§ 114.9 9 CFR Ch. I (1–1–12 Edition)

of the suspension is given to the li- on the Internet at (http:// censee. www.aphis.usda.gov/animallhealth/ vet biologics/vb forms.shtml). (Approved by the Office of Management and l l Budget under control number 0579–0013) (b) Special outline. An outline describ- ing the preparation of a component of [39 FR 16869, May 10, 1974, as amended at 48 a biological product or an operation FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, 1991; 75 FR 20773, Apr. 21, 2010] performed in the preparation of a bio- logical product may be required if such § 114.9 Outline of guide- special outline could be referred to in lines. Outlines of Production to eliminate Each Outline of Production shall be repetition. Each special outline shall prepared in accordance with the appli- be identified by number and shall not cable directions provided in this sec- be used until accepted and filed by Ani- tion. mal and Plant Health Inspection Serv- (a) General requirements. (1) All copies ice. of each Outline of Production or spe- (c) Outline of Production for anti- cial outline or revised pages of either serum, antitoxin, and normal serum shall be prepared on heavy paper shall be written according to the fol- (8.5″ x11″) of a type receptive to perma- lowing: nent stamp ink. OUTLINE GUIDE FOR PRODUCTION OF ANTI- (2) The name of the biological prod- SERUM AND ANTITOXIN AND NORMAL SERUM uct (or component), the establishment license number, and the date prepared License No. Name of Product Date shall appear on a front cover page and I. Serum animals. A. Species, conditions, each page of the Outline of Production age, and general health. B. Examination, preparation, care, quar- or special outline. The name of the li- antine, tests, and treatment of animals be- censee (or foreign manufacturer) shall fore injections are started. appear on the front cover page. C. Holding, handling, exercising, and moni- (3) The pages shall be numbered in toring the condition of animals after injec- the upper center. At least 11⁄2 inch mar- tions are started. gin shall be left at the top of the first II. Antigens. A. Composition and character page and a 2 inch margin at the bottom of the antigen. 1. Micro-organisms. of each page for the Animal and Plant 2. Source and date of accession of each Health Inspection stamp. micro-organism. (4) Amended pages shall be numbered 3. Strains. the same as those being superseded. 4. Proportions of each micro-organism and They shall bear the date prepared and strain. refer to the date on the pages being su- B. Identification methods used for each perseded. If one replacement page su- micro-organism and frequency with which these methods are applied. persedes more than one page, the new C. Virulence and purity of cultures or anti- page shall indicate same, but if several gen and the determination and maintenance replacement pages are added to super- thereof. Range of subcultures or passages to sede one page, the page number fol- be used in production. lowed by letters shall be used. D. Attenuation, if any, before use for pro- (5) The last page of both copies of ei- duction purposes. ther a new or a completely rewritten E. Character, size, and shape of containers Outline of Production and each page used for growing micro-organisms. F. Media used for stock, seed, and antigen revised separately shall be signed in cultures (composition and reaction of). May the lower left corner by the authorized refer to a special outline by number. representative of the licensee (or for- G. Preparation of the antigen or toxin and eign producer). Stamped or facsimile toxoid. Complete and full description of each signatures are not acceptable. step and its manner of accomplishment and (6) A summary of changes shall ap- number these steps in sequence. Include all pear on an attached page and refer to tests for each antigen, and the specifications each page, paragraph, or subparagraph for character, identity, virulence, concentra- tion, and standardization. being changed. III. Immunization of animals. A. Outline (7) Transmittal forms shall be used fully with special attention given to the fol- for the original and subsequent revi- lowing: sions. Transmittal forms are available 1. Character and dose of the antigen.

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2. Method and frequency of injections. I. Composition, etc., of the product. A. Micro- 3. Time required for immunization or organisms used. Give the isolation and pas- hyperimmunization. sage history. 4. Preliminary bleedings and tests, if any, B. Source and date of accession of each to ascertain of serum. micro-organism. 5. All other similar matters, including C. Strains. treatments between bleedings. D. Proportions of each strain. B. Period of time elapsing between last in- II. Cultures. A. Brief description of methods jection and first bleeding; and between bleed- of identifying each micro-organism and the ings. frequency with which these methods are ap- C. Technique of bleeding operations; vol- plied. ume of blood collected at each bleeding; and B. Virulence and purity of cultures and the period of rest. determination and maintenance thereof. IV. Preparation of the biological product. A. Range of subcultures or passages to be used Describe fully and show each step of prepara- in production. tion from the first bleeding to the comple- C. Composition and reaction of media used tion of the preserved product in bulk con- for seed and production cultures. Include the tainers prior to filling of final containers. source of eggs, tissue, or cell cultures, and B. Composition of the preservative and the tests to determine that eggs, tissues, and proportions used. Indicate at which step of cells are free of contamination. production, and the method used in adding D. Character, size, and shape of containers the preservative. used for growing cultures. C. Agglutination and complement-fixation E. Storage conditions of seed cultures. titers and the methods of their determina- F. Methods of preparing suspensions for tions. seeding or inoculation. D. Disposition of unsatisfactory biological G. Technique of inoculating (1) seed media; products and infective materials not used in (2) production media. Titer or concentration production. of inoculum, and the volume of medium for E. Assembly of units to make a serial; vol- each size and type of culture container. ume of the average serial; and the volume of H. Period of time and conditions for incu- the maximum serial. bation and degree of temperature used for V. Testing. Indicate the stages in the prepa- each micro-organism or group of micro-orga- ration of the biological product at which nisms. samples are collected. Refer to all applicable I. Character and amount of growth; obser- Standard Requirements. Outline all addi- vation as to contamination of growth. tional tests in detail and state minimum re- J. Method of attenuation, if any, before quirements for each satisfactory test. used for production purposes. A. Purity. III. Harvest. A. Handling and preparation of B. Safety. cultures and media (including eggs) before C. Potency. removal of micro-organisms or tissues for D. Other tests. production purposes. VI. Post preparatory steps. B. Minimum and maximum period of time A. Form and size of final containers in elapsing from time of inoculation until har- which the product is to be distributed. vest. B. Methods and techniques of filling final C. Technique of harvesting micro-orga- containers. Volume of fill for each size final nisms or tissues (specify) for production pur- container. poses. C. Collection, storage, and submission of D. Specifications for acceptable harvest representative samples. Indicate at which material. steps in the production these samples are E. Handling of discarded material not used taken. in production. D. Expiration date based on the earliest F. Include any additional pertinent infor- date of harvest and the date of the last satis- mation. potency test. IV. Preparation of the product. Describe E. Use, dosage, and route of administration fully and show each step of preparation from for each animal species for which it is rec- harvest of antigen containing tissues or pro- ommended. duction cultures to the completion of the F. Include any additional pertinent infor- finished product in final containers. In de- mation. scribing the preparation of the product, em- (d) Outline of Production for vaccines, phasize the following: A. Method of inactivation, attenuation, or bacterins, antigens, and toxoids shall be detoxification. written according to the following: B. Composition of preservative, adjuvant or stabilizer, and proportions used stated in OUTLINE GUIDE FOR VACCINES, BACTERINS, such a manner that the concentration can be ANTIGENS, AND TOXOIDS calculated; stage and method of addition. License No. Name of Product Date C. Method and degree of concentration.

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D. If product is standardized to give con- 7. Method and technique of filling and seal- centration of antigen, show procedures and ing of final containers. calculations. 8. Amount material per dose or doses in E. 1. Assembly of units to make a serial (il- final container. lustrate by example). III. Testing. Indicate the stages in the prep- 2. Volume of average serial. aration of the biological product at which 3. Volume of maximum serial. the samples are collected. Refer to all appli- 4. Any other pertinent information. cable Standard Requirements. Outline all ad- F. Volume of fill for each size vial. Type of ditional tests in detail and state the min- vial if unusual. imum requirement for each satisfactory test. G. Method and technique of filling and A. Purity. sealing of final containers. B. Safety. H. Desiccation, including moisture control. C. Potency. Give maximum percent moisture. D. Any other tests. I. Amount of antigenic material per dose E. Include any additional pertinent infor- or doses in final container. mation. V. Testing. Indicate the stages in the prepa- IV. Post preparatory steps. A. Form and size ration of the biological product at which the of final containers in which the product is to samples are collected. Refer to all applicable be distributed. Standard Requirements. Outline all addi- B. Collection, storage, and submission of tional tests in detail and state the minimum representative samples. Indicate at which requirement for each satisfactory test. steps in the production these samples are A. Purity. taken. B. Safety. C. Expiration date based on the earliest C. Potency. date of harvest and the date of the last satis- D. Moisture, if desiccated. factory potency test. E. Any other tests. D. Use, dosage, and route of administration VI. Post preparatory steps. A. Form and size of final containers in which the product is to for each animal species for which the bio- be distributed. logical product is recommended. B. Collection, storage, and submission of (f) Outlines of Production for diag- representative samples. Indicate at which nostic test kits based on antigen-anti- steps in the production these samples are body reactions, and other diagnostics taken. C. Expiration date based on the earliest whose production methods are ame- date of harvest and the date of the last satis- nable to description as described herein factory potency test. If applicable, give the shall be written according to the fol- date of lyophilization. lowing requirements: D. Use, dosage, and route of administration for each animal species for which the bio- OUTLINE GUIDE FOR DIAGNOSTIC TEST KITS logical product is recommended. License No. Name of product Date (e) Outlines of Production for aller- genic extracts shall be written accord- Introduction ing to the following: Provide a brief description of the kit as fol- lows: OUTLINE GUIDE FOR ALLERGENIC EXTRACTS 1. Principle of the test (ELISA, latex ag- License No. Name of Product Date glutination, etc.). 2. Antigen or antibody detection test. I. Composition of the product. A. Source and 3. Sample(s) used for testing (serum, whole type of raw material. blood, tears, etc.). B. Weight/volume concentration. II. Preparation of the product. A. Describe 4. List reagents, references, and equipment fully and show each step of preparation to included. the completion of the finished product in 5. Identify materials obtained under split true containers. In describing the prepara- agreements. tion of the product, emphasize the following: 6. General description of test interpreta- 1. Method of extraction. tions and their limitations, including fol- 2. Composition of preservative, adjuvant or lowup tests. stabilizer, and proportions used; stage and I. Antibody Components method of addition. 3. Method and degree of concentration. A. Production of polyclonal antibody com- 4. Standardization of the product. ponents. 5. (a) Assembly of units to make a serial. 1. If purchased, list suppliers, criteria for (b) Volume of average serial. acceptability, and describe all tests per- (c) Maximum serial. formed after receipt to determine that speci- 6. Volume of fill for each size vial. fications have been met.

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2. If produced in-house, describe the spe- d. Describe all characterization procedures cies, age, weight, conditions, and general and include the expected reactivity of all ref- health of all animals used in antiserum pro- erence monoclonal antibodies. duction. a. Preinjection considerations: II. Antigen Preparation Describe the examination, preparation, A. Identify the microorganism(s) or anti- care, quarantine procedures, and treatments gen being used. If previously approved Mas- administered before immunization(s). De- ter Seed virus, bacteria, or antigen derived scribe all tests used to determine suitability therefrom is used, provide pertinent infor- for use. Describe the preparation of any mation on the testing performed, and details standard negative serum(s) collected prior to immunization. of dates of United States Department of Ag- b. Immunization of animals. riculture confirmatory tests and approval, as i. Describe the character and dose of the appropriate. antigen; if adjuvant is used provide details B. Describe all propagation steps, includ- on its preparation. If commercial product is ing identification of cell cultures, media in- used include its true name as shown on the gredients, cell culture conditions, and har- label, the manufacturer, serial number, and vest methods. For antigen produced in eggs, expiration date. give the egg source, age, and route of inocu- ii. Describe the method and schedule for lation. If cell lines are being used, give dates immunizations. of testing and approval as specified in 9 CFR iii. Describe the method for harvesting and 113.52. evaluating the immunization product, in- C. Describe procedures used for extracting cluding tests for acceptability. and characterizing the antigen. iv. Provide number and intervals between D. Describe the method used to standardize harvests, volume obtained, and any other the antigen. pertinent information. E. If the antigen is purchased, identify the B. Production of Monoclonal Antibody supplier and describe the criteria for accept- Components. able material, including all tests performed 1. Hybridoma components: by the producer and/or the recipient to deter- a. If hybridoma components are purchased, mine acceptability. list suppliers and criteria for acceptability; if tests are performed after receipt, describe III. Preparation of Standard Reagents fully. b. If hybridomas are prepared inhouse, A. Describe the positive and negative con- identify the antigen(s) used, describe the im- trols included in the kit. If purchased, list munization scheme, and the species of ani- suppliers and criteria for acceptance. mal used. B. Describe the preparation and standard- c. Identify the tissue of origin, and the pro- ization of the conjugate(s). If purchased, list cedures for harvesting, isolating, and identi- suppliers and criteria for acceptance. fying the immune cells. C. Describe the preparation and standard- d. Describe the source, identity, and the ization of the substrate(s). If purchased, list product secreted (light or heavy chain) by suppliers and criteria for acceptance. the parent Myeloma Cell Line. D. Identify buffers, diluents, and other re- e. Summarize cloning and recloning proce- agents included in the kit. The preparation dures, including clone characterization and of these components may be described in this propagation, if appropriate. section or in filed Special Outlines. f. If appropriate, describe procedures for establishing and maintaining seed lots. IV. Preparation of the Product g. Describe any other pertinent tests or Fully describe methods used to standardize procedures performed on the hybridoma cell antigens, reference standards, positive con- line. trol serum, negative control serum, and 2. Antibody production: standard reagents from production/purchase a. Describe the production method. If pro- to completion of finished product in final duced in cell culture, animal serum additives containers, including the following: must conform to 9 CFR 113.53. If produced in animals, describe fully including husbandry 1. Composition and quantity of preserva- practices and passage procedures. tive in each. b. Provide the criteria for acceptable 2. Method of filling, plating, or attaching monoclonal antibody, including tests for pu- the antigen or antibody component to a solid rity. phase. c. Describe all tests or other methods used 3. Minimum and maximum acceptable fill to ensure uniformity between production volumes for each final container of reagent lots of monoclonal antibody. Include all re- included in the kit. action conditions, equipment used, and reac- 4. The disposition of unsatisfactory mate- tivity of the component. rial.

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V. Testing ological product, when prepared as rec- Refer to all applicable standard require- ommended for use, shall not exceed the ments. amounts listed in this paragraph: Pro- A. Purity. vided, That in the case a desiccated bio- Describe all tests of the kit for purity or logical product is to be used with an in- specify the exemption as provided in 9 CFR definite quantity of water or other 113.4. menstruum, the determination shall be B. Safety. In vitro products are exempt from safety based on 30 ml. per 1,000 dose vial or tests. equivalent. C. Potency. (2) Except as prescribed in paragraph Provide details of tests used to determine (c) of this section, only one antibiotic the relative reactivity of the kit including shall be used as a preservative in a bio- minimum requirements for a satisfactory logical product. The kind and max- test. Reference standards and control serum used for this purpose should be identified by imum amount per ml. of such anti- unique codes or lot numbers. biotic shall be restricted to: Amphotericin B ...... 2.5 mcg. VI. Postpreparatory Steps Nystatin (Mycostatin) ...... 30.0 units A. Describe the form and size of final con- Tetracyclines ...... 30.0 mcg. tainers of each reagent/component included Penicillin ...... 30.0 units in the kit. Streptomycin ...... 30.0 mcg. B. Describe the collection, storage, and Polymyxin B ...... 30.0 mcg. submission of representative samples. Refer Neomycin ...... 30.0 mcg. to 9 CFR 113.3(b)(7). Gentamicin ...... 30.0 mcg. C. Specify the expiration date. Refer to 9 (c) Permitted combinations: CFR 114.13. (1) Penicillin and streptomycin. D. Provide details of recommendations for use, including all limitations, qualifications, (2) Either amphotericin B or nys- and interpretation of results. tatin, but not both, may be used with E. Submit confidentiality statement iden- one of the other antibiotics listed in tifying specific parts of the outline con- paragraph (b) of this section, or with a taining information, the release of which combination of penicillin and strepto- would cause harm to the submitter. mycin, or with a combination of poly- (Approved by the Office of Management and myxin B and neomycin. Budget under control number 0579–0013) (3) The maximum amount of each an- [39 FR 16869, May 10, 1974, as amended at 48 tibiotic in a combination shall be the FR 57473, Dec. 30, 1983; 56 FR 20124, May 2, amount prescribed for such antibiotic 1991; 56 FR 66784, Dec. 26, 1991; 75 FR 20773, in paragraph (b) of this section. Apr. 21, 2010] (d) Antibiotics used in virus seed stock purification are not restricted as § 114.10 Antibiotics as preservatives. to kind or amounts provided carryover Antibiotics are authorized for use as into the final product is controlled and preservatives for biological products if specified in outlines of production. used within the limitations as to kinds and amounts prescribed in this section. [39 FR 16869, May 10, 1974, as amended at 56 (a) When an antibiotic or combina- FR 66784, Dec. 26, 1991] tion of antibiotics, with or without a fungistat is to be used in the prepara- § 114.11 Storage and handling. tion of a biological product, the kind(s) Biological products at licensed estab- and amount(s) of each shall be speci- lishments shall be protected at all fied in the outline for such product in times against improper storage and such a way that the concentration in handling. Completed product shall be the final product may be calculated. kept under refrigeration at 35 °to 45 °F. Except as may be approved by the Ad- (2 °to 7 °C.) unless the inherent nature ministrator, only those individual anti- of the product makes storage at a dif- biotics or combinations of antibiotics ferent temperature advisable, in which listed in paragraphs (b) and (c) of this case, the proper storage temperature section shall be used. shall be specified in the filed Outline of (b) Permitted individual antibiotics: Production. All biological products to (1) The antibiotic level of a specified be shipped or delivered shall be se- individual antibiotic in one ml. of a bi- curely packed.

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