u I ~ T I N MARIETTA ENERGY SYSTEMS LIBRARIES

3 4456 02773b3 3

ORNL-6355

Expedient Antibiotics Production Final Report

interagency Agreement DOE 1457-1457-A 1 and FEMA EMW-84-E-1737, Work Unit 241 1K APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED

Printed in the United States of America. Available from National Technical Information Service U S . Department of Commerce 5285 Port Royal Road, Springfield, Virginia 22161 NTlS price codes-Printed Copy: A 1 0 Microfiche A01

This report was prepared as an account of work sponsored by an agency of the UnitedStatesGovernment. NeithertheUnitedStatesGovernment norany agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United StatesGovernment or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of theUnitedStatesGovernment or any agency thereof.

l a . REPORT SECURITY CLASSIFICATION

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3 DISTRIBUTION 1 AVAILABILITY OF REPORT

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4 PERFORMING ORGANIZATION REPORT NUMBER(S)

Unlimited
S MONITORING ORGANIZATION REPORT NUMBER(S)

ORNL

-

6345
6b OFFICE SYMBOL (If dpplicable)

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7a. NAME OF MONITORING ORGANIZATION

6a NAME OF PERFORMING ORGANIZATION

Oak Ridge National Laboratory

N /A -., -7b ADDRESS (City, Stan, and Zli

u1I lllulul IIulllIlUIl
MARTIN MARIETTA ENERGY SYSTEMS LIBRARIES

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8a. NAME OF FUNDING / SPONSORING ORGANIZATION Federal Emergency 8b OFFICE SYMBOL (If d p p k d b k )

3 4 4 5 6 0277363 3

9 PROCUREMENTINSTRUMENT~OENTIFICATION NUMBER

Management Agency

Interagency Ag. EMW-84-E-1737 (FEMA) 1457-1457-A1 (DOE)
10 SOURCE OF FUNDING NUMBERS
PROGRAM TASK No.
WORK UNIT

2411K

I

9

I

I

ACCESSION NO

19

I

EXPEDIENT ANTIBIOTICS PRODUCTION
1 2 PERSONAL AUTHOR(S)

I
14 DATE OF REPORT (Year, Month, Day)

Paul R. Bienkowski, Charles H. Byers, and Douglas D. Lee
13a. TYPE OF REPORT

Final
16 SUPPLEMENTARY NOTATION

13b TIME COVERED FROM 1985 TO

1988

April 1988

17

FIELD

I

COSATI CODES GROUP 1 SUB-GROUP

18 SUBJECT TERMS (Continue on reverse

if

necessary and rdentl+ by block number)

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I
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Sulfa, Penicillins, Tetracyclines, Bacitracin, Cephalosporins, Antibiotics, Antibiotic Manufacturing, Antimicrobials, Emergency Production of Antibiotics (cont'd

19 ABSTRACT (Continue on reverse if necessary and Identify by block number)

~UNCLASSIF~ED~UNLIMITED SAME AS RPT 0

0 OTIC

USERS

Unclassified
22b TELEPHONE (Induck Area Code) 22c. OFFICE SYMBOL
I

22a NAME OF RESPONSIBLE INDIVIDUAL

I

1

00 FORM 1473,84 MAR

83 APR edition may be used until exhausted. All other editions are obsolete.

SECURITY ClASSlFlCATlON OF THIS PAGE

Antibiotic Manufacturers, Antibiotic Purification and Separation, Alternate Antibiotic Production Sites, Fermentation Equipment, Disease Control, Pharmaceutical Industry

19.

ABSTRACT (continued)

I
I

particular antibiotic product. The fermentation section requires specialized equipment for operation in a sterile environment which is not usually available in other industries. The emergency production of antibiotics under austere conditions will be feasible only if a substantial reduction in the complexity and degree of separation and purity normally required can be realized. Detailed instructions were developed to assist State and Federal officials who would be directing the resumption of antibiotic production after a nuclear attack. Initially, all plant managers should be contacted (using the lists provided in this report) to determine if their facility has been destroyed or damaged. If a plant has been damaged, the manager should determine the feasibility of making repairs, the potential capacity of the repaired plant, and the type of antibiotic that can be produced. If enough production cannot be realized from undamaged and slightly damaged plants, the plants from the list of alternate emergency production sites must be contacted, determination of the plant status made, and the managers informed of impending conversion to antibiotic production. If alternate sites must be used, a team of skilled personnel must be assembled to convert the plant to antibiotic production in the austere environment.

SECURITY CLASSIfICATION OF T H I S P A C E

DE-AC05-840R21400 . D. Category UC-41 Chemical Technology Division EXPEDIENT ANTIBIOTICS PRODUCTION P. f o r the U. DEPARTMENT OF ENERGY under Contract No. DOE 1457-1457-A1 formerly 40-1457-84 Work Unit No. R. H. Approval does not signify that the contents necessarily reflect the views and policies of the Federal Emergency Management Agency. DC 20472 Interagency Agreement. 'I Prepared by the OAK RIDGE NATIONAL LABORATORY Oak Ridge. FEMA EMW-84-E-1737.S. Tennessee 37831 operated by MARTIN MARIETTA ENERGY SYSTEMS. Lee Date Published Approved for public release: - ?lay 1988 Distribution unlimited Prepared for the FEDERAL EMERGENCY MANAGEMENT AGENCY Washington. Byers D. Bienkowski C.ORNL-6355 Dist. Inc. 2411K "This report has been reviewed in the Federal Emergency Management Agency and approved f o r publication.

iii

CONTENTS

LISTOFTABLES LISTOFFIGURES

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V

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vii xi xi .xi xi xii xvii

EXECUTIVE SUMMARY Objective . Background . Procedure . Conclusions ABSTRACT

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1.
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INTRODUCTION

1
3
5

DEFINITION OF AN ANTIBIOTIC

ANTIBIOTICS SELECTED FOR EXPEDIENT PRODUCTION
3.1 3.2 3.3 3.4 3.5

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Penicillins . . Cephalosporins Tetracyclines . Bacitracin . . Sulfonamides .

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14 14 16 19
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ANTIBIOTIC MANUFACTURERS

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5.

ANTIBIOTIC PRODUCTION TECHNOLOGIES . GENERAL DESCRIPTION 5.1 5.2 5.3 5.4 5.5

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Raw materials and utilities . . . . . . . . . . . . . Fermentation . . . . . . . . . . . . . . . . . . . . Separation and purification . . . . . . . . . . . . . Analytic Procedures/ Quality Control . . . . . . . . Advanced Technologies . . . . . . . . . . . . . . . .

25 27 28

30
30 33 33 33 44

6.

EXPEDIENT PRODUCTION OF ANTIBIOTICS

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6.1 Alternate production sites
6.2 6.3

. . . . . . . . . . . . . Conversion to antibiotic production . . . . . . . . . Single Cell Proteins . . . . . . . . . . . . . . . .
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7.

ALTERNATE SOURCES OF ANTIBIOTICS

47
49

8 . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . .

8.1 Cited References . 8.2 General References 8.3 Penicillins . . . . 8 . 4 Cephalosporin . . .

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49 52
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57

iv

8.5 Tetracyclines . . . . . . . . . . . 8.6 Bacitracin . . . . . . . . . . . . 8 . 7 Sulfonamides . . . . . . . . . . . 8.8 Separation and Purification . . . 8.9 Preservation . . . . . . . . . . . 8.10 Sterilization . . . . . . . . . . . 8.11 Analytical Methods . . . . . . .
APPENDIX A APPENDIX B APPENDIX C APPENDIX D APPENDIX E APPENDIX F APPENDIX G APPENDIX H APPENDIX I APPENDIX J APPENDIX K APPENDIX L

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58 59
60 63 63 64
60

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Production of Penicillin Antibiotics

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65

Production of Cephalosporin Antibiotics . . . . . . . 87 Production of Tetracycline Antibiotics Sulfonamides

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103

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111
117

Bacitracin Production . . . . . . . . . . . . . . . Organic Product Fermentations . . . . . . . . . . . Enzyme Production Technology

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133 139 145

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Single Cell Protein Production

Inoculum Preservation . . . . . . . . . . . . . . . Sterilization Equipment

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151
161

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Separation and Purification . . . . . . . . . . . .

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High Performance Liquid Chromatography

Supercritical Fluid Extraction . . . . . . . . . . . . . .

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V

LIST OF TABLES

Number

Page

1. U.S. antibiotic production 2.
3.

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6

Antibiotics produced in the U.S. (by classification) . Dosage forms and dosage recommendations for expedient antibiotics . . . . . . . . . . .

1 1

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12
20 26

4. Risk assessment for antibiotic producers in the U . S . assuming three different attack scenarios . . . . .
5.
6.

Growth media for penicillin

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shake flask and factory

Risk assessment for enzyme producers in the U.S. assuming three different attack scenarios . . .

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34

7.

Risk assessment for fermentation ethanol producers in the U.S. assuming three different attack scenarios

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36
40
48
68

8.

Other candidates for conversion to antibiotic production Alternate antibiotic production sites outside of the continental U.S. . . . . . . . . . . . . . . .

9.

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A-1. Raw materials needed for penicillin production A-2. Alternate materials for defined medium for penicillin production . . . . . . . . . .

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68
71

A-3. Specific uptake rates for nutrients during a penicillin fermentation. . . . . . . . . . . . . . . . . . . . . .
A - 4 . Carbon distribution into penicillin, mycelium, and maintenance energy . . . . . . . . . . . . . . .
A - 5 . Maximum and real yields of penicillin from glucose

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73 73 78

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A-6. Traditional penicillin purification
B - 1 . Cephalosporin production media.

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90

B - 2 . Carbon utilization for cephalosporin production

91
94 95
97

B - 3 . Clinical characteristics of common cephalosporin
antibiotics

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B-4. Characteristics of cephalosporins of clinical interest
B - 5 . Microorganisms which produce various cephalosporins

. . . . L 3 . . . .vi Number Page E. . SCF related physical properties of selected antibiotics . . . . . . . . . . 170 177 178 183 L 2 . . K 1 . . HPLC assays of antibiotics . . . . 122 122 . Solubility o f bacitracins in various solvents E. . . . . . . . . . . . Precipitating agents for bacitracin . Chromatographic parameters for tetracyclines .2 . . . . . L. . .1 . . . Suppliers of oxygen probes used in fermenters . . . . .1 .

. . . . For convenience. . . . . . . . . . . Antibiotic producer locations superimposed on U. . . . . Structure of Tetracycline showing substitutions to get related antibiotics . . . . . . . .vii LIST OF FIGURES Number PaRe 7 8 10 15 17 1. . Procedure and flowsheet for beginning a penicillin fermentation. 3. .7 . map with two types of nuclear attack . A-3. . . . . . . . . process 22 7. . .6 . . . . . . . . . . . . . . . . . . . . . A . 4. . . . . . . . . . . 76 77 79 81 A . . . . extraction solvent (isoamyl acetate). . . .S. . . . . . . map with two types of nuclear attacks . . . . . . . . . . Structure of Bacitracin A . . . . . . . Penicillin purification process of Gist-Brocades . . pH o f penicillin G between water and solvents . . Enzyme producer locations superimposed on U. . . . Fermentation ethanol producer locations superimposed on U . . Chemical structures of some semisvnthetic penicillins . . . . . Chemical synthesis of 6-aminopenicilanic acid (6-APA) A . . . 2. . . Structure of sulfonamides 5. . .S. . S . . . . . Distribution coefficients vs. . . . . 29 9. . . . . . 70 72 A-2.5 . . Block diagram for a general antibiotic production . 24 8. . . . . . . . . .8 . . . . . Typical concentration profile of fed-batch penicillin fermentation. . . . . Traditional three-stage extraction of penicillin . . . . . . . . . . . Chemical Modification to penicillin G to produce semisynthetic penicillins . . . . . . . 6. . . . . . . . . . A . . . . . . . . . . . . . . . . . 38 10. . . . . . . most of the H atoms in the formula have been omitted . . General production scheme for penicillin production . . 82 . . . . . 39 A-1. . Partition of penicillins as a function of the pH of the . . Structures of Cephalosporin C and Penicillin G produced in antibiotic fermentations . map with two types of nuclear attack . 74 A-4. . .

. . Common stirred-tank batch fermenter for ethanol production F . . F-4. Structures of many of the cephalosporin antibiotics . . . . . . . . . . . . . . . . Therapeutically important sulfa drugs and sulfones E-1. Biosynthetic pathway for the formation of cephalosporin B-3. Common commercial enzyme process . .3 . . Purification equipment for anhydrous ethanol production G-1. 93 96 100 102 106 107 c-1. . . F-1. . . . . . . . G . . . . . . . PaRe 84 85 92 .viii Numb er A-9. . . . . . . . Equipment used in the production of ethanol from corn stover . . . B-4. B-2. .2 . . . . Intracellular enzyme purification f l o w sheet G . . . . . .2 . . 136 137 . . . . . . . . . Alternate enzyme recovery processes . . . . . . Production scheme for oxytetracycline . Block diagram of the Hutchinson. . . . 138 H-1. . . MN. D-1. . Chemical structures of some semisynthetic cephalosporins B-1. . . . . . . . . . . . . . . Cephalosporin C purification from broth by adsorption . . . . . c-2. . . . . . . . . . . . . . . 129 130 131 F . . . Schematics for recovery and concentration of cephalosporin C B-5. . 114 120 Bacitracin production flow and equipment scheme . . E-2. . . . Conversion of penicillin G into 7-aminodeacetoxycephalosporanic acid (7-ADCA) . Production chart for chlortetracycline . . . . . HPLC chromatogram of purified bacitracin 121 124 128 . . . . . SCP production facility . . . . . . . 142 1-1. .3 . . . . . . . Block diagram for the production of bacitracin E-3. . . . . . . . 149 . . A-10. . . . . . . . . . . . Chemical deacylation of Cephalosporin C . . . . . . .The effect of drying temperature on survival of freeze driedcells. Purification equipment for 95% ethanol distillation .

. Continuous steam sterilization of medium feed 5. . . . . . . . . . . . Continuous sterilization using heat exchangers . . Schematic diagram showing the many types of chromatography that are in use . . . . L. . . . K. . . . . . .5 L. . . . 174 175 176 L2 . . . . 181 . . . . . . . . . 168 . . . 178 .2 Pane 154 154 157 . . K2 . . . . . . 158 164 165 K1 . . . . . . . . . . . .3 . . .ix Number J. Geometric configuration for agitator placement in a typical fermenter . . . . . . Agitator shaft seal for a fermenter . . .l 5. . .5 . . Sample chromatograms for the separation of cephalosporin C Schematic diagram of continuous rotating annular chromatograph .3 Process chromatograph flow diagram . . . . . . . . . Schematic diagram for supercritical fluid extraction using GO2 . . . . . . . Piping diagram for fermenter air filtration . .l . . . . . Survival curves for sterilization of spores . . . . L4 . . Mechanical shaft seal for fermenter agitator . . . . . . . . . . 5.3 . . . . .4 . Typical piping arrangement for sampling and inoculation K. . 166 167 K4 . . . . . . . . . . . . . . . . . . Standard fermenter diagram . . . . . . . . L. . . . . . . . . . .

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modification We also propose and of antibiotic production equipment. PROCEDURE The approach used to obtain the necessary information and develop the desired instructions for the expedient production of antibiotics involve the following: 1. identify several antibiotics for emergency production based clinical need and efficiency of production and provide documentation on the production of the antibiotics. at the same time. conditions under which epidemic diseases and large numbers of injuries occur. . processes that would allow expedient production of broad-spectrum antibiotics under the austere conditions following a nuclear attack. BACKGROUND Meaningful preparedness for catastrophic disasters which would cause mass casualties includes both development of capabilities for reducing the impact of the hazard. and steps to deal with the effects that cannot be prevented. The availability of broad-spectrum antiOne purpose of this project is to on biotics could be of major importance to the survival and recovery of the threatened population.xi EXECUTIVE SUMMARY OBJECTIVE It is desired to determine the most efficient methods for meeting the antibiotic needs of the population for the period beginning 30 days after a nuclear disaster. material. A catastrophic disaster could destroy production capabilities for antibiotics and create. based on need and production capabilities. Select antibiotics for emergency production.

sulfamethizole. making recommendations as to how these facilities could be used for production of antibiotics. widely bacitracin (as a topical antibiotic). Transportation is can be simplified and dosage control for oral antibiotics is feasible outside of sterile hospital locations. Capsule. and are suspension forms provide enhanced storage stability and decreased need for sterility. tablet. Identify alternative sources of antibiotics. and quality control required for injectable forms. etc. and available). materials. an alternative if cephalexin is not able to be produced). The expedient dosage form should be oral. Identify potential emergency production sites which lie outside of the risk areas. purity.xii 2. Recommend modifications in current production technology and functional substitutions for certain basic equipment and facilities which would allow rapid conversion of emergency production sites to antibiotic manufacturing. and processing steps for the selected antibiotics. Document current U. 5. potential capacities. 3. Document current production technologies including equipment. Antibiotics chosen are penicillin V and amoxicillin (from penicillin G)(and combined with clavulanic acid. cephalexin (a cephalosporin). and sulfanilamides (sulfamethazine. 6. sulfathiazole.) which chemically produced. and procedures for expedient contacting of plant managers after a nuclear attack. 4. and and oxytetracycline prescribed (broad-spectrum. Several large antibiotic-manufacturing facilities lie outside of the risk areas and probably will not be damaged. antibiotic production sites.S. and purification methods Fewer and simpler separation used. . widely tetracycline tolerated. CONCLUSIONS 1.

determine the Company headquarters status of warehoused should be contacted to antibiotics. estimates b. and accurate materials a.xiii 4. a.. All existing antibiotic-manufacturing facilities should be surveyed immediately after an attack. Charlotte. d. Undamaged facilities should be operated at maximum capacity for a selected antibiotic. Evaluate and determine the best separation method for retrofit under austere conditions. Provide State and Federal personnel with a retrofit design at once-could also serve as the model for the other retrofits which might be needed. A "model" retrofit design to an existing plant (e. Design protocols for the conversion of undamaged non-antibiotic production sites to antibiotic production must be determined. SCP plant) should be made. Boston. MN. 8. St. San Francisco. Rapid assessment should be made of damaged facilities to determine repair feasibility. 6.to 8-week working inventory of finished products ready for formulation. b. manpower. Designated possible expedient antibiotic-production sites . Provide State and resource required. The outcome of this model design would be: Federal personnel with very of time. 7. Louis. c. Louisville. and Atlanta have major distribution facilities. Establish methods of planning for conversion. Dallas. Distribution facilities for these companies located in major cities throughout the country also may have substantial inventories of antibiotics. Seattle. 5. Pharmaceutical companies often maintain a 4 . subject to feedstock and solvent availability. Miami. Several large antibiotic-manufacturing facilities lie just within the risk areas and may sustain only minor damage.to 16-week inventory of finished products and a 4 .g. Kansas City. Chicago. penicil- lin V production at the Hutchinson.

12 . etc. A l s o someone who is familiar with the biological needs of the fermentation organisms and the growth and fermentation of microorganisms biologist. Many strains . Someone with plant construction or modification experience is also desirable to help plan the modifications and implement the des ired changes. 9 Personnel who are familiar with a chosen emergency antibioticproduction plant must be brought together. highly a complex specialized. Primary alternate sites must include an in-place aerobic fermentation system and should contain some existing separation/ purification equipment which could be adapted to one of the antibiotic production schemes.xiv should be surveyed. Impure products are generally acceptable only for oral use. and their management should be informed of impending conversion to antibiotic production. may also be used. plant operators. Included are the operating engineer.) is necessary. Other methods that produce a usable product in one or two steps. A listing of technical experts both within and outside of the military should be assembled and kept current. 11 Antibiotic production aerobic couples a relatively section small. and chemical and biochemical engineers. fermentation with multistep separation/purification section. 10 To assure the availability of personnel who are adequately trained in the use of the equipment and the process. Expedient produc- tion at alternate sites will only be feasible if there is a significant simplification in the separation and purification sections of the process. This may be accomplished by reducing the number and complexity o f the separation steps by using new separation methods. such as filtration followed by spray drying. thereby producing an impure product. molecular microbiologist. The specialized microorganisms used in the various antibiotic fermentations must be procured and kept viable. consideration should be given to training military personnel who have a background in this area. (such as a biochemist.

General utility requirements for antibiotic production include plentiful steam for sterilization and for solvent recovery and purification. Electricity is required to run the pumps. They could be used with caution in an emer- 15. Consideration should be given to importation of the necessary antibiotics from these locations. extractors. and chilled water machinery. if the most efficient strain is not selected and used. 17. etc. but lower in purity. Basic analyt- . 16. organic acids. and responsible funding agencies should be identified. 6-APA. but. filter aid and precoats for broth filtration. Small volume antibiotics with specialized uses (e. finished bulk chemicals (sulfur. Consideration should also be given to stockpiling some specialized equipment. steep lactose). Many veterinary antibiotics are identical to human drugs. 14. A source of chilled water is needed to remove heat from the fermenters and to provide cooling for the separation processes that often operate at 0 to 3°C.xv of an organism can be used to produce a given antibiotic.g. The raw materials necessary for fermentations are generally available. Separation/purification requires organic solvents (amyl or butyl acetate. and other critical supplies. Substantial antibiotic capacity is available in Mexico and Puerto Rico. and various inorganic chemicals for pH control and growth. lincomycin for serious bacterial infections in penicillin-allergic patients) should be stockpiled. agitation equipment. nitrogen corn liquor or soybean meal). mixers. and various salts and minerals for salting-out procedures and pH control. 1-butanol).). 13. The desired strains of the organisms should be stock- piled by freeze drying or alternate long-term storage techniques that would allow the cultures to be immediately available to expedient producers. plant capacity can be substantially reduced (by ten to one hundred times). gency. cen- trifuges.. sucrose. and They include carbohydrates sources (such (like as glucose.

These technologies include high performance liquid chromatography (HPLC) using continuous annular chromatography. and membrane separation methods. supercritical fluid extraction. Several advanced separation technologies hold great promise for making vast reductions in the separation/purification resources required to produce purified antibiotics. . 18.xvi ical and microbiological laboratory facilities are necessary for process control and quality assurance.

and the managers informed of impending conversion . otics under austere conditions will be feasible only if a substantial reduction in the complexity and degree of separation and purity normally required can be realized. but generic. Plants that manufacture antibiotics in the continental United States. Detailed instructions were devel- oped to assist State and Federal officials who would be directing the resumption of antibiotic production after a nuclear attack. and Puerto Rico have been identified along with potential alternate sites such as those where SCP. If a plant has been damaged. enzyme. This investigation revealed that a typical antibiotic-manufacturing facility is composed of two main sections. Cephalexin (a cephalosporin). and a bibliography of the pertinent material was compiled. determination of the plant status made. the manager should determine the feasibility of making repairs.xvii ABSTRACT The literature on the manufacture. all plant managers should be contacted (using the lists provided in this report) to determine if their facility has been destroyed or damaged. and sulfonamide (chemically produced) were identified for emergency production. the plants from the list of alternate emergency production sites must be contacted. and clinical uses of antibiotics was reviewed. (1) a highly specialized. Five antimicrobial drugs. Bacitracin (topical). Initially. penicillin V and G (and amoxicillin with clavulanic acid). If enough production cannot be realized from undamaged and slightly damaged plants. and the type of antibiotic that can be produced. fermentation unit and (2) a multistep. tetracycline and oxytetracycline. and fermentation ethanol are produced. the potential capacity of the repaired plant. complex separation and purification unit which is specific to a particular antibiotic product. separation and purification. The fermentation section requires specialized The emergency production of antibi- equipment for operation in a sterile environment which is not usually available in other industries. Mexico. Detailed process flow sheets and process descriptions have been derived from the literature and documented.

I f alternate sites must be used.xvi i i to antibiotic production. . a team of skilled personnel must be assembled to convert the plant to anti- biotic production in the austere environment.

R. Such determinations z r e quite cnmplex and beyond the scope of the present study. treatment o f wounds and control o f communicable diseases. The extent to which antibiotic capacity becomes critical in a given postattack environment depends on many factors. Among the variables are: 1. This increased demand will arise from two sources. depending on the extent o f the population destroyed. or increase. Byers D. Lee 1. D. problems in the control of communicable diseases will rise sharply due to disruptions in the food and water supplies and sewage treatment systems. 3. Bienkowski C. The antibiotic requirements per survivor relative to preattack per capita demands. 2. some of which are interdependent. The current antibiotic-manufacturing sites .1 EXPEDIENT ANTIBIOTICS PRODUCTION P. remain constant. At the same time. and The sizes of surviving antibiotic stockpiles. This report documents the clinical uses of the various antibiotics and recommends antibiotics for expedient production in a postattack environment. INTRODUCTION It is expected that the per capita demand for antibiotics in the postattack environment will rise sharply although total demand may decrease. H. It is anticipated that a large portion of the current antibiotic production capacity will be destroyed because most antibiotic- manufacturing facilities lie within probable nuclear attack zones. The relative size of population losses and antibiotic capacity losses.

These include in decreasing order of desirability: damaged but rebuildable antibiotic production facilities. By coupling this study with mathematical models which predict possible post attack environments for assumed attacks. various possible methodologies and recommendations can be explored by FEMA planners. this report develops the methodologies necessary for expanding antibiotic capacity in a post attack environment.2 are identified along with descriptions of the manufacturing technologies for the selected antibiotics. In summary. The extent to which these recommendations are implemented will be determined by the actual environment in existence after a nuclear attack. msg and other molecular biology product makers. . brewers and fuel ethanol producers. animal and plant antibiotics producers. enzyme. Commercial production technologies for the selected antibiotics are documented along with recommendations on how these processes can be modified to allow for conversion of alternate production sites to antibiotic production. citric acid producers. single cell protein producers. A highly specialized fermentation section requiring sterile conditions is coupled to a very large separation and purification system which requires large volumes of solvents and a great deal of energy. and farm fermentation units. Possible sites for producing antibiotics under austere conditions are also identified. the dairy industry. The processes for manufacturing antibiotics are very difficult to reproduce at alternate facilities due to the unique nature of antibiotic production. large hospitals and universities with fermentation research facilities.

With the exception of the penicillins and cephalosporins which are produced by molds.' terial infections.' Other plants. which varies not only for the species but also the strain of the infecting organism and other conditions. The sulfonamides were originally active against a wide range of bacteria.' Antibiotics are in general ineffective against typical virus diseases except for the tetracyclines which are sometimes effective against large viruses. all major classes of antibiotics are derived from bacteria. DEFINITION OF AN ANTIBIOTIC Antibiotics are a class of antimicrobial chemicals which are produced by microorganisms (mainly the saprophytic molds and bacteria o f the soil). However current antibiotics are now more potent than the sulfonamides.3 2. Each antibiotic has its own characteristically selective "spectrum" of potency against various microorganisms. The sulfonamides are still the choice medication in several common bac- . but an increasing incidence of resistance in bacteria formerly susceptible to the s u l fonamides has decreased their effectiveness (a single mutation in the bacteria allows some organisms to develop resistance). antimicrobial substances are produced by the higher Some are synthetically produced such as p-aminosalicylic The sulfonamides were the first major acid and the sulfonamides. broad spectrum antimicrobial drugs. These substances will inhibit the multiplication o f various microorganisms or may even destroy these organisms by either interfering with cell wall development and/or metabolism. The term "broad spectrum" is applied to antibiotics which are effective against a wide range of bacteria.

.

They often cause chronic infections after puncture or burn wounds and are often resistant to penicillin but susceptible to cephalexin. They include antibiotics which are taken in capsule or tablet or suspension form-penicillin V.3 Figures 1 and 2 show the general structures of the cephalosporins. and tetracyclines. Table 1 gives the 1980 and 1985 figures for antibiotic production in in use.) or by semisynthetic means. A combin- ation of clavulanic acid (a suicide inhibitor of /I-lactamaseenzymes) with amoxicillin (called augmentin) gives similar or better coverage than cephalexin in many cases. coli. Once a manufacturing process is developed for one of these classes of antibiotics. different growth media. their share of production capacity and dollar value means that there are many plants producing or capable of producing them. penicillins. all of the others are then produced from penicillin . For example while there are many penicillins it is a viable alternative to cephalexin. the cephalexin derivative of cephalosporin C . precursors. only three are produced naturally by fermentation (penicil- lin G . etc.5 3. many similar antibiotics can be produced by either very minor changes in the process (i. 0. Penicillins. Also selected is the topical antibiotic Also. tetracycline and oxytetracycline. ANTIBIOTICS SELECTED FOR EXPEDIENT PRODUCTION The following antimicrobial drugs have been targeted for expedient production. If the production capabilities are available (it requires the same equipment as penicillin. some Pseudomonads and Bacteroides pathogens. the amoxicillin derivative of penicillin G . and the sulfonamides which are produced chemically.) organisms such as E. cephalexin. the United States. and V). bacitracin. and tetracyclines were selected for their widespread clinical applications because they are effective in common respiratory and gastrointestinal tract infections and some common opportunistic infections.e.. but uses a different organism). Opportunistic infections are caused by gram positive and gram negative (G+ and G .

246 4122 771 86.884 (International Trade Commission 1 9 8 0 ) Antibiotic U.S.211.488 10. 252. C ephalo sp o r ins Penicillins Tetracyclines All Others TOTAL .6 Table 1 .497.082 44 8 32. 280. Production ( $ Millions) 1 9 8 5 1. antibiotic production Antibiotic Cephalosporin Penicillin V & G Penicillin G for medicinal All other uses Semisynthetic Penicillins Amoxicillin Amp ic i11 in All Others Tetracyclines Sulfonamides Production (1000 l b s ) 977 6514 2750 3764 1768 323 964 481 6562 4841 Sales 1000 lbs) 970 1 97 4 Value ($1000) 32. 754. S . 2. U .

Structures of Cephalosporin C and Penicillin G prodwed in antibiotic fermentations.7 ORNL D W G 8 7 . 1.4 7 0 COOH CEPHALOSPORIN C PENICILLIN G Fig. .

Structure of Tetracycline showing substitutions to get related antibiotics. .OH Y OH DIFFERENT ANTIBIOTICS .CONH.4 7 1 . 0 OH 0 R. 2. ARE DIFFERENT IN Rl - R2 H H H OH TETRACYCLINE CHLORTETRACYCLINE BROMTETRACY CLINE OXYTETRACYCLINE H CI Br H Fig. AND R.8 ORNL DWG 8 7 .

Because the sulfonamides are produced chemically and do not require the complex and specialized equipment necessary for the fermentation antibiotics. The other natural penicillins are not therapeutically useful. However. it is desirable to have one topical antibiotic for their treatment.9 G by enzymatic (penicillin acylase) (Fig. Table 3 is a list of the various antibiotics and the generally recommended dosages for a course of treatment with the antibiotics. The sulfonamides were the first broad spectrum antimicrobial drugs. penicillin 0 are produced in commercial quantities. This can be used to estimate the total antibiotic required if the affected population can be estimated.4 Also listed are the approximate requirements to treat a million patients per month at the recommended treatment rate. Penicillins act by specific inhibition of bacterial cell wall synthesis. and it has a wide range of clinical applications. to a small extent. to the point where they do not have the same potency as the fermentation antibi0tics. 3 ) and/or chemical reacThe other reaction shown in Fig.1 PENICILLINS Over 100 different penici ins have been produced by natural fermentations. . Due to overuse. tion which penicillin resistance. 3.l They are still useful and the drugs of choice for urinary tract infections (UTI) and some ear infections. they would require less production resources in a postattack environment. The sulfonamides also represent a family of related drugs. penicillin V I and. The clinical activity of these three classes of antibiotics covers a "broad spectrum" of uses (Table 2). the effectiveness of these drugs as "broad spectrum" antibacterials has been reduced over the years as organisms built up resistance. Because of the possibility for a large number of burns and skin wounds. 3 is the deactivation reacis used by penicillin resistant organisms to confer tions. coming into widespread use in the 1930s. Bacitracin is the most common topical antibiotic. only penicillin G .

10 ORNL DWG 88-236 H 0 PENICILLIN G COOH I PENICILLIN ACYLASE C P H ENY LAC ET1C AC ID 6 -AM IN 0 P EN IC ILLA N IC AC ID CHEMICAL R EACY LATION SEMISYNTHETIC PENICILLIN Fig. to penicillin G to produce . 3. Chemical modification semisynthetic penicillins.

G- 0-Lactam 4. Phenethicillin Propicillin. 12. Cephaloridine. Sulfasalazine Sulfaquinoxaline. Cefadroxil Cefamandole.13. Piperacillin.6. Fennenta 10. 6. fbbott Laboratories 2. Inc. Rolitetracycline Methacycline.5. 15. Beecham Laboratories 5.17 Penicillin chrvsonenum G+. Sulfadiazine Sulfanitran. G- Polyketide 2.9. Antibiotics produced in the United States (by classification) ANTIBIOTIC --CEPHALOSPORINS-Cefaclor. Cephradine --SEMISYNTHETIC PENICILLINS-Amoxicillin. Nafcillin.13. Cefonicid. Mafenide Acetate. 11. SmithKline Beckman Corp.13 Penicillichrvsonenum G t O-Lactam 4. Sulfachlorpyradi zine Uti. Cloxacillin. G-. 17. 8. Merck & Co.8. Bristol-Myers Company 7. Sulfonamide 14.13. acr emoni um ear. Sulfisoxazole Sulfamethazine. Augmentin.9 11. Cefoxitin Cephalexin. subtilis Acetyl Sulfisoxazole.. Pfizer.27 Streptomyces SP. Inc. Inc 16. Inc . Oxytetracycline ---BACITRACIN--- PRODUCER ORGANISM ACTIVITY TYPE 3. 4. Inc. The Upjohn Canpany . Sulfathiazole.1 1 Table 2 . Pivampicillin. Tetracycline Demeclocycline.6.11. Bacampicillin. Cephalosporium skin bone. Hoffmann-La Roche Inc. Rickettsia. Oxacillin. Methicillin. Napp Chemicals. respiratory Streptomces 0-Lactam SP. 13. G+. Doxycycline Minocycline. Carbenicillin Ticarcillin. Dicloxacillin. American Cyanamide 3. Sulfabenzamide. IMC Chenical Group 11. Flucoxacillin ---NATURAL PENICILLINS--Penicillin V Penicillin G ---TETRACYCLINES-Chlortetracycline. Eli Lilly and Company 9. Mycobacteria Mycoplasma 10 Bacillus licheniformis B. Sulfamethizole.8. Cyclacillin Epicillin. Inc. Cephapirin Cephacetrile. Cephalothin Cefuroxime. Ampicillin. Salsbury Laboratories. Sulfadimethoxine Sulfachloropyrazine.15 Chemical Reaction PRODUCING CCMPANIES 1. Sulfacetamide. Anheuser-Busch.11 G+. Sulfonamide.6. 14. Burroughs Wellcome Co. Biocraft Laboratories. Ear 2.

2-2 m i l ufd 0.6-3.000 - Sulfamethoxazolel trimethoprim needed per l o 6 treatedlmonth treated lmonth = 16. u/d 10 d Oral syrup 0.8-2og 1-14g 10-2og 5-2Og 1-14g 14-40g 16-22.100 5.5 g / d 0.000 k g (tablets) 33.000 .300 = = Penicillin V required per lo6 treatedlmonth 33.4-1.800 k g (tablets) 40.000 = 4.000 1.000 k g (capsules) 20.000 k g (syrup) = 10.000 = Su 1fameth i z o le needed per 10 treated /month = 14.25-2 g/d 0.6 g/d 2-5 times/d as needed Penicillin G required per l o 6 treatedlmonth = 80.75-1.200 2.375.2-158 2.000 k g (tablets) 22.25-2 g/d 2-4 g/d 10 d 10 d 4-7 d 7-10 d 10-14 d 10 d Sulfarnethizole or Sulfamethoxazole/ trimethoprim Bacitracin 1.5-2 g/d 0. Antibiotic Penicillin G DosaRe form Dosage forms and dosage recommendations for expedient antibiotics Dosage Time Total given 80-375g 17-50g 8-50g 10-33g 3.000 kg (capsules) 20.2-15g 2.000 5. u/d 10-15d (powdered for reconstitution)(l mil u=60Omg Pen G sodium) Oral tablets 1-3 mil.000 k g (injection) 20.000 k g (syrup) Augmentin-Amoxicillin required per Clavulanic acid Cephalexin required per 1' 0 lo6 treatedlrnonth = = treatedlmonth = - 15.000 k g (capsules) 40. acid 1-4 g/d 0.000 kg (syrup) 14.3-33s 5.000 kg (tablets) 3.3-1.4g 4-16g 14000 ultube Tetracycline A C l Oxytetracycline 1-2 g/d 0.400 k g (tablets) 16.000 k g (tablets) = 8.6gld 0.5 g/d 0.75-1.5-4 g/d 1-2 g/d 0.600 10.000 k g (syrup) 20.000 50.000 7.000 k g (syrup) Amoxi c i 11in required per 10 tr eatedlmonth 5.800 1.8g Injection 5-15 mil.000 - Oxytetracycline needed per l o 6 treatedlmonth = 10.000 .375 g/d clav.6-2 mil u/d 0.000 kg (capsules) 15.200 2. 0 0 0 kg (injection) 40.5 g/d amox 10 d 10 d 7-10 d 7-10 d 7-10 d Penicillin V Amoxicillin Amoxicillin + Clavulanic acid (Augmentin) Cephalexin + 0.50.000 5.5-3 mil u/d 10 d Oral tablets Oral syrup Oral capsules Oral syrup Oral tablets Oral syrup 0.1-15g 5.25-2 g/d Oral capsules Oral syrup Oral capsules Oral syrup Inject ion Oral capsules Oral syrup Injection Oral tablets Oral tablets Oral syrup Topical 10 d 10 d 7-10 d 7-10 d 4-7 d 10-40g 5-40g 7-20g 1.000 kg (injection) = 17.000 = 14 x B a c it r ac in required per 10 l o 9 units .000 - 15.000 = 3.000 k g (syrup) 1 4 .000 k g (syrup) Tetracycline required per 1' 0 treatedlmonth = = = 1.12 Table 3.

and is converted to cephalexin or other active forms which are therapeutically important. while not harming resting cells.to 200. they act by inhibiting bacterial cell wall (peptidoglycan) synthesis. final fermentation broth concentrations are much lower (1. However. All three penicillins are produced by the same fermentation process with the constitution of the growth media (the added precursor) determining All of the natural penicillins are available in which is p r ~ d u c e d . the yield is strongly dependent on the strain which is used. ~ dry. strain selection. 0 .' Separation and purification often constitute 90% or more of the steps (especially in the production of injectable antibiotics) in an antibiotic plant. 3. over 20 therapeutically useful semisynthetic penicillins are derived from penicillin G . While many organisms can be used to produce penicillin.13 and kill only growing cells. and they have low toxicity. and its stability in solution is rather limited. In a manner similar to the penicillins. and they kill growing cells but not resting cells.2 CEPHALOSPORINS Cephalosporins are economically and therapeutically as important as the penicillins. ear.000-1 fermenters.0 to .bacteria. active against respiratory. Penicillin V.g Natural cephalosporin C is the main fermentation product.6.7 In general. Cephalosporin C is produced by the same fermentation process used for the penicillins but utilizes different growth media and producer organisms. the concentrations of However. crystalline form and are stable for several years under controlled conditions. However. and bone infections caused by G+ and G . Oxygen transfer and the specific culture used are quite important. 0 0 0 . These drugs are broad-spectrum antibiotics. with proper antibiotics in the fermenter are very low. penicillin can be produced in concentrations of up to 40 g/1 which is 1 or 2 orders of magnitude greater than many other antibiotics. Penicillin G is mainly administered parenterally in solution form. and G are produced with the submerged culture process in 4 0 . making the concentration in the fermentation broth very important. but is not very therapeutically active. skin.

as well as rickettsias. Like the penicillins and economic and cephalosporins.l1-I3 3. there are about 20 different organisms which The yield is low. leptospiras. mycoplasmas.4 BACITRACIN Bacitracin is used as a topical antibiotic. the separation Bacitracin is stored as and purification process is fairly simple. spirochetes. l 5 For animal feed uses (as a growth promoter).14 4 .3 TETRACYCLINES Tetracyclines are an important class of broad-spectrum antibiotics that are active against both gram-positive and gram-negative bacteria. 3. where binding of aminoacyl-tRNA to the ribosomal A-site is inhibited.l 4 While it is very effective when taken internally. the tetracyclines are produced by an aerobic fermentation process except that the organism is a bacteria instead of fungi. Tetracyclines act as inhibitors of protein synthesis with the site of action being the 30s ribosome. 0 g/L) and include many similar compounds resulting in a more The active cephalo- difficult separation and purification process. Bacitracin is produced by conventional aerobic submerged culture fermentations like those of the other antibiotics. and it has broadspectrum antibiotic effects against gram-positive and normal streptococcus and staphylococcus skin infection-producing bacteria. 4 . which separation and purification control the process depends upon the use. There are three natural and six semisynthetic tetracyclines of therapeutic importance. Many different strains of bacteria can be used. with maximum published produce tetracyclines. and chlamydias." sporins (cephalexin or others) are normally administered orally in capsule form.0 g/l. . The structure of bacitracin is shown in Fig. The most effective The extent to produces a fermentation broth concentration of 9 g/l. fermentation broth concentrations of only 2. Presently. a fine white powder with good long-term stability. this use is severely restricted due to problems with toxicity.

c CF . 0 c-c 05c . . most of the H atoms in the formula have been omitted. .” pp 6 6 5 . “The Bacitracins: Properties. For convenience. New York.15 ORNL DWG 88-282 2 CYSTEINE 3 \C-C / LEUCINE OH \ 1 c . Inc. Structure of Bacitracin A. C 0 c’ N-#-~-~-N% I c’ \ / N \ CLN \ 9 PHENYLALANINE Fig. 4.c--C+N\ C%N / \SHC C ‘ NH2 C’ \ C O ’ 4 5 \C GLUTAMIC ACID 12 ASPASAGINE N.H2 $ I ISOLEUCINE cC ‘’ 4 . 0 I 7 / .. O*C x C I 7 8 ORNITHINE ISOLEUCINE C oOL\ 10 HISTIDINEC c. ’c. J. ISOLEUCINE C C ’.. Biosynthesis.6 9 4 in Biotechnologv of Industrial Antibiotics ed E. and Fermentation. I c -c N OH * . 0\\ %N’c-cAc. 1986. Vandamme. Source: 9 . 11 ASPARTIC ACID HO\C c\ LYSINE / NX 6 $ I \ ti c\C 4 0 w N C-C-C-NH. Fqdyshov. Marcel Dekker.

R group in the para position. quent.” . However. N H . and they exhibit long-term storage stability when they are stored in light-resistant containers. serious or fatal reactions are infre- Most adverse reactions appear to be due to hypersensitivity and increase with increasing dosage.16 3.) and an .16 are normally administered in capsule form. Several examples are shown in The antibacterial properties are derived from the drug’s Sulfonamides ability to disrupt the microorganism’s metabolism. Adverse effects of sulfonamides are numerous and may involve nearly all organ systems. 5.S 0 . Fig.5 SULFONAMIDES Sulfonamides are a family of chemically produced anti-infective agents containing a benzene ring with a sulfonamido group ( .

17 ORNL DWG 88-235 / H H. .N -(->%--N 0 II \R SU LPHAN I LAM IDE WHERE R I : S I = C -NH. (5) NH2 J-J S (7) H WHERE ( 1 ) IS SULFAMETHYLDIAZINE (2) IS SULFAQUANIDINE (3) I SULFATHIAZOLE S (4) I SULFAMETHAZINE S (5) I SULFATHIADIAZOLE S (6) I SULFAQUINOXALINE S (7) IS SULPHANllAMlDE (SULFONAMIDE) F i g . 5. Structure of sulfonamides.

.

" The TR-82 risk areas were used in this analysis. Cyanamid's facility These include: American facility in in Hannibal. . The difference should not be significant since most antibiotic plants are located in highly industrialized areas assumed at risk in both documents. which is at the edge of the potential blast zone centered on West Lafayette. along with their telephone numbers. and Salsbury Laboratories' plant in Charles City. Indiana. Iowa. Many facilities in the risk areas are near the edge and may sustain only minor damage. As can be seen from Fig. for example. 6 . Table 4 lists all of the current manufacturing facilities. some of these production sites lie outside the risk areas and may not sustain damage. North Carolina.19 4. Missouri. ANTIBIOTIC HANITFACTURERS The first step to be taken by emergency managers after a nuclear attack is to contact the antibiotic-manufacturing facilities for a damage assessment. The analysis here may be a little more conservative (pessimistic). Indiana. Lilly's Clinton. Burroughs Wellcome's plant in Greenville. Eli Lilly has a very large plant south of Lafayette. The (current) NAPB-90 planning base was not available when this analysis was done.

Cephalosporins Cephalosporins Penicillin V. NY 13221 Onandaga XXXX xxxx Semi-synthetic Penicillins 444 2012 i d sls 919 248-3000 G r e e n v i l l e . Tenn.G f i Semi-Synthetic Penicillins 307 E .O.O. I L 60016 . N J 07407 B r i st o 1-My e r s Company P. N J 07463 Bergen XXXX xxxx Semi .20 Table 4. 27709 E l i L i l l y and Company 317 261-2000 317 474-1430 I n d i a n a p o l i s . Westchester XXXX Sullivan Somerset **** 615 764-5141 201 469-5200 B r i s t o l . NC 27834 Pitt ___ ___ Acyclovir. WV 26190 Bound Brook. 37620 Biocraft Laboratories. I L 60064 American Cyanamid Company One Cyanamid P l a z a Wayne. Box 4755 S y r a c u s e . I L 60064 910 235 1584 Lake xxxx ___ **** EES 1400 S h e r i d a n Rd. Risk assessment for antibiotic producers in the U. 46206 Fermenta ----XXXX ___ xxxx ___ 215 436-7500 West C h e s t e r . NY 13221-4755 Burroughs Wellcome Co. I N 47842 L a f a y e t t e . trimethopr i m 3030 C o r n w a l l i s Road Research T r i a n g l e Park N. N J 08854 XXXX XXXX xxxx **** B r i s t o l . Union S t . North Chicago. I N 47808 Vigo xxxx ___ xxxx Bacitracin Des P l a i n e s . assuming three different attack scenarios A N T I B I O T I C PRODUCER C M A Y HEADQUARTERS O PN TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY ATTACK ATTACK ==BLAST +*=FALLOUT ****=BOTH ANTIBIOTICS PRODUCED TELM ==HIT ---=Clear ==HIT ---=Clear ---=Clear (TR-82) Abbott L a b o r a t o r i e s 800 323-9065 North Chicago. NY 10965 Beecham L a b o r a t o r i e s 501 F i f t h S t . 315 432-2000 S y r a c u s e . S. N J 07470 201 831-2000 Hannibal.s y n t h e t i c Penic i l l i n s Elmwood Park. Inc 92 Route 46 201 796-3434 Waldwick. McCarty I n d i a n a p o l i s . P 19832 A 902517 ___ 510 E . Box 1808 West C h e s t e r . P. t9J 63401 130 400 W i l l o w I s l a n d . I N 47902 Marion Vermillion Tippecanoe Chester XXXX ___ _-_ -__ **** Penicillin V.C. TN 37620 Piscataway. I N D . P 19380 A IK Chemical Group 666 Garland P l a c e 312 296-0600 T e r r e Haute. I N 46285 C l i n t o n . N J 08805 Marion Pleasants Somerset ___ XXXX XXXX ___ Tetracyclines Tetracyclines Sulfa Tetracyclines Semi-synthetic Penicillins ___ **** P e a r l R i v e r .

NC 27542 Johnston --- 08540 616 323-4000 224 465 Kalamazoo. CT 06340 T e r r e Haute. . Lodi. Squibb & Sons P . N 3 07065 Albany. M I 49001 Kalamazoo The Upjohn Company 7000 P o r t a g e Road x)x x( --- xxxx EES . Mo 63116 Lodi. 201 574-4000 126 E . Box 4000 Princeton. VA 22827 D a n v i l l e . Thienn lmy c i n Cephalexin riboflavin none Sulfa --- --XXXX -__ 314 353-7000 Napp Chemicals. New York. NY 10017 Hoffmam-La Roche I n c . 42nd S t . P 19428 A Montgomery --- xxxx Cephalosporin P h i l a d e l p h i a . S t r e p t o Tetracyclines. e t c Kalamazoo. Louis. I n c . I A 50616 Floyd -. I N 47808 Milwaukee. (Continued) ANTIBIOTIC PRODUCER COMPANY HEADQUARTERS TELEPHONE/ MANUFACTURING E A T I O N COUNTY INDUSTRIAL MILITARY XXXX=BLAST +*=FALLOUT ****=BOTH ---=Clear ANTIBIOTICS PRODUCED TELEX ATTACK ATTACK ==HIT ---=Clear XXXX=HIT ---=Clear Merck h C o . N J 07644 xxxx --_ )(xxy 199-T Main S t . Inc 800 247-1833 Charles C i t y .- Sulfa 2000 Rockford Rd. New J e r s e y 07110 Salsbury Laboratories. C h a r l e s C i t y . N . W I 53212 New London Vigo Milwaukee XXXX 235 E . P 17821 A Dougherty Rockingham Montour S t . J . N J 07110 Warren Essex 133 417 xxxx xxxx --- ----- Nutley. Louis Bergen XxXx --_ -__ --_ xxxx --- Su I f a . O . 609 921-4000 Kenly. 201 773-3900 134 649 xxxx xxxx 212 573-2323 Groton. I A 50616 SmithKline Beckman Corp. M i c r o b i a l Products Sulfa Sulf a xxxx _-_ **** 201 235-5000 B e l v i d e r e . GA 31705 E l k t o n . Lincoln Ave.21 Table 4. R . Rahway. 340 Kingsland S t . Michigan 49001 . N J 07823 Nutley. e t c . N3 07644 P f i z e r Inc. P 13101 A E . 1500 S p r i n g Garden S t r e e t 215 854-4000 83 4487 Conshohocken. xxxx )(xxx ------- **** Mw( Pen. T e t r a . I n c St.

. 6. Antibiotic producer locations superimposed on U. S.22 ORNL D W G 87-472 Fig. map with two types of nuclear attacks.

For these reasons.GENERAL DESCRIPTION Antibiotic-manufacturing plants contain two main processing segments. Distillation columns are used f o r solvent recovery and puri- fication. Operating conditions specify large volumes of sterile air. The equipment includes centrifuges and filters for separating the microbes from the broth which contains the antibiotic. If the antibiotic is the injectable type. pumps. the orally administered antibiotics are recommended for production during the period which requires expedient antibiotic production techniques. energy-intensive separation and purification section. with very strict quality control. This equipment must all be operated in a sterile manner. the purification is very exacting and extensive. etc. separation and purification section generally employs The standard equipment normally found in the chemical processing industries. heat exchangers. . Solvent extractors (such as the Podbielniak extractor. a sterile environment and growth media. heat removal is critical). 7 ) . be purified and recycled. the first centrifugal unit to gain commercial acceptance during its use in the separation of penicillin) are used to separate antibiotics from the fermentation broth. it accounts for only a relatively small portion of the processing steps required for antibiotic production (approximately 10% of total steps are fementation related) (see Fig. with no by-products or foreign matter.23 5. agitators. ANTIBIOTIC PRODUCTION TECHNOUXIES . air and fluid filters.) is necessary. in turn. The separation and purification sections use large volumes of solvents which must. low temperature cooling water or chilled water (in the penicillin fermentation for example. Figure 7 does not show the solvent purification section which usually requires distillation columns. These sections can contain a large number of s t e p s . usually by energy intensive distillation. similar in s i z e and function to a small oil refinery. because the antibiotic has to be pure. etc. While the fermentation section generally occupies about 50% of the space and accounts for about 50% of the equipment costs. 1 9 Highly specialized fermentation equipment (including fermenters. a highly specialized fermentation section and a large.

t - - MOTHER LIQUOR OF THIRD CRYSTALLIZATION PURE C R Y S T A L S FERMENTATION PROCESS EQUIPMENT Fig. VACUUM DRYING THIRD CRYSTALLIZATION . WASH WATER I LIQUID WASTE FIRST EXTRACTION EXTRACT # SOLVENT 4 1 BUFFER SOLUTION SECOND EXTRACTION ' SPENT SOLVENT I 4 v LIQ 'ID WASTE C - I I I - I THIRD E x TR A C T I 0N I FIRST CONCENTRATION \ MOTHER LIQUOR / * SECOND C RZ A T l A L Ll YST ON C - SECOND CONCENTRATION P \ * I FIRST CRYSTALLIZATION < DISSOLVE AND DEcOLORING ACTIVE C A R B O N AND 4 SOLVENT \ J * FIRST CRYSTALS SECOND CRYSTALS \ / > 1 PRODUCT . Block diagram for a general antibiotic production process.24 ORNL D W G 87-469R INCINERATION 4 CAKE 4 FILTRATION F I L T E R AID. . 7 .

and bases.000 liters per batch. portionately. The growth medium is quite important and specific to the particular organism used and the antibiotic desired. 50. The specific chemicals required are dependent on the particular antibiotic being produced. because although many different organisms can produce a particular antibiotic. C O ~ S L L T ~ ~ ~ O ~ depends The exact dramatically on the particular antibiotic which is being produced and is covered in the appropriate appendices. For example. Large amounts of cooling and chilled water are required to control the large amount of heat evolved during aerobic fermentations and . The time necessary would also increase pro- The most efficient organisms for each type of anti- biotic are discussed in the appropriate appendices. are also important in order to produce the desired type o f antibiotic. electricity. energy. purified water. and steam. and the end use (whether formulated for oral delivery or for injection) (see appropriate appendices). Table 5 lists the com- ponents of a typical growth media for penicillin in a shake flask or factory. such as phenylacetic acid for penicillin.000 kg of penicillin would require about 6 . cooling and chilled water.5 If the concentration were only 4 g/liter (still high compared 6 3 fermentation batches would be required. the yield and final fermentation broth concentration are strongly dependent on the particular organism used. to the wild strains). The energy and carbon sources required by the organisms along with other nutrients are provided by the growth media. at 40 g/liter broth penicillin concentration. the desired purity. Most antibiotics are separated by filtration from the fermentation broth and further purified by solvent extraction which requires organic solvents.1 R A W MATERIALS AND UTILITIES The most important raw materials are the organisms themselves. acids. 3 fermentation batches of 200. The antibiotic precursors. and raw material requirements. greatly increasing the separation and purification.25 5. Utility requirements consist of sterilized air. Growth media for the other types of antibiotics are given in the appropriate appendices.

26

Table 5. G r o w t h media for penicillin - shake flask and factorya Corn-steep liquor medium Calam and Hockenhull's medium

Component

Main carbohydrateshake-flask--lactose production-- continuous glucose Other carbohydrates Glucose Starch Non-reducing polysaccharides Specific precursors Phenethylamine and other precursors Phenylacetic acid Organic Acetic Citric Lactic acids acid acid acid

3.0-4.0

3.0

0.0-0.5
variable

1.0 1.5

variable Continuous supply for production

0.05

0.05
0.5

0.25 1.0

Main nitrogen source Amino acids, peptides, amines Ammonium sulphate Other nitrogen sources Ammonia Ethylamine Total nitrogen Total solids

Variable

0.5
Variable
0.3

0.15-0.2
8.0-9.0

0.2 8.5

aD. Perlman, "Chemically defined media for antibiotic production," Ann. N. Y. Acad. Sci. 139 258-269 ( 1 9 6 6 ) .

27

because the optimum fermentation temperature is usually near ambient. Consumption of electricity is high due to the large amount of agitation required for oxygen transfer in the aerobic fermentations (5 to
40 hp/1000 gal) and for the production of chilled water.

For those

antibiotics which are recovered by solvent extraction, large steam consumptions result from the purification of the extracting solvents by distillation for recycle back to the purification section.

5.2

FERMENTATION

Operating an antibiotic fermentation unit requires that special consideration be given to culture preservation, growth media, air and media sterilization, oxygen transfer, and temperature control in the fermenter. Equipment that is capable of being sterilized and maintained in aseptic condition during operation is also essential. Many different cultures can be used to produce a specific antibiotic; however, the fermenter yield (units of antibiotic per unit fermenter volume), which helps determine plant capacity, varies dramatically with the specific culture used. Long-term preservation of Three

an efficient culture is vital to antibiotic manufacturing.

methods used for culture preservation: subcultivation, low-temperature storage, and freeze drying (see Appendix I)." Each company

jealously guards its own production organisms as trade secrets. The growth medium is an important part of an antibiotic fermentation, influencing the yield and, in some cases, the specific antibiotic which is produced (penicillin G , V , or 0 depending on the precursor ~upplied).~ The growth medium can be rather complex and is different for each antibiotic produced. growth medium for penicillin. Antibiotics can be destroyed or inactivated by some microorganisms, and/or microorganisms can invade the fermentation broth and disrupt the pure culture fermentation. See Table 5 for a typical

In both cases a substantial

reduction in yield will result. To prevent contamination, a sterile environment must be maintained. Fermentations require large volumes

of air which must be sterilized to remove bacteria which have particle sizes down to 0.5 /I. There are three commercial methods for air

sterilization: (1) heat treatment (whole air supply maintained at
2100 " C ) ,

(2) filtration through fibrous media and filtration through

granular media (see appendix J).

In order to maintain an aseptic environment, specialized equipment is required for the fermenter, transfer pumps, etc, that can be easily cleaned and sterilized. The culture must be increased in

size several times before a commercial fermenter can be charged (see Fig. 8 ) . " Steam and Each one of these steps requires a sterile transfer. chemical sterilization methods are used for in-place Appendices J and K cover specialized Antibiotic fermentations are

sterilization of equipment."

equipment and sterilization methods.

aerobic with optimum operating temperatures in the 20 to 3 0 ° C range. Oxygen transfer in the fermentation broth can be a controlling variable with vigorous agitation required. Aerobic fermentations generate a great deal of heat which results in demands for chilled water for cooling.

In addition, the vigorous agitation produces foaming pro-

blems, which often cannot be dealt with using standard antifoam chemicals because they cause downstream separation problems.23

5.3

SEPARATION AND PURIFICATION
All antibiotic fermentations are generic in nature (i.e., they

can all use the same equipment).

Different antibiotics are produced

by using different cultures and growth media, while some alterations in operating conditions are required for optimum performance.

The

separation and purification sections o f an antibiotic plant can differ substantially, depending on the antibiotic which is being produced. The specific separations required for the antibiotics selected

for expedient production are delineated in the appropriate appendices.

In general, filtration, centrifugation, extraction, adsorption, and
crystallization are involved. Some veterinary antibiotic are produced by spray drying the filtration supernatant without the usual extensive

29
ORNL D W G a a - 2 5 3

PRODUCTION FERMENTER CARBON, NITROGEN PHENYLACETIC ACID

3

I

-c

FILTER
5

LYOPHILIZED SPORES

INOCULUM CULTURES

03

C3

FILTRATE

PREFERMENTERS MYCELIUM

C

'/ PURIFICATION

Fig. 8 . fermentation.

22. 5 ADVANCED TECHNOLOGIES Three relatively new and potentially very powerful separa- tion/purification methods for antibiotics are (1) chromatography (particularly continuous annular chromatography). These liquid chromatography and supercritical fluid extraction (see Appendix L). Two recent developments in separation technology have the potential to substantially reduce the size of the of separation/purification section are high performance antibiotic-manufacturing plants. The major toxic reaction from impure antibiotics is an allergic reaction to the intermediates and degradation products and is much more common. (2) supercritical fluid (SCF) extraction and ( 3 ) r n e m b r a n e ~ . spray drying may be an attractive alternative allowing production of a dry product for oral use (impure products cannot be used for injection).4 ANALYTIC PROCEDURES/QUALITY CONTROL Chromatography and fluorescence detection are standard methods for assaying antibiotics. the major toxic effect of the pure antibiotics is an allergic reaction and is quite rare. and recently for the tetracy- Thin-layer paper and gel chromatography are used for HPLC assays are particularly bacitracin and the tetracyclines. 5 . ~ ~ .2’ important for the B-Lactam antibiotics in order to detect impurities from degradation products and intermediates which are quite similar to the active antibiotic and result in toxic reaction in humans. modular units which can separate specific antibiotics easily and inexpensively.29 When given in clinical dosage. with ultraviolet light as the detector source.28. to analyze for the penicillins clines. with reduced purity requirements.~the size of the If ~ antibiotic-manufacturing process is to be substantially reduced. 5.24 Under austere conditions.30 purification steps required for human antibiotics. this reduction will have to come in the separation and purification . High-performance liquid chromatography (HPLC) uses regular and reverse phase and ion exchange packings. These new methods may become increasingly important in the next three to five years as single-step.25g26 and cephalosporins.

5 When.41 These new technologies not only have the potential for a vast reduction in the size of the separation equipment. The new technologies offer much greater selectivity which should translate into a substantial reduction in the number of processing steps required. Chromatography.31 sections of the plant which represent well over half o f the total plant size. although some penicillins and cephalosporins are separated commercially using HPLC and/or membranes. in 1943. Most of this work is still in the developmental stages. These new separation technologies could have a similar impact on the separation and purification of antibiotics that submerged culture fermentation had on the fermentation end of the process. antibiotic plants were very inefficient because surface culture fermentations were used.5 . they will greatly simplify such a conversion within the next 3 to 5 years and beyond. While these new technologies could not be used to retrofit an antibiotic plant if a national disaster occurred in the near future. It would be possible to make good estimates of the impact of either annular chromatography or SCF extraction on the antibiotic process in a retrofit situation because most of the pertinent information is available in the literature. SCF extraction. and membrane technologies are relatively new in regard to their application to the antibiotic purification process. In the 1 9 4 0 s . one 500-gallon fermenter using the submerged culture process outproduced all of the large surfaceculture antibiotic plants that were operating in England. but also in the total number of processing steps. Normal antibiotic separations require between 40 and 60 processing steps. the submerged culture process was demon- strated at the USDA Peoria Labs.j9' 4 0 Several European patents have been filed on the use of SCF extraction for antibiotic separation and purification.

.

42 Figures 9 and 10 illustrate the proximity of these plants to the nuclear attack zones. it is anticipated that insufficient antibiotic-production capacity will remain intact to meet the needs of the population. and breweries. Some These include enzyme. possible candidates for conversion are listed in Table 8.33 6. alternative facilities will have to be converted to antibiotic production. The first expedient production measure to be taken should be a damage assessment of existing antibiotic-manufacturing facilities and a determination of feasibility o f timely repair. For this reason. and other polymers) makers.on broth and purification to an acceptable level for the required end use. a plant which is to be converted .2 CONVERSION TO ANTIBIOTIC PRODUCTION Antibiotic production is a two-step process: (1) production of the antibiotic via fermentation of the raw materials in the growth media. 6.2 and B-12. and fermentation ethanol producers. single-cell protein producers. the dairy industry.1 ALTERNATE PRODUCTION SITES The most promising candidates for conversion to antibiotic production are animal or plant antibiotic producers. single cell protein producers. enzyme or molecular biology product production plants. The fermentation section of the process is quite specialized. If antibiotic- production capacity is still insufficient. and would be very difficult to retro-fit to a plant without fermentation capability. molecular biology product (mono sodium glutamate (MSG). universities. citric acid producers. Large hospitals with research facilities. EXPEDIENT PRODUCTION OF ANTIBIOTICS Based on the TR-82. and farm ethanol fermentation units may provide additional candidates. xanthan. 6. Tables 6 and 7 list the locations and telephone numbers of these plants." Industrial Emphasis and Military Emphasis attacks. mushroom producers. vitamin B . and (2) separation of the antibiotic from the fermentati.

OH 44122 Pharmacia P-L Biochem. 414 225-2600 Milwaukee. W I 53701 Passaic Dane XXXX xxxx --- _-_ _-_ **** xxxx 607 335-2111 Norwich. W I 53202 Novo L a b o r a t o r i e s . Milwaukee.. CT 06897 Gist-Brocades U A I n c . NY 13815 Chenango _-_ 17 Eaton Ave. OH 44128 980718 Cuyahoga XXXX --- **** Cleveland. assuming three different attack scenarios E N N Z P E PRODUCER TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY ATTACK XXM(=BLAST +++=FALLOUT ****=BOTH ---. CT 06897 Fairfield XXXX ___ 59 Danbury Road Wilton. 414 271-6755 Juneau. 61650 Peoria xxxx -_- 709 N.Clear C M A Y HEADQUARTERS O PN TELEX ATTACK ==HIT ---=Clear XXXX=HIT ---=Clear (TR-82) Amber L a b o r a t o r i e s 4 3 3 E. .- 319 264-4211 Muscatine. Biochem. B a r t l e t t Ave. I n c . Ill.O. I n c . Box 22400 216 765-5000 Cleveland. P. SC 29556 W i lliamsburg -.E. P . Po Box 196 D a n v i l l e .Corp. W I 53039 Dodge --_ _-- -__ Milwaukee. Box 932 E l k h a r t . I A 52761 46 8497 F 4 Fermentation P r o d . 203 762-2401 Wilton. O . 964 385 704-527-9000 K i n g s t r e e . Norwich. NY 13815 U. S Po Box 241068 C h a r l o t t e .Inc. W I 53202 Milwaukee XXXX ___ xxxx 2202 N . N J 07015 Madison. 414 347-7464 TP Peoria. W I 53202 Miles L a b o r a t o r i e s . P e o r i a . I N 46515 Norwich Eaton Pharmaceut 219 264-8111 C l i f t o n . I A 52761 Muscatine --- 1600 Oregon St Muscatine. Michigan S t .S. NC 28224 Grain Processing Corp. Water St. I n c . 61650 Merck Chern.34 Table 6. Risk assessment for enzyme producers in the U. I l l . PA 17821 201-574-4000 D a n v i l l e . PA 17821 Montour --- . Mfg. S . Div.

La J o l l a . I L 60901 Kankakee --- ___ ___ PO Box 511 Kankakee. (Continued) ENNZYME PRODUCER CCMPANY HEADQUARTERS TELEPHONE/ MANUFACTLlRING LOCATION COUNTY INDUSTRIAL MILITARY ATTACK ==BLAST +++=FALLOUT ---=Clear TELEX ATTACK ---=€lear XXXX=HIT ---=Clear XXXX=HIT ****=BOTH Atomergic Chemetals Corp. N J 07080 Anheuser-Busch. One Busch P l a c e S t . 516 433-4900 Westbury. W I 53212 Milwaukee XXXX ___ xxxx 4253 N. I1 60501 American Hoechst C o w . CA 92037 San Diego XXXX ___ **** 10933 Torry Pine RD. NY 11803 SmithKline Beckman Corp. Nassau XXXX ___ **** 14 4612 Plainview. Dynamics Corp. 714-871-4848 Carlsbad. M I 49001 Revlon. I n c .35 Table 6 . C a l i f . 314 577-2000 S t . M I 49001 224 465 Kalamazoo XXXX --_ XXXX 7000 Portage Road Kalamazoo. PO Box 347 Argo. Milwaukee. NY 11590 CFC I n t e r n a t ' l I n c . C a l i f . W I 53212 Chem. 3001 Hadley Road 414 332-3545 Milwaukee. K. I L 60901 Accurate Chem. 201 753-5000 S . NY 11590 800 645-6264 14 4617 201-894-4000 Argo.Corp. 92008 San Diego XXXX ___ *+ 6200 E l Camino Real C a r l s b a d . 92008 The Upjohn Co . P o r t Washington Ave. Louis. N J 07080 003 447 Somerset )(xxx _-_ **** South P l a i n f i e l d . I L 60501 Nassau xxxx --_ +*** 300 Shames Drive Westbury.&Sci. P l a i n f i e l d . 619 450-9600 La J o l l a . 212-572-5000 Kankakee. Louis. 63118 S a i n t Louis XIOM XXXX xxxx . CA 92037 P f i z e r Inc. NY 11803 100 F a i r c h i l d Ave.516 349-8800 Plainview. 616 323-4000 Kalamaroo. M3 63118 Inc.

I A 52732 Clinton XXXX __- --- a 600 Three F i r s t N a t i o n a l P l a z a Chicago. assuming three different attack scenarios FERMENTATION ETHANOL COMPANY HEADQUARTERS TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXXX=BLAST +++=FALLOUT ****=BOTH ---=Clear (TR-82) Capacity (million galiyr) TELEX ATTACK ATTACK ==HIT ---=Clear ==HIT ---=Clear Archer D a n i e l s Midland Co. OH 45680 Lawrence XXXX --_ --_ --_ xxxx XxxX -__ 60 < 10 Alcohol Energy Carp. TN 37774 Decatur. 703 528-9091 S t a l e y .36 Table 7 . W I 5320 South P o i n t E t h a n o l I n c . CO 80741 Reston. I A 52761 Midwest S o l v e n t s Co. C o r p . C o . Inc. E . Risk assessment for fermentation ethanol producers i n the U. Michigan S t 217 423-4411 Loudon. I L 62525 Georgia. GA 30303 Grain P r o c e s s i n g Corp 1600 Oregon S t Muscatine. 2200 E a s t Eldorado S t Decatur. S t a l e y MFG. I L 62500 P e o r i a . South P o i n t . I L 62525 Amber L a b o r a t o r i e s 4 3 3 E . NC 27355 Campo.(CCFC) 319 242-1141 C l i n t o n . CO 81029 Merino. I A 52761 hscatine --- --- --_ EO 913 367-1480 309 353-3990 Atchison. NE A t l a n t a . KS 66002 Nabisco Brands. I L 61600 Bellingham. VA 22090 Randolph Baca Logan Fairfax --- ___ xxxx XXXX -__ <lo < 10 **** --_ **** < 10 . 1300 Main S t --_ ___ xxxx xxxx )220 xxxx --- xxxx _-_ 6 --_ 319 264-4211 46 8497 Muscatine. I L 60602 Pekin Energy Co 312 269-5300 309 346-1121 Pekin. I L 62525 4 1 4 271-6755 Juneau. I n c . WA 98225 Linn Macon Peoria Whatcorn xxxx _-- --- FO Box 1470 Decatur. I A 52413 Decatur. h'I 53039 Milwaukee. Bornhoft. K S 66002 Pekin. Paul A. I L 61554 Tazewell _-_ )cxxx xxxx EO Po Box 10 Pekin. Smith Bowman D i s t . Baca Food and F u e l Coop.S. I L 61554 Atchison Tazewell --XXXX --_ --_ --_ xxxx 22 9 Atchison. I L 61554 A.P a c i f i c Corp 133 P e a c h t r e e S t . 319 398-0600 217 424-5200 309 673-7828 404 521-4000 Cedar Rapids.

S . I n c . I n c . Spudcohol Syncorp. 21 < 10 TX 76661 .= 1 0 Xaven Alcohol D i s t i l l e r y Southern D i s t i l l e r i e s C o . Food and Energy. CA 05237 Van Buren. I n c . Bailey Minnehaha Baca Van Buren Lancaster Kennebec Simpson Taylor Falls Bates Fresno Houston Bingham Crawford San Joaquin XXXX Crawford <lo < 10 <lo z . SD 57060 Walsh. NE 68430 Litchfield. Colorado Gasohol I n c . I n c .= 10 . KY 42134 Lenox. White Flame F u e l s . I A 52620 Roca. F r a n k l i n . I n c . Muleshoe. Inc. I n c . M 64723 I Selma. E. Coburn E n t e r p r i s e s . I D 83262 Roberta. E c o l o g i c a l Energy. TX 79347 Sherman. CA 93662 Ashford. --- __**** < 10 --- __- .= 10 Amsterdam. Crystal Fuels. I A 50851 Marlin. Montana. (Continued) FERMENTATION ETHANOL COMPANY HEADQUARTERS TELEPHONE/ MANUFACTURING LOCATION COUNTX INDUSTRIAL MILITARY ATTACK ATTACK XXXX=BLAST +++=FALLOUT ****=BOTH ---=Clear Capaclty (million gal/yr) TELEX XXXX=HIT ==HIT ---=Clear ---=Clear (TI-82) Charmel Energy Co. GA 31078 Lockeford. c10 < 10 <lo U.37 Table 7 . I n c . 10 <lo c 10 ME 04350 Kentucky A g r i c u l t u r a l Energy Coop Lenox Grain F u e l s . CO 81090 Bonaparte. AR 72956 <10 . Marlin Car Care A . AL 36312 P i n g r e e . Gasohol Corp.

. Enzyme producer locations superimposed on U.S. map with two types of nuclear attack.38 ORNL D W G 8 8 . 9 .2 8 9 NUCLEAR ATTACK ON MILITARY FACILITIES ENZYME PRODUCERS - 0 Fig.

S. Fermentation ethanol producer locations superimposed on U. .39 OmNL O W G 8’ 1129 NUCLEAR ATTACK WITH EMPHASIS ON INDUSTRIAL F A C ~ L I T ~ E S NUCLEAR 4 T T I C K ON MILITARY FACILITIES FERMENTATlON ETHANOL PROOUCEss SMALL FERMENTATWN ETHANOL ~ PLANTS I P R O B I B L I CLOSED) ~ 0 Fig. 10. map with two types of nuclear attack.

St.E. Corp. Po Drawer 10710 Jefferson. Heinz Co. Pittsburgh. E4 01760 2 Eartlett Chemicals Inc. 617-655-5805 N a t i c k . Pfizer. PA 15230 Allegheny XXXX __- **** Standard Brands.. S. 5 5 4 4 Oakdale Rd. 212-573-2323 Terre Haute. Inc. Louis. MO 63147 Middlesex XXXX __- +++ 504-734-1971 Jefferson. CT 06340 235-T E. 784 Carle Place. LA 70181 Mallinckrodt. 42nd St. LA 70181 Jefferson XXXX -_- xxxx 800-325-7155 St. Groton. GA 30080 Cobb XXXX 30080 American International Chemical. Mo 63147 800-325-8888 St. New York. PA 15230 412-237-5757 Pittsburgh. CA 92643 Orange XxxX --_ **** Citric acid producers Miles Laboratories 219-262-7453 Elkhart. IN 47808 New York. Louis.. CA 92643 212-759-4400 714-680-1000 Fullerton. GA 404-696-6711 Snyrna. Louis XXXX XxxX xxa Gallard-Schlesinger Chemical Mfg. NY 10017 Brooklyn. NC New London XXXX _-_ **** Vigo Kings Brunswick xxxx x(x )x )(xxx ----_-_ xxxx XXXX xxxx . Inc. NY 11514 Pfizer Chemical Div. IN 46515 Mayo Chemical Co. IN 46515 Elkhart --- Citro-Tech Products Po Box 932 Elkhart.40 Table 8. 525 Madison Ave. Natick. Inc. NY 11514 Nassau xxxx _-_ **** Minenla Ave 516-335-5600 Carle Place. 2nd & Mallinckrodt sts. Srnyrna. J. Central St. Valencia Dr. NY Southport. Inc. ~~~ ~~~~ Other candidates for conversion to antibiotic production OTHER BIOPRODUCT MAKERS COMPANY HEADQUARTERS TELEPHONE/ TELEX MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXXX=BLAST ATTACK ATTACK +++=FALLOUT XXXX=HIT ==HIT ****=BOTH ---=Clear ---=Clear ---=Clear (TR-82) Acetic acid (vineKar) producers H. MA 01760 209 W. Fullerton. NY 10022 Beatrice/Hunt-Wesson Foods 1645-T W.

New York.Chem. N J 07110 201-235-5000 Nutley. I n c . D a n v i l l e . . P 17821 A Montour Merck h C o . C l e v e l a n d . NY 10017 212-573-2323 Milwaukee. OH 44122 Cuyahoga xxxx ___ xxxx S t a u f f e r Chemical Co. 199 Main S t . Div. OH 44122 216-831-0200 Cleveland. OH 43216 Franklin Ashland Chemical C o . . CT 06881 203-222-3000 Westport. N J 07644 201-773-3900 Lodi. I n c 414-347-7467 PO Box 2219 Milwaukee.Div. N J 08852 Merck 6 C o . Nutley. N J 07110 Essex . I n c . 3737-T Park E a s t Dr. 340 Kingsland S t . W I 53201 Milwaukee xxxx _-_ xxxx Milwaukee. Cowles Chem. W I 53201 614-889-3333 Columbus. Inc E l k t o n . Food I n g r e d i e n t s Nyala Farm Road Westport. N J Monmouth 08852 Monrmuth J u n c t i o n . PO Box 2219 xxxx xxxx xxxx Columhus. VA 22827 Rockingham ___ **** Hoffmann-La Roche I n c . OH 43216 Napp Chemicals. PA 17821 201-574-4000 D a n v i l l e . (Continued) OTHER BIOPRODUCT MAKERS CCMPANY HEADQUARTERS TELEPHONE/ TELEX MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXXX=BLAST *=FALLOUT ****=BOTH ---=Clear (TR-82) ATTACK ==HIT ---=Clear ATTACK ==HIT ---=Clear Gluconic and Klutamic a c i d producers PMP Fermentation P r o d . VA 22827 201-574-4000 E l k t o n . . 201-297-0100 PO Box 125 Monmouth J u n c t i o n . W I 53212 Milwaukee xxxx xxxx ___ ___ xxxx **** Rhone-PoulencInc. CT 06881 Fairfield xxxx Vitamin B-2 o r B-12 producers Pfizer Inc. N J 07644 Bergen xxxx _-- xxxx S t a u f f e r Chemical C o .41 Table 8 . Lodi.

Hercules P l a z a Wilmington. San Diego. . 201-457-8000 Piscataway. Wayne. 619-292-4900 302-594-6500 W i h i n g t o n . 1ndust.Chem. N J 08852 Monmouth XXXX --_ **** H o m u t h J u s c t i o n . CA 92123 Merck 6 C o .Prod. NJ 08854 Somerset XXXX --- **** Piscataway. Kelco Div. DE 19894 Merck & Co. (Continued) OTHER BIOERODUCT MAKERS TELEPRONEI MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY ATTACK ATTACK ==BLAST +++=FALLOUT ****=BOTH ---=Clear CCMPANY HEADQUARTERS TELM ==HIT ---=Clear ==HIT ---=Clear Xanthan and o t h e r uum producers American Cyanamid Co. . NJ 07470 201-831-2000 Wayne. BOO-T C e n t e n n i a l Ave. . OK 74447 619-292-4900 Q k m l g e e . I n c . DE 19894 New C a s t l e XXXX --- **** San Diego. D i v . I n c ... . POB 125 201-297-0100 Monmouth J u n c t i o n . OK 74447 Okmulgee XXXX --- --- Rhone-Poulenc I n c . Okmulgee. CA San Diego XXXX --_ **** 8355-T Aero D r .42 Table 8 . Chem. N J 08852 . N J 08854 Hercules I n c . Kelco Div. I n c . N J 07470 Pharmacia.

Spray-drying and veterinary antibiotic industries. The requirements are (1) the organism to produce the antibiotic. and it is a readily available technology requiring only a large source of clean.). hot. (5) fermenter agitation and cooling and chilled water capacity (with either internal coils or vessel jacket). injection quality antibiotics could be produced at these locations. and is specific to the antibiotic being produced. ( 4 ) sterile air supply.43 to expedient antibiotic production must have a sterilizable fermentation section in place. etc. and (6) basic instrumentation for process control (oxygen tension. equipment in an aerobic fermentation unit should be able to produce any of the desired antibiotics. the degree of purity required varies with the application and delivery form of the antibiotic. pressure. SCP. Spray-dried antibiotics could only be given as oral anti- biotics because of the lack of complete purity and some adverse reactions could be expected. For the conversion to be feasible under the constraints of the time limits involved. In addition. The separation and purification section of an antibiotic plant is usually large (of comparable size and cost to the fermentation section). contains many steps. is commonly used in the enzyme. Impure products can be made by spray-drying the filtered broth. Reduced-purity products can only be tolerated in antibiotics which are taken orally. ( 3 ) ability to sterilize all equipment. dry gas and a large cylindrical vertical chamber into which the material can be sprayed in the form o f small droplets. If existing antibiotic-producer plants are intact or easily repaired. The most likely way to accomplish this is to make substantial reductions in the purity of the final antibiotic product. (2) sterile conditions for culture trans- fers. a substantial reduction in size and complexity of the separation and purification unit must be achieved. however. pH. temperature. The major retrofit required to convert an existing fermentation plant to antibiotic production is the addition of a separation and purification section. The specific fermentation equipment required The for the production of the different antibiotics is similar. thereby greatly simplifying the production procedure. .

while most other fermentation industries deal with the much simpler mixed culture fermentations.35-43 These new separation methods could substantially reduce the size and complexity of an antibiotic plant without sacrificing product quality. especially filters and centrifuges for separating cells from the media. The equipment necessary to operate at the subambient conditions of antibiotics plants ( 0 25°C) is also present. The production of antibiotics is highly aerobic requiring large quantities of compressed sterile air. Both the antibiotic and SCP fermentation processes deal solely with pure cultures. and often ultrafiltration units for producing very pure macromolecular products (like antibiotics). and quality assurance analyses required during the production of sterile products. Some separation equipment is also available. The SCP plant located at Hutchinson. nutrients.3 SINGLE-CELL PROTEINS Facilities that produce single-cell protein are probably the best candidates for conversion to anitbiotic production following a nuclear disaster. and SCP plants have this capacity. media.000-gallon) state-of- . spray.drying facilities to produce a dry stable product.44 Other possibilities for reducing the size and complexity of expedient separation and purification equipment involve employing either HPLC or supercritical fluid (SFC) extraction to perform the separation and purification from the filtered or unfiltered fermentation broth. has been identified as a top candidate for conversion to antibiotic production after a national disaster. 6. both HPLC and SCF extraction are presently in the developmental stage for antibiotic separations. This plant is outside any of the nuclear attack zones and contains two 75. These plants also have all of the necessary laboratory facilities required for chemical. These plants have fermentation facilities which operate under highly aerobic and sterile conditions to grow microorganisms which are similar to those used to produce antibiotics. microbiological. Minnesota. biochemical. The sterilization equipment is in place for sterilizing the air.700-liter (20. and the downstream separation processing equipment. However.

If chilled water has to be produced by refrigeration. it represents a large energy requirement. and a constant source o f 1 0 ° C cooling water is present. The surrounding area can provide most of the raw materials required. The cooling water source is quite important because of the large chilled water requirements in antibiotic plants. . Detailed information on the Hutchinson SCP plant is given in Appendix H.45 the-art fermenters.

.

ALTERNATE SOURCES OF ANTIBIOTICS Under conditions when it becomes impossible to produce enough antibiotics in the postattack environment. telephone numbers of the four antibiotic manufacturers in Puerto Rico and the Mexican manufacturers. There is substantial antibiotic-manufacturing capacity in Table 9 lists the headquarters and Mexico and in Puerto Rico. and spectinomycin are used for the treatment of some severe infections in penicillin allergic patients. lincomycin.47 7. any case several low volume antibiotics which have unique properties should be stockpiled. . Small planes can carry significant quantities of dry crystalline antibiotics. Antibiotics can be stored for many years in bulk crystalline form at low temperatures. tobramycin. For example. Consideration should be given to importing antibiotics from these locations after a nuclear attack. two additional alternatives exist. conIn sideration should also be given to building stockpiles of antibiotics stored in a manner which will ensure a shelf life of many years. In planning for a nuclear attack.

V. Cephalexin Clindamycin. Amphotericin b Mexico Orsabe. NJ 08540 ( 6 0 9 ) 921-4000 Humacao 00661 Nystatin. de C. Synthetic Penicillins Tetracyclines F e m i c S . EES. Fermentaciones Syntasis. Ixapalapa. y Cuernavaca. de C. Squibb h Sons P. IL 60064 1400 ( 8 0 0 ) 323-9065 ( 9 1 0 ) 235-1584 Barceloneta 00617 Erythromycin and its salts Eli Lilly and Company 307 E. R. S . S. McCarty Indianapolis. A . MI 49001 ( 6 1 6 ) 323-4000 Arecibo 00612 E. Mexico Synthetic Penicillins .O. Mexico Gentamicins Mexico Cephalosporin C Synthetic Penicillins Cephalosporin C . IN 46206 ( 3 1 7 ) 261-2000 Carolina 00630 Mayaguez 00708 Tobramycin sulfate Erythromycin estolate. Lincomycin The Upjohn Company 7000 Portage Road Kalamazoo. A . Mexico Quinonas de Mexico Mexico City. Alternate antibiotic production sites outside of the continental U. Box 4000 Princeton.S Antibiotic producer company headquarters Telephone/ telex Manufacturing location Antibiotics produced F'uerto Riw Abbott Laboratories Sheridan Road North Chicago. Beneficiadora e Industrializadora S .V. A . A . Mexico Synthetic Penicillins Carretera MexicoLaredo.48 Table 9.

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"Tetracycline Production Using Cottonseed Endosperm Flour. 12. User. R. B. Kane. Minieri. J. 1 9 7 1 . Sokol. August 2 2 . 4237 ( 1 9 8 6 ) . May 2 2 . "Production of the Antibiotic Tetracycline by Fermentation." British Patent # 7 7 5 1 3 9 . S . Lu. R. A." British Patent # 7 7 5 1 3 9 December 1 0 . LePetit. (10) 1 6 8 1 ( 1 9 7 5 ) . "Bacitracin. A. 43 ( 7 ) 1 4 8 8 ( 1 9 5 1 ) . 13. C . J.S. P. H. 2. B. 1 9 5 4 . "Production of Tetracycline and Substituted Tetracyclines. J . F. Sobin. 10. 15. Gourevitch and J. and M. "Production of the Antibiotic Tetracycline by Fermentation. U. "Process for Producing Tetracycline by Fermentation. Margineanu. 1 9 4 9 . PATENTS 8. Wasserman and T." Progress in Industrial Microbiolonv. 1 9 5 7 . H. A. J. G .59 6. A. Firman. Lein. Patent # 2 . W. 1 9 5 3 . Lein. "Process for the Isolation and Purification o f Tetracycline. 11. 1 9 5 3 . "Bacitracin! Product of Biochemical Engineering. 16." Journal o f Pharmaceutical Sciences. "Separation and Quantitative Determination of Impurities in Tetracycline. 1 93-150 (1964). "Terramycin and It's Production. M. McGhee and J. S . 9. . A . 1 9 7 2 . Gavrilescu. H. US Patent # 2 7 6 3 5 9 1 December 7 . C . Dudley. 18 0 7." US Patent #2 7 1 2 517 March 3 . "Process for the Production o f Tetracycline. Hatch." Journal of the American Chemical Society. 1954. W. Willekens. Bennett. Pal. Hickey. 2 4 3 January 1." British Patent #1 2 8 6 9 2 9 June 3 0 . 1 9 5 7 ." US Patent # 2 7 3 4 0 1 8 September 2 8 . Melna. "Process for the Preparation o f Tetracycline and Chlortetracycline. "On the Total Synthesis of Tetracyclines. 7 7 6 ." US Patent #2 5 1 6 0 8 0 November 2 8 . C. 8. H. Ionescu and N. P." Industrial And Eneineerine Chemistry. C." British Patent #1 3 6 8 6 6 8 . and M. Shepard. 64 G. 14. J. Its Manufacture and Uses. S . Hunt and J.6 BACITRACIN 1. E. A . Finlay and J.

and W. Longnecker. Perlman. Soc. Scholhofer. 8. R. Advancing Fronts in Chemistry: Chemotherapy Reinhold Publishing Co. 4. New York 1979. Diedrich. Astrakhantsen. M. J ." German Patent #831 880 February 1 8 . 2 135 (1947). Spangenberg." Science. C. 8 3. Part 1.. Shridhar and K. 8." J. Patent #180 656 January 1 0 . M. W. A .A New Approach. Tsurooka." Pharmazie. 12. C. Academic Press. Narang.60 3.S.. Braeuniger and K. 2. J .. 5." Analvtic Chemistry. J. 1952. Maemoto. J ." Farmatsiva. 'I 10. Bennett. 2 343 (1954). 9. Second Edition." British #336 512 January 1 7 . "Sulfonamides. PATENTS 8. H. H . "Refining of Sulfonamides. Microbial Technology Volume 1. New York (1946). "Water-Soluble Sulfonamide Preparations. D. . Lapiere. "Sulfonamide Solutions for Chemotherapeutical Use. "Analytical Study of Sulfonamides. I. "Separation of Sulfonamides by Countercurrent Distribution.7 SULFONAMIDES 1. Matzke. "Sulfa Drugs. 24 195 (1954). W. 705 (1984). Indian Chem. 1955. . H. P.( 4 ) 12 (1945). Peppler and D. 1952. "Studies in Sulphonamides . S . 30. Dohrn and P. "Simplified Partition Chromatographic Procedures." German Patent #840 241 May 19. 21(11) 1402 (1949). H.8 SEPARATION AND mTRIFICATION G. G ." U. Sisson. Pharm. Sprent. 1930. Dodd.. J. . "Sulfonamide Derivatives. 33 (5) 305 (1956). Aitiba and M. F. 1. Powers. August 21. 6. "Aid to the Pharmaceutical Industry by VNIKRFl During the Patriotic [Second War] War. AIChE. Japan Patent #4077 11. 1953. H. N. Begovich and W. u.

K a l y a n p u r . F. J. Skea.93. Technol. 6. H. Cantwell. 14. M. N." J . "Chromatographic Purification of Beta-Lactam Antibiotics. Dwyer. W. J . 23 ( 6 ) . M. and M. 1 0 103 1 (1975). Dwyer. " AIChE Symposia Series. 8 J. M." American Laboratory 136." Developments in Industrial Microbiolow. 4 4. G . Ferment. Kalyanpur. 1 (3) 143 (1972). Kobayashi. "Filtration and Separation in Antibiotic Manufacture. J. "The Application of Electrophoresis for Separation of Tetracycline and polyene antibiotics." Journal o f Chromatographv. N.. "High Performance Chromatography as a Process T o o l . 12. A. Dwyer. Dzierzynska. Jurand. Lowy and J. "A Simple Method for Purification o f Sodium Penicillin from Penicillinic Acid Contaminant. I. Skea. Chatterjee. M. 82 (250) 120 (1987). Calderone. 5." Filtration and Separation 165 May/June 1984 11." Annual Reports on Fermentation Processes. 205 (1971). R." World Biotech. 8. "Isolation of Cephalosporin C From Fermentation Broths Using Membrane Systems and HighPerformance Liquid Chromatography. R. Knox and J." Dissert. . "Process Scale-up of a p-Lactam Antibiotic Purification by High-Performance Liquid Chromatography. Report. and M. 3. 13. Skea. Pharrnacol. 9. 8 (9) 100 (1984). Siwak. 8 November 1984. 651 (1984). 3L6 133 (1984) . and M. Cazes. W. J . May (1985). "Process Separations of Antibiotics by HPLC Process Systems." SA Filtration. Journal of Chromatopraphv. Inman. "Design and Performance of a Preparative-scale HPLC. Itamihira. H. M. Tanifuchi and T. 26 455 (1985). 10. Findeisen and W. (1985). C ." Pharmaceutical Technolopv. 7. D. Dwyer. Siwak.61 2. Sienko." Hindustan Antibiotics Bull. "Removal of Organic Solvent from Antibiotics with supercritical Carbon Dioxide. "Separation o f Tetracyclines by HighSpeed Liquid Chromatography. Colin. . "Chromatographic Purification of Semisynthetic BetaLactam Antibiotics. 65 (1) 671 (1987). Pharm. "Isolation of Cephalosporin C from Fermentation Broths Using Membrane Systems and HPLC.

D. N. Sisson. 16. 2 ( 2 ) 7 3 ( 1 9 8 6 ) . New York. and M. 180 April 1 9 8 2 . Caldwell. Separation and Purification. Larson and M. L. Sunshine. J . New York. 3 r d edition. G. C. Scott. Shih. N. Morecombe. M. H. Proc. Cohen. S . 23. "Large-Scale Liquid Chromatography 5 3 October ( 1 9 8 3 ) .62 15. "High-Efficiency Preparative Scale ReversedPhase High-Performance Liquid Chromatographic Purification of I4C-Labelled Antibiotics. 1 9 8 7 . and W. M. D." Anal." Industrial Chemist 8 (8) 16 (1987). Regosz. L. 33 26. "Process Scale Liquid Chromatography. R. Donohue. 28. King. C. Meyers. "High-Performance Liquid Chromatography as an Aid to the Rational U s e of Antimicrobial Agents. K. A . Walsh. Weiss. J. Scherr. 18. Weissbeager. W. 33 27. G . D. ChromatoFraDhv." Fluid Phase Equilibria 295. 21. Watson. J. E. 25. January 1 9 . E. Advances in Applied Microbiology." J . Uri. FL. M. Chromatography 389 389 ( 1 9 8 7 ) . 17. Snavely. I. R."Chem. and El L. T. Engr. J . J . Rasmussen. . Ikonomov and M. 20." Solubility Data Series. V. I. Academic Press. "Evaluations of Supercritical Fluid Extraction in the Pharmaceutical Industry. Mascone. (1986). (1987). P. J . "Separations are key to Biotech Scale-up. F. Molnar. "Preparative Low Pressure Chromatography o f Antibiotics on a Column o f Diol-Bonded Silica Gel. . "Supercritical Phase Behavior: The Entrainer Effect. Boca Raton." Biotechnolow Progress. R.( 4 ) 305. S . Spence. 24. CRC Press. D." m. Platt. "Antibiotics: 1 j3-Lactam Antibiotics. B . Stewart. Volume 11. 1 6 381 2 22. McIntosh." J. Chromatography 386 325 (1987). D. P. 19. Pergamon Press. Perlman. Tomlinson and A . New York (1985). C. ( 1 9 7 9 ) . Perry and A. ( 1 9 7 8 ) . C. M. D. John Wiley and Sons. Paul. and H." Acta Microbiologica Hungarica. System. J . "Novel Fermentation Procedures for the Production and the Isolation of Antibiotics. (1976). Handbook of Analytical Toxicolow. ( 1 9 6 9 ) . K. J . E..

E. W.63 29. Dinel. 2 ( 2 ) 169 (1980). "Antibiotic Purification Process. 8. Behme. Hockenhull." Purification of Fermentation Products. "Separations and Quantitative Determination o f Impurities in Tetracycline. J. Guze. H. Bellamy. R. R. "High-pressure Liquid Chromatography for Quantitation of Antimicrobial Agents. Zarembo. 2. J." The Journal of Antibiotics. Ayotte. C. and J . Whitby. 14 P. New York (1981). M. D. K. 5. B. R. "Production of Antibiotics by Fermentation. L.9 1. "The Preservation o f Cultures. B." Progress in Industrial Microbioloev. G. (1959). Coenen. Maitra. Pines. "Reverse Phase High Speed Liquid Chromatography of Antibiotics. 30. S . Muggleton. 32. D." Advances in Amlied Microbiology. K. Ausman. Holmes. Black. Schotz.10 STERILIZATION 1. Kundsin. B. PRESERVATION 8. ." Progress in Industrial Microbiologv. 31. 1 (9) 1 542 ( 1 9 7 7 ) 3." Essays in A m l i e d Microbiologv. B." Reviews of Infectious Diseases. C. B. 1 49 . and R. Walter. Wildfeuer. 34 (7) 836 (1981). A. "Activity of Antibiotic Admixtures Subjected to Different Freeze-Thaw Treatments. 191 ( 1 9 6 2 ) . "Preservation of Foods and Drugs by Ionizing Radiations. T. 34. 411 John Wiley and Sons. D r w Intelligence Clinical Pharmacy. 1976. D. S. M. J. Willekens. D. Yoshikawa. G. 37 ( 1 9 6 3 ) . J . 1983. W. E. E. PATENTS 33. White and J ." Journal of Pharmaceutical Sciences. L.( 5 ) 353 (1980). 6 4 (10) 1681 ( 1 9 7 5 ) ." German Patent #33 18 194 May 19. "Isolation and Purification of Antibiotics. C." Drug Intelligence and Clinical Pharmacy." US Patent #3 9 8 3 108 September 2 8 . 4. and L. 156 (1985). L. . D. T. Cherry and S. W. "Approaches to Cephalosporin C Purification from Fermentation Broth. Kemp. ir. "The Sterilization of Air.

11 ANALYTICAL METHODS H. 8. C. Boca Raton.N I R Spectroscopy. "Modern Trends in Steam Sterilization." J . B. (1975). M. . "HPLC of Antibiotics.V I S . April. "Improvement of Chemical Analysis of Antibiotics: Determination of Eight Tetracyclines using Thin-Layer and High-Performance Liquid Chromatography. 82 (1986). E. Baker. Wilkinson and L. D. P. Sunshine." J . Richter. "Applications o f Remote Sensing Using Fiber Optics and W . FL. 8. Sunshine. Lirl.h Perform. 1 1 (1959). Anderson." Chromatography. 0 R. E. Herold and J . P. "Fermentation Equipment. FL (1979). R. "Analysis of Various Classes of Drugs by Capillary Supercritical Chromatography. Ikai. G . 28 (1987). Sci. G . R. R." J . Schirmer and A. Later. Liquid Chrorn. Chromatography Clin. Gargus. A . W ." Progress in Industrial Microbiolony 5 231 (1964). Benn. Fiedler and A. "Separation of Amino Acids and Antibiotics by Narrow-Bore and Normal-Bore High Performance Liquid Chromatography with Pre Column Derivatization. L . 386 229 (1987). "Protected Fermentation." Advances in Apulied Microbiology.. "Critical Evaluation o f LC with W . Ristuccia." HiF." J . Sampson and R. 393 285 (1987). Kawamura. R. D. Handbook o f Analytical Toxicology. CRC Press. 1 231 (1971). Chrorn. Uno and M. G. Ryall and D. K. CRC Press. N.V I S Photodiode-Array Detection in Pharmaceutical Analysis. Boca Raton. Lab. Chrom. 9. Plaga. Necasek. E. I. 24 249 (1986). Methodology for Analytical Toxicology Volumes I and 111. Solomons. P . H. Knowles and M. Yamada. I . Chrom. Y. Oka. "Liquid Chromatographic Assays of Antimicrobial Agents.64 M. Radzik." Advances in Applied 4 Microbiology." American Laboratory December 30 (1986). 1 (2 and 3) 241 (1987).

APPENDIX A Production of Penicillin Antibiotics .

.

PRODUCTION OF PENICILLIN ANTIBIOTICS The production of penicillin begins from a master stock of the production organism from which vegetative mycelium is prepared in a series of inoculum development steps to increase the concentration of fungal mycelium. tration of 500 kg/m3 solution with a glucose feed rate of 1. 5 to 2 4 . 5 . It is very important that the culture still be i n the unin- hibited growth phase ( l o g growth) when the transfer i s done.1 and A . The initial mycelium concentration in the fermenter The other conditions include broth temand a continuous feed glucose concenis 1 to 2 kg dry weight/m3. glucose utilization is less efficient and enzyme levels and penicillin production are lower.5 to 1. The present fermentation process is a fed-batch process o r a repeated fed-batch process (a portion of the broth is withdrawn at intervals).0 to 2. and the carbon dioxide must be kept below 4% to prevent strong inhibition during penicillin production. dense pellets are formed. The spore concentration is only An inoculum volume important in the first shake flask inoculation.0 VVM) at 300 kPa ( 4 3 psia) pressure with a fermenter pressure of 135 to 170 kPa ( 1 9 . calcium carbonate as a buffer to prevent low pH values. pH of 6 . o f 5 to 10% of the production fermenter (at about 20 kg mycelium dry weight/m3 product from the inoculum reactor) is required to give a high specific penicillin production rate in the production fermentation. A critical spore concentration (=5 x lo9 spores/m3 ) occurs above which more filamentous mycelium are formed. 5 psia) and an oxygen uptake rate of 25 to 50 m o l / m 3 broth per hour.67 Appendix A.5 . A fermenter of 1 5 0 to 200 m3 is used at about 80% full ( 1 2 0 to 160 m3 o f broth) with 3. enzyme levels are high and penicillin production is good in the production reactor.to 4-kW/m3 power input. and various inorganic salts as in Tables A . an organic nitrogen source. Oxygen is supplied by air flow at 30 to 60 m3/m3 broth per hour (0.2 . The presence of about 1% carbon dioxide appears essential for germination of spores. perature of =25"C. Below this concentration. The inoculum development is carried up through shake flasks and then into an agitated vessel with a medium containing sufficient fermentable carbohydrate.

Pentoses. Acetic acid o r acetate. dextrins. amnonium sulfate.3100-4200 kg) Material @antity (approximate) Used for Glucos e 2. NaH2m4. . Raw materials needed for penicillin production (120-160 m3 batch-yielding 28 kg Pen G Na salt/m3 (-20. and ammonia.05 up up up up up up up up up up up up to to to to to to to to to to to to 160 kg 1600 kg 1280 kg 1280 kg 800 k g 128 kg 64 kg 8 kg 1300 g 9600 g 800 g 8000 g Alternate carbon sources--Lactose.4 0.05 0.5kg/m3 broth per hour(200 hr -56. etc.06 0-0.000 gal) A m y l or butyl acetate - - Activated charcoal Potassium or sodium acetate Isopropyl or butyl alcohol Sulfuric or phosphoric acid Table A-2.68 Table A-1.000 kg/batch Fermentation Fermentation Fermentation Fermentation Fermentation Fermentation Fem e n tation Fermentation Filtration Solventextraction Purification Crystallization Crystallization pH adjustment Phenylacetic acid -0-6kg/m3 500 kglbatch Acetic and/or Lactic acid Amino acids. disaccharides (maltose.amines Amnonia 40-50kg/m3 5000 kg/batch Corn-steep liquor solids Antifoam-consumable oil Variable by continuous feed Minerals for defined medium Filter-aid 3 0 .4 0 .01-0.04-0. Precursor Sources--1-phenyl-n-decane and 1-phenyl-n-dodecane. peptides. and molasses. Ethanol.4 0 m3 ( 3 0 .) Alternate nitrogen sources--Cottonseed meal.). Pharmamedia.Penicillim mycelium.005-0.000 units per ml) ~~ - . (Ethanol or fatty oils cause a higher oxygen demand and heat generation than glucose or other sugars. starch hydrolysates.8 0.25-0.01-0. Hexoses. plyols.000-25.4-8 0-5 0. K ~ m 4 CaC03 @so4 7n20 FeS04"7H20 /Fe(NH4)2(S04)2 ZnS04'7H20 CuS04 5H20 MnS04' 4H20 0-1 0-10 0-8 0. brans with phytic acid and phosphorus.01 0.005 corn4 CaC12 NOTES: 7n20 0-0. Alternate materials for defined medium for penicillin production Component Concentration (kg/m3) Batch requirement (kg) Na2S04 (NH4)2SO4 NH4N03 o r KN03 ~2PO4. 0 0 0 L/8-10.

and the specific growth rate. maintaining the optimum glucose and nutrient feed rate during the fermentation. and the metabolic heat and the mechanical heat generated by agitation and aeration must be removed from the reactor. Specific oxygen uptake rates and carbon dioxide evolution Temperature control is rate are also associated with productivity. carbon dioxide evolution.8 kg/m3 per hour optimum) and a total fermentation duration of 180 to 220 hours.1 and A . fermentation. limited mycelial growth rate. and shown in Fig. and having the appropriate precursor affect productivity.2 in a typical concentration profile of the fedbatch penicillin fermentation with some of the control parameter profiles shown.69 kg/m3 broth per hour (1. based on dissolved oxygen. Gist-Brocades‘ state-of-the art process is described The first step of the separation is below. 4 4 Several control algorithms for the carbon feed have been developed.4 and A . and maintenance energy during a for penicillin G and V from glucose. The uptake rates for the other nutrients besides carbon (glucose) are shown in Table A . this becomes more difficult as the viscosity of the mycelial mass increases during the f e r m e n t a t i o n . pH. and mycelial mass during the rapid growth phase ending at the optimum mass limit of about 80% of the maximum. the growth curve. Shown in Fig.1 shows the overall process.23 Productivity in the penicillin fermentation depends upon the genetic capability of the culture. important for obtaining the maximum production rate of penicillin. biomass concentration. A . However.3 . starting at the optimum glucose concentration for the fermentation. A .5 give some of the common yields mycelium. 2 ’ 5 * e ’ 3 5 . and the distribution of glucose into penicillin.2 give the approximate requirements for the chemicals needed for the fermentation. Tables A . Product Recovery Processes The recovery of penicillin from a fermentation broth takes about 15 hours. the filtration of the mycelium from the broth on a rotary vacuum filter using filter aid or precoats and on which the mycelium is . Tables A .3 . Also. Figure A .

E T C . S P O R E S T O INOCULUM P R O D U C T I O N FERMENTOR CLARIFIER ACTIVATED CARBON ROTARY VACUUM FILTER SOLVENT EX T R A C T I O N CARBON TREATMENT T O CHEMICAL MODIFICATION --1 I I DRYER W CRYSTALLIZER BLENDER I OUALITY ASSURANCE TESTING BULK S T E R I L E P R 0D U C T 'i Fig. A-1.70 ORNL D W G 8 7 .4 2 0 FEED-SUBSTRATES. . General production scheme for penicillin production. SEED V E S S E L LABORATORY CULTURE.

growth and penicillin production. .013 Precursor (Phenylacetic acid) a D. in mmol/g E Nutrients and precursor mycelium dry weight/h) ~~ Carbon (sugar) Oxygen Nitrogen (as NH.029 0. BioenE.71 Table A-3." Biotechnol. Specific uptake rates for nutrients during a penicillin fermentationa ~ ~~ Specific u take rates (at p=O. "Quantitative physiology of Penicillium chrvsoPenum in penicillin fermentation.O15 hr . R y u and J.12 0.) Phosphorus (as PO. For maintenance.006 0.60b 0.) Sulfur (as 0. D. . 22 289-298 ( 1 9 8 0 ) . Hospodka.33 1. Y.

72 ORNL DWG 88-288 100 - I I I 1 I I I I I I I I I I I 0 I . D. /' cn I z 3 t U 40 a U m U I - 20 a 0 20 40 60 80 100 120 140 160 FERMENTATION TIME (h) Fig. Bioeng. /. 22 2 8 9 -2 9 8 ( 1 9 8 0 ) . 60 / . 0 /. Hospodka. A-2. Typical concentration profile of fed-batch penicillin fermentation. Source: D." Biotechnol./. . ' /. Y.e*- - d DISSOLVED OXYGEN (% saturation) W ./' cn U C 0 a cu 80 /. "Quantitative physiology of Penicillium chrvsoqenum in penicillin fermentation. Ryu and J . PENICILLIN ( g / L x 1 0 ) .

and maintenance energy. Hospodka. Inc. mycelium. 22 2 8 9 . J . Bioeng.048-0. J.057-0. Y. C . Carbon distribution into penicillin. W. M.410-1.667 ( u er g pen G-Na g g lucose) - Maximum from model calculation Model calculation Experimental 0. Maximum and real yields of penicillin from glucosea Method Units pen G or V (per mg glucose) 700-2198 Mole pen G or V (per mole glucose) 0. Table A-5. "The Penicillins: Properties.. Marcel Dekker. and P.1 4 9 in Bictechnclonv cf Industrial AntibiotLcs ed E. and Fermentation. 1 9 8 6 .73 Table A-4.2 9 8 ( 1 9 8 0 ) . New York. "Quantitative physiology of Penicillium chrvsogenum in penicillin fermentation.061 0. van der Beek.e.12 0. Vandaxr.024-0. Hersbach." Biotechnol.115 aG.207-0. van Dijck.029-0. M. .064 0. D. P. Biosynthesis.a Percentage of carbon fed that is consumed for Penicillin Mycelium Maintenance 6 25-29 20-28 65-69 61-70 Remarks 140 hr fermentation 10-11 2 0 0 hr fermentation aminoadipic acid recycled 16 27 57 aminoadipic acid discarded aD.32 95-200 80-212 0." pp 45 . Ryu and J .

2 3 7 t ACID TO SOLVENT RECOVERY DISTILLATION I TO SOLVENT RECOVERY SALT J 00 cx3 .74 ORNL D W G 8 8 .

and drying. Spent solvents are processed for solvent purification by washing. and temperature. and the extractor. distillation. resulting in a 9 9 . and sterility to minimize chemical and enzymatic degradation of the penicillin in the pumps. or other volatile solvent and then treated with fresh solvent on a horizontal vacuum belt filter and dried with warm air.75 also washed. 0 . penicillin concentration. The crystals are washed and predried with anhydrous isopropyl alcohol. and penicillin G or V is crystallized as the salt from the solvent phase. 5 4 0 L/h (26. Penicillin is extracted in the acid form from the fil- trate under very carefully controlled conditions of temperature. The crystals are separated on a rotary vacuum filter. The extractant is amyl acetate in a continuous countercurrent multistage centrifugal extractor at pH 2. Potassium or sodium acetate is added to the solvent.5 . 5 % pure penicillin. filtered on a precoated rotary vacuum filter. pH value. vessels. Following extraction.0 and at 0 to 3°C. A . and dilute sulfuric or phosphoric acid are used to control the pH. A problem of this method is that the components of mycelium will also be extracted and will contribute impurities that must be removed during the purification steps. butyl alcohol. The water phase and the organic are fed to the extractor at a 4:l ratio. and washed with solvent. the penicillin-containing solvent is treated with a slurry of activated charcoal. Table A . pH. Centrifugal contactors such as the Podbielniak extractor with throughputs up to 9 8 .6 describes the steps in the traditional purification process and Fig. with critical parameters including potassium or sodium concentration. Partition of penicillin between organic Demulsifying and water phases is shown in Figures A-4 and A .5 to 3. using short residence times to prevent degradation of the penicillin under the acid conditions of the extraction.6 shows the stages in a typical extraction.000 gal/h) are used. It is also possible to extract the whole broth from the fermenter with the solvent without going through a filtration step. agents are used to prevent emulsion formation and to obtain a high separation efficiency.

IZec~-.er-. 1947). Fig. F e d e r .0 I 2.76 ORNL DWG 88-280 50 1 I I I I I 1 20 10 5 1 05 . A-4.6 I 32 .S. 0.0 I 5.8 PH I 4.6 60 . Source: R. (June. of Penicillin--Diostribution Coefficients and VaDor-Liauid Eauilibria. I 3. Distribution coefficients vs. Thesis. . pH of penicillin G betxeen water and solvents. L. M.05 2. Polytechnic Institute of Brooklyn.1 0.4 I 5.

and C.1 1 2 3 4 PH 5 6 7 8 Fig. .’ pp 3 9 . Chemical Engineering Progress Symposium Series. J . 1970. V o l . No. Source: M. New York. Dunn. A-5.0 1.77 100 10. American Institute o f Chemical Engineers. G. Souders. 100. “The Recovery o f penicillin by extraction with a pH gradient. Partition of penicillins as a function of the pH of the extraction solvent (isoamyl acetate).o 0. 6 6 . L. Pierotti.4 2 in The history o f uenicillin uroduction.

penicillin m r e soluble in aqueous phase tomm (260OOgal~) Crystallization Further purification and stabilization Tanks and gravitational separators Vacuum o r w m . rotary vacuum d r m filter to 1 4 8 m2 (1600 f t ' ) Extraction Separation of penicillin from other soluble components Continuous.78 Table A-6.pKa. penicillin more soluble in organic phase: when pFI. multistage countercurrent extractors Differential extraction: when pE>pKa.a i r driers Via addition of sodium or potassium ions Drying Stabilization Anhydrous solvent . ~~~ Traditional penicillin purification Step Purpose Equipnent Basis Size Filtration Separation of mycelia from penicillincontaining broth Continuous.

79
ORNL D W C HH ? H I

FRESH SOLVENT

L E A N BUFFER

ADDITIVES

H,SO,

9
?
FILTERED PENICILLIN BROTH

?
STAGE 1

? *

w

STAGE 2

7 1
TO SOLVENT RECOVERY STAGE 3
4
I

J -1

SOLVENT

?

SPENT BUFFER

Fig. A-6. Traditional three-stage extraction of penicillin. S. Queener and R. Swartz, "Penicillins: Biosynthetic and Source: semisynthetic," pp 35-122 in Economic microbiology, V o l . 3. ed A. H. Rose. Academic, New York, 1979,

Three types of indirect assay methods have been used for penicillin quality control. They the iodometric techniques, mercury These have been

imidazole and the ferric hydroxamate methods.

improved through the years s o that automatic analyses can be done. Direct ultraviolet absorption is used to detect penicillins as they are separated on reverse-phase HPLC columns containing octyl and octadecyl bonded material as the stationary phase.

HPLC is able to

separate the side-chain precursor, penicillin, and penicillin byproducts or degradation products in one analysis. Because o f this,
it is a fast, easy to learn, reliable method for quality assurance.

Immobilized penicillinase can also be used in combination with a pH electrode or a thermistor to measure the concentration of the

penicillin in the fermenter or fermentation broth. Modification of Penicillin G to Produce Semisynthetic Penicillins and Cephalosporins. The semisynthetic penicillins are produced with 6-aminopenicilanic acid (6-APA) as the starting material by chemical or enzymatic deacylation o f penicillin G . Some cephalosporins can be produced

using penicillin G as the starting material. The chemical synthesis
o f 6-APA is shown in the Fig. A - 7 .

The enzymatic route is usually

accomplished using acylase enzymes bound to supports and operating continuously in columns to convert the penicillin G to 6-APA. The 6-APA is converted by chemical acylation with appropriate acid chlorides and results in some of the structures shown in Fig. A - 8 . Enzymatic methods are also available to convert the 6-APA to the semisynthetic penicillins, but such methods cannot compete economically with the chemical conversion.
as

Other semisynthetic penicillins such

oxacillin, cloxacillin, dicloxacillin, and flucloxacillin also Clavulanic acid (produced

exhibit lactamase inhibitory properties.

along with cephalosporins by Streptomyces clavuligerus) and olivanic acid (produced by Streptomyces olivaceus) are produced by fermentation and are potent irreversible (suicide) inhibitors of B-lactamases but have no or very weak antibiotic function on their own. They are used

in conjunction with one o f the other semisynthetics such as amoxycillin to give a much broader spectrum product
so

that the

penicillin

81
ORNL DWC 88-238

PCI.

6-AMINOPENICILIANIC ACID

Fig. A-7. Chemical synthesis of 6-aminopenicilanic acid (6-APA). Source: G. J . M. Hersbach, C. P. van der Beek, and P. W. M. van Dijck, "The Penicillins: Properties, Biosynthesis, and Fermentation," p p 4 5 - 1 4 0 in Biotechnologv o f Industrial Antibiotics ed E. J . Vandamme, Marcel Dekker, Inc., New York, 1986.

a2
ORNL OWG 00-240

R -

NAME PROPICILLIN

OXACILLIN

CI CLOXACILLIN

CI DlCLOXAClLLlN

CI FLUCLOXACILLIN m C H 3

6
OCH,

M ETlClLLIN

AMPICILLIN

AMOXYCILLIN

NH2

!/\/

CH

-

CAR BENIC I LLIN

COOH

Fig. A-8. Chemical structures of some semisynthetic penicillins. G . 3. ?1. H e r s h m h , C. P. ~ l a n der Beek, and P. W . M. van Dijck, "The Penicillins : Properties, Biosynthesis, and Fermentation," pp 45-140 in Biotechnology of Industrial Antibiotics ed E. J . Vandamme, Marcel Dekker, Inc., New York, 1986.
Source:

A D C A ) in about a 70% yield by oxidation to its sulfoxide. Fig.83 is effective even against bacteria which are normally resistant to the j3-lactarn antibiotics such as certain resistant Gram-positive bacteria and Gram-negative bacteria including Pseudomonas sp.9 .1 0 . A . and subsequent deacylation as shown in Fig. thetic cephalosporins which can be formed by this route are shown in . conversion of the sulfoxide to the corresponding deacetoxycephalosporanic acid with transient protection of the carboxyl The semisyn- group. A . Penicillin G can also be converted to 7-aminodeacetoxycephalosporanic acid ( 7 .

cephalosporanic acid (7-ADCA)." pp 45-140 in Biotechnology of Industrial Antibiotics ed E. C . Conversion of penicillin G into 7-aminodeacetoxySource: G . Hersbach. New Y c r k . M.9 .C 0 II NHlZ2c.).C. Marcel Dekker.. W. and Fermentation. OH ACID 0 II OeC H O ' 7-AMINODEACETOXYCEPHALOSPORANIC Fig.SI(CH3) CH3 ROH 0 CH2. . "The Penicillins: Properties.0 .84 ORNL DWG 88-252 CH2. J.: PENICILLIN G 0 //c \ 0 OH I o 0 I PYRIDINE HBr 80% >90% PCI5 I I + (CH. P. Inc. M. van der Beek. Biosynthesis. 1385. J. van Dijck. Vandamme.SiN =C I . A . and P.

C . Chemical structures of some semisynthetic cephalosporins. W.CH2 -N. A-10. Vandamme. J . Source: G. and F s r i i i e n t a t i o n . J . Hersbach.n ed E. van Dijck. 1986. M. P. New York.i. /--/ 0 k-d 0 11 N-C -NH OH I CH3 CEFOPERAZONE OH 0 I CH3 CEFAMAN DOLE CEFADROXIL I CH. CH3 CEFOTIAM CEPHRADINE NH ' ( -T 0 JH . "The Penicillins: Properties. Biosynthesis. Marcel Dekker.85 ORNL DWG 88-244 0 CH3 . M. and P.!N N O " 'OH I H CE FATRlZl NE Fig. Inc.. van der Beek.2 'C C - S . . -N . " pp LtJ-ILtu in S i o t e c h n o l o ~ vo f I n d u s t r i a l Antibiotics I C 11.CH.

.

APPENDIX B Production of Cephalosporin Antibiotics .

.

l' The production . meat meal." Structurally. and ammonium acetate used. Figures B-2 and B-3 illustrate the general pathway.8 to 4 .4 provide information on the clinical activity of the cephalosporins against a wide variety of organisms. Unlike the penicillin fermentation. B-3). 0 g/L depending upon the particular cephalosporin produced and the organism employed.2 volumes per volume of medium per minute. One advantage of this process is that the penicillins can . and Table B .9 (5 to 7. Within these classifications. The fermenter is normally operated at 22°C and a pH of 6.5 provides a listing of some of these organisms and the particular cephalosporins which they produce. Cephalexin was one of the first cephalosporins produced and i s one of the most often used. The dif- ferences are in the growth media. It has now come off of patent restricCephalexin is produced by tions and is available in generic form. PRODUCTION OF CEPHALOSPORIN ANTIBIOTICS Cephalosporins are produced by a fermentation process which is very similar to that used in the production of penicillin. the cephalosporins are very similar to the penicillins and several are being produced commercially from penicillin G by biosynthetic means. chemically splitting of the cephalosporin C to make 7-minocephalosporanic acid (7-ACA) and then a chemical reacylation (see Fig. Cephalosporins are produced by classes of microorganisms known as fungi and actinomycetes.media is a complex media with corn steep liquor. sucrose. Several cephalosporin producing organisms (C-2237 and C-3009) can produce the antibiotic while growing on a paraffin-based growth media as shown in Table B . microorganism used and the final fermentation broth concentration of the antibiotic. a large number of strains have been found which produce the antibiotic.8 to 1.2 .3) with an air rate of 0. glucose. Table B-1 lists four typical growth media for cephalosporin fermentations taken from the patent 1iterature. the final fermentation broth concentration is quite low. The overall fermen- Figures B-1 and B-2 give the chemical structure of some clinically important cephalosporins while Tables B3 and B .89 Appendix B. tation takes about 114 hours. 0.

0 4. .575 6.0 3.0 2. Washington.0 6.4 ----- ----0.45 0. bLard oil is rarely used now as it causes problems in the separation and purification steps.45 0.3 0.0 2. Component Cephalosporin production mediaa Percent Medium B Medium C Medium D Medium A Peanut meal Soybean meal Beet molasses Methyl oleate Lard oilb Sodium sulfate Ammonium sulfate Methionine Calcium carbonate Calcium sulfate Yield: pg Cephalosporin/ml 4.0 2. Wick. "Laboratory Evaluation of Cephalosporin Antibiotics.4 6. American Institute of Biological Sciences. E.0 0. A .4 0. (1975).90 Table B-1. Preston and W. continuous carbohydrate feed is now commonly used.2 ___ ___ 0.0 4.1 ___-0.575 4.0 2.45 0.0 3.45 0.2 --- --3240 ___ 3560 4190 3920 aD. D.0 0.575 3.2 --0.0 3." DeveloDments in Industrial Microbiologv Volume 16.575 6.C.

A 2% mixture in the medium and 1% fed at 72. +. C12-22. A. E. DCPC-Deacetylcephalosporin C .4% fA mixture o f one part acetic acid and two parts sodium acetate. C14=27.5%.91 Table B-2. "Laboratory Evaluation of Cephalosporin Antibiotics.C.3%." DeveloDrnents in Industrial Microbiologv Volume l6. Carbon utilization for cephalosporin productiona Strainb Carbon Source Conc. very good. (1975). u. % Growth' Cephalosporins producedd (Pg/ml) 120 25 15 85 160 25 C-2237 n-Paraffine Sodium acetatef Soy bean oil G 1ycero1 Glucose 8 5 +++ U 5 5 8 +++ +++ U+ -__ 110 5 10 60 5 35 120 C-3009 n-Paraffind Ethanol Sodium acetatee Soy bean oil Glycerol Glucose 8 5 +++ +++ U 5 5 5 8 18 15 lo 6 8 +++ +U ----- 3 15 5 15 5 5 30 +++ 50 15 aD. C13=49. American Institute of Biological Sciences. poor growth. Preston and W. D. CPC-Cephalosporin C.9%. good. and 120 hours. Washington. C15=0. carneus C-2237 cSymbols. 96. +++. Wick. eComponent. . bP. dDACPC-Deacetoxycephalosporin C . persicinus C-3009 and P.

E.92 ORNL DWC 00-270 ONa CEPHALOTHIN SODIUM CEPHRADINE N=N N=C I / ’ O I. Wick.NH2 II CEPHALORIDINE CEFOXlTlN .C -/IN . i C . 6 (1975). ONo / 0 -S -6 ’s ’ ’CHS o”c‘oo CEPHALEXIN CEFAZOLIN SODIUM -- ’ - 7 C H .C . American Institute of Biological Sciences. . Source: D. H I H I 1 H I ‘N-cHZ-c-N-c . H H CH. Structures of many of the cephalosporin antibiotics. 0 C I ”c ‘CH2OH I 0 0 . A. “Laboratory Evaluation of Cephalosporin Antibiotics. - H O I1 H H I C N S C C.C.S\ 1 H C-H 1 ’S . Preston and W .C I L I 0 H CHZO I.C . .C-N. 1 . D.C N C C ONHS 0’ C 0 C CHs CEPHALOGLYCIN Fig. 0 I ’ .“ Developments in Industrial Microbiolom. Washington. B-1.C-N\ c .s Cc I H c-H C \CH.

(CH2)s C O* 0 21 7 . C?S t -- 4H d -(L- a -AMINOAOIPYL)-L-CYSTEINYL-D-VALINE \ OH ISOPENICILLIN N 0 OH PENlCLLlN N __ . C 0 1 I-CH.(CH2)3 -C -N II H . .CH.(CH2)s I -C 0 II . 0 CH20H \ OH 0 ' 'hACH.-L 0 DEACETYLCEPHALOSPORIN C c -(CH2)s c NH f 1 .CH.a -AYINOADIPIC ACID HO \ "2 /CHJ \ ___c C-C-CH 0 D / C . C Og OH OH ' r o// OH OEACETOXCEPHALOSPORIN C ACETYL CoA . Biosynthetic pathway for the formation of cephalosporin.CHI d . - - HZN\ C .CHs OH CEPHALOSPORIN C Fig.N H )~ I no NH.SH HO NH2 02\ OH a -AMINOADIPYL)-L-CYSTEINE 0 II SH cH- 4 H20 d -(L- 0 c .s. N\.. .N .( c H ~. / \\ cL.'.93 ORNL DWG 88-24! L-a -AMINOADIPIC ACID ACTIVATED L.c .CH.NH-C- -L 02 HZN\ .~ " . 0 0 I 0 .. 0 . B-2.

++++ ++ = moderately e f f e c t i v e : + = l i t t l e effect. S h i g e l l a s p . . K l e b s i e l l a rmeumoniae. D . . E . tt+ = e f f e c t i v e . Clinical characteristics of common cephalosporin antibioticsa C h a r a c t e r i st i c b Cephalothin Cephaloridine Cephaloglycin Cephalexin Cefazolin In v i t r o a c t i v i t y a g a i n s t gram+ c o c c i ( e x c e p t group D s t r e p ) In v i t r o a c t i v i t y against cephalosporinasen e g a t i v e gram negC In v i t r o activity against cephalosporinaseproducing gram neg d In v i t r o activity a g a i n s t Pseudomonas tt +++ fttt +++ tt ft +++ ++++ ++ ++ + _- Route o f a d m i n i s t r a t i o n R e l a t i v e peak concent r a t i o n i n mouse blood a f t e r 20 mgJkg Relative duration Parenteral Parenteral Oral Oral Parenteral ++ +tt + +*+ t+++ + +tt ++ ++ ++* aD. bSymbols: (1975). c o l i . E. . d e . = very e f f e c t i v e . Salmonella s p . S e r r a t i a s p .g. E n t e r o b a c t e r sp. " Developnents i n I n d u s t r i a l Microbiology Volume 16. . g . P r o t e u s m i r a b i l i s . "Laboratory Evaluation o f Cephalosporin A n t i b i o t i c s . Wick.. C . f = marginal: -- = no e f f e c t .94 Table B-3. American I n s t i t u t e of B i o l o g i c a l S c i e n c e s . P r e s t o n and W. Indole-producing Proteus s p .. 'e. A . Washington.

pg/ml) Method of administration against the following: Injection Oral S. Characteristics of cephalosporins of clinical interest ~ Name Specific activity (minimum inhibitory concentration.9 0.4 9 + + + + 17 13 0.aureus Pseudomonas Cefamandole Cefazolin Ce foxit in Cephalexin Cephaloglycin Cephaloridine Cephalothin Cephap irin Cephace trile Cephradine + + + + + 0.8 0.6 2 5 a 20 .95 Table B-4.8 2 2 6 9 5 2 2 + 4 0.

0- I1 C- CH3 Fig. . B-3.96 ORNL DWC 88-243 .C . Chemical deacylation of cephalosporin C.C .x(J R1-c-NHnJ (CH3)3SICI BASE R1. COOH COOSl(CH3) 2 PCI 1 1 L?JcH2-R2 R OH COOSI(CH3)3 R. = R.= 0 OR H I HOOC .(CH2)3 I CHZ R.NH I O O / CH2-R2 0 / CH2-R.= .= .H 0 R.

. Scopulariopsis sp. C. minims. Str. polyaleunn. Paec . acrezzwnium. An. An. polyaleurum. Microorganisms which produce various cephalosporins1 Compound Microorganism Cephalosporium acremonium. Emericellopsis sp. Paec. peruviana. Ar. C. Paeciiomyces persicinus. Str. Spiroidium fuscum. Arachnomyces minims. e. Res. Clavuligerus Str. persicinus. Cephalosporin C Deacetylcephalosporin C Mutants of C. clavuligerus. Streptoqces l i p m i i . carneus. Str. Lab. Str.. Kanzaki and Y. Str. peruviana. minims. acremnium Mutnats of C. C. rochei. Str. fuscum. c.. chrysogenum. halstedii. Str. carneus Mutants of C. sp. Sp. Str. lipmanii Str. 'Source: . chartreusis. fuscum. Diheterospora chlanydosporia. lactamdurans Str. Anixiopsis peruviana. 2 (2) 324 (1975). polyaleurum. Paec. carneus. "Recent Progress in Cephalosporin Fermentations. F'ujisawa. clavuligerus Str. viridochromgenes Same Deacetoxycephalosporin C A 16886 A A 16884 A Cepharrycin C or A16886 B Cephamycin B Cephamycin A F1 as Cephamycin B producers."J. Mutants of C.97 T a b l e B-5. At-. fimbriatus. cimamnensis. Sp.Takeda. Paec. griseus. Paec . acrenwnium. acrenwnium A l l strains which produce cephalosporins c2 Penicillin N T. persicinus.

98 Table B-5.v a l i n e and . cysteine. acremonium C . acremonium 6 . (Continued) 6-Aminopenicillanic acid C. /I-hydroxyvaline.( L .a m i n o a d i p y l ) . acremonium (Dimer of 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine) Peptide S 2 (Disulfide of methanthiol) Mutants of C. valine and glycine) Tetrapeptide P 1 C.and glycine) Peptide S 1 Mutants of C. acremonium Tripeptide P 3 (6-(L-a-aminoadipyl)-L-cysteinyl-D-valine) Tetrapeptide P 2 C.a .D . cystein.c y s t e i n y l .L . acremonium (a-aminoadipic acid. acremonium (a-aminoadipic acid.

Fig. B . L-(+)cysteine (precursor).40 This is not as important for the oral product as for the products for injection. lead tetraacetate. followed by adsorption of the filtrate on activated carbon. ion exchange resins. including conventional solvent extraction. however. pyridine. phosgene. t-butanol. and eluting the resin with a salt solution at a pH of from 5. toluene. Among the more common are4' . ~ t 1 . The general procedure is to filter the fermentation broth at acidic pH (5 . Frequently. removal of the adsorbed antibiotic by contacting the carbon with a mixture of water and a polar organic solvent.5-10. contacting the eluate with an anion exchange resin.46 Product recovery Cephalosporins are extracellular fermentation products. Woodward et library. 4 ~ The step-by-step syntheses is given by and is available in any chemistry department A mathematical model for the fermentation of cephalosporin C is given by Matsumura et a1.O) .4 gives a general process flow sheet for the recovery and purification of cephalosporins. allowing a bulk filtration to be used to separate the solid matter from the antibiotic containing fermentation broth. Recovery from the filtrate is difficult due to the low concentration of product and the need to remove high molecular weight (1000 MW) biological compounds which can lead to allergic and toxic manifestations when the drug is administered. Cephalosporins can also be produced chemically. and sodium acetate). Many different separation and purification schemes are employed.7 4 " C . Some steps require temperatures of . Many different precipitations are possible. all of these compounds can be found in quantity at many university chemistry departments and could be used to serve the needs of a small area.99 be produced at a lower cost than the cephalosporins. liquid chromatography with conventional resins and salting out procedures. the large-scale commercialization of the process is not feasible because of the operating conditions. and expensive and toxic chemicals are required (such as benzene. However. the carbon adsorption steps are replaced with a precipitation step.0. methylene chloride. while other steps occur at 120°C.

Kalyanpur. of Cephalosporin C from Fermentations Broths Using Membrane Systems in and High-Performance Liquid Chromatography. W . RECOVERY PROCESS FROM FERMENTATION BROTH I BYPASS REVERSE OSMOSIS MODULE CHILLED WATER D PERMEATE CONCENTRATE RETURN REVERSE OSMOSIS CONCENTRATION OF CEPHALOSPORIN C Fig. 26 4 5 5 (1985). PIGMENT REMOVAL CON :ENTRATE CONCENTRATE n PURIFIED ANTIBIOTIC U I HPLC CEPHALOSPORIN C. . Skea and M. B-4. "Isolation sporin C. Schematics for recovery and concentration of cephaloSource: M. Siwak." DeveloDments Industrial Microbiology.100 ORNL D W G 88-233 -h FERMENTER MEMBRANE FILTER ULTRA FILTER REVERSE OSMOSIS PERMEAT E _____) CONCENTRATE CELL HARVEST CELL WASHING CONCENTRATE PROTEIN.

Sodium-2-ethyl hexanoate will precipitate the sodium salt of N-derivatized cephalosporins from solvents. 4. Insoluble derivatives such as the N-2. .101 Crystallization of the potassium or sodium salt from purified aqueous solution of the cephalosporin by concentration and/or addition of large volumes of a miscible solvent. The zinc salt (also copper. lead. nickel. and manganese) can be crystallized from purified aqueous solutions. iron. Figure B . cobalt.5 gives a typical flow diagram for the purification of cephalosporins using ion exchange resins.4 dichlorobenzoyl cephalosporin and tetrabromocarboxybenzoyl cephalosporin are crystallized as the acid from aqueous solutions. cadmium.

156 (1985)." Purification of Fermentation Products. Cephalosporin C purification f r m bro'h by adsorption. . B-5. Wildfeuer.2 4 2 20% ACETONE PYRIOYLACETATE CEPHALOSPORIN C NON-IONIC RESIN XAD 2 HP-20 ANIONEXCHANGE RESIN IRA 6 8 ELUATE I ELUATE * NON-IONIC RESIN ADSORPTION CEPHALOSPORIN C BROTH (EXCHANGE H ' F O R INORGANIC CATIONS) c CATIONEXHANGE RESIN IRC a4 c ANIONEXCHANGE RESIN IRA 9 4 - ANIONEXCHANGE RESIN IRA 6 8 CEPHALOSPORIN C ADSORPTION ELUATE ELUATE I ELUATE TO ANION-EXCHANGE ADSORPTION Fig. E. Source: M. "Approaches to Cephalosporin C Purification from Fermentation Broth.102 ORNL DWG 8 8 .

APPENDLX c Production of Tetracycline Antibiotics .

.

2 to 0 . C .105 Appendix C. Oxygen must be provided for oxygen transfer and aeration rates of 1 volume of air per volume of fermenter per minute ( W M ) are customary.to 150-d working volume with three open turbines that are 1450 to 2100 mm in diameter with maximum speeds of 80 rpm and a power input of 300 kW (400 Hp) at the shaft (up to 3 kW/m3). allowed to grow for 18 to 20 hr. glucose or molasses. PRODUCTION OF TETRACYCLINE ANTIBIOTICS Tetracycline antibiotics production utilizes preinoculation. The culture is monitored for pH. and vegetable oil (0. Chloride ions serve as precursors of chlor-tetracycline. About lo5 to lo6 spores/cm3 are inoculated into the first submerged culture fermentation in the preinoculation tank and allowed to grow for 24 to 26 hours. inoculation. 2. followed by the second sporulation in a stationary culture on millet in flasks. and then this material (about 5 % of the fer- mentation vessel volume) is added to the fermentation vessel with the nutrients and allowed to grow for 100 to 200 hr with tetracycline production. 3 % ) .5% wt/vol. The first step requires 7 to 10 days. and the second 14 days.2%). 0. Oxygen supply continuity is very important because interruption of the oxygen supply for more than 10 min results in the stopping of tetracycline production. a buffer (calcium carbonate. inorganic salts (NaC1 and K H 2 P 0 . C-2. The processing scheme is shown in Fig. All growth takes place at 2 9 ° C . Strains are kept either in a freeze-dried or liquid-nitrogen state as spore stock.7%). and fermentation tanks similar to those of penicillin. Oxygen enriched air provides faster growth and higher yields. residual sugar. A culture is started by sporulation on an agar slant. and the procedure is shown in Fig. . ) . The contents of the first fermentation are transferred to the inoculation tank. and benzylthiocyanate is added as an inhibitor o f undesirable metabolic pathways during lack o f oxygen. 1. The fermenter can have a 100. organic nitrogen sources (soybean meal or corn-steep liquor.1 . The medium for tetracycline production includes sucrose.

Source: D. New York (1959). Production scheme for oxytetracycline. . Hockenhull. Interscience Publishers Inc.. J .106 ORNL D W G 0 7 . C-1.4 7 3 AMMONIUM SALT f- 1 Fig. D. The Fermentation of the Tetracyclines.

Hockenhull. New York (1959).2% (NH4)2P04.2 2-10% Inoculum 60-65 hr 28 " C 1% sucrose.05% Asparagine. Production chart for chlortetracycline.2 . 1.0 Purification from the clear broth after removal of the mycelium Fig. Interscience Publishers Inc. 0. 0.5% CaC03 I 24 hr Same as for shake culture Prefermenter 5% Inoculum 19-24 hr pH 5. 0.107 Inoculum Preservation (spores on agar slant or in soil) Agar Plates Spores as Inoculum Shake Flask 2% Meat extract. 0. Source: D.2%KH2PO4.8-6. 1% Gulcose.00033% CuS04*5H20 0. 3% Sucrose. 0.005% ZnS04-7H20. D.1% CaC03. 0.2-6. 0.5% K2HP04. J .025% MgS04'7H20. The Fermentation o f the Tetracyclines. 0.3% Agar 2% Corn steep liquor (50% solids).00033% MnC12-4H20 pH 5. . C . 0. 1% corn steep liquor..

The pH. 4. and ZnSO. precipitation of Ca2+ with ammonium oxalate. and extraction of the metabolite with an organic solvent. extraction of liquid fractions . based on salting out (NaC1) the antibiotic from the aqueous to the organic phase (1-butanol). Adsorption on diatomaceous earth or active charcoal with subsequent chromatography or selective extraction. Several methods have been used to recover and purify tetracyclines and some of these are listed below. Aeration ranges up to 0. 3. concentration). Tetracyclines are precipitated as complexes with alkaline earth metal compounds or with primary and secondary alkyl amines.” 1. Product Recoverv and Purification Isolation methods for tetracyclines must take into account their amphoteric nature and the possibilities of their polymerization or rearrangement.8 medium vol/min and oxygen overpressure o f up to 0. Precipitation (dry salt) process based on precipitation the antibiotic from dilute aqueous solution of aryl azosulfonic acid dyes. 2. usually one o f the methylalkyl ketone type. oxygen content of the medium (at least 20% satu- ration) and the CO. Nutrients are sterilized at 120°C for 40 min or at 140°C for 1 min with C and N sources sterilized separately.108 respiration (CO.Fe(CN). 5. The most frequently used extraction agent is 1-butanol. and morphology. Direct mash extraction based on solubilizing the antibiotic by acidification.. precipitation of ballast compounds with K. Extraction from acid or alkaline medium. addition of quaternary ammonium compounds as carriers. This method is also suited for refining a crude Oxytetracycline is purified by combining the release of oxytetracycline into the medium by acidification. increase of biomass in concentration and in volume. in the exhaust air are monitored continuously. Solvent extraction o f the antibiotic with salt. owing to its suitable partition coefficient and economic availability. product.1 MPa are used.

which yield nonhygroscopic preparations. Particularly efficient is crystal- lization from boiling solvents.109 with butyl acetate. while sulfate anions have the opposite effect. using both regular and reverse phase packings and ion exchange packings. thin-layer chromatography. Residual amounts of antibiotic in the mother liquor are increased by oxalate and chloride anions. hydrochlorides) or bases. such as lower alcohols. Further purification is carried out by crystallization as salts (e. During the fermentation and isolation process. Crystallization is most efficiently performed at 2 ° C for 3 hours.g. or aliphatic ethers of ethylene glycol. Methacycline. After adjusting the filtrate with EDTA.. and precipitation of Ca2+ with oxalic acid. and citric acid. Methacycline is made by a three-step synthesis and is isolated as a sulfosalicylate which is converted to a hydrochloride of methacycline by crystallization from an acid mixture of acetone and methanol in a 62% yield. Na. paper chromatography. ketones. Using this method. and gel chromatography can be used for assay. has been used for analyses of the tetracyclines.SO. Analytical Methods Chemical assays of tetracyclines based on metal complexes utilize the characteristic absorption of fluorescence of the complex formed. the crystalline Sase of oxytetracycline is obtained.27 . doxycycline.. Recently. rninocycline and rolitetracycline can be prepared. Polarographic assay of tetracyclines is based on their reduction in a boric acidsodium borate buffer.48 Semisynthetic tetracyclines are prepared from oxytetracycline by chemical synthesis. HPLC. one can analyze the principal tetracyclines both in biological material and in commercial preparations.

.

APPENDIX D SulfonaInides .

.

nation of acetanilide. The processing steps necessary to produce purified sulfonamides are common to the organic chemical and dyestuffs industries and are described briefly below. The acetyl product is treated with aqueous ASC is obtained by chlorosulfo- alkali to the free amino compound. $ /c-c. c-c . and there are over 5. Figure D .49 .-Cl + 0 B RNH.1 presents the chemical structure of the most important sulfonamides. C-. The sulfonamides came into common usage in the mid 1 9 3 0 ' s and are still produced in quantity today. and azo and nitro derivatives may be yellow. From the base compound. Assays for the sulfa drugs are usually done by titration with nitrous acid and paper or thin-layer chromatography. SULFONAHIDES The sulfonamides (sulfa drugs) represent a large class of antimicrobial drugs which are produced by chemical reaction. The drugs are white powders melting above The sulfas are 15OoC. sparingly soluble is cold water but are somewhat soluble in alcohol or acetone.113 Appendix D. base S Ic-C\ 0 CH.-C-NH-C\ . many clinically active derivatives have been produced. -C-NH-C. C-S-NHR c-c 0 Extra amine or base is used to neutralize the hydrochloric acid freed in the reaction.16 The usual method of sulfonamide preparation is the reaction of N-acetylsulfanilyl chloride (ASC) with the appropriate amine. CH.000 different sulfonamide derivatives.

.114 ORNL DWG 88-275 NAME STRUCTURE SULFANILAMIDE H2N @SO*-NH~ SULFAPY RlDlN E -- “0 / H SULFATHIAZOLE SULFADIAZINE -“I -“g /H /H SULFACHLOROPYRADAZINE -“a / H CI N-N SULFADIMETHOXINE 0 -CH3 / H SULFAETHIDOLE -N rsTC2H5 N-N CH3 SULFAMETHAZINE -N$3 / H SULFAMETHAZOLE -NrsTcH3 N-N SULFAMETHOXAZOLE -N?rH3 N-0 Fig. New York (1978). Source: EncycloDedia of Chemical Technology. ed -. Third Edition. Therapeutically important sulfa drugs and sulfones. D-1. Kirk-Othmer. Interscience.

N b N-N' OCH. SU LFAMETER - < @ OCH. SULFAMOXOLE SULFAPHENAZOLE SULFAPYRAZINE -Nb HN / SULFAQUINOXALINE SU LFlSOMIDI N E SULFISOXAZOLE SU LFACYTINE 0 .115 ORNL DWG 88-276 NAME STRUCTURE SULFAMETHOXYPYRIDAZINE H2N @ S 0 2 .

H H3 N’-ACETYLSULFISOXAZOLE -N 0’’ SUCCINYLSULFATHIAZOLE --N@!502 0 -N / H v’p N PHTHALYLSULFATHIAZOLE :’ O@ N / H v’p N SALICYLAZOSULFAPYRIDINE k-J MAFENIDE / CH2@ SO. .116 ORNL DWG 88-277 NAME STRUCTURE SULFACETAMIDE SULFAQUANIDINE H3 Lyf ‘CCH3 .NH. NH2 DAPSONE ACEDAPSONE NH2 PRONTlSlL .

APPENDIX E Bacitracin Production .

.

1 and E . Table E . BACITRACIN PRODUCTION Bacitracin has been produced using several different types of media. carried out at 30 to 3 7 " C . E - 3 shows the flow scheme and gives the media compositions used in the various steps of the process. for 24 to 30 hr. zinc bacitracin. The bacitracin-producing strains of Bacillus licheniformis are kept on agar slants or in the spore form in dry sterile sand. Recently. but nickel. E .2 . Bacitracin production levels are on the order of 400 to 600 IU/ml where 7 4 IU (intemational units) corresponds to 1 mg of the Second International Standard of bacitracin or 5 to 9 g/liter. monitored by continuous pH and 0 measurements. or continuous sterilization. Zinc bacitracin is used primarily as an animal feed-grade After fermen- product and is probably the most simple separation. They include (1) synthetic media containing amino acids as carbon and nitrogen sources. but this is not commercial. primarily zinc.119 Appendix E. licheniformis immobilized in polyacrylamide gel. salts such as methylene disalicylic acid.14 Recovery and Purification o f Bacitracin from Fermentation Broth Bacitracin is prepared by direct precipitation form the clarified fermentation broth by salts of divalent cations. and Table E-2 lists some precipitation agents for bacitracin. tation. and alkylbenzene sulfonic acid . bacitracin production process is shown in Fig.1 gives the solubility of bacitracin. hydroxymethane sulfonic acid. manganese and cobalt have also been used. lignin sulfonic acid. and ( 3 ) high-producing media with soybean meal and starch or dextrines/sugars as nutrients. A suitable amount of the culture is used to inoculate shake flasks of tryptone or peptone broth. (2) rich media containing beef extracts or tryptone broth. and bacitracin F in various solvents. The fermenters are equipped with an agitator and a The medium is sterilized either by batch The fermentation process is usually The fermentation is jacket for steam or water. The Fig. bacitracin has been produced by cells of B. . An intermediate fermenter is usually used as a seed tank for the main fermenter.

$FzR FERMENTER STERILE AIR 0 0 SEED TANK BUTANOL STERILE AIR INTERMEDIATE CENTRIFUGAL BUTANOL VAPOR RECOVERY CONCENTRATOR SPENT BEER TO STRIPPER CAKE T O TREATMENT Y h t BACITRACIN PRODUCT Fig. Bacitracin production flow and equipment scheme. . Source: H. Peppler and D. Second Edition.1 . J. New York (1979). Academic Press. E . Microbial TechnoloEy Volume 1.120 ORNL DWG 88-283 A SOY BEAN MEAL STARCH - I STEAM c 0 0 STERILE AIR SEED FERMENTER MASH COOKER - I ___) FERMENT E R MEDIA c. Perlman.

0.121 I Inoculum Preservation 1 Shake culture I (spores on agar slant I 4 X 1 L Erlenmeyer flask with 200 m l tryptone or or peptone culture medium I 18-24 hr at 37°C Same as for shake culture 6 hr at 37°C 1 with intensive J Prefermenter (3000 aeration L) 5% Soy meal. ionexchange c-hromatography hr at n b For use i animal feeds: .S04 0.2% Sucrose. 0.2 . I Growth to log phase Production Fermenter 30 5% Soy meal.2% cam3 7. 0. E .2% cam3 . Block diagram for the production of bacitracin.SO4.2% (NH4).2% (NH4). 2. Extraction with n-wztanol. Furification a For pharmaceutical uses: . concentration of the aqueous phase. Spray drying of the whole femtation broth Fig. 1.4% Sucrose. extraction of the organic phase with buffer. .

4 >15.122 Table E-1.24 16.2 Acetone 0.a Ammonium molybdate Ammonium rhodanilate Azobenzene p-sulfonic acid Benzoic acid Divalent metal ions (Zn. "The Bacitracins: Properties.) Furoic acid Methylene disalicylic acid Molybdic acid Phosphomolybdic acid Phosphotungstic acid Picric acid Picrolonic acid Salicylic acid a $3. bThe solubility of bacitracin (pharmaceutical grade) . etc. New York. Fr$yshov.. and bacitracin F (oxidized bacitracin) were tested at 25°C.1 11. --- 0.Butanol 4. 1986.08 Methanol >40.3 0. and Fermentation.005 10. J . .0 1. "The Bacitracins: Properties. New York.002 0. and Fermentation. Marcel Dekker. Inc. Acetonitrile 0. Solubility of bacitracins in various solventsa Solubili tyb (mg/ml ) Solvent Bacitracin Zinc Bacitracin Bacitracin F Petrolether (bp 100-120°C) Petrolether (bp 40-60°C) Carbontetrachloride Toluene -Diethylether 0.1 0. Vandamme. 0.007 0.07 _0. Biosynthesis. 1986. Marcel Dekker.68 0.006 Chloroform Pyridine 17. Table E-2.06 17.002 0." pp 665-694 in Biotechnology of Industrial Antibiotics ed E. Inc." pp 665-694 in Biotechnology of Industrial Antibiotics ed E.2 a $3. 0.005 Water >40.07 11. J . Frayshov. Precipitating agents for bacitracin.. Vandamme. zinc bacitracin (pharmaceutical grade).009 _- 0.37 0. Biosynthesis.09 >15.

or ion exchange followed by solvent extraction is practiced. A chromatogram is shown in Fig. and then dried by drum or spray drying. primarily zinc. centrifugation. Assay methods for bacitracin include microbiological (in which inhibition of a test organism is compared to a standard bacitracin). bacitracin is concentrated by filtra- tion. Bacitracin can be adsorbed/precipitated on solid carriers like bentonite and celite. isoelectric focusing. and pulse polarographic techniques. E - HPLC has been used for monitoring the fermentation broth during fermentation and during the purification process. The precipitation of bacitracin is accomplished with divalent metal ions. After precipitation. electrophoresis. solvent extraction from the clarified fermentation broth with butanol. or evaporation. 4.123 have been used for precipitation of the bacitracin salts. For pharmaceutical-grade bacitracin.50 . paper and thin-layer chromatography. The assay method of choice is HPLC using reversed-phase packing and programmed gradient elution or an isocratic system.

UV ABSORBANCE ( 2 5 4 nm) r C I I m >8 2 -B '1 SUBSTITUTED BACITRACINS BACITRACIN A 0 z C 0 r C 3 m OXIDIZED BACITRACIN A .

APPENDIX F Organic Product Fermentations .

.

activated carbon and ion exchange columns. and glass.scale antibiotic fermentation. because during an ethanol fermentation. and fermentations occur at a higher temperature (35°C vs 23 to 25°C). The purification steps include filtra- tion. Separation/purification equipment will gener- ally not be adequate because the ethanol process only includes filtration and distillation and azeotropic distillation (benzene) equipment. broth viscosity changes very little. and centrifugation (which readily adapt to antibiotic purification needs). The heat exchange and agitation equipment may not be adequate for full. crystallization. oxygen is required for proper growth. . and 4 ) . F-1) is used to produce a fermentation product which must be separated and purified before use (Figs. The ethanol fermentation is fundamentally differSome ent. The vinegar product is filtered using filter aids and In fining agents for purification. Organic acids Acetic acid is now often produced in a submerged-culture batch fermentation using a stainless steel fermenter with a high-speed agitator on the bottom of the fermenter for high efficiency oxygen transfer. because it is essentially an anaerobic process. F-2. and the aeration equipment may be in place in the fermenters to allow for the sparging that is required for an antibiotic fermentation. the submerged-culture citric acid process. aeration and agitation requirements are similar to antibiotic production needs. precipitation. ORGANIC PRODUCT FERMENTATIONS Ethanol process The process for production of fermentation ethanol is similar in its initial steps to antibiotic production in that a stirred-tank batch fermenter (similar to Fig. 3 .127 Appendix F. though. Some distillation towers could possibly be used as extraction columns to obtain some separation. Gluconic acid is also produced in a sub- merged fermentation at 30°C using air sparging in batch mode.or plastic-coated or 316 stainless steel fermenters are used (adaptable to sterilization and antibiotics fermentations).

////.. Inc. Biochemical Enpineering. Source: S. . Aiba. Fig. F . New York (1973). F.///////. Academic Press.//. E.128 ORNL D W G 88-296 MOTOR DRIVE FOAM colOLlNG C I BAFFLE PLATES FLAT-BLADE TURBINES SPARGER STERILE AIR INLET ////// l///~/.1 ./!///\///////. and N. Millis. Humphrey./. Common stirred-tank batch fermenter for ethanol production. A.

WATER CORN STOVER i 1 b L . Mavituna. F-2. stover. . Nature Press. Biochemical Engineering and Biotechnology Handbook. Atkinson and F. London (1983).129 O R N L D W G 88-293 H2S04. VITAMINS AN A E R 0 B IC WASTE D lQ E S T 10N YEAST Equipment used i n the production o f ethanol from corn Fig. Source: B.ACID PRETREATMENT ENZYME REACTOR t SUQARS FERMENTER ENZYME PRODUCTION EXCHANGER I li RESDUAI SOLIDS C E N T R IF U Q E 4 1 FILTER 0 < a < n STERILIZER ETHANOL PROD U C T l O N SOLWS - ADSORPTION OR ION E X C H A N Q E MINERALS.

25% ETHANOL .130 ORNL D W G 8 8 .3 0 1 FUSEL OILS ~ FUSEL OILS t .

131 ORNL D W G 88-300 RECYCLE I 12. Mavituna. . F-4. Biochemical Enpineering and Biotechnologv Handbook.5% ETHANOL 75. London (1983). Atkinson and F. Nature Press. Source: B.1% BENZENE a U DECANTER * 96% ETHANOL * 96% Fig. Purification equipment for anhydrous ethanol production.

stirred fermentation followed by high-speed centrifugation. sparged fermenters. The fermentation of glutamic acid and penicillin are very similar. aerobic. evaporation. and Zanflo. but very little heat exchange. which is very oxygen-transfer-dependent. centrifugation. and both fermentations can and do use the same fermentation and product-recovery equipment. and activated carbon adsorption. dextran. jacketed. and drying. drying.1 2 ) is produced in a submerged. and filtration or adsorption and elution (all very adaptable and necessary for most antibiotic production procedures). are produced in batch fermentations followed by precipitation. Glutamic acid is another organic acid produced by fermentation. Cyanocobalamin (vitamin B . extrusion. The fermentation of micro- organisms utilizes stirred. Another separation method uses an extraction with phenol or other solvent and precipitation or crystallization. and leaching. heavy duty agi- tation due to the high viscosity of the broth as the fermentation proceeds. and combination of these. chemical reaction. followed by centrifugation. Riboflavin (vitamin B-2) is produced by fermentation performed at about 29°C with agitation and aeration in a jacketed fermenter. . crystallization. solvent extraction. ion exchange. Extracellular polvsaccharides such as xanthan gum. heat treatment to release the product.132 Purification equipment includes filtration. The product is recovered by heating the broth followed by centrifugation. The fermentations require oxygen.

APPENDIX G Enzyme Production Technology .

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EnzyKes are usually 1 to 2 orders of magnitude larger than antibiotics. chromatography. giving rise to the different separation schemes used for enzymes such as ultrafiltration.2 . and dialysis. and the filtration and purification equipment for different enzymes. but only the liquid submerged process is applicable for the discussion of antibiotic production in enzyme equipment. compared to the multiple-stage. G .3 detail the various equipment including the fermenter and associated culture starting tanks. and very little change would be required for the plant to produce certain of the antibiotics on short notice. sterile air supply. G . ENZYME PRODUCTION TECHPiOLOGY Enzyme production is based on the fermentation of a microorganism to produce the complex enzyme which is then purified by various processing steps to yield a stable preparation. The basic differences are in the various solvent extraction requirements and equipment. . and G . multiple-solvent processing required for most antibiotic production schemes.1 . Semisolid or liquid media are used.135 Appendix G. The process flowsheets shown in Figs. sterilization equipment. feed tanks. which in an enzyme process involve only a single. The separation and purification equipment is very much the same as that found in an antibiotic production facility.or two-stage extraction.

London (1983).I36 ORNL D W Q 88-291 FERMENTER C EN TR IF UQE FILTER E N ZY ME SOLUTION C IP IT AT IO N S OLV E N T S O L V E N T P R E C IPlTAT IO N CHROMATOGRAPHY CENTRIFUGE FINAL PRECIPITATION VACUUM ENDOCELLULAR E N ZY ME P R OD U C T Fig. flow sheet. Source: B. . G-1. Biochemical Engineering and Biotechnology Handbook. Intracellular er?zFe purificatio.. Nature Press. Mavituna. Atkinson and F.

6-2. Nature Press. Mavituna. . London (1983). Atkinson and F. Alternate enzyme recovery processes. Biochemical Engineerinv and Biotechnology Handbook.137 ORNL D W G 88-290 FERMENTER f EXCHANQER HEAT h 1c STORAGE o o L PRrTREATMENT ~ ~ ~ !\ 1 'Lp 1 DECANTER 1 60 ___) EVAPORATOR H EA T EXCHANGER E C IPlT AT10 N FILTER PR E S S BACTERIAL FILTER SPRAY DRIER1 PRODUCT ' I Fig. Source: B.

ETHANOL I t r-+ I I MICROBIAL E N Z Y M E S IT IFERMENTER. London (1983). Comon commercial e n z p e process. .138 ORNL D W Q 88-292 S E P A R A T I O N I P U R I F IC A T I 0 N FILTER PRESS 11 PLANT EXUDATE = ACETONE. Source: B. Mavituna.T T R A Y DRIER EVAPORATOR HEAT EXCHANGER wuu rh D R Y CRUDE PRODUCTS VACUUM FREEZE DRIER SALT W FR A C T l O N A T E D PRODUCTS S T AND ARDlZE D DIL UT E Lla ID C 0 N C E N T R A T E D PR O D U C TS LIQUID P R O D U C T S D R Y P R O D U C T S Fig. G-3. Nature Press. Atkinson and F. Biochemical Engineering & Biotechnology Handbook. I 1 8 CENTRIFUGE ~ R INGR E DIE N T S I-+.

AP N I H P E DX Single C e l l Protein Production .

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Minnesota. Figure H . The entire plant operates under aseptic condi- tions with facilities for steam sterilization and clean-in-place (CIP).000-gal continuous pressurized state-of-theart fermenters.0 million lbs/yr of SCP for human consumption. C a n d i d a u t i l i s ) and utilizes a pure culture continuous fermentation under sterile conditions. diagram of this manufacturing facility. many major oil companies began researching the possibili t y of using microorganisms to upgrade refinery streams with the aim of reducing the cost of refining. facilities for air compression and sterilization. produces 15. SINGLE-CELL PROTEIN PRODUCTION Single-cell proteins are various groups of microorganism that have been considered for food or feed use. The main components of the plant consist of facilities for raw materials storage and preparation. The separation system uses centrifuges to separate the microorganisms from the fermentation broth and a large spray dryer for drying the final product. nutrient sterilization. fermentation with two 20. and a separation and product recovery system.141 Appendix H . thus saving a great deal of energy. including algae. bacteria. The heat transfer facilities consist o f the fermenter cooling baffles. The resulting protein-rich biomass (yeast and bacterial cells) might serve as a source of animal feed or even as a potential feed source to supplement agriculturally produced human foods. plate and frame heat exchangers for the cooling system.l presents a block In addition a 540-unit Romicon ultrafiltration unit is in . and higher fungi. In the early 1 9 6 0 s . a chilled water system for fermenter and product cooling. and tri-pipe exchangers for the pasteurization system. Certain groups of microorganisms are capable of selectively consuming specific components from these streams which could then be returned to the refinery for further processing. The Pure Culture Products SCP plant in Hutchinson. The chilled water system utilizes 10°C-year-round well water for a major portion of the cooling duty. The dried cells of these organisms are collectively referred to as single-cell proteins (SCP). molds. yeasts. The plant has FDA approvals for the product (torula yeast.

SCP production .142 ORNL D W G 8 8 . MN.+ -I . facility.* U N I- 2 cT w e AGITATED AERATED FERMENTERS 3O0C. Block diagram of the Hutchinson. H-1. () I 4 RECYCLE PASTEURIZ A TI0 N SPRAY DRYER STERILIZATION WASTE L ULTRAFILTRATION UNIT DRIED PRODUCT Fig.7 4 5 INOCULUM SYSTEM 1 10°C WELL WATER CHILLED WATER COMPRESSED . 2 atm - PRODUCT COOLING STORAGE c SEPARATION SYSTEM (CENTRIFUGES) I-.

. The Hutchinson plant was designed to also serve as a research facility and thus has a complete fully equipped laboratory with state-of-the-artanalytical capabilities that would be required for antibiotic production.143 place for the recovery of macromolecules (like antibiotics) from the autolyzed fermentation broth. The plant is adjacent to a small river The surrounding farming area which would facilitate waste removal. The process is highly exothermic and operates at 30 " C with 1 0 ° C chilled water circulation for temperature control. would be capable of providing nost of the raw materials required for antibiotic production. The fermenter is sterilized with 1 2 1 " C . 15-psig steam and has operated for up to 75 days under aseptic conditions while maintaining a pure culture. The facility can a l s o be operated in a batch mode as required for various antibiotics production processes. The fermentation system operates under highly aerobic conditions with hydraulic drives and turbine agitators to ensure high oxygen transfer rates.

.

APPENDIX I Inoculum Preservation .

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For a spore crop. growth times for cultures are Standard Lyophilization is the preferred method for long- Lyophilized cultures Frozen cultures Bacteria Actinomycetes Fungi Refrigerated cultures Bacteria Actinomycetes Fungi 4-10 days 4 . The objective of preservation is to maintain strains as long as possible without cell division. or on a solid substrate in 2. The total spore culture is suspended with a surface- . and lyophilization (freeze-drying) with the addition of protective agents such as skim milk or sucrose. The three most common techniques for preservation are low temperature (2 to 6 " C ) .4 8 hours 1-5 days 1-7 days 4 . rice. a second series of shake cultures is made in more flasks from the first flask.147 Appendix I. frozen (-18°C or- 8 0 ° C in freezers o r . Not only their survival but also their capability for product formation must by preserved. on agar. or barley. term storage. with sterile aeration and daily shaking.2 4 hours 1-3 days 1-5 days Following the first flask culture. INOCULUM PRESERVATION The preservation of production strains of antibiotic-producing organisms over a long period is a basic requirement for a practical fermentation.1 9 6 ° C under liquid nitrogen). the culture can be cultivated in specially devised liquid media. and they frequently degenerate during successive transfers. peat.2 o The preserved culture is revived by growth in a shake flask liquid culture or on solid medium for spore formation. High-yielding strains have often been damaged in primary metabolism during the strain selection process.to 10-liter glass vessels for 8 to 24 days on bran.

possible.3 0 ° C . teminduction or repression Culture trans- growth. the nutrient medium. the cells will suffer extensive damage. fermenter precultures must be made. after which the rate should be as fast as possible down to the storage temperature which should be below -55°C. inoculum age. Thawing should be as rapid as The cultures should be kept down to small volumes and in containers which allow for rapid heat transfer. glucose (10%) or sucrose (10%) to the suspending medium will aid cell survival. perature of During all steps. Simple apparatus are available for preservation of cultures by freeze drying.0-ml thin glass ampules are recommended). storage.148 active agent like Tween 80 and transferred to the fermenter. (2. BCG versus the temperature of the freeze drying. and Stokes Div. HETO. medium should be kept low. Labconco. from companies like Virtus . of Pennwalt. @ Then. . and phenomena must be controlled for optimum production. depending on the volume of the production fermenter. The freezing rate should be about l"C/min down to -2O"C. The electrolyte content of the suspending The addition of glycerol (10 to 20%). Temperature fluctuations should be avoided during If the storage temperature is permitted to rise and fall above -2O"C. Figure 1-1 shows the survival of a culture of of Mycobacterium tuberculosis var. The freeze drying should take place at a constant temperature below . The temperature vs time profile is important to minimize damage to the cells in both the freeze and thaw processes. fers to the larger fermenters should be made while the culture is in the log growth phase to minimize culture lag time at the next stage.

"The Preservation of Cultures. Source: P." Progress in Industrial Microbiologv. 1-1. . 4 191 (1962). Muggleton. W.149 70 I I I Y 60 - de 50 40 /* - 2 2 3 > 30 a 20 10 0 TEMPERATURE (OC) Fig. The effect of drying temperature on survival of freeze dried c e l l s .

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APPENDIX J Sterilization .

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including bacteriophage and large viruses (0. Figure J . especially activated carbon and alumina. The efficiency will depend on the bed depth. The bed depth should be at least 2.5 to 7. and asbestos/cellulose fibers. the filtration bed is taken off line and sterilized with dry heat. and degree of contamination of the air. filtration through The most fibrous media. Amor. medium.5 ft/s). and up to 2 to 3 m deep for some commercial fermentations). Only three methods have been applied to the sterilization of air in commercial fermentation processes. and granular media. The fiber diameter should be below 6 microns. Many kinds of fibrous material can be packed into the filtration vessels.5 cm (usually more. Filters can be regenerated every 2 to 5 hours by sterilizing with Itdry''heat or steam at 160 to 180°C f o r 2 hours.153 Appendix J. These are heat. Sterilization of air implies the complete removal or destruction of all viable particles ranging in size down to the smallest bacterial cell. slag wool. glass wool. commonly used method t o clean intake sir is filtration through beds of fibrous material such as glass wool and slag wool.7 to 100.0% after 2 to 5 hours of operation. This will produce filtration efficiencies in the range of 99. air velocity. the bed is sterilized only before the The filtration mechanism for either granular fermentation begins. but they usually are regenerated only at the start of each batch along with the fermenter. Particle sizes are typically in the 3 0 to 50-mesh range with filtration bed pressure drop up to 10 psi.5-micron). after every 2 to 5 hours o f operation. . can be used to sterilize air.g the more common are cotton wool. and process equipment. absolute filter media. STERILIZATION This appendix will discuss three areas of sterilization which are vital to the successful operation of an antibiotic plant-the sterilization of air. In some plants.1 shows the general process flow sheet for piping a fermenter with an air filter. and it should be designed to produce a superficial velocity in the bed of 15 cm/s (0. In other plants. Granular material.

-u r c e . " . .ORNL D W G 88-246 STEAM INLET AIR FiLTER i EXHAUST FERMENTER P i p i n g diagram for fermenter air filtration.J-1. "The S t e rilization of Ai r S oP r o z r e s s in Industrial M i c r o b j D l o L y . B . K f i x p . G . C h e r r y and S . 4 37 ( 1 9 6 3). i). Fig.

The medium and equipment are normally sterilized with heat by using steam. Continuous sterilization can be done by injection of Sterilization with steam injection steam or by using heat exchangers. gives the two most common methods for controlling a unit. and (5) settling or gravitational effects.3 fermentation brcth medium is normally sterilized in the fermenter before introduction of the inoculum. (Figure 5 . minutes is sufficient. giving a probability of finding one viable spore in 1000 sterilizations if each started with lo5 spores.3 ) is done by injecting steam into the nutrient solution . ( 3 ) direct interception of a particle by the bed material.155 or fibrous bed is a combination of several mechanisms operating simultaneously: (1) diffusion effects due to Brownian motion. Sterilization of the main fermenter is sterilization accomplished with 145°C steam with a total turn around time of 10 hours. a 3-second exposure is sufficient. The alternative cycle of 115°C for 30 minutes is only marginally adequate due to an F. there is an approximate exponential relation by which the time taken to kill microorganisms decreases as the temperature to which they are exposed increases. Sterilization with steam is a time vs temperature Usually 121°C for 15 optimization. it is often not applied for economic reasons. The Figure J . Factor of 7. Sterilization of air by heating is technically feasible and is probably the most certain method. (2) electrostatic attraction between particles and the bed material. however. At temperatures as low as 170 to 200"C. ( 4 ) interception as a result of inertial forces acting on the particle and causing it to collide with the bed material.5 (unit of lethality in the food industry equivalent in minutes at 121°C of ail heat considered with respect to its capacity to destroy spores o r vegatative cells of a particular organism) giving the probability that 50% of the 1000 batches above would have a viable spore. Continuous sterilization of culture medium is practiced for adding nutrients to ongoing fermentations and for heat sensitive nutrients. as shown in Figure 5 .2 which presents several timesurvival curves for steam sterilization. At 300°C.

. 5 .8 1. D . Russell. Hugo and A . -. Survival curves for sterilization of spores.2 0. P h a r m a c e u t i c a l M i c r o b i o l o g v . B .CHARACTERISTIC OF M A N Y BACTERIAL SPORES C = STEEP INITIAL CURVE F O L L O W E D B Y S H A L L O W E R CURVE A T LONGER TIMES ACTIVATION = O C C U R S WHEN SPORES ARE GERMINATED A T SHORTER TIMES (MOIST H E A T O N L Y ) Fig. B l a c k w e l l S c i e n t i f i c .CHARACTERISTIC OF M O S T ORGANISMS B = CURVE WITH SHOULDER F O L L O W E D B Y LINEAR REGION .0 HEATING TIME A T 115OC (min) RADIATION D O S E (Mrad) A = LINEAR C U R V E . S o u r c e : W .ORNL D W G 8 8 .4 0.2 .6 0. London ( 1 9 7 7 ) .2 9 4 SO' ----rT-R A DiA T ION 100 2 0 + 0 10-1 c c3 U LL 10-2 z I 5 v) > 10-3 10-4 10-5 0 10 20 30 40 50 0 0.

3 . CONTINUOUS STERILIZER CONTROL SYSTEM Fig. R. Baker." P r o g r e s s in Industrial Microbiolonv 5 231 ( 1 9 6 4 ) . 5 . "Modern Trends in Steam Sterilization. Continuous steam sterilization of medium feed. C . Wilkinson and L. Source: G.157 ORNL D W G 88-250 STEAM CONTROL VALVE TEMPERATURE TRANSMITTER/ CONTROLLER STEAM NOZZLE MEDIUM - -----1 I I I HOT MEDIUM CLOSED LOOP CONTINUOUS STERILIZER CONTROL SYSTEM FLOW CONTROLLER 7 I MEDIUM TRANSMITTER TEMPERATURE TRANSMITTER/ CONTROLLER STEAM NOZZLE - HOT MEDIUM FEEDFORWARD-FEEDBACK. .

4 . and the whole process takes only 3 to 4 minutes.158 where the temperature is quickly raised to 1 4 0 ° C and maintained for 4 to 120 seconds. The added moisture is then removed by vacuum. the process is sensitive to changes in viscosity of the medium and to pressure variations. however. The procedure for using heat Over 90% of the energy input is Some recovered. cleaning agents and the heat exchangers are resterilized. deposits do form on the heat transfer surfaces and are removed by . exchangers is shown in Figure 5 .

"Modern Trends in Steam Source: Sterilization." ProEress in Industrial Microbiology 5 231 (1964). R.159 ORNL D W G 88-251 CLEANING CYCLE c *STERILE MEDIUM TO FERMENTER t - I MEDIUM RESERVOIR n -i - HEA TER x STEAM Fig. Continuous sterilization using heat exchangers. . Wilkinson and L. C. Baker. 5-4. G.

.

APPENDIX K Equipment .

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While fermenters are constructed from many different materials. oil. however. agitator seals. aseptic metering pumps. a jacket or coil for heating and cooling. Specifically included are the main fermenter and auxiliary piping. electromagnetic (mechanically linked to open and close) gear. K .2 illustrates a typical piping arrangement for sampling and inoculation of a fermenter employed in the production of antibiotics. there is an advantage to locating the stuffing box at least partially within the fermenter to gain better heat transfer. The stuffing box is usually packed with graphite-asbestos and lubricated with silicone. Three areas of instrumentation which are crucial to the operation o f an antibiotic plant are dissolved oxygen concentration. the flow is not continuous and the metering accuracy is not great. Oxygen transfer is very important in antibiotic fermentations. Basic instrumentation consists of temperature. Peristaltic pumps will provide aseptic operating conditions. for aseptic operation. It is essential. or antifoam oil. This is usually accomplished with a mechanical seal (Figs. or rotary sleeve pumps are recommended. and specialized instrumentation. pH. mineral Since it is difficult to sterilize the packing. and an air filter (see appendix J). and this is normally accomplished with mechanical agitation. to seal the agitator shaft as it enters the fermenter. Aseptic metering pumps present some problems for long-term operation. Figure K . an air sparger. K-4 and K . and dissolved oxygen measurement and control devices.1 ) usually consists of an upright cylindrical tank fitted with four baffles. A conventional fermenter (Fig. Figure K . and . pH.5 ) .163 Appendix K. Either valved pumps with the valves operated by cams and springs. 316 stainless steel clad vessels are almost always used in the antibiotics industry.3 illustrates several geometric configurations for a typical fermenter. EQUIPMENT Antibiotic production equipment This section addresses the specialized equipment required for an antibiotics plant. a device for mechanical agitation.

5 H/D=0.O-2.O-2. Standard fermenter d i a g r a . .33) (UNGASSED) Z / T = l . 0 8 .25 Wi/D= 0. W/T= 0 . 1 2 B/D =1 .50 (.2 4 7 ANTlF RIENTS COOLING COIL OR JACKET D / T = 0.0 .2 Fig.0 K-1.164 ORNL D W G 8 8 .0 L/D=0.5-1.30-0.

SAMPLE/DRAIN - HARVEST LINE STERILE FERMENTER INOCULATION FROM SEED TANK K-2. ” I STEAM + I - INOCULUM LINE I 4 I STERILE AIR PRESSURE GAUGE +CU-D VENT SAMPLE PORT/DRAIN 0 Fig.165 ORNL DWG 88-249 STEAM STERILE AIR FERMENTER I1I 6 I SEED VESSEL STERILE SA h. . Typical piping arrangement for sampling and inoculation.PLI N i SAMPLE i FERMENTER / M I .

5D I D t Q. .5D f Z IS UNGASSED D I T = 0. Geometric configuration for agitator placement in a typical fermenter.5D t f 1 3D It”” 2.33 Fig.5D 0. K-3.166 ORNL D W G 8 8 .2 4 0 fI T Z I T =2 =1 f I 2.

New York (1973). Humphrey. A .4 . Inc. F. Agitator shaft seal for a fermenter. E.. Source: S . A i b a .167 ORNL D W G 88-297 / AGITATOR SHAFT F i g . . Biochemical EnyineerinR. and N. K . Academic Press. Millis.

Humphrey. A .. Aiba. Biochemical Source: S . N w York ( 1 9 7 3 ) . E . K-5. Mechanical shaft s e a l for fermenter agitator. Academic P r e s s . M i l l i s .168 ORNL D W G 88-295 BEARING HOUSING CONDENSATE Yi ''''' ' ' ' ' . ' . and N . F .. . EnJzineering. I n c .- '" FERMENTER HEAD 3 J A G I TATOR SHAFT Fig. e .

as indicated by the following equation: where : N . Table K . oxygen partial pressure in the influent gas or the fermenter head pressure. . The importance of pH control has been well The microorganisms can established in antibiotic fermentations. a = Rate of 0 transfer by the fermenter.) The pH-sensing electrodes that are inserted directly into a recirculating l o o p outside of the fermenter are used to measure the pH of the medium. .temperature. In antibiotic processes. aeration rate. Overall 0 transfer coefficient. Total interfacial area of gas bubbles in the fermenter.1 gives a list of the manufactures. (NaOH) with metering pumps. . P. K. Dissolved 0 concentration in the medium.SO. it is desired to control the dissolved oxygen concentration at a set level. = = = = P . The control of pH is and/or bases achieved by the automatic addition of acids (H. secrete metabolites which will make drastic changes in the pH of the medium leading to nonproductive conditions. . Partial pressure of 0 in the influent gas. There are many excellent oxygen probes on the market. Control of the dissolved oxygen concentration at a given level can be achieved by altering the agitation rate. Temperature measurement and control are important and can be accomplished with conventional equipment plus the addition of chilling equipment for those locations which do not have cold (lO°C) ground water for cooling. .

5663-28 -. B Polarographic Yes Honeywell Technology Inc Polarographic No Polarographic No 0.100% Beckman 777 Polarographic Yes 0 . Electronic I n s t r Ltd.100% Lee Scientific 100 55145-01. Inc Polarographic Yes 0.100% 0100% 5-5663-00 Tngold Electrodes. A15A Galvanic No 0-14% U n i o n Carbide 1101 Polarographic No 0-l5ppm E.5 0 mg. Sargent & Go.-02 P016OL.I70 . J . S-38640-10B Galvanic No 0 . Delta Scientific 75 Galvanic ? 0. Table K-L. Suppliers of oxygen probes used in fermenters ComD any Ccir-Psrmer Model No.116mm.- Steam Sterilizsble Yes Yes Oxygen Ra-n=- Galvanic Polarographic 0. Hg PO. 0 /liter . H.100% .

APPENDIX L Separation and Purification .

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preparative chromatography and supercritical fluid extraction (SCF). although some penicillins and cephalosporins are separated commercially using chromatography. L-4. SEPARATION AND PURIFICATION This section will discuss two relatively new and potentially very powerful separation/purification methods for antibiotics. Tables L-1 and L . and it is difficult to scale up due to the flow dynamics in the narrow bore columns which are required. These problems were solved with the introduction of the annular chromatograph depicted in Fig.1 illustrates the many different types of chromatography available. it causes dilution of the product. thus making the process continuous and of much greater utility from a processing . L .51 This device uses the same exact resins which are used in HPLC columns except that now the material is packed into the narrow rotating annulus. Figure L . Most of this work is still in the developmental stage. These tables give the necessary infor- mation on the operation of the HPLC for many different antibiotics. The rotation is timed to be in phase with the feed rate s o that each component inthe feed exits the adsorption bed at a specific point. High Performance Liquid ChromatoEraDhv Figure L . the reduction will have to come in the separation and purification section of the plant which represents a large portion of a typical plant.3 for the purification of cephalosporin C (the most difficult separation for many antibiotics is the removal of derivatives and other antibiotics). If the size of the antibiotic manufacturing process is to be substantially reduced. A sample chromatogram is given in Fig.2 presents a flow sheet for a commercial process for separating p-lactam antibiotics (i.e. The disadvantages o f HPLC are that it is a batch process. Chromatography and SCF extraction are relatively new separation techniques being applied to the antibiotic process.2 summarize much of the work done on separation and purification of antibiotics using HPLC. penicillins and cephalosporins) using high performance liquid chromatography (HPLC)..173 Appendix L.

showing types of . GAS-SOLID COLUMN A OPEN BED LIQUID-SOLID BONDED-PHASE EXCLUSION GEL.FILT R A T ION Fig. Schematic diagram chromatography that are in use.QRNL D W G 88-255 CHROMATOGRAPHY GAS -LIQUID A \$ . L-1.PE R ME ATlO N A the many GEL.

l?. Sienko. "Process Source: Scale-up of a /?-Lactam Antibiotic Purification by High-Performance Liquid Chromatography. Cantwell. A. L-2. Calderone. Process chromatograph flow diagram. R . 316 133 (1984). aid ?f. ." Journal of Chromatographv.175 ORNL DWO 81-474 WASH SOLVENT CRUDE CEFONlClD DIAFLTRATON MOBLE PHASE J DETECTOR SATURATOR ONEXCHANGE CARBONFLTER ULTRAFLTER WASTE COLLECTED FRACTION Fig.

176 ORNL D W G 8 8 .5) P.5 pg 5-METHYCEPHALOSPORIN C. 4. 0. L-3. RANGE: 2 5 4 nm.08 AUFS CHART SPEED: 5 mm/min FLOW RATE: 1.2 9 8 U. . 1.5 Fg DEACETYLCEPHALOSPORIN C.V. cephalosporin C . 16 min DEACETOXYCEPHALOSPORIN C.5 p g CEPHALOSPORIN C.5186.5 ml/min COLUMN SIZE: 2 x 2 5 0 mm PACKING: p NH2 (VARIAN AEROGRAPH) S 0 LV E N T: HO Ac-C H 3 0 H-CH 3C N-H 2 0 (2 1417. 2.I.S.: 3400 2 INJECT 1. 2. 1.0 p g Sample chromatogram for the separation of Fig. 1. 3.

silane 0.5:l) Poor column efficiency Poor reproducibility Poor partition efficiency Octadecyl 0 . 1. . . 0.O.B. O m EDTA pH 5.4u NaC10. 0. I j HC10.9u l NaC10. u HC10.0025N citric acidBonded on acetonitrile (85:15 v v /) Partisil (18~) .2 O Adsorption Chromatography Aqueous 0.08Ij HC10.Olu EDTA 1% isopropyl alcohol (PH 9. . .8) Poor column efficiency Very poor efficiency Broad overlapping peaks Good separation 0 . m citric acid.4u K. Chromatographic parameters for tetracyclines Chromatographic system and column Ion Exchange Support Mobile phase Comments Zipax SCX 0.005Ij citric acidacetonitrile ( 2 : l ) Liquid-liquid Partition Chromatography ReversedPhase Partition Chromatography Corasil Coated with Polyethylene glycol 400 Dioxane-pentane (1. 0.O. 0.177 Table L-1.

C .0 Reverse phase C-18 Cefuroxime Dimethylformamide 95-100 Acetic acidmethanol-water Partition/ W absorption 1.5 Reverse phase Cephr adine None 92-100 Ammonium carbonate c-18 Ampicillin/ Arnoxicillin Penicillins Perchloric acid --_-_ MethanolPhosphate buffer C-8 Partition/ W absorption 0.0 ammonium acetate Reverse phase MethanolPhenylsilane Partition1 W absorption 10. 0 5 t j phosphoric acid Erythro.178 Table L-2. _ . "Stationary Mode ot Mobil phase MethanolSodium acetate Phase c-18 Separation Sensit: Detection (mIiJt * 'i - CxtractI on methanolsodium acetate Trichloroacetic (X) Cef amandole --- Partition/ W absorption 0.0 Reverse phase Partition/ W absorption 0.8 is Octyl silane bonded phase . HPLC assays of antibiotics Recovery Llrli.nycin Diethyl ether 96+ Acetronitrilewater C-18 Partition/ U Fluorometry 0 .1 8 45% 0 .5 Reverse phase . 0 5 t j phosphate C-18 Acetonitrile Tetracyclines 55% acetonitrile1 C .I .5 Reverse phase Partition/ UV absorption Reverse phase Partition/ UV absorption Reverse phase 0 .0 Reverse phase Cephalexin Methanol =loo% Methanol-water C-18 Partition/ UV absorption 0. 1 Reverse phase Ammonium acetate- is Octadecyl silane bonded phase.0 Reverse phase c-18 ___ ammonium acetate 97f Cephalothin Methanolacetic acid Partition/ W absorption 1. \ Cephaloridine Trichloroacetic acid Cephalothin Trichloroacetic acid Dimethylformamide 98 MethanolPhenylsilane Partition/ W absorption 2.3 Reverse phase Cefazolin 85+ MethanolAcetic acid Pnenylsilane Partition/ W absorption 1.

.4 . L .179 ORNL D W G 74-1263814 GAS 1 OVERPRESSURE ELUENT INLET STATIONARY INLET DISTRIBUTOR INLET PRESSURE SEAL FEED INLET CONTINUO FEED STREAM ROTATING ANNULAR CHROMATOGRAPH ELUENT STREAMS \ SEPARATED CONSTITUENTS STATIONARY ELUENT WASTE COLLECTION ELUATE EXIT i PRODUCT COLLECTION ANNULAR BED Fig. Schematic diagram of continuous rotating annular chromatograph.

These same data can be used with the aid of mathematical models developed at ORNL for evaluating the performance of other annular chromatographs with these same resins. is low. these solubilities can increase by one or two orders of magnitude.41 Carbon dioxide is the solvent of choice because of its availability. however. Several large-scale pilot units of the annular chromatograph are available at ORNL. The separation and purification of antibiotics with SCF extraction has been patented in Germany. nontoxicity. The use of an annulus for the resin instead of a packed bed eliminates the scale-up problems with Sed compression as the column diameter is increased.standpoint. it would be possible during a national emergency to skid-mount an annular chromatography system on a truck and simply drive it to any emergency production facility. Recently SCF extraction w a s applied commercially to the decaffination of coffee and has received widespread interest in the pharmaceutical industry because this separation technique can be performed at low temperatures (20 to 60°C). Because these units have a very high capacity. Figure L-5 presents a crude flow sheet for the process. Supercritical Fluid Extraction The solubility of solids in SCF was first discovered over 100 years ago. and proper thermodynamic properties. There is a wealth of experimental data in the literature on the selectivity of resins for antibiotic separation which has been evaluated in HPLC columns. The advantages are SCF extraction operates like distillation or liquid/liquid extraction but at lower temperatures and with milder less toxic solvents. obvious.52 Annular chromatography has the potential to effect a vast decrease in the size and complexity of the separation and purification of antibiotics on a scale comparable with the great advances made in the 1940s by the submerged culture process for the fermentation of antibiotics. The solubility of most antibiotics in SCF CO.35 Recently the Oak Ridge National Laboratory (ORNL) has demonstrated that annular chromatography can concentrate products in addition to separating them.53 . if 5 to 10% of a polar second solvent is added.

using CO. Schematic diagram for supercritical f l u i d extraction . .181 ORNL DWG 88-254 PURIFIED ANTIBIOTIC 4 EXPANSION WASTE BROTH 'POLAR ORGANIC MAKE-UP HIGH PRESSURE HIGH PRESSURE PUMP Fig. L-5.

The major advantages would be a reduction in the number of processing steps and the elimination of the large and expensive solvent recycle system. the transport properties of S C F ' s are closer to those of the gaseous state than the liquid state. the solubility of penicillin V in ethanol is over twice what it is in water (the major constituent of the fermentation broth). Recently. A solvent residue may be left on the product thus requiring further purification. penicillin V would be concentrated in the SCF phase.182 A unique property of this process is that the dissolved substance may be separated by decreasing the fluid density by either decreasing the pressure or increasing the temperature. giving rise to enhanced diffusivities and viscosities which will minimize any mass transfer problems in the extraction. The best solvents can often be easily identified from thermodynamic data which is rapidly becoming available in the open literature. Many different SCF solvents are possible as extraction agents. a German patent has been issued for the separation and purification of antibiotics using supercritical fluids. with ethanol distributing between the two phases.which is quite common in the antibiotic industry. An exact prediction of the final result can be made if the thermodynamic data are used to tune a mathematical model known as an equation of state. .3 . often requires a difficult crystallization. In addition. The process can be easily staged s o as to provide enhanced recovery with a small expenditure of energy. Conventional liquid-liquid-extraction. a distillation and/or use of a second solvent. the extract is separated by reducing the solvent density to the gaseous state. For example.35g42 The pressures required are in the same range as the high pressilre steam which is found at most industrial manufacturing facilities. In the supercritical region ethylene and water are immiscible. With SCF extraction. thereby leaving very little solvent on the product. The key point is that a small change in temperature or pressui2 will result in a large chan5e in solvent density and thus a large change in solubility. It is reasonable to assume that if a SCF mixture of ethylene and ethanol were used for fermentation broth extraction. from Table L .

4 182.97 K salt TETRACYCLINE >20.42 9.0 172.4 444.0 0.0 0.0 1.5 >20.0 124.65 1.0 >20.95 BACITRACIN 1421.85 8.0 K salt PENICILLIN G (Benzylpenicillin) Na salt 388 .0 10.035 0.0 OXYTETRACYCLINE 4 6 0 .0 263.3 >20.3 .1 aSolubility in g/1 at 2 9 4 .5 1.0 215.O 334. SCF related physical properties of selected antibiotics Antibiotic Molecular Weight Melting Point ("C) Water Solubility in Ethyl Acetate >20.183 Table L .0 >20.0 >20.02 0.7 CEPHALORIDINE 399.7 17.1 CEPHALOTHIN Na salt 382.4 160.0 >20.0 372 .0 9.3 0 1 "C .60 0.0 E thano1 20.40 0.4 404.O 215.0 0.0 9.047 17.0 PENICILLIN V (Phenoxymethyl penicillinic acid) 350.0 0. 0 CEPHALOSPORIN C 415.34 356.

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National Emergency Training Center Federal Emergency Management Agency ATTN: Learning Resource Center Washington. A. H. G . W. Federal Emergency Management Agency ATTN:Ralph B. 22. ORNL RC 27. C . DC 20472 46-85. R.O. R. 23. Defense Technical Information Center Cameron Station Alexandria. Watson Central Research Library ORNL-Y-12 Technical Library Document Reference Section 24-25. 11. Laboratory Records 26. Oak Ridge. EXTERNAL DISTRIBUTION 28. Los Alamos Scientific Laboratory ATTN : Documenty Library Los Alamos. 18. Office of Assistant Manager for Energy Research and Development. 17. 16. 20. V. L. D. C.185 ORNL-6355 Dist. C. Office of Scientific and Technical Information. Strandberg V. 13-15. D. Swisher Washington. Box E. M. P. TN 37831 31-42. ORNL Patent Section 1-5. Vaughen J. 6-10. D. T. S . Bienkowski Byers Chester Donaldson Lee Reichle Scott Stewart 19. Oak Ridge. DC 20472 45. Federal Emergency Management Agency ATTN: Librarian Washington. TN 37831 29-30. D. NM 87544 . DOE. 21. 12. VA 22314 43-44. C . DC 20472 86. ORO/DOE P. Category UC-41 INTERNAL DISTRIBUTION G . Laboratory Recores.

186 87. J . CA 90401 88. 1 1000 Brussels BELGIUM 90.W. Canadian Defence Research Staff ATTN: Mr. Ministry of Social Services 11 Spartis Street Athens GREECE . PA 15260 89. Direction de la Securite Civile Ministere de 1’Interieur 18 Rue Ernest Cognac 92 Levallois Paris.. Washington. Director Civilforsvarsstyrelsen Stockholmsgade 27 2100 Copenhagen 0 DENMARK 92. Secretaire d‘hdministration Ministere de 1’Interieur Direction Generale de la Protection Civile rue de Louvain. The RAND Corporation ATTN : Document Library 1700 Main Street Santa Monica. Janet Schlarb University Center for Social and Urban Research Risk Analysis & Emergency Management Program 16th Floor Cathedral of Learning University of Pittsburgh Pittsburgh. Ray Brown 2450 Massachusetts Ave. DC 20008 91. FRANCE 93. Ms. Bundesministerium des Innern Graurheindorfer Strasse 198 5300 Bonn 1 FEDERAL REPUBLIC OF GERMANY 94. N.

Stato Maggiore Difesa Civile Centro Studi Difesa Civile Rome ITALY 97. 8 Madrid-8 ESPANA Civil Defense Administation Ministry of Interior Ankara TURKEY 101. Almannavarnir Rikisins Reykjavic ICELAND 96. Esch Grande-Duche de LUXEMBOURG 98. B. Seccion de Estudios y Planificacion c/Evaristo San Miguel. Directeur Organisatrie Bescherming Bevoling Ministry of Interior Schedeldoekshaven 200 Postbus 20011 2500 The Hague THE NETHERLANDS 99. 57 1200 Lisbon PORTUGAL Jefe. The Head of Sivilforsvaret Sandakerveien 12 Postboks 8136 Oslo dep Oslo 1 NORWAY 100. 102. Servicio National de Proteccao Civil Rua Bela Vista a Lapa. Home Office Scientific Advisory Branch Horseferry House Dean Ryle Street London SWlP 2AW ENGLAND .187 95. Directeur de la Protection Civile Ministere de 1’Interieur 36 Rue J . 103.

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S. Walmer E. Washington. DC 20310-2200 William Gillen Emergency Preparedness Manager DHHS 3B 10 Hubert Humphrey Bldg. CA 94603 Federal Emergency Coordinators 116 Craig Alderman. GA 30333 Bruce Pelton Emergency Coordinator U. Chuck Wilton Scientific Services. .Rm 4-81 Rockville. Public Health Service 5600 Fishers Lane .F 3 8 Atlanta.S. N.M S . DC 20230 Thomas P. Joanne Shore IEAL 2600 Virginia Ave.E. Department of Commerce Office o f the Secretary Room 5044 HCHB Washington. MD 20857 117 118 119 120 . Washington. DC 20037 114. DC 20201 Kent Gray CDC ERCG 1600 Clifton Road. Suite 101 Bailey's Crossroads. N. 200 Independence Avenue. Reutershan Emergency Coordinator U. Strope Center for Planning and Research 5600 Columbia Pike.W. Inc 517 East Bayshore Redwood City. S.189 113.W. VA 22041 115. Director. M s . Jr.. Emergency Planning Office of the Secretary o f Defense Department of Defense Washington.

O. Given distribution as shown in Category UC-41. Box 27322 S-102 54 STOCKHOLM. CANADA 125 126 127-284. 2 Italia Tel. Mrs. (0481) 83632 Dr. West Ottawa KlAOW6 Ontario. Ontario K1A OL3 CANADA 123. Thomas Ritchey National Defence Research Institute Defence Analysis Department P. Lars Clausen Soziologisches Seminar d e r Christian-Albrechts-Universitat Olshausenstrasse 40/60 D-2300 Kiel FEDERAL REPUBLIC OF GERMAXY 122. West Ottawa KlAOW6 Ontario. . CANADA 124 Carlo Pelanda Istituto di Sociologia Internazionale I34170 Gorizia . Sweden Lloyd Swick Principal Consultant for Pub 1ic Protec t ion Emergency Planning Canada Gillin Building 141 Laurier Avenue. F. Dr. Lorraine Davies Director.Via Malta. Emergency Services Medical Services Branch Health and Welfare Canada 11th F l o o r Jeanne Mance Building Tunrrey ' s Pasture Ottawa. Prof.190 OVERSEAS 121. Ross Lunn Office of International Affairs Emergency Planning Canada Gillin Building 141 Laurier Avenue.

Antibiotics plants i n the United States. Five antimicrobial drugs. cephalosporin. separation and purification. ce halosporin. 1457-1457-AI Interagency Agreement: FEMA No. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. EMW-84-E-1737. DOE No. and fermentatton ethanol plants were identified. EMU-84-E-1737. Bacitracin. R Bienkowski. Antibfottcs plants i n the United States. and a bibliography of the pertinent material was compiled.m a n u f a c t u r i n g plants contain a generic fermentation unit with specialized sterile equipment and an antibiotic-specific separation and purification unit. a m n a m i d e f were identified for :mergencx production. Alternate sites require a team of skilled personnel to convert to antibiotic production Ln the austere environment. and clinical uses of antibiotics was reviewed. A n t i b i o t i c . and sulfonamide-. C. Bienkowski. Eyers. and a bibliography of the pertinent material was compiled. EXPEDIENT ANTIRIOTICS PRODUCTION -- 1988 Unclassifted April 1988 200 pages 1457-1457-A1 The literature o n the Fanufacture. C. enzyme. Bacitracin. R. The literature o n the manufacture. H. Bacitracin. were identified for emergency production. A n t t b i o t i c . DOE No. Alternate sites require a team of skilled personnel to convert t o antibiotic production i n the austere environment. penicillin V and G . Alternate sites require a team of skilled personnel to convert to antibiotic production in the austere environment. TN 37831 5 Interagency Agreement: FEMA No. and Puerto Rico and potential alternate sites such as single cell protein. A n t i b i o t i c . separation and purification. Bienkowski. Alternate sites require a team of skilled personnel to convert t o antibiotic production in the austere environment. Mexico. and clinical uses of antibiotics was revtewed. EMW-84-E-1737. Oak Ridge National Laboratory. DOE No. DOE No. Oak Ridge. tetracycline. TN 37831 Interagency Agreement: FEMA No. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. D Lee . Five antimicrobial drugs. TN 37831 Interagency Agreement: FEMA No. and Puerto Rico and potential alternate sites such as single cell protein. and Puerto Rico and potential alternate sites such as single cell protein. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. cephalosporin. tetracycline. penicillin V and G . enzyme. Antibiotics plants in the United States. Antibiotics plants I n the United States. Feasible emergency production will require a substantial reduction in the complexity and degree of separation and purity normally required. and fermentation ethanol plants were identiEied. EXPEDIENT ANTIRIOTICS PRODUCTION -. R. Bacitracin. 1457-1457-A1 The literature on the manufacture. by P. Lee . and clinical uses of antibiotics was reviewed. D. separation and purification. A n t i b i o t i c . Lee . a n d a bibliography of the pertinent material was compiled. tetracycline. enzyme.14 7-A 1 The ltterature o n the manufacture. and D D. Feasible emergency production will require a substantial reduction in the complexity and degree of separation and purity normally required. and a bibliography of the pertinent material was compiled. C. Lee Oak Ridge National Laboratory. and Puerto Rico and potential alternate sites such as single cell protein. were LdentiEied f o r emergency production. Ryers. D. Feasible emergency production will require a substantial reduction i n the complexity and degree of separation and purity normally required. and D. were identified for emergency production. and sulfonamide. Oak Ridge. penicillin V and G . enicillin V and G .m a n u f a c t u r i n g plants contain a generic fermentation unit with specialized sterile equipnent and an antibiotic-specific separation and purification unit. Byers. Oak Ridge. separation and purification. Yexico.Unclassified EXPEDIENT ANTIRIOTICS PRODUCTION -. 11. 11. enzyme. and clinical uses of antibiotics was reviewed. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. Bienkowski. R. Mexico. EXPEDIENT ANTIRIOTICS PRODUCTION -- 1988 Unclassified April 1988 200 pages by P . Five antimicrobial d s . . 1457 . and fermentation ethanol plants were identified.m a n u f a c t u r i n g plants contain a generic fermentation unit with specialized sterile equipoent and an antibiotic-specific separation and purification unit. 11. Oak Ridge.m a n u f a c t u r i n g plants contain a generic fermentation unit with specialized sterile equipment and an antibiotic-specific separation and purification unit. Mexico.1988 April 1988 200 pages by P. Five antimicrobial drugs. and D. tetracycline. Oak Ridge National Laboratory. cephalosporin. EMW-84-E-1737. and sulfonamide. and D. TN 37831 Oak Ridge National Laboratory. Feasible emergency production will require a substantial reduction in the complexity and degree of separation and purity normally required. Eyers.1988 April 1988 200 pages by P. C. and fermentatton ethanol plants were identified.