in an effortto eliminate the troublesome centrifugation and maintainani essentially closed production system.The protective antigen is a rather labile substance

elaborated only under carefully controlled conditions. Thesatisfactory results obtained in production of this sub-stance in 300-liter lots may justify attempts to produceother labile biologicals on a large scale by bioengineeringtechniques.

APPL. AIICROBIOL.

ACKNOWLEDGMENTS

The authors are indebted to R. Wilson, C. Newman, andtheir staffs at Merck, Sharp and Dohme, and to ClarenceF. Lehman (Sp-4, Chemical Corps, U.S. Army) for as-sistance.The portioni of this work conducted at AMerck, Sharp

and Dohme was supported by a contract with the U.S.Army Biological Laboratories, Fort Detrick, Frederick,Md.

LITERATURE CITED

BRACHMAN, P. S., H. GOLD, S. A. PLOTKIN, F. R. FEKETY, M.WERRIN, AND N. R. INGRAHAM. 1962. Field evaluation of ahuman anthrax vaccine. Am. J. Public Health 52:632-645.

HANSEN, A. 1953. Preparation of aluminum hydroxide gel. WorldHealth Organ. Tech. Rept. Ser. 61:64-65.

MICGANN, V. G., R. L. STEARMAN, AND G. G. WRIGHT. 1961. Studieson immunity in anthrax. VIII. Relationship of complementfixing activity to protective activity of culture filtrates. J.Immunol. 86:458-464.

NEISH, A. C. 1952. Analytical methods for bacterial fermentations.Natl. Res. Council Can., Rept. 46-8-3, 2nd rev.

PUBLIC HEALTH SERVICE REGULATIONS. 1961. Biological Products-Title 42-Part 73, July 28, 1961. U.S. Department of Health,Education, and Welfare, Washington, D.C.

PUZISS, M., AND G. G. WRIGHT. 1959. Studies on immunity inanthrax. VII. Carbohydrate metabolism of Bacillus anthracisin relation to elaboration of protective antigen. J. Bacteriol.78:137-145.

PUZISS, M., AND G. G. WRIGHT. 1963. Studies on immunity inanthrax. X. Gel-adsorbed protective antigen for immuniza-tion of man. J. Bacteriol. 85:230-236.

SCHLINGMAN, A. S., H. B. DEVLIN, G. G. WRIGHT, R. J. MAINE,AND M. C. MANNING. 1956. Immunizing activity of alum-precipitated protective antigen of Bacillus anthracis in cattle,sheep, and swine. Am. J. Vet. Res. 17:256-261.

THORNE, C. B., AND F. C. BELTON. 1957. An agar-diffusion methodfor titrating Bacillus anthracis immunizing antigen and itsapplication to a study of antigen production. J. Gen. Micro-biol. 17:505-516.

WRIGHT, G. G., T. GREEN, AND R. KANODE. 1954. Studies on im-munity in anthrax. V. Immunizing activity of alum-precipi-tated protective antigen. J. Immunol. 73:387-391.

WRIGHT, G. G., M. PUZISS, AND W. B. NEELY. 1962. Studies on im-munity in anthrax. IX. Effect of variations in cultural condi-tions on elaboration of protective antigen by strains of Ba-cillus anthracis. J. Bacteriol. 83:515-522.

on Decem

ber 28, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from