STANDARD OPERATING PROCEDURE - United States … · This standard operating procedure will be used when inspecting ... Biology of Microorganisms, Third Edition ... (May 1988). Health

STANDARD OPERATING PROCEDURESOP NO.: GLP-C-04 Page No.: 1 of 73

Title: COMPLIANCE REVIEW OF BIOTECHNOLOGY FACILITIES

Revision: 1 Replaces: Original Effective: 01/04/99

1. PURPOSE

To provide a standard procedure for conducting a Good LaboratoryPractice (GLP) Standards compliance inspection at biotechnology laboratoriesconducting studies that have been submitted and accepted by the Agency insupport of applications for research or marketing permits for bioengineeredpesticide products regulated by EPA [Sections 3, 4, 5, 18, and 24(c) of theFederal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended] orpursuant to testing consent agreements, test rules, and pre-manufacturingrequirements [issued under Sections 4 and 5 of the Toxic Substances ControlAct (TSCA)].

2. SCOPE

This standard operating procedure will be used when inspectinglaboratories and field sites that have conducted testing of bioengineeredtest substances under FIFRA and TSCA. Throughout this document, the term"bioengineered test substance" or simply "test substance" refers to thebioengineered substance under study, which may have been either abioengineered organism (i.e., microorganism or transgenic plant) or asubstance produced by the bioengineered organism. In addition, portions ofthis SOP apply to auditing studies that have been conducted by biotechnologylaboratories. In these sections, the term "inspector" has been replaced with"auditor".

3. OUTLINE OF PROCEDURES

Organization and Personnel

Preparation of Bioengineered Test Substance

! Characterization of Donor and Recipient Microorganisms andTransgenic Plants

! Materials and Methods for Producing the Bioengineered TestSubstanceCharacterization, Production, and Handling ofBioengineered Test Substance

! Characterization of Bioengineered Test Substance! Production of Bioengineered Test Substance! Handling

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Studies of the Bioengineered Test Substance

! Study Protocol! Test System Care! Efficacy! Infectivity/Pathogenicity/Toxicity

Facilities and Equipment

! Facility Design! Equipment

4. REFERENCES

Auditing Efficacy Studies (SOP No. GLP-DA-06). U.S. EnvironmentalProtection Agency. June 15, 1991.

Auditing Field Studies (Analytical Chemistry) (SOP No. GLP-DA-01). U.S.Environmental Protection Agency. February 1, 1991.

Auditing Residue and Environmental Fate Studies (Field Portions) (SOPNo. GLP-DA-4) U.S. Environmental Protection Agency.

Bergev's Manual of Determinative Bacteriology, Eighth Edition (1975).R.E. Buchanan and N.E. Gibbons (eds). The Williams & Wilkins Company,Baltimore.

Biochemistry, Second Edition (1981). Stryer, Lubert. W.H. Freeman andCompany, New York.

Biology of Microorganisms, Third Edition (1979). Brock, Thomas D.Prentice-Hall, Inc., New Jersey.

Biosafety in Microbiological and Biomedical Laboratories, Second Edition(May 1988). Health and Human Services, Public Health Service, Centersfor Disease Control and National Institutes of Health. HHS PublicationNo. (NIH) 88-8395.

Biotech Inspection Outline (Draft). Food and Drug Administration, Officeof Regulatory Affairs, Office of Regional Operations, Division of FieldInvestigations (HFC-130). August 1988.

Biotechnology at Work. Glossary of Terms. Industrial BiotechnologyAssociation. August 1992.

Biotechnology at Work. What Is Biotechnology. Industrial BiotechnologyAssociation. 1989.


Biotechnology Facilities Standard Operating Procedures Support Document

Biotechnology Compensation and Benefits Survey. 1992. Radford Associatesand Alexander & Alexander Consulting Group.

Biotechnology: The Science and the Business (1991). Moses, V. and R.E.Cape. Harwood Academic Publications, New York.

Characterization of the Biotechnology Industry. Submitted by ICFIncorporated to the Environmental Protection Agency, Office of ToxicSubstances. March 13, 1987.

Conducting a Field Site GLP Compliance Inspection (SOP No. GLP-C-01).October l, 1990. U.S. Environmental Protection Agency

Current Protocols in Molecular Biology, Volume 1 (1993). Ausubel, F.M.,R. Brent, R.E. Kingston, D. D. Moore, J.G. Seidman, J.A. Smith, and K.Struhl (eds). Current Protocols.

Elements of Microbiology (1981). Pelczar, M.J., Jr. and E.C.S. Chan.McGraw-Hill Book Company, New York.

Federal Insecticide, Fungicide and Rodenticide Act (FIFRA): GoodLaboratory Practice Standards: Final Rule 40 CFR Part 160.Environmental Protection Agency. Federal Register, August 17, 1989, 54FR 34052 - 34074.

Good Developmental Practices for Small Scale Field Research withGenetically Modified Plants and Micro-Organisms (A Discussion Document).Organization for Economic Co-Operation and Development, Group ofNational Experts on Safety in Biotechnology. March 1990.

Good Laboratory Practices Inspection Manual. U.S. EnvironmentalProtection Agency, September 1993.

Guidelines for Research Involving Planned Introduction in theEnvironment of Genetically Modified Organisms. U.S. Department ofAgriculture, Office of Agricultural Biotechnology. Document No. 91-04.March 1992.

Guidelines for Research Involving Recombinant DNA Molecules: Notice.National Institutes of Health, Department of Health and Human Services.Federal Register, May 7, 1986, 51 FR 16958 -16985.

Opportunities in Biological Science Careers ( 1990). Winter, Charles A.VGM Career Horizons, Illinois.

Opportunities in Biotechnology Careers (1991). Brown, Sheldon. VGMCareer Horizons, Illinois. Pesticide Assessment Guidelines SubdivisionG: Product Performance. Environmental Protection Agency, Office ofPesticide Programs. November 1982.



Pesticide Testing Guidelines Subdivision M: Microbial and BiochemicalPest Control Agents. Environmental Protection Agency, Office ofPesticides And Toxic Substances (H-7501C). July 1989.

Physical Biochemistry. Applications to Biochemistry and MolecularBiology, Second Edition (1982). Freifelder, D.M. W.H. Freeman andCompany, New York.

Plant Biotechnology (1989). Kung, Shain-dow and Charles A. Arntzen(eds).Butterworth Publications, Massachusetts.

Plant Biotechnology: Comprehensive Biotechnology Second Supplement(1992). Fowler, M.W., G.S. Warren, and M. Moo-Young (eds). PergamonPress Inc., New York.

Plant Cell and Tissue Culture (1991). Stafford, A. and G. Warren. OpenUniversity Press, UK.

Plant Tissue Culture: Applications and Limitations (1990). Bhojwani,S.S. (ed). Elsevier, New York.

Points to Consider in the Preparation and Submission of TSCAPremanufacture Notices (PMNs) for Microorganisms. U.S. EnvironmentalProtection Agency, Office of Toxic Substance, Chemical Control Division,Program Development Branch. July 23, 1990.

Points to Consider in the Production and Testing of New Drugs andBiologicals Produced by Recombinant DNA Technology (Draft). Food andDrug Administration, Center for Drugs and Biologics, Office of BiologicsResearch and Review. April 10, 1985.

Principles of Genetics, Eighth Edition (1991). Gardner, E.J., M.J.Simmons, and D.P. Snustad. John Wiley & Sons, New York.

Short Protocols in Molecular Biology, Second Edition (1992). Ausubel,F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, andK. Struhl (eds). John Wiley & Sons, New York.

Supplement to the Points to Consider in the Production and Testing ofNew Drugs and Biologicals Produced by Recombinant DNA Technology:Nucleic Acid Characterization and Genetic Stability. Food and DrugAdministration, Center for Biologics Evaluation and Research. April 6,1992

Toxic Substances Control Act (TSCA): Good Laboratory Practice Standards;Final Rule [40 CFR Part 7923. Environmental Protection Agency. FederalRegister, August 17, 1989, 54 FR 34034 - 34050.



5.0 SPECIFIC PROCEDURES

5.1 ORGANIZATION AND PERSONNEL

Since the biotechnology field is very specialized, it isparticularly important that qualified individuals hold the key positionson the team performing the biotechnology study. Individuals contributingto a biotechnology study, particularly those in positions ofresponsibility such as the study director, need to have the appropriateeducation, training, and experience. The inspector will be concernedwith many of the same general personnel issues with biotechnologystudies as with other types of studies submitted under FIFRA and TSCA.For instance, regardless of the type of study, the inspector shouldevaluate whether a facility performing a study has a staff of sufficientsize and whether their qualifications are appropriate and documented.

For guidance on personnel and organizational issues to addressduring the inspection, the inspector should refer to the SOP for"Conducting a Field site GLP Compliance Inspection" (SOP No. GLP-C01,Section 5.1.2) and the Good Laboratory Practices Inspection Manual, U.S.Environmental Protection Agency, September 1993.

5.2 PREPARATION OF BIOENGINEERED TEST SUBSTANCE

For studies submitted under FIFRA and TSCA, the bioengineered testsubstance may be a bioengineered microorganism or a transgenic plant.Methods for characterization of the donor and recipientmicroorganism/plant and preparation of the bioengineered test substance,and the subsequent issues of concern to the inspector, will be dependenton whether the resulting test substance is a microorganism or a plant.Specific elements the inspector should consider regarding the materialsand methods used by the laboratory in the preparation of test substancesare addressed below.

5.2.1 CHARACTERIZATION OF DONOR AND RECIPIENT MICROORGANISMSAND TRANSGENIC PLANTS

Microorganisms

The donor and recipient. microorganisms must have a taxonomicidentification and a description of the genotypic and phenotypiccharacteristics. To evaluate the laboratory's characterization ofthe donor and recipient microorganisms, the inspector should checkthe storage conditions, records, and SOPs for handling the donorand recipient organisms. Questions for the characterization of thedonor and recipient microorganisms include the following:

! How does the laboratory assure that it has a pureculture? What IS the frequency of this test?



! How are the organisms stored? Are storage conditionsadequate to ensure the stability of the organism?

! Does the laboratory have a documented history of thebacterial strain?

! Does the laboratory have an SOP for doing the taxonomicidentification?

! Does the SOP address identification of genotypic andphenotypic characteristics?

NOTE: Genotypic characteristics include a determination of thegenetic material such as genetic maps of chromosomes and plasmids;genetic transfer capabilities such as transformation, transduction,or transfection; presence of transposable elements, insertionsequences, plasmids, phages, and phage cross-infectivity.

Phenotypic characteristics include culture requirementsand characteristics; nature and degree of pathogenicity, virulence,infectivity, or toxicity to humans, other animals, plants, ormicroorganisms; life cycle including sexual/asexual reproductioncycle and dormant stages; appearance; antibiotic resistance;resident antibiotic production; survival/persistence; known controlagent~; and biological control characteristics.

! For donor and recipient organisms from external sources(i.e., purchased or obtained from another laboratory),has the laboratory received a letter of identification(i.e., certificate) from the supplier?

- Does the facility test the purchased organism uponarrival to certify its identity and purity?

- What are the shipping conditions for the organisms?- Does the facility have records of receipt?

Plant Characterization

The selection of plant(s) to be used will depend on severalfactors including whether the plant can be reproductively isolatedand whether or not it is likely to persist in an environment thatis not cultivated (i.e., outside the test plot). The inspectorwill need to check the storage conditions, documentation and SOPsfor characterizing and preparing the plants and/or plant cells.

! How and where are the plants and plant cells stored?! Is there documentation of the biology of the reproductive

potential of the plant (e.g., flowers, pollinationrequirements, and seed characteristics) being used in thestudy?



! Does the laboratory have a documented history ofcontrollable reproduction with lack of dissemination?Can the plant be established in an environment that issimilar to the field test site?

5.2.2 MATERIALS-AND METHODS FOR PRODUCING THE BIOENGINEEREDTEST SUBSTANCE

The inspector should check the storage conditions (includinglocation) and records for materials used in producing thebioengineered test substance. The inspector should also checkdocumentation regarding methods used in producing the bioengineeredtest substance.

Materials

! If DNA is obtained from external sources (i.e., purchasedor obtained from another laboratory), what are theshipping conditions? Was the DNA tested upon arrival? Howis the DNA stored?

! If a restriction endonuclease (or any other enzyme) isobtained from external sources, how does the laboratoryassure that the enzyme is neither contaminated norinactive? How frequently is it tested? How is the enzymestored?

! For other chemicals or substances used as reagents,buffers, or growth media, how does the laboratory assurethe quality of the materials?

! Does the laboratory use distilled, deionized water toprepare all reagents and buffer solutions?

! Is the laboratory using standard methods for thepreparation of reagents, buffer solutions, and media?

Methods

! Does the laboratory have SOPs:

- For isolating and identifying DNA being insertedinto the recipient organism or plant cell?

- For constructing the vector with the inserted DNA?- For verifying the DNA insertion in the vector?



- For the method being used to introduce theDNA/vector into the recipient organism?

(1) Transformation, transduction, conjugationfor microorganisms

(2) Agrobacterium-based plant transformation,particle acceleration, electroporation,microinjection for plants

- For measuring the success of the insertion of thegenetic material?

- For determining the stability of the inserted DNAin the recipient microorganism or plant?

! How does the laboratory validate the methods that ituses?

! Who constructed the vector and performed the proceduresin preparing the bioengineered microorganism ortransgenic plant?

! If protoplasts (for transgenic plants) are being used,are standard tissue culture procedures used?

5.3 CHARACTERIZATION, PRODUCTION, AND HANDLING OF BIOENGINEERED TESTSUBSTANCE.

5.3.1 CHARACTERIZATION OF BIOENGINEERED TEST SUBSTANCE

The GLP regulations require that the test substance beanalyzed for identity, strength, purity, and composition, asappropriate for the type of study. This section addresses analysisof the bioengineered test substance for several characteristicsincluding identity, purity, and stability. The auditor willreview the identity, purity, and stability information and datathat have been included in the study. Section 5.5 will addressinfectivity, pathogenicity, toxicity and efficacy testing.

Identity

Methods for determining the identity of the bioengineered testsubstance may be genotypic and phenotypic or physicochemical. Thegenotypic and phenotypic characterization includes the followingitems:



! Genotypic. Characterization/expression of the introducedgenetic material in the organism, such as DNA fragmentsize, presence of coding/non-coding sequences, geneticmaps of chromosomes or plasmids, type and number ofrepeat sequences, insertion sequences or transposableelements, and the presence of lysogenic bacteriophage;

! Phenotypic. Characteristics include culture requirementsand characteristics; nature and degree of pathogenicity,virulence, infectivity, or toxicity to humans, otheranimals, plants, or microorganisms; appearance;antibiotic resistance; and survival/persistence.

The type of questions the auditor might ask with respect tothe genotypic and phenotypic characterization are as follows:

! Does the laboratory have SOPs for identification ofgenotypic and phenotypic characteristics?

! Were appropriate methods and instruments used in thegenotypic and phenotypic characterization of thebioengineered test substance?

! Was DNA sequencing conducted? If so, what method wasused? Did the laboratory use a commercial kit orautomated sequencer?

! If gel electrophoresis was done, was the type ofelectrophoresis appropriate for the size of DNAfragments? For agarose gels, was the percentage ofagarose appropriate for the size of the DNA fragments tobe separated? Were solutions, reagents, buffers, etc.adequately labeled (including the expiration date)? Wasthe person responsible for running the gels knowledgeableabout the electrical parameters?

! Has the laboratory identified any unusual morphological,biochemical, or resistance characteristics that aredifferent from the classic description of the organism?If so, have these been documented?

! Were all data available for review by the auditor?

If the desired product is a protein produced by the organism,the identity may have been determined through physicochemicaltests. The type of questions the auditor might ask with respect tothese analyses are as follows:

! Does the laboratory have SOPs for identifying/determiningpurity of the protein?

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! Are the SOPs of adequate scope and detail (if reviewed bythe auditor)?

! If gel electrophoresis was done, was the type ofelectrophoresis appropriate for the protein size? Weresolutions, reagents, buffers, etc. adequately labeled(including the expiration date)?

! Were appropriate methods and instrumentation used in theidentity testing?

! If ion exchange chromatography was done, how was the ionexchange gel chosen? Was the pH range where the proteinis stable used? Was it appropriate for the size of theprotein being separated? Was the gel equilibrated? Wereany solutions, reagents, buffers, etc. adequately labeledwith an expiration date?


! Did the analytical data from the identity tests show anycontamination of the test substance?

NOTE: Protein identity can be determined by amino acidcomposition analysis, partial sequence analysis, peptide mapping,polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing,or high performance liquid chromatography. These methods aredescribed in the support document.

Purity

In many cases, the purity of the test substance will bedetermined concurrently with the identity testing. For example, ifthe laboratory is performing DNA sequencing and the resulting datashow additional DNA fragments that should not be present, then thebioengineered microorganism may not be pure. Likewise, if thelaboratory is performing antibiotic resistance testing and themicroorganism shows sensitivity where there should be resistance,then the test substance may be contaminated. In addition toreviewing information from the identity tests, the .~auditor shouldalso consider these questions:

! Does the laboratory assure that it has a pure culture?

! Was purity testing done on each batch of the testsubstance used in a study?

! Were precautions taken to prevent and control viral,bacterial, mycoplasmal, or other contamination?

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Stability

Using records and data generated by the laboratory, theauditor should confirm that the test substance was analyzed forstability. The test tor stability is specific or unique to the typeof bioengineered test substance and should be specified in an SOPand/or in the study protocol. The auditor should interview studypersonnel (as necessary) and review documentation to ensure thatthe tests were conducted as specified in the SOPs and/or studyprotocol. The auditor should ask the following questions:

! Did the laboratory test the stability of the testsubstance?

! Did the laboratory test to determine the reversion rateor rate of plasmid loss from the host cell? How was therate determined? Did the laboratory resolve any problemsrelated to this?

! Was testing done at a point in the study such that anystability problems were identified early enough to avoidan adverse effect on the study?


5.3.2 PRODUCTION OF BIOENGINEERED TEST SUBSTANCE

Since biotechnology studies commonly involve large populationsof bioengineered organisms, rather than individual organisms, afacility will have procedures for propagation of the organism.

Microorganisms

Once the bioengineered microorganism has been prepared,it will need to be propagated/grown into a cell population.When the cell population has reached a sufficient size, themicroorganisms are harvested (i.e., extracted from the growthmedium). If the desired product is a substance produced by themicroorganisms (e.g., a protein), rather than the cellsthemselves, the desired product will need to be recovered fromthe microorganisms. The bioengineered test substance (eitherthe microorganisms or the substance they produce) will then beused for testing. The facility should have SOPs in place forboth propagation and harvesting and should document compliancewith the SOPs.

Elements that should be considered during the inspector'sevaluation are the following:

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! Does the laboratory have SOPs for propagation/growth ofthe bioengineered microorganism?

! Do they contain information such as the growth medium,physical conditions, nutrients, and expected generationtime?

! Do the SOPs set out the specific equipment to be used(e.g., type of bioreactor) and the appropriate operatingparameters (e.g., pH, temperature, effective mixing,viscosity, oxygen and CO2 concentrations)?

! Are the equipment and conditions specified in the SOPappropriate for the level of containment needed for thestudy? For information on containment levels, theinspector should refer to the guidelines in Biosafety inMicrobiological and Biomedical Laboratories (May 1988)

! Does the laboratory have SOPs for harvesting thebioengineered microorganisms?

! Do the SOPs contain guidelines for separation andpurification?

! Has the separation procedure been validated?

! Has the purification system been validated?

! How are parameters for the separation and purificationsystems monitored?

! Do the systems yield a uniform product? Are testsconducted to determine uniformity?

! Does the laboratory have SOPs for waste recovery anddecontamination of any wastes remaining after separationand purification?

! If the bioengineered microorganism is produced in morethan one batch (i.e., consecutive batches, or concurrentbatches in more than one bioreactor), what are thecriteria for pooling these batches for subsequentportions of the study (i.e., application and testing)?.

5.3.3 HANDLING

The inspector should ensure that the facility has proceduresin place for handling the bioengineered test substance. (Note: Thebioengineered test substance refers to the bioengineered organism,the product generated by the bioengineered organism, or a

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transgenic plant.) These procedures should address properidentification, storage, and distribution of each batch/aliquot ofthe test substance. To evaluate the laboratory's handlingprocedures, the inspector may interview laboratory personnel,examine SOPs, observe laboratory practices, and review substancecontrol logbooks. The following are issues to guide the inspectorin evaluating the facility's compliance for handling of thebioengineered microorganism or transgenic plant:

! Does the laboratory have SOPs for handling thebioengineered test substance?

! Are these SOPs adhered to and any deviations properlyauthorized and documented?

! How is the test substance stored?

! Do storage conditions appear to be adequate forpreserving the test substance?

! Are storage procedures designed to limit the potentialfor contamination or degradation of the substance?

! How are samples/batches distributed?

! How is the test substance transported to the test site?

! Are appropriate containers used so as to preserve thecharacteristics (especially purity) of the testsubstance?

! Does the laboratory document the receipt and distributionof the test substance?

! Does the documentation contain complete information,including who received each sample/batch, when, and inwhat quantity?

! Are all samples/batches of the test substance properlyidentified throughout storage and distribution?

! Are handling conditions specific to the test substanceclearly identified, such as temperature conditions orlength of storage time before reversion oftransformation?

! Does the laboratory retain reserve populations ofmicroorganisms or plants?

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5.4 STUDIES OF THE BIOENGINEERED TEST SUBSTANCE

The TSCA and FIFRA GLP Regulations include specific requirementsfor the protocol for and conduct of a study submitted under TSCA andFIFRA. The GLP Regulations require that each study have an approvedstudy protocol in place and that all changes to the protocol beexplained, documented, and signed and dated. The regulations alsorequire that the study be conducted in accordance with the approvedprotocol and that data generated during the study be recorded andmaintained properly. This section addresses the items the auditor shouldevaluate with regard to the study protocol, conduct of the study, fieldsites for testing, test system care, and the efficacy and infectivity,pathogenicity, toxicity of the bioengineered test substance. For generalquestions regarding the study and test system, the auditor should referto SOP GLP-DA-06 "Auditing Efficacy Studies."

5.4.1 STUDY PROTOCOL

The studies of bioengineered test substances may also besubject to FDA or USDA guidelines. In these cases, the auditorshould determine if the study has been conducted in accordance withthe applicable FDA or USDA guidelines (see Section 4.0, References,for complete titles).

Field Tests

! Does the protocol describe the experimental design,including methods for the control of bias? Are theobjective for the field test clearly set out?

! Does the field test protocol describe test plotpreparation, the spacing of plants, and the growingcycle?

! Does the protocol specify the method of application/routeof administration of the test substance and the reasonfor its selection? Are the application/dosage level andthe frequency of application specified? Are thereprovisions for controlling the unintended release orspreading of the test substance during application?

! Does the protocol specify sampling locations?

! Does the protocol describe the procedures for packagingand transporting samples from the field site to thelaboratory for processing? Were these procedures adequatefor ensuring that samples remained segregated anduniquely identified (e.g., labeling of samplecontainers)? Are procedures specified for samplepreservation?

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! Does the protocol describe how unused test substance,field test samples and materials are to be disposed ofafter analysis and/or use?

Field Sites

! Were the conditions of the field test site documented,including a description of the site (e.g., size,proximity to other test sites), its location, soilcharacteristics (e.g., texture, pH, cation exchangecapacity), and site history (i.e., past uses)? Did thesite conditions meet the requirements of the protocol?Was the historical agricultural practice for the testsite documented?

! Were the test sites maintained in accordance with thespecifications of the protocol (e.g., application offertilizers/pesticides other than the test substance,tilling, weeding)?

! Before use in the study, was the test system acclimatizedto the environmental conditions specified for the tests(e.g.~ gradual reduction in humidity from cultureconditions to testing conditions)? Did theacclimatization period appear to be adequate?

! Was the field equipment (e.g., balances, applicationequipment, and field analytical equipment) inspected andcalibrated before use? Were the precision and accuracy ofthe test equipment verified? Was the equipmentdecontaminated between sampling events?

! Do the study records contain a description of themeteorological conditions (e.g., air and soiltemperatures, relative humidity, wind direction andvelocity, rainfall) that existed during the study?

! Were containment measures taken to prevent contamination?Was the reproductive isolation of plants ensured? Formicroorganisms, was the gene transfer capabilitymonitored?

! Were sample locations recorded with the analyticalresults or on a site map?

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5.4.2 TEST SYSTEM CARE

The GLP Regulations define the test system as “any animal,plant, microorganism, chemical or physical matrix, including butnot limited to, soil or water, or components thereof, to which thetest, control, or reference substance is administered or added forstudy." In accordance with the Regulations, the test system shouldbe cared for to ensure that the integrity of the study is notcompromised by unhealthy or diseased test subjects, contaminationfrom other species or test systems or from the feed, soil, water,or bedding. The auditor should review study notes and observations,interview study personnel, and visit test sites to evaluate whetherthe study implemented proper procedures for test system care. Foranimal and plant test systems, the questions the auditor wouldevaluate are found in other relevant SOPs. Specific elements theauditor would evaluate related to microorganism test systems are asfollows:

! If the test system was a cell culture (i.e.,microorganisms) and was purchased, from an outsidesource, were the microorganisms characterized? Was aletter of identification (regarding the characterization)received from the supplier? Were records of thecharacterization in the study files?

! How were the microorganisms stored? Were the storageconditions appropriate to ensure stability of themicroorganisms?

! Was the cell culture tested for contamination beforebeing used in the study?

5.4.3 EFFICACY

Efficacy, or product performance, is a measure of all aspectsof a product's effectiveness and usefulness. Efficacy of the testsubstance is affected by the activity of the test substance itself,as well as by application methods, the type of target pestorganisms, dosage rate and frequency, duration of use, otherenvironmental conditions, and the use of other pest controlsubstances or methods. Initial efficacy testing is generallyconducted in the laboratory, in greenhouses, or on small fieldplots. If the results of the small-scale testing indicatesufficient product effectiveness, large-scale testing in thelaboratory, field, and through simulated-use tests is subsequentlydone. The auditor should consider the following elements inevaluating the efficacy testing:

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! Were efficacy tests done to confirm support for anyproposed product labeling claims?

! Was the testing done at various dosage levels? Didtesting include the dosage levels corresponding with theproposed use?

! Did the testing program determine the effective exposurerange and the minimum effective dosage for the usesinvolved?

! Did the testing program evaluate the effectiveness of thetest-substance under different application methods?

! Did the testing program consider the effects of usingdifferent application schedules and the time ofapplication relative to the time of planting, stage ofgrowth, and time Of harvest?

! If the test substance was formulated into a mixture andis intended to be used as such, was the efficacy of anyother active ingredients also determined (from eitherexisting data or through the testing program)?

! Were the number and type of samples taken described? Didsamples allow for analysis of all the characteristics ofthe test population that are to be evaluated?

! Did testing procedures include the evaluation of controlor reference substances? Were the results of the producttests compared against the results from similarmeasurements done on the control or reference substancesso that product performance could be evaluated against abaseline? Were containment practices used to keep controlor reference substances free from contamination with thetest substance or other substances?

! If testing indicated poor or inconsistent productefficacy, were subsequent tests and statistical analysisdone to further evaluate the same responses?

! Were tests done under multiple meteorological conditions(e.g., dry, rainfall, humidity, temperature, sunlight) todetermine product effectiveness under climatic conditionsexpected to occur during product use?

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! Were tests done under various soil conditions (e.g.,moisture, pH, texture, fertility) and soil treatments(e.g., fertilizers, irrigation) so that effects of thesevariables on product efficacy could be determined?

5.4.4 INFECTIVITY/PATHOGENICITY/TOXICITY

Through examination of records and data generated by thelaboratory, the auditor should confirm that the bioengineered testsubstance was tested for infectivity/pathogenicity/toxicity. Allnotebooks, worksheets, scratch sheets, notes, computer printouts,calculations, graphs and tables pertaining to the test should beconsidered raw data and subject to examination. If possible, theauditor should also interview the scientists, and, if appropriate,the technicians who performed the tests . The following series ofquestions are based on the Pesticide Testing Guidelines SubdivisionM: Microbial and Biochemical Pest Control Agents (July 1989) andare only guidelines for laboratories. However, the questions shouldhelp the auditor focus on specific points or aspects of the testsunder review.

! Was the test substance tested for infectivity,pathogenicity, and toxicity?

! Did the laboratory identify the test methods by name orby reference?

! How did the laboratory select the animal that was tested?Was the animal determined to be disease free?

! Was the technique used to quantify the dose described?

! Was the technique used for application, treatment ordosing described?

! Were the appropriate dose or strength, vehicle, andvolume used?

! Was a control group used?

! What was the observation period for the test? How was itdetermined?

! How often did the laboratory examine the animals? Was theexamination comprehensive (e.g., skin and fur, eyes andmucous membranes, respiratory system, circulatory system,diarrhea, lethargy, salivation, etc.)? Were theobservations adequately documented?

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! Was the technique used to determine the clearance of thetest substance described?

! Was the technique used to enumerate the test substance inbody parts (i.e., tissues, organs, and body fluids)described? What body parts were used for the analyses?Were recovery values, detection limits, and sensitivitylimits determined for each technique used?

! Was a gross necropsy of all animals done?

Cell culture tests

! How was the cell culture selected? Was the cell lineadequately identified?

! Was the most permissive host system used?

! Were the appropriate controls used?

! Was a cell transformation assay done?

! Were the procedures used for toxicity evaluationdescribed?

5.5 FACILITIES AND EQUIPMENT

5.5.1 FACILITY DESIGN

In accordance with the FIFRA and TSCA GLP Regulations, thefacilities used for biotechnology studies must be sufficient toenable the proper conduct of the study. The facilities must provideappropriate space, environmental conditions, containment,decontamination areas, and support systems (e.g., air, water) forthe study being conducted. Facility design should take into accountthe Biosafety controls required for the organisms used in thestudy, the need for maintaining a controlled environment for thedevelopment of the bioengineered organism and should have testingfacilities that adequately provide for a controlled environment andseparation of test systems. The facility also needs to haveadequate areas for receipt and storage of the host Organism, anystocks of plants and soils used in the study, and the bioengineeredtest substance, as well as facilities for waste disposal.Additionally, the facility needs to include sufficient space forarchives that allow for the storage and retrieval of raw data andcell cultures by approved personnel.

The inspector needs to evaluate both the laboratory facilitiesand any separate facilities, such as greenhouses and field sites,that are used for testing the bioengineered test substance. This

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evaluation requires a walk through inspection of all areas of thefacility and the field plots so the inspector can make a directassessment of the adequacy of the facilities. Specific elementsthat should be considered during the inspector's evaluation are thefollowing:

! Are the laboratory and testing facilities appropriate forwork with infectious agents or potentially infectiousmaterials?

! Are any outdoor testing facilities, such as field sites,of sufficient design (layout, size, and location) tosupport the testing conditions of the study?

! Are these facilities sufficiently segregated from othertesting areas to prevent any cross-contamination?

! Are indoor testing facilities designed to provideadequate separation of test systems?

! Do the facilities, particularly those used for testing,allow for adequate control of environmental conditions,such as temperature, humidity, ventilation, and lighting?For instance, does the facility have proper ventilationso that air flows from areas of low contamination toareas of higher contamination and is complete aircontainment and decontamination provided?

! Are environmental conditions monitored using appropriateinstruments? Are these conditions as specified in theprotocol for the ongoing study?

! Is an ongoing record kept of the environmentalconditions, noting any deviations from those intended forthe study?

! Are there appropriate and sufficient facilities for thereceipt and storage of both the host organism and testsubstance, as well as for the other materials (e.g.,soil, feed) used in the study? Are these areas separatedfrom one another and from other areas of the facility?

! Do the storage facilities provide environmentalconditions (e.g., temperature, moisture) to maintain thepurity, strength, and stability of the bioengineered testsubstance?

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! Does the facility provide the necessary containment forthe appropriate biosafety level to protect persons andthe environment both in and around the study facility?

! Are rooms and testing facilities designed to provide abarrier to the unintended release, particularly throughthe atmosphere, of any microorganisms if a spill orapplication accident were to occur?

! Are decontamination facilities separated from the otherareas of the facility?

! Does the laboratory have procedures for collecting anddisposing of contaminated plants, soils, and other

! Does the laboratory have decontamination procedures forcontaining or killing bioengineered organisms and hostorganisms?

! What types of support systems are available? For example,what is the source of water? Are water suppliessufficient? How are water conditions regulated?

5.5.2 EQUIPMENT

The types of equipment commonly used in a biotechnologylaboratory will vary based not only on the types of biotechnologyprocesses and organisms used, but also on whether the equipment isused during the development and processing of the microorganism orplant, during application of the test substance to the test system,or during testing. Types of equipment commonly used includebioreactors, air compressors, sterilization equipment, productrecovery systems (e.g., centrifuges, cell disrupters), wasterecovery and decontamination equipment, sampling and analysisinstruments, safety equipment (e.g., biosafety cabinets, protectiveclothing), equipment for transporting biological materials (e.g.,sealed containers), and environmental control equipment.

Since many of the questions will be similar to those asked inother laboratories submitting studies under TSCA or FIFRA, theinspector should refer to "Conducting a Field Site GLP ComplianceInspection" (SOP No. GLP-C-01, Section 5.1.2).

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/s/____________________________ 01/04/99Reviewed by: Daniel Myers DateChemist/Inspector

/s/____________________________ 01/04/99Approved by: Francisca E. Liem DateChief, Laboratory Data Integrity Branch

/s/____________________________ 01/04/99Approved by: Rick Colbert DateDirector, Agriculture and Ecosystems DivisionU.S. Environmental Protection AgencyOffice of Enforcement and Compliance AssuranceOffice of Compliance

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GLOSSARY

Bacteriophage: a virus that lives in and kills bacteria: also called "phage"

Biochemical: the product of a chemical reaction in a living organism

Chemoheterotroph: an organism that derives energy and carbon from theoxidation of preformed organic compounds

Chemolithotroph: an organism that uses carbon dioxide as its principal sourceof carbon for growth and obtains its energy by the oxidation of inorganiccompounds

Chemoorganotroph: an organism that obtains its energy by the oxidation oforganic compounds, which are also its principal sources of carbon

Dicotyledon: any plant of the class Magnoliopsida, all having two cotyledons

Dioecious: having the male and female reproductive organs on differentindividuals

Explant: to transfer living tissue for culture in an artificial medium

Flagellate: having flagella (relatively long, whiplike parts of certainbacteria or protozoans that provide locomotion and produce a current in thesurrounding fluid)

Fermentations: an anaerobic process of growing microorganisms to producevarious chemical or pharmaceutical compounds

Genotype: genetic make-up of an organism

Gram-Negative/Gram-Positive Cell: cells distinguished from each other by thecomposition of cell wall. Gram-negative cell has a multilayered, complexcell wall; gram-positive ¢ell has a cell wall consisting of a single layerthat is often much thicker than the wall of a gram-negative cell.

Immunology: study of all phenomena related to the body's response toantigenic challenge (i.e., immunity, sensitivity, and allergy)

Lysis: rupture of a cell that results in loss of the cell's contents

Medium: a substance containing nutrients needed for cell growth

Morphology: a branch of biology that deals with the structure and of anorganism at any stage of its life history

Motility: capable of or exhibiting spontaneous motion

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Oxidation: a chemical reaction that increases the oxygen content of acompound

Peritrichous: of bacteria, having a uniform distribution of flagella on thebody surface

Phage: see "bacteriophage"

Phenotype: observable characteristics resulting from interaction between anorganism's genetic make-up and the environment

Phototropism: movement of a part of a plant toward or away from light

Physiology: the study of the basic activities that occur in cells and tissuesof living organisms by using physical and chemical methods

Plasmid: a small circular form of DNA that carries certain genes and iscapable of replicating independently in a host cell

Sporangium: a case or envelope in which spores are formed

Spore: a single-celled or multi-celled, asexual reproductive or resting bodythat is resistant to unfavorable environmental conditions and produces a newvegetative organism when the environment is favorable

Striated: marked with minute lines, bands, grooves, or channels

Transposable Elements: mobile DNA sequences that change positions onchromosomes; also called "transposable genetic elements" or simply“transposons"

Vector: the agent: (e.g., plasmid or virus) used to carry new DNA into a cell

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Appendix A

Standard Operating Procedure for ConductingGLP Compliance Inspections of Biotechnology Facilities

Inspection Checklist

PART I -INSPECTOR CHECKLIST (2)

PART 11-AUDITOR CHECKLIST (8)

(Suggested for use with the GLP FIFRA/TSCA compliance checklist)

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Biotechnology FacilityInspector ChecklistOriginal: March 3/94

Facility:_____________________________Insp. Init.:_____________Date_________

PART I - INSPECTOR CHECKLIST

General Instructions/Information1. For any “No” answers, provide an explanation in Remarks column.2. Remarks can be continued in the “Comments” section on the back of each page.3. Places a line through and missing item. For example, “...name/signature”.4. Section numbers refer to the corresponding sections in the SOP.

Section 5.2 Preparation of Bioengineered Test Substance Yes No N/A Remarks

Section 5.2.1 Characterization of Donor and RecipientMicroorganisms and Transgenic Plants - Microorganism

How does the laboratory assure tat it has a pureculture? What is the frequency of this test?

How are the organisms stored? Are storageconditions adequate to ensure the stability ofthe organism?

Does the laboratory have a documented history ofthe bacterial strain?

Does the laboratory have an SOP for doing thetaxonomic identification?

Does the SOP address identification of genotypicand phenotypic characteristics?

For donor and recipient organisms from externalsources (i.e., purchased or obtained from anotherlaboratory), has the laboratory received a letterof identifications (i.e., certificate) from thesupplier?

- Does the facility test the purchasedorganism upon arrival to certify itsidentity and purity?

- What are the shipping conditions for theorganisms?

- Does the facility have records of receipt?

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Facility:____________________Insp.Init.:______________________Date_________

Section 5.2.1 Characterization of Donor and RecipientMicroorganisms and Transgenic Plants - PlantCharacterization

How and where are the plants and plant cellsstored?

Yes No N/A Remarks

Is there documentation of the biology of thereproductive potential of the plant (e.g.,flowers, pollination requirements and seedcharacteristics) being used in the study?

Does the laboratory have a documented history ofcontrollable reproduction with lack ofdissemination? Can the plant be established inan environment that is similar to the field testsite?

Section 5.2.2 Materials and Methods for Producing theBioengineered Test Substance - Materials

If DNA is obtained from external sources (i.e.,purchased or obtained from another laboratory),what are the shipping conditions? Was the DNAtested upon arrival? How is the DNA stored?

If a restriction endonuclease (or any otherenzyme) is obtained from external sources, howdoes the laboratory assure that the enzyme isneither contaminated nor inactive? Howfrequently is it tested? How is the enzymestored?

For other chemicals or substances used asreagents, buffers, or growth media, how does thelaboratory assure the quality of the materials?

Does the laboratory use distilled, deionizedwater to prepare all reagents and buffersolutions?

Is the laboratory using standard methods for thepreparation of reagents, buffer solutions, andmedia?

Section 5.2.2 Materials and Methods for Producing theBioengineered Test Substance - Methods

Does the laboratory have SOPs:- For isolating and identifying DNA being

inserted into the recipient organism orplant cell?

- For constructing the vector with theinserted DNA?

- For verifying the DNA insertion in thevector?

Facility:______________________Insp.Init.:______________________Date_________

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Section 5.2.2 Materials and Methods for Producing theBioengineered Test Substance - Methods (continued)

- For the method being used to introduce theDNA/vector into the recipient organism?

(1) Transformation, transduction,conjugation for microorganisms(2) Agrobacterium-based planttransformation, particle acceleration,electroporation, microinjection for plants

- For measuring the success of the insertionof the genetic material?

- For determining the stability of theinserted DNA in the recipient microorganismor plant?

How does the laboratory validate the methods thatit uses?

Who constructed the vector and performed theprocedures in preparing the bioengineeredmicroorganism or transgenic plant?

If protoplasts (for transgenic plants) are beingused, are standard tissue culture proceduresused?

Section 5.3 Characterization, Production and Handlingof Bioengineered Test Substance

YES NO N/A REMARKS

Section 5.3.2 Production of Bioengineered TestSubstances - Microorganism

Does the laboratory have SOPs for thepropagation/growth of the bioengineeredmicroorganism?

Do they contain information such as the growthmedium, physical conditions, nutrients, andexpected generation time?

Do the SOPs set out the specific equipment to beused (e.g., type of bioreactor) and theappropriate operating parameters (e.g., pH,temperature, effective mixing, viscosity, oxygenand CO2 concentrations)?

Are the equipment and conditions specified in theSOP appropriated for the level of containmentneeded for the study? For information oncontainment levels, the inspector should refer tothe guidelines in Biosafety in Microbiologicaland Biomedical Laboratories (May 1988)

Does the laboratory have SOPs for harvesting thebioengineered microorganisms?

Facility:___________________Insp.Init.:______________________Date_________

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Section 5.3.2 Production of Bioengineered TestSubstance - Microorganisms (continued)

Do the SOPs contain guidelines for separation andpurification?

Has the separation procedure been validated?

Has the purification system been validated?

How are the parameters for the separation andpurification systems monitored?

Do the systems yield a uniform product?Are tests conducted to determine uniformity?

Does the laboratory have SOPs for waste recoveryand decontamination of any wastes remaining afterseparation and purification?

If the bioengineered microorganism is produced inmore that one batch (i.e., consecutive batches,or concurrent batches in more than onebioreactor), what are the criteria for poolingthese batches for subsequent portions of thestudy (i.e., application and testing)?

Section 5.3.3 Handling YES NO N/A REMARKS

Does the laboratory have SOPs for the handlingthe bioengineered test substance?

Are these SOPs adhered to and any deviationsproperly authorized and documented?

How is the test substance stored?

Do storage conditions appear to be adequate forpreserving the test substance?

How are samples/batches distributed?

Are storage procedures designed to limit thepotential for contamination or degradation of thesubstance?

How is the test substance transported to the testsite?

Are appropriated containers used so as topreserve the characteristics (especially purity)of the test substance?

Does the laboratory document the receipt anddistribution of the test substance?

Does the documentation contain completeinformation, including who received eachsample/batch, when, and in what quantity?

Section 5.3.3 Handling (continued)

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Are all samples/batches of the test substanceproperly identified throughout storage anddistribution?

Are handling conditions specific to the testsubstance clearly identified, such as temperatureconditions or length of storage time beforereversion of transformation?

Does the laboratory retain reserve populations ofmicroorganisms or plants?

Section 5.5 Facilities and Equipment YES NO N/A REMARKS

Section 5.5.1 Facility DesignAre the laboratory and testing facilitiesappropriate for work with infectious agents orpotentially infectious materials?

Are any outdoor testing facilities, such as fieldsites, of sufficient design (layout, size, andlocation) to support the testing conditions ofthe study?

Are these facilities sufficiently segregated fromother testing areas to prevent any cross-contamination?

Are indoor testing facilities designed to provideadequate separation of test systems?

Do the facilities, particularly those used fortesting, allow for adequate control ofenvironmental conditions, such as temperature,humidity, ventilation, and lighting? Forinstance, does the facility have properventilation so that air flows from areas of lowcontamination to areas of higher contaminationand is complete air containment anddecontamination provided?

Are environmental conditions monitored usingappropriate instruments? Are these conditions asspecified in the protocol for the ongoing study?

Is an ongoing record kept of the environmentalconditions, noting any deviation from thoseintended for the study?

Are there appropriate and sufficient facilitiesfor the receipt and storage of both the hostorganism and test substance, as well as for theother materials (e.g., soil, feed) used in thestudy? Are these areas separated from oneanother and from other areas of the facility?

Do the storage facilities provide environmentalconditions (e.g., temperature, moisture) tomaintain the purity, strength, and stability ofthe bioengineered test substance?

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Does the facility provide the necessarycontainment for the appropriate biosafety levelto protect persons and the environment both inand around the study facility?

Are rooms and testing facilities designed toprovide a barrier to the unintended release,particularly though the atmosphere, or anymicroorganisms if a spill or application accidentwere to occur?

Are decontamination facilities separated from theother areas of the facility?

Does the laboratory have procedures forcollecting and disposing of contaminated plants,soils and other materials?

Does the laboratory have decontaminationprocedures for containing or killingbioengineered organisms and host organisms?

What types of support systems are available? Forexample, what is the source of water? Are watersupplies sufficient? How are water conditionsregulated?

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Facility:____________________Insp.Init.:______________________Date_________

PART II - AUDITOR CHECKLIST

General Instructions/Information1. For any “No” answers, provide an explanation in Remarks column.2. Remarks can be continued in the “Comments” section on the back of each page.3. Places a line through and missing item. For example, “...name/signature”.4. Section numbers refer to the corresponding sections in the SOP.

Section 5.3 Characterization, Production, and Handlingof Bioengineered Test Substance

Yes No N/A Remarks

Section 5.3.1 Characterization of Bioengineered TestSubstance - Identity

Does the laboratory have SOPs for identificationof genotypic and phenotypic characteristics?

Were appropriate methods and instruments used inthe genotypic and phenotypic characterization ofthe bioengineered tests substance?

Was DNA sequencing conducted? If so, what methodwas used? Did the laboratory use a commercialkit or automated sequencer?

If gel electrophoresis was done, was the type ofelectrophoresis appropriate for the size of DNAfragraments? For agarose gels, was thepercentage of agarose appropriate for the size ofthe DNA fragments to be separated? Weresolutions, reagents, buffers, etc. adequatelylabeled (including the expiration data)? Was theperson responsible for running the gelsknowledgeable about the electrical parameters?

Has the laboratory identified any unusualmorphological, biochemical, or resistancecharacteristics that are different from theclassic description of the organism? If so, havethese been documented?

Were all data available for review by theauditor?

Does the laboratory have SOPs foridentifying/determining purity of the protein?

Are the SOPs of adequate scope and detail (ifreviewed by the auditor?)

If gel electrophoresis was done, was the type ofelectrophoresis appropriate for the protein size? Were solutions, reagents, buffers, etc.adequately labeled (including the expirationdate)?

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Facility:_____________________Insp.Init.:_____________________Date___________

Section 5.3.1 Characterization of Bioengineered TestSubstance - Identity (continued)

Were appropriate methods and instrumentation usedin the identity testing?

If ion exchange chromatography was done, how wasthe ion exchange gel chosen? Was the pH rangewhere the protein is stable used? Was itappropriate for the size of the protein beingseparated? Was the gel equilibrated? Were anysolutions, reagents, buffers, etc. adequatelylabeled with an expiration date?


Did the analytical data from the identity testsshow any contamination of the test substance?

5.3.1 Characterization of Bioengineered Test Substance - Purity

Does the laboratory assure that it has a pureculture?

Was purity testing done on each batch of the testsubstance used in the study?

Were precautions taken to prevent and controlviral, bacterial, mycoplasmal, or othercontamination?

5.3.1 Characterization of Bioengineered Test Substance - Stability

Did the laboratory test the stability of the testsubstance?

Did the laboratory test to determine thereversion rate or rate of plasmid loss from thehost cell? How was the rate determined? Did thelaboratory resolve any problems related to this?

Was testing done at a point in the study suchthat any stability problems were identified earlyenough to avoid an adverse effect on the study?


Facility:_____________________________Insp.Init.:___________Date___________

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5.4 Studies of the Bioengineered Test Substance YES NO N/A REMARKS

5.4.1 Study Protocol - Field Tests

Does the protocol describe the experimentaldesign, including methods for the control ofbias? Are the objective for the field testclearly set out?

Do the field test protocol describe test plotpreparation, the spacing of plants, and thegrowing cycle?

Does the protocol specify the method ofapplication/route of administration of the testsubstance and the reason for its selection? Arethe application/dosage level and the frequency ofthe application specified? Are there provisionfor controlling the unintended release ofspreading of the test substance duringapplication?

Does the protocol specify sampling locations?

Does the protocol describe the procedures forpackaging and transporting samples from the fieldsite to the laboratory for processing? Werethese procedures adequate for ensuring thatsamples remain segregated and uniquely identified(e.g., labeling of sample containers)? Areprocedure specified for sample preservation?

Does the protocol describe how unused testsubstance, field test samples and materials weredeposed of after analysis and/or use?

5.5.1 Study Protocol - Field Sites

Were the conditions of the field test sitedocumented, including a description of the size(e.g., size, proximity to other test sites), itslocation, soil characteristics (texture, pH,cation exchange capacity), and site history(i.e., past uses)? Did the site conditions meetthe requirements of the protocol? Was thehistorical agricultural practice for the testsite documented?

Were the test sites maintained in accordance withthe specifications of the protocol (e.g.,application of fertilizers/pesticides other thanthe test substance, tilling, weeding)?

Before use in the study, was the test systemacclimatized to the environmental conditionsspecified for the tests (e.g., gradual reductionin humidity from culture conditions to testingconditions)? Did the acclimatization periodappear to be adequate?

5.4.1 Study Protocol - Field Sites (continued)

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Was the field equipment (e.g., balances,application equipment, and field analyticalequipment) inspected and calibrated before use? Were the precision and accuracy of the testequipment verified? Was the equipmentdecontaminated between sampling events?

Do the study records contain a description of themeteorological conditions (e.g., air and soiltemperatures, relative humidity, wind directionand velocity, rainfall) that existed during thestudy?

Were containment measures taken to preventcontamination? Was the reproductive isolation ofplants ensured? For microorganisms, was the genetransfer capability monitored?

Were sample locations recorded with theanalytical results or on a site map?

5.4.2 Test System Care

If the test system was a cell culture (i.e.,microorganisms) and was purchased from an outsidesource, were the microorganisms characterized? Was a letter of identification (regarding thecharacterization) received from the supplier? Were records of the characterization in the studyfiles?

How were the microorganisms stored? Were thestorage conditions appropriate to ensurestability of the microorganisms?

Was the cell culture tested for contaminationbefore being used in the study?

5.4.3 Efficacy

Were efficacy tests done to confirm support forany proposed product labeling claims?

Was the testing done at various dosage levels? Did testing include the dosage levelscorresponding with the proposed use?

Did the testing program determine the effectiveexposure range and the minimum effective dosagefor the uses involved?

Did the testing program evaluate theeffectiveness of the test substance underdifferent application methods?

Did the testing program consider the effects ofusing different application schedules and thetime of application relative to the time ofplanting, stage of growth, and time of harvest?

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If the test substance was formulated into amixture and is intended to be used as such, wasthe efficacy of any other active ingredients alldetermined (from either existing data or throughthe testing program)?

Were the number and type of samples takendescribed? Did samples allow for analysis of allthe characteristics of the test population thatare to be evaluated?

Did testing procedures include the evaluation ofcontrol or reference substance? Were the resultsof the product tests compared against the resultsfrom similar measurements done on the control orreference substances so that product performancecould be evaluated against a baseline? Werecontainment practices used to keep control orreference substance free from contamination withthe test substance or other substances?

If testing indicated poor or inconsistent productefficacy, were subsequent tests and statisticalanalysis done to further evaluate the sameresponses?

Were tests done under multiple meteorologicalconditions (e.g., dry, rainfall, humidity,temperature, sunlight) to determine producteffectiveness under climatic conditions expectedto occur during product use?

Were test done under various soil conditions(e.g., moisture. PH. Texture, fertility) and soiltreatments (e.g., fertilizers, irrigation) sothat effects of these variables on productefficacy could be determined?

5.4.4 Infectivity/Pathogenicity/Toxicity

Was the test substance tested for infectivity,pathogenicity/toxicity?

Did the laboratory identify the test methods byname or by reference?

How did the laboratory select the animal that wastested? Was the animal determined to be diseasefree?

Was the technique used to quantify the dosedescribed?

Was the technique used for application, treatmentor dosing described?

Were the appropriate dose or strength, vehicleand volume used?

Was a control group used?

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What was the observation period for the test? How was it determined?

5.4.4 Infectivity/Pathogenicity/Toxicity (continued)

How often did the laboratory examine the animals? Was the examination comprehensive (e.g., skin andfur, eyes and mucous membranes, respiratorysystem, circulatory system, diarrhea, lethargy,salivation, etc.)? Were the observationsadequately documented?

Was the technique used to determine the clearanceof the test substance described?

Was a gross necropsy of all animals done?

Was the technique used to enumerate the testsubstance in body parts, (i.e., tissues, organs,and body fluids) described? What body parts wereused for the analyses? Were recovery values,detection limits, and sensitivity limitsdetermined for each technique used?

5.4.4 Infectivity/Pathogenicity/Toxicity- Cell culture test

How was the cell culture selected? Was the cellline adequately identified?

Was the most permissive host system used?

Were the appropriate controls used?

Was a cell transformation assay done?

Were the procedures used for toxicity evaluationdescribed?


Support Document for the Standard Operating Procedure

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March 1994

for Conducting GLP Compliance Review of BiotechnologyFacilities

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March 1994


TABLE OF CONTENTS

5.1 Organization and Personnel .......................................4

5.2 Preparation of Bioengineered Test Substance ......................45.2.1 Taxonomic Identification ...............................4

Characteristics of Some Commonly Used Microorganisms ...55.2.2 Materials and Methods for Preparing the Bioengineered

Products ...............................................7Materials ..............................................7Methods ................................................7

5.3 Characterization,Production, and Handling of Bioengineered Microorganism .........................................11

5.3.1 Characterization of Bioengineered Organism ............11Methods ...............................................11

5.3.2 Production of Bioengineered Organisms .................14Bioengineered Microorganisms ..........................14Plants ................................................17Waste Recovery and Decontamination Techniques .........19

5.4 Studies of the Bioengineered Test Substance .....................20Containment Issue .....................................20Microorganisms ........................................20Plants ................................................21Application Issues ....................................23Analysis of Study Control Plots .......................24

5.5 Facilities and Equipment ........................................255.5.1 Facility Design ..................................255.5.2 Equipment ........................................28

Glossary of Disciplines ..............................................32

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5.1 ORGANIZATION AND PERSONNEL

Since the biotechnology field is particularly specialized andinvolves the application of many emerging technologies, individualsconducting a biotechnology study need to have specific qualifications.The education, training and experience of personnel in key positionsshould include a significant amount of work with bioengineered organismsor in closely related scientific disciplines, such as organic chemistryor toxicology. Standard positions found in biotechnology laboratoriesand facilities and the appropriate qualifications for individuals inthese positions are described in the document Biotechnology Compensationand Benefits Survey, 1992, which may be obtained from the ScientificSupport Branch on request. This information should assist the inspectorin determining if a biotechnology study is led and staffed by adequatelytrained individuals.

The positions discussed above require degrees in scientificdisciplines related to biotechnology. A listing of these disciplines andtheir definitions are provided in the Glossary of Disciplines. Chemistsin a biotechnology laboratory usually have experience in one or more ofthe following areas: biochemistry, synthetic chemistry, organicchemistry, formulation chemistry, analytical chemistry, physicalchemistry, polymer chemistry, peptide chemistry, and toxicology.Biologists in the Principal Scientist position are generallyknowledgeable in one or more of the following fields: agronomy, botany,entomology, forestry, cell biology, microbiology, immunology, membranebiology, biochemistry, histology, and genetics.

5.2 PREPARATION OF BIOENGINEERED TEST SUBSTANCE

5.2.1 Taxonomic Identification

Taxonomy is the study concerning the appearance and structureof a plant or animal, its proper classification and name in theplant or animal kingdom, how it differs from other organisms, anda description of its surroundings. Traits to verify, taxonomicclassification include morphological, biochemical, immunological,and physiological characteristics.

The taxonomic identification of a microorganism involves theuse of standard tests to determine the individual characteristicsof the microorganism which collectively lead to the exactidentification. A complete taxonomic identification can be done ina stepwise fashion for an unknown bacterial culture. A summary ofthe steps are as follows:

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1. Start with a pure, uncontaminated culture.

2. Determine the energy requirements. These are defined throughisolation and culture methods that will indicate whether theorganism is phototrophic, chemolithotrophic, orchemoheterotrophic.

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3. Examine living cells by phase contrast microscopy and describethe morphological characteristics (i.e., rod., coccus, vibrio,spiral, spirochete, filament, sheath, etc.) ExamineGram-stained cells by bright-field microscopy and determinewhether the organism is Gram positive or negative.

4. Examine cells for spores, stalks, prosthecae, or otheridentifying characteristics. The examination for spores shouldbe done carefully using different culture media to inducesporulation.

5. Examine for motility in wet mounts and determine whether theorganism is polarly or peritrichously flagellated.

6. Examine colonies or mass growth for pigments or other uniquecharacteristics.

7. Test for oxygen requirements.

8. Test the dissimilation of glucose or another simple sugar todetermine whether the microorganism is oxidative orfermentative.

9. Complete any additional tests necessary based on the resultsof the previous tests.

The SOP used by the lab for taxonomic identification would containsome or all of these tests to confirm the type of organism being used.For example, since the laboratory should know the identity of theorganism (e.g., if from external sources, through the letter ofidentification), then only specific tests would need to be done toverify the identification.

Characteristics of Some Commonly Used Microorganisms

Three microorganisms that are frequently used in the preparation ofbioengineered microorganism or transgenic plants are Agrobacteriumtumefaciens, Bacillus thuringiensis, and Escherichia coli. The commonand unique qualities of these organisms are discussed below.

Agrobacterium

This genus includes organisms of similar morphology that causetumor like growths of plant tissues in dicotyledonous plants,resulting in the called crown gall and hairy root. The diseaseresults from infection by the bacteria that enter the plant througha wound or break in the plant's outer protective layer.

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The organisms are small, short rods, motile by means offlagella arranged either peritrichously or subpolarly .Agrobacterium is related to Rhizobium morphologically, in itsability to infect plants, and in its genomes. Agrobacteriumcontains a plasmid called the Ti (for Tumor inducing) plasmid thatcontains the trait for tumor formation. ritrichously or subpolarly.The percentage of DNA base composition ranges from 58 to 63.5 G-C.

Characteristics. Non-sporing; Gram-negative; growth oncarbohydrate-containing media usually accompanied by copiousextracellular, polysaccharide slime; colonies non-pigmented andusually smooth, tending to become striated with age;chemoorganotrophic; aerobic, but able to grow under reduced oxygentensions in plant tissue; optimum temperature 25-30 degreesCentigrade; optimum pH range 6.0-9.0.

Bacillus

This genus is a heterogeneous group that can be considered anassemblage of closely related organisms. The organisms arerod-shaped, that are usually motile, and possess peritrichousflagella. The percentage of DNA base composition varies from 30 to50 G-C and studies on nucleic acid homologies by hybridization andgenetic transformation also support considerable geneticheterogeneity. Members of the genus are easy to isolate from soilor air and are among the most common organisms to appear when soilsamples are streaked on agar plates containing various nutrientmedia. Many Bacilli produce extracellular hydrolytic proteins thatbreak down polysaccharides, nucleic acids, and lipids, permittingthe organisms to use these products as carbon and energy sources.In addition, many Bacilli produce antibiotics, includingbacitracin, polymyxin, tyrocidin, gramicidin, and circulin.

A number of Bacilli are insect pathogens. They work by forminga crystalline protein during sporulation that is deposited withinthe sporangium, but outside the spore. The action of this toxin(i.e., the crystalline protein) causes fatal diseases of mothlarvae such as the silkworm, cabbage worm, tent caterpillar, andgypsy moth.

Characteristics. Rod-shaped 0.3-2.2 by 1.2-7.0 um; heat-resistantendospores not repressed by exposure to air; majorityGram-positive; chemoorganotrophic; metabolism strictly respiratory,strictly fermentative or both respiratory and fermentative, usingvarious substrates; catalase formed by most species.

Escherichia

This genus almost universally inhabits the intestinal tract ofhumans and warm-blooded animals. The microorganisms in this groupare generally non-sporulating rods, nonmotile or motile byperitrichously occurring flagella, and are facultative anaerobes.

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They have relatively simple nutritional requirements and theyferment sugars to a variety of end products. The percentage of DNAbase composition is about 50 G-C.

Characteristics. Straight rod-shaped cells 1.1-1.5 by 2.0-6.0 um;Gram-negative; chemoorganotrophic; metabolism respiratory andfermentative; motile by peritrichous flagella or nonmotile;colonies on nutrient agar may be smooth (S), low convex, moist,shiny surface, entire edge, gray and easily emulsified in salinesolution or rough (R), dry and not well emulsified.

Biotechnology Facilities StandardOperating Procedures Support Document

5.2.2 Materials and Methods for Preparing the BioengineeredProducts

Materials

The materials used in biotechnology laboratories are verydiverse ranging from simple neutralization buffers to the Northernhybridization solution. A listing of the various reagents andbuffer solutions used in molecular biology protocols can be foundin Short Protocols in Molecular Biology Second Edition - ACompendium of Methods from Current Protocols in Molecular Biologypublished by Greene Publishing Associates and John Wiley & Sons,199'. For example, the technique for minipreps of plasmid DNA (aslisted in this reference) requires the following materials:

! LB medium (tryptone, yeast extract, NaCI, NaOH)containing the appropriate antibiotic

! Glucose/Tris/EDTA (GTE) solution! NaOH/SDS solution! Potassium acetate solution, pH 4.8! 95% and 70% ethanol.

Methods

Bioengineered Microorganisms

There are several methods that can be used to introduce DNAfrom one organism into another. These methods include:

! Transformation. The process in which free DNA is inserteddirectly into a competent recipient cell

! Transduction. The transfer of bacterial DNA from onebacterium to another through a temperate or defectivevirus

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! Conjugation. Transfer of genetic information from onecell to another by cell-to-cell contact.

For example, one commonly used process is genetic transferthrough the transformation process. In this process, recombinationinvolves (1) the insertion of a fragment of genetically differentDNA derived from a donor microorganism into a smallself-replicating chromosome such as a plasmid (i.e., the vector)and (2) the introduction of the recombinant plasmid into arecipient or host microorganisms where the vector will replicate.An example is the in vitro synthesis of an E. coli plasmidcontaining one gene of Drosophila. The recombinant DNA (orrecombinant plasmid) is transferred into a host E. coli cell whereit is replicated to produce many identical copies for subsequentbiochemical analyses. The various processes for construction ofrecombinant


DNA including transformation and transduction are summarized in thePrinciples of Genetics Eighth Edition by Gardner, simmons, andSnustad, 1991.

The specific molecular biology methods used in preparing andanalyzing bioengineered organisms are described in Short Protocolsin Molecular Biology Second Edition - A Compendium of Methods fromCurrent Protocols in Molecular Biology published by GreenePublishing Associates and John Wiley & Sons, 1992. The methodscompiled in this book cover the following areas:

! Escherichia Coli, Plasmids, and Bacteriophages

! Preparation and analysis of DNA

! Enzymatic manipulation of DNA and RNA

! Preparation and Analysis of DNA

! Construction of recombinant DNA libraries

! Screening recombinant DNA libraries

! DNA sequencing

! Mutagenesis of Cloned DNA

! Introduction of DNA into mammalian cells

! Analysis of proteins.

These are all important techniques that may be used inbiotechnology laboratories. For example, DNA sequencing is an

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important tool in verifying the insertion of the DNA gene into thevector, (DNA sequencing and protein analysis are discussed in moredetail in Section 5.4.) The inspector is referred to the resourceslisted above for information if s/he finds that more detailedinformation is necessary for a particular inspection

Transgenic plants

There are several methods that can be used to producetransgenic plants. These methods are described below.

Agrobacterium-based Plant Transformation

Using this method, the transfer of genetic material into theplant occurs through Agrobacterium Ti plasmid-mediatedtransformation. The Ti plasmid which is in the cytoplasm of


the bacterium is transferred into the cytoplasm of the plant'scells in an area infected with Agrobacterium. After the transfer,the new genes overproduce a plant hormone known as cytokinin. Theoverproduction upsets the plant's normal metabolism and results inthe formation of galls (i.e., tumors) that support theproliferation of bacteria in the plant walls.

This transformation method is done either with tissue explantsor protoplasts co-cultivated with A. tumefaciens cells harboringthe Ti plasmid. The easiest procedure involves co-cultivating sterile leaf discs or root sections with A. tumefaciens for a fewdays, and then transferring the inoculated explants intoselection/regeneration medium. The selection/regeneration mediacontains (1) an antibiotic that kills the Agrobacterium and (2) theappropriate antibiotic (e.g., kanamycin, hygromycin) to select forthe transformed plant cells.

The Ti plasmid can be used to transport other genes insertedinto the plasmid DNA through recombinant DNA techniques. Thistechnique has been successfully used in transforming dicotyledonousplants such as tobacco, tomato, potato, petunia, and sunflower. TheTi plasma mediated system has been used to produce insectresistance in tobacco using the Bacillus thuringiensis crystalprotein toxin gene. One disadvantage of this method is limitedrange of plants to which this method can be applied.

Particle Acceleration

This method, which is also known as particle bombardmentinvolves coating the DNA onto tiny particles that are thenaccelerated into intact plant cells with the intent of integrating

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the DNA into targeted plant cells. An example of the method is theUse of an electric discharge apparatus to propel DNA-coated goldparticles at rapidly growing soybean tissue taken from immatureseeds. The transformation is stable and the foreign genesintroduced into the soybean tissue are expressed in progeny plants.This system has several advantages:

! It only uses the DNA that the scientist wants toincorporate into the plant

! It can be used for any plant species

! It is not dependent on many parts of tissue cultureregeneration that are time and labor intensive.

Electroporation

This method uses a short pulse of high-intensity electriccurrent to disrupt cell membranes and render them temporarilypermeable to DNA molecules. The DNA molecules cross into the plantcell's cytoplasm and become part of the cell's genetic code.Electroporation requires the use of protoplasts which are singleplant cells with their walls removed through the use of enzymes. Inthis method, once the cells have been prepared, hundreds ofthousands of cells can be treated at the same time. This system hassome disadvantages compared to the other methods:


! Lengthy tissue culture manipulations are necessary andmany cells are lost in the regeneration steps

! In many cases, the transformation is short-lasting (i.e.,the gene alteration only persists for a short time).

Microinjection

This method involves the direct physical injection of DNA intothe cytoplasm or nucleus of the target cell using a specialmicromanipulator and fine glass micropipettes. This process must becarried out under a microscope and involves the use of protoplasts.This method requires considerable expertise and is labor intensive,since the DNA must be injected into each plant. cell individually.In addition, it involves considerable effort in tissue culturemanipulations.

5.3 CHARACTERIZATION, PRODUCTION, AND HANDLING OF BIOENGINEEREDMICROORGANISMS

5.3.1 CHARACTERIZATION OF BIOENGINEERED ORGANISM

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Methods

Techniques commonly used in characterizing the bioengineeredmicroorganism (DNA sequencing) and the test substance protein(protein analysis) are described in the next sections.

DNA Sequencing

Genotypic characterization of bioengineered genetic materialmay require DNA sequencing. Most DNA sequencing techniques arebased on polyacrylamide gel electrophoresis, which allows theresolution of oligonucleotide that differ in length by a singlebase. This level of resolution may be obtained betweenoligonucleotide of up to 300 to 500 bases.

Two widely used DNA sequencing techniques are dideoxy (Sanger)sequencing and chemical sequencing. These differ primarily in themechanism used to generate a "ladder" of oligonucleotide of varyinglength for use on the gel.

Dideoxy sequencing uses a DNA polymerase enzyme to synthesizea complementary copy of a single strand of the DNA template beingsequenced. A short oligonucleotide is attached to the DNA templateto serve as a primer for annealing the synthesized DNA to thetemplate. The oligonucleotide is then labeled with aradioactively-labeled base. At this stage, one protocol calls fora round of DNA synthesis before sequencing (thelabeling-termination protocol) while the other protocol does not(the Sanger protocol). The Sanger protocol is most reliable forshort oligonucleotide and the first few bases of longeroligonucleotide. The labeling-termination protocol is best forgenerating sequence information for longer oligonucleotide. At thispoint, the reaction mixture is divided into four aliquots, one eachfor the A, G, T, and C reactions. Each


aliquot receives a mixture of the bases and a single type ofdideoxy base (e.g., dATP). The DNA polymerase synthesizes acomplement until it is halted by the addition of the dideoxy base.In this way, synthesized oligonucleotide of varying length areproduced, each ending with a single type of base. Each reactionmixture is loaded on a separate lane on the polyacrylamide gel.Electrophoresis then resolves the various complementaryoligonucleotide based on length. The DNA sequence can then bedetermined by finding the lane in which each successively longeroligonucleotide appears.

Dideoxy sequencing can be done with a number of radiolabels.If radiolabeled dATP is used in the reaction mixture, 35S or 33P may

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be substituted for the standard 32P label. 35S has the advantages ofsharper autoradiographic bands, lower radiation energy, and longerstorage life of labeled DNA. 33P combines some of the advantagesof each radiolabel. Alternatively, labeling may be done through a5'-end-labeled primer. The label may be 32P or 35S. This approach hasbeen found particularly effective with large, double-stranded DNAtemplates.

Unlike dideoxy sequencing, which is achieved through DNAsynthesis, chemical sequencing (or Maxam-Gilbert) is achievedthrough cleavage of the oligonucleotide being studied. Fouraliquots of a 3'- or S'-end-labeled oligonucleotide are subjectedto four separate chemical reagents that cleave the DNA at one ortwo specific bases. The four reaction mixtures are loaded andresolved via electrophoresis in the same manner as the dideoxysequencing technique.

Dideoxy sequencing is more rapid than chemical sequencing andcan achieve excellent electrophoresis band resolution. Chemicalsequencing eliminates problems that DNA polymerases encounter withcertain DNA compositions and structures. Chemical sequencing isbest for small oligonucleotide.

Other related techniques include: substitutingchemiluminescent detection for autoradiographic detection,multiplex sequencing through the use of probes specialized for aparticular sequence (such as an insertion sequence, transposon, orpossible repeat sequence), automated sequencers, and thermal cyclesequencing (involving repeated rounds of denaturation, annealing,and synthesis to generate a sequencing ladder).

Protein Analysis

Protein identity may be determined through a number ofanalytical techniques. Among the useful techniques are amino acidcomposition analysis, partial sequence analysis, peptide mapping,polyacrylamide gd electrophoresis (PAGE), isoelectric focusing, andhigh performance liquid chromatography (HPLC).

Amino acid composition analysis involves the determination ofthe number of each amino acid present in a peptide. This type ofanalysis provides no information as to the sequence of amino acidsin the peptide. A standard procedure is to hydrolyze the peptideinto its constituent amino acids. The amino acids may then beseparated by ion-exchange chromatography or HPLC.

Reaction of the amino acids with a compound such as ninhydrin,fluorescamine, or orthophthalaldehyde yields a colored orfluorescent derivative that may be quantified via aspectrophotometer.

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The standard method for sequence analysis of peptides is theEdman degradation. This method uses sequential drivitization andidentification to determine the sequence of amino acids in thepeptide. Phenyl isothiocyanate derivitized the amino terminal ofthe peptide. The derivitized amino acid is then liberated, leavinga shortened peptide. The derivitized amino acid may be identifiedchromatographically; alternatively, the shortened peptide may beanalyzed for amino acid composition, revealing the identity of theremoved amino acid by comparison to prior composition. The entireprocedure is then repeated on the shortened peptide. Several roundsof Edman degradation will elucidate the sequence of the entirepeptide.

Since sequence analysis may be difficult on entire proteins orlengthy peptides, peptide mapping may be necessary. Cleaving theprotein into smaller peptides allows sequencing of the shortersegments. The next step is to construct a map showing how theshorter peptides fit together to form the protein. Cleavage isachieved at specific sites in the protein by chemical or enzymaticmethods. The resulting peptides are separated chromatographically.To determine the order of peptide segments, a different chemical orenzyme is used on the protein to create overlap peptides. These peptides are cleaved at different sites, and theirsequences can be used to establish the order of the first set ofpeptides.

Polyacrylamide gel electrophoresis may be used to confirm theidentity of a protein. The protein may be isolated through thistechnique and then compared to a standard protein. Severalapproaches are available to achieve this. One-dimensional gelelectrophoresis simply separates proteins based on their mobilitythrough the voltage applied across the gel. Gradientelectrophoresis adds the additional separator of a gradient ofincreasing concentration of polyacrylamide in the gel, causinglarger molecules to move slower than small ones.Isoelectricfocusing is based on the fact that proteins contain bothpositively and negatively charged functional groups.

since a protein's chargedepends on the pH, a proteinmoving across a gel that has apH gradient will cease movingwhen it reaches the pH where itis uncharged.

Proteins may also be identified through High PerformanceLiquid Chromatography. The retention time are elution volume of theunknown protein may be compared to that of a known standard, thusidentifying the unknown. Reversed-phase, ion-exchange,size-exclusion, or hydrophobic-interaction chromatography columnsmay be used to separate proteins, depending on the type of proteinsto be analyzed.

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5.3.2 PRODUCTION OF BIOENGINEERED ORGANISMS

Bioengineered Microorganisms

Propagation/Growth of the Organism


Growth of microorganisms occurs in two ways: by the growth ofindividual cells in the absence of cell division, which basicallyis an increase in the size and weight of the individual cell, andby the growth of a population of cells, which is an increase in thenumber of cells as a result of cell growth and division. sincemicroorganisms are so small and the products they generate (e.g.,proteins) are generally produced in very small quantities, studiesinvolving these substances are performed using populations ofcells. The Master Cell Bank (MCB) is a collection of the cells ofuniform composition stored in aliquots under defined conditions,from which all the subsequent cell banks are made. In many cases,a single host cell containing the expression vector is cloned togive rise to the MCB.

To achieve the desired population of organisms for the study(either to be used themselves or to be used to produce the testsubstance if it is a protein), a suitable medium for growth isnecessary. The medium for growth is chosen based on the nutrientsit provides, the generation time achieved (i.e., the time it takesfor the population to double), and the controls for purity anduniformity of product. The culture medium must be properly preparedand sterilized, and conditions during growth need to be carefullymonitored and controlled.

Bioprocess technology is used to propagate the microorganismsto produce the desired populations for the study. The termfermentation is often used interchangeably with bioprocessing,although fermentation is actually the subset of bioprocessingtechnology. that is conducted without oxygen. Such anaerobicbioprocessing uses yeasts and bacteria to yield products in theabsence of oxygen. Aerobic bioprocessing requires oxygen andbacteria to produce the desired products which are usually morecomplex than those produced by anaerobic bioprocessing. The resultsof bioprocessing are an increase in the size of the organismpopulation, the generation of proteins (including enzymes), and theproduction of secondary products such as ethanol and methane.

The first step in bioprocess technology or fermentation is toprepare a seed culture so that the organism can be propagated. Toincrease the volume of the seed culture so that a sufficient cellmass is achieved, the seed culture is incubated and transferred tosuccessively larger containers in a sterile manner. Parameters such

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as pH, oxygen, and the level of nutrients in the growth mediumshould be monitored to ensure successful growth. The seed cultureis grown in this manner until a sufficient population is producedfor introduction to a bioreactor.

A bioreactor provides a durable, controlled, asepticenvironment for growth of the bioengineered microorganism. In orderto support such growth, a bioreactor must contain sufficientnutrients and oxygen to fulfill the metabolic needs of themicroorganism. A substrate serves as the growth medium for thebioengineered microorganism in the bioreactor. Substrates containcarbohydrates in the form of sugars and refined feedstocks,starches, or cellulosic materials. Substrates are pretreated andsterilized before entry into the bioreactor and prior toinoculation with a bioengineered microorganism. Sterilization ofthe substrate can be accomplished in several ways, including batchsterilization and continuous sterilization. Continuoussterilization can consist of heat treatment, filtration,irradiation, or chemical sterilization. To meet the oxygen needs ofthe bioengineered microorganism, sterilized air is supplied to thebioreactor. The air


supply is filtered to remove other microorganisms and contaminants.If high volumes or concentrations of air are necessary, such as foracetic acid formation, the air supply is also compressed beforeentry into the bioreactor.

The laboratory should have specific protocol addressing thetype of bioreactor used [e.g., mechanical or non\mechanical (whichboth provide agitation), plug flow (which may provide agitation),and immobilization], the operating parameters (e.g., pH,temperature, mixing, oxygen concentration), and nutrients toachieve a desired generation time for microorganism growth.Operating parameters should be carefully monitored and controlledsince changes in temperature, pH, pressure, agitation, oxygen andCO2 concentration, moisture, liquid flow rate, exhaust gases, foam,and nutrients all impact the growth of the microorganism.

Growth of microorganisms occurs in four phases, identified asthe lag phase, exponential phase, stationary phase, and the deathphase. Upon inoculation of the growth medium, a period of time, thelag phase, passes before growth begins. The length of the lag phasedepends on the growth history of the inoculum, the composition ofthe medium, and the size of the inoculum. A period of exponentialgrowth follows the lag phase. During the exponential phase, thenumber resulting in no net growth. During the stationary phase,growth has slowed due to the exhaustion of an essential nutrient inthe medium and/or accumulation of a toxic metabolic product. The

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stationary phase is eventually followed by the death phase at whichpoint cell lysis or another cause of loss of cell viabilitysurpasses growth.

Separation and Purification Techniques

Once the desired amount of product is generated (either asubstance produced by the organisms, such as a protein, or theorganisms themselves), harvesting is done and the product isrecovered and purified. If the desired product is a protein orother constituent of the microorganism, the organisms may be killedby heat, chemicals, or mechanical disruption. Processing may alsoinvolve solid-liquid separation, centrifugation, filtration, celldisruption, precipitation, liquid-liquid extraction, orchromatography to concentrate or purify the desired products andfreezing or drying to facilitate handling and storage. [f themicroorganisms themselves are the desired product, filtration andcentrifugation are generally used to separate and concentrate theorganisms. Cell disruption is avoided as it negatively impactsmicrobial viability. Brief descriptions of each of these techniquesare included below. Several of these techniques may be used insuccession to achieve product recovery.

! Solid-liquid Separation. This technique may be used at severalpoints during product recovery, including to separate thebiological material from the broth in the bioreactor and toremove the cell debris after cell disruption. Separation maybe accomplished using chemical, physical, or biologicalmethods. Methods include coagulation, flocculation, and pHadjustment (chemical); heating, freezing and thawing insuccession, and freezing and stirring (physical); and the useof enzymes to achieve aggregation, and cell aging or geneinsertion to increase floccillation (biological).

! Liquid-liquid extraction. Use of this technique is generallylimited to the purification of intracellular and extracellularproducts. The method consists of selective removal of thedesired substance from a liquid mixture through the additionof a solvent (e.g.. benzene, methyl ethyl ketone) in which thedesired product is soluble. The mixture is then agitated, thesolvent/product portion is removed, and the solvent isseparated from the product through precipitation, solventevaporation, or steam stripping.

! Cell disruption. In some cases, the desired product isintracellular (i.e., it is produced within the bioengineeredmicroorganism and is not subsequently excreted into thesurrounding environment). To recover the intracellularmaterial, the cell walls must be disintegrated by physical,chemical, or enzymatic methods to release the material. Cell

disruption can be achieved through both mechanical(e.g., high speed bead/ball mills. high pressure,

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homogenizers ) and non-mechanical (e.g., enzymaticlysis, chemical lysis, heat treatment) methods;Cell disruption techniques are generally conductedat temperature below 15oF (-9oC) to preventdestruction of the desired products.

! Precipitation. Precipitation is often used as an earlypurification step for proteins. Precipitation converts asoluble protein to an insoluble form, through techniques suchas pH variation and salting-out. Although precipitation is notas effective as many other purification methods, it hascontinued to be used after the development of other moreeffective techniques because precipitation methods can handlelarge quantities of crude material in continuous operation.

! Centrifugation. This solid-liquid separation method isgenerally used to isolate viable cells from the bioreactorbroth. Intracellular and extracellular products (e.g.,proteins) are usually not heavy enough to be separated fromliquid broth through centrifugation. Separation bycentrifugation is relatively quick and free of operationalproblems compared to filtration methods, but can lead to cell denaturation andthe production of aerosols that must then be contained.

! Filtration. Filtration can be used to segregate microorganismsand intracellular and extracellular products from bioreactorbroth. There are two general types of filtration systems,membrane filtration and open air filtration systems that useplate and frame presses and rotary drum filters. Rotary drumfilters (either vacuum or pressure-operated) provide theadvantage of continuous operation, but since they are open-airsystems they are only useful when containment requirements arenot strict. Membrane filtration relies on the use of a porousmembrane to filter media of different sizes and is alsoreferred to as microfiltration or ultrafiltration depending onthe size of the pores. Membrane filtration can be used forcell harvesting, removal of cell debris, and concentration ofintracellular and extracellular products.

! Chromatography. This method can be used to separate lowconcentrations of intracellular and extracellular products,generally proteins. Types of chromatography used are affinity,hydrophobic, ion-exchange, and gel filtration. ton-exchange isoften used for protein purification and affinity forbiologically active substances. All types of chromatographyrely on physicochemical interactions between the dissolvedcomponents of a mixture and a stationary phase (a solid or aliquid supported on a solid that is contained in a packedcolumn).

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After product recovery, freezing and drying are used forpreservation of the product during storage and for ease oftransportation and handling. Freezing is generally preferredsince it does not encourage the formation of aerosols thatmust then be contained. To prevent ice crystals fromdestroying cells during thawing, either glycerol is added whenviable cells are frozen or ice crystals are sublimed duringslight warming without thawing. Spray dryers and drum dryersare used to remove water from the product and operate attemperatures low enough to avoid product damage. Freeze dryingand vacuum-tray drying are often used for small volumes ofmaterials that are in liquid or paste form.

Plants

The propagation/growth of transgenic plants and bloengineeredmicroorganisms are similar since in both cases the desired result may beproduction of the organism itself or the generation of compounds ofinterest produced by the organism. However, if the test substance is thetransgenic plant itself, production of the test substance involvesregeneration of the whole plant, as opposed to production of single-cellmicroorganisms. Compounds of interest may be produced by a cell culturesimilar to that used for microorganisms, or may be produced byregenerated cell tissue. The regeneration of cell tissue involves someof the complexities. since plant regeneration requires extensive growthand subsequent tissue variation, it is more complex than microorganismproduction.

Propagation and Harvesting of Test Substance from Plant Cells

lf the desired product is a compound of interest (e.g., a protein)generated by bioengineered plant cells, the techniques used to attainthe product are similar to those employed with microorganisms. Forpropagation/growth of the plant cells, various types of bioreactors haveproven effective. The most commonly used bioreactor is the conventionalstirred tank reactor, which is a type of mechanical bioreactor.Bioreactor technology has been successfully applied for growing largevolumes of plant cells. Growth can be achieved using either asingle-stage or two-stage process. The single stage (or first stage ofthe two-stage process) is used for the production of biomass. In thesecond stage of the two stage process, growth conditions are set toencourage generation of the desired product. The single stage process isapplicable to the production of biomass, primary metabolites, or someenzymes. The two stage process is generally used when generation of thedesired product results from tissue or organ development rather thanfrom cell growth and division.

Another technique used to generate desired products from plant cellgrowth is immobilized cell technology. With immobilized cell systems,plant cells are entrapped within a gel matrix. The technology

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immobilized plant cells continue to generate products while spent matrixis removed for product extraction. However, because of difficulties inachieving release of the product into the medium, low productivity, andgenetic instability, immobilized cell technology has not been used forcommercial applications. A third technique for generating desiredproducts from plant cells 15 the growth of regenerated plant tissue. 'This growth may be in the form of plant callus, hairy roots, or othertypes of tissue. All or parts of the tissue are then harvested forextraction of the product. In general, recovery of the desired productusually involves partial harvesting of a continuously growing culture,followed by water removal and concentration. After concentration isachieved, absorption and partition/extraction are generally used tocomplete product recovery. Product recovery &m regenerated plant tissueencounters some of the problems associated with regeneration of wholeplants (discussed below) in that a small amount of the desired productis contained in a large amount of biomass or water. However, productrecovery from plant cell biomass is less difficult than recovery fromwhole plant biomass since rigorous treatments to break down tissues andwoody, waxy structures are not needed.

Regeneration of Whole Plants

In many cases, the desired products are plants regenerated frombioengineered plant cells. New plants develop in two ways: embryogenesisand organogenesis. Organogenesis involves the successive formation ofshoots and then roots from plant cell and tissue cultures. Inembryogenesis, shoot production and root formation occur simultaneouslyin a coordinated manner. Both methods can bring about plantletformation.

Organogenesis can be used to generate whole plants from roots orshoots through clonal propagation. A cell mass or callus can be inducedto undergo organogenesis through alteration of the ratio of twohormones, auxin and cytokinin. A high auxin/cytokinin ratio inducesproduction of roots, while a low ratio induces production of shoots.Within two to three weeks, transformed callus tissue grows on the cutsurfaces of leaf disks or root sections and begins to differentiate intoshoots. The shoots are excised and placed into root-inducing medium.After root formation, the roots or shoots can be transferred to growthmedia (e.g., soil) for regeneration of whole plants. The resultingtransgenic plant is usually obtained within five to six weeks of theco-cultivating process.

Somatic embryogenesis involves replicating the process ofreproductive embryogenesis by using media to cause somatic tissue toacquire totipotency, or the ability to regenerate an entire plant.Generally, the proliferation of proembryogenic masses (a stage leadingto embryo formation) can be induced by the presence of auxin. A cultureof recurrent embryogenesis may be produced, where embryos do not maturebut instead produce successive cycles of embryos, through the use of avery high initial auxin concentration followed by maintenance on lowlevels of auxin. Active embryos may be obtained by placing these

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cultures on an auxin-free medium. Growth is then induced in a sucroseculture. Desiccation of the plantlets before growth may be necessary tocreate vigorous plants after growth. Following this growth period,plantlets are acclimatized to climatic conditions by slow, progressivereduction in humidity from their water-saturated cultures.

Waste Recovery and Decontamination Techniques

Bioengineered Organisms

Wastes generated during the production of the bioengineered testsubstance must be decontaminated before disposal. These wastes includecontaminated air and gases, as well as solid and liquid wastes. Air andgaseous waste streams are generally treated through one of the followingmethods:

! Filtration. Filtration is accomplished through eitherhigh efficiency particulate air (HEPA) filters ormembrane filters used in series to decontaminate vent orexhaust gases.

! Incineration. Incineration may be used independently oras a supplement to filtration and is generally used forsmall volume gas streams. Automatic safety devices shouldbe used with incinerators to protect against problemsresulting from power failures and overheating.

! Irradiation. Irradiation involves exposing the wastematerials to x-rays, ultraviolet rays, or other ionizingradiation to decontaminate them.

For liquid wastes, treatment is either chemical or thermal.When liquid wastes are of limited volume, chemical treatment isoften used. However, since proteins present in liquid wastes candeactivate the sterilant used in chemical treatment, thermalsterilization may be more appropriate for wastes involvingbioengineered microorganisms. Solid wastes are usually sterilizedby autoclaving and may then be incinerated.

5.4 STUDIES OF THE BIOENGINEERED TEST SUBSTANCE

Tests with microorganisms generally involve large populations,rather than a single organism. Therefore, during field tests, thebioengineered microorganisms will come in contact with one anotherand with microorganisms from the surroundings. The test system designshould incorporate containment measures to prevent contamination andhorizontal gene transfer, but complete containment cannot always beattained. Certain characteristics of the microorganism (e.g., dispersal,survival, and multiplication; interactions with other microorganisms;and potential for gene transfer), test site characteristics (e.g.,meteorological conditions, physical layout), and experimental practicesused at the test site will all impact containment of the microorganism.

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Plants

Plants used in biotechnology field studies are generallydomesticated, can be reproductively isolated, and are not likely topersist in a non-cultivated environment. Containment is affected by:

! Certain characteristics of the plant [e.g., the biology of thereproductive potential of the plant, history of controllablereproduction of the plant

! Nature of biological vectors used in transferring DNA to theplants (if this application technique is used)]

! Test site characteristics (e.g., meteorological conditions,physical layout-proximity of plants to one another) and

! Experimental practices used at the test site.

Vector Considerations

When biological vectors are used to create transgenic plants,measures should be taken to ensure that the vector does not remaincapable of acting as an infectious agent. The vector should eitherbecome biologically inactive or be eliminated from the transgenicplant. If the vector does not naturally become biologicallyinactive, it is necessary to either eliminate the vector from theplant or inactivate it after the transformation of the plant hasoccurred. To assist with this process, the DNA used in developingthe transgenic plant should possess the following characteristics:

! thoroughly characterized and unlikely to be transmittedafter entering the plant

! Its donor plant should be of the same or a closelyrelated species as the recipient plant

! Transferred from non-pathogenic prokaryotes ornon-pathogenic lower eukaryotic plants

! Come from plant pathogens only if the DNA sequence forproduction of disease or damage in plants has beenremoved.

Field site Considerations

Field experiments should be designed so that plants arereproductively isolated from sexually compatible plants outside thetest site, bioengineered microorganisms are not released into theenvironment outside the test site, and the plants used will notcause unintended, uncontrolled adverse effects.

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Some current experimental practices that are used to maintainreproductive isolation in plants are the following:

! Spatial Separation. The distance the field should be fromany field containing plants of the same species (i.e.,sexually compatible plant populations) depends on thebiology of the plant species involved. Self-pollinatedspecies with fragile pollen do not need to bewidely-separated to achieve reproductive isolation.However, open pollinated species that have hardy pollenmay need to separated by more than several miles toachieve reproductive isolation.

! Removal of Reproductive structures. For some dioeciousplants, removing the male or female reproductivestructures may preserve reproductive isolation in plantsgrown in close proximity to compatible plants. Forexample, mechanical detasseling in seed corn productionremoves the pollen-producing male flowers and preventsgenetic transfer.

! Use of Cytoplasmic Male Sterility Trait. By incorporatingthis trait into a plant, production of viable pollen isprevented and the plant is biologically andreproductively isolated.

Examples of Methods for Reducing Population Levels of Microorganisms in theEnvironment4

Habitat Immediate1 Short-term2 Long-term3

Free-Living

FumigationFloodingChemicals

FumigationFloodingChemicals

Erosion ControlSoil Amendments

FumigationFlooding

Erosion ControlSoil Amendments

Plants Burning (Eradication)QuarantineTillage

ChemicalsBiological ControlIrrigation/flooding

Insect vector controlMachinery sanitationRun-off water control

Solarization (cover withplastic

Breeding forresistance

Biological controlQuarantineChemicals

Crop rotation Cultivar rotation

Irrigation/floodingHeat treatment

Soil solarizationInduced resistanceMeristem/tissue

cultureInsect vector control

Weed/natural hostcontrol

Erosion control

Breeding forresistance

Biological ControlCrop rotation

Cultivar rotationSoil amendments

Weed/natural hostcontrol

Erosion control

1 Treatment effective within hours to several days.2 Treatment effective within weeks to 3 years.3 Treatment effective after more than 3 years.4 Table is from Good Developmental Practices for Small Scale Field Research with

Genetically-modified Plants and Micro-organisms, Organization for EconomicCo-operation and Development, March 1990, as taken from the Annual Review ofPhytopathology. 27:551-581.

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! Temporal Isolation. By growing the plants at a time that willproduce flowers either earlier or later than those forcompatible plants in close proximity, genetic transfer andpollen dissemination is limited or eliminated. Dispersion ofpollen can also be eliminated by placing bags or otherphysical barriers over plant flowers.

! Early Harvesting. If seed is not required for the field test,plants might be harvested before flowering. This method canachieve reproductive isolation in plants that are difficult toisolate (e.g., plants that are insect-pollinated).

Application Issues

The equipment used in applying the test substance for the studyshould be similar to equipment that would be used in real worldapplications so that effects related to application can be simulated.Methods of application include aerial, irrigation system, directedsprays, and subsurface soil application. Some specific considerationsfor each application method are discussed below:

! Aerial Application. Many variables can affect the efficacy ofa test substance or increase damage to crops/plants in thetarget area when aerial application is used. These variablesinclude dosage, spray volume, maximum height from nozzles totarget, type of aircraft (fixed wing or helicopter)~ groundspeed, nozzle type and pressure, wind velocity and direction,and relative humidity. Therefore, field efficacy andcrop/plant phytotoxicity data should be generated to supportclaims to be made on the proposed label.

! Irrigation System Application. When an irrigation system isused, data on efficacy and crop/plant phytotoxicity, yield,and quality should be developed for multiple plots within atreated field so that pesticide distribution can be evaluated.In order to make such evaluations, data taken for theindividual plots should be reported separately rather thanaveraged together. Data should also include soil texture,percent soil organic matter, relative soil moisture conditionat application, acre-inches of water applied, andprecipitation quantities in the week after application. If anoverhead sprinkler irrigation system is used, test plotsshould be situated at several nozzle positions, at the extremeends of the spray area, and in areas where sprinkler patternsoverlap. If a surface irrigation system (flood and furrow) isused, test plots should be located both where the treatedwater enters the field and at the lower end of the field.

! Directed Sprays. In a directed spray system, sprays aredirected toward or away from certain sections of the testplot. When this type of application method is used, the study

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should include an indication of nozzle arrangements andorientations, and the extent of spray contact with soil orplants.

! Subsurfase Soil Application. Subsurface soil applicationinvolves the application of the test substance directlybeneath the soil surface. If this application is used, studydata should include detailed information on equipment. Forexample, for injection equipment, the nozzle spacing, depth ofoperation, nozzle pressure, speed of operation, and volume ofliquid or gas applied per unit area should be recorded.

Analysis of Study Control Plots

To make efficacy determinations, the results derived from testplots treated with the test substance should be compared with the dataobtained from evaluating untreated control plots/plants. The controlplot(s) should receive the same treatments as the test plots, except forapplication of the test substance. Similar treatments should includeequal application of pesticides (other than the test substance),irrigation, tilling, and meteorological conditions. The data collectedand analysis for both the test plots and the control plots should be thesame. If the test substance is a herbicide, both test and control plotsshould be evaluated for weed species present, density of weedpopulations, and weed vigor.

5.5 FACILITIES AND EQUIPMENT

5.5.1 FACILITY DESIGN

The design of a facility that handles infectious agentsprovides secondary containment to protect persons and theenvironment outside the laboratory from exposure to infectiousmaterials. There are three types of laboratory designs that providedifferent levels of containment. The type of laboratory that isappropriate for a particular biotechnology study is generallydetermined by the nature of the study and the degree of biosafetynecessary. The inspector should review the study protocol todetermine what level of biosafety and type of laboratory wereintended for the study. To assess the appropriateness of theseelements, the inspector should use the information below (developedfrom the CDC-NIH guidelines) and further refer to the informationsupporting the guidelines in Biosafety in Microbiological andBiomedical Laboratories (May 1988). The three types of laboratoriesare described below:

! Basic Laboratory. These facilities are generallyappropriate for Biosafety Levels I and 2 (describedbelow). Basic labs are also used for biotechnologystudies where there is a minimal level of hazard andstudy personnel can achieve sufficient protection from

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the implementation of standard laboratory practices. Theorganisms used in the study are not associated withdisease in healthy adults.

! Containment Laboratory. Containment labs qualify asBiosafety Level 3 facilities. These facilities aredesigned with protective features to allow for thehandling of hazardous materials in a way that preventsharm to the study personnel and the surrounding personsand environment. Containment labs may be freestandingbuildings or segregated portions of larger buildings, aslong as the laboratory is separated from public areas bya controlled access zone. Containment labs also have aspecialized ventilation system to regulate air flow.

! Maximum Containment Laboratory. These laboratories areBiosafety Level 4 facilities. Maximum containment labsare designed to provide a safe environment for carryingout studies involving infectious agents that pose anextreme hazard to laboratory personnel or maycaus¢,serious epidemic disease. These facilities havesecondary barriers, including sealed openings into thelab, air locks, a double door autoclave,- a separateventilation* system, a biowaste treatment system, and aroom for clothing change and showers that adjoins thelab. Maximum containment labs are usually located inindependent buildings, but may also be in a separate,isolated portion of a larger building.

The four bio-safety levels referred to in the above laboratorydescriptions consist of three elements: laboratory practices andtechniques, safety equipment, and laboratory facilities. The firsttwo elements are considered primary containment since they provideprotection within the laboratory to personnel and the immediateenvironment. The third element, the design of the facility itself,is considered secondary containment since it gives protection topersons and the environment outside of the facility. Importantcharacteristics of each of the biosafety levels are summarizedbelow. Unless superseded by a more stringent criterion, thecharacteristics of a biosafety level apply to all successive levels(from I to 4). These characteristics, along with the training andexperience of the study personnel and any special conditions in theoperation of the laboratory or the organisms involved, should beconsidered in determining an appropriate biosafety level. For moredetailed descriptions of the biosafety levels, the inspector isreferred to the CDC-NIH guidelines, which can be obtained from theScientific Support Branch on request.

! Bio-safety Level 1. The organisms involved in the studyare defined and characterized strains of microorganismsthat are of minimal hazard and are not known to causedisease in healthy human adults. Although access to the

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lab may be restricted by the lab director, the facilityis generally not closed off from the rest of thebuilding. Most work is conducted on open bench tops andspecial containment equipment is usually not needed. Thelab is designed to facilitate cleaning, with spacebetween equipment and cabinets and bench tops that areimpervious to water and resistant to solutions. Eachlaboratory has a hand washing sink and fly screens forany windows that open. Lab personnel should beknowledgeable in lab procedures and supervised by ascientist trained in microbiology or a related science.Decontamination of work surfaces should be done daily andafter spills, and all contaminated wastes should bedecontaminated before disposal. Personal safetyequipment, such as lab coats or uniforms, should be wornand hands washed before and after handling viablematerials. Procedures are performed in a manner thatlimits the creation of aerosols. Any contaminatedmaterials that will be decontaminated at another locationare transported in a durable leak proof container that issealed before removal from the lab.

! Bio-safety Level 2. Work done under Biosafety Level 2involves organisms of moderate potential hazard. Many ofthe characteristics of this level are the same as thosefor Bio-safety Level 1; the differences and additionalguidelines are indicated here. For Bio-safety Level 2,laboratory access is limited while work is beingconducted and only persons informed of the potentialhazards of the lab environment and meeting any otherentry restrictions developed by the lab director areallowed entry. Biological safety cabinets (Class I or II),are used for containment when procedures with a highpotential for treating infectious aerosols (e. g.,centrifuging, blending) are conducted and when h I e hconcentrations or large volumes of infectious agents areused. (The inspector is referred to Appendix A of theCDC-NIH guidance for a description of bio-safety cabinetclassifications.) Lab personnel are trained in handlingpathogenic agents and are under the direction of skilledscientists. An autoclave is available for Use indecontaminating infectious wastes. Before leaving thelab, personnel either remove any protective clothing andleave it in the lab or cover it with a clean coat. Skincontamination with infectious materials is avoided andgloves worn when such contact is unavoidable. Spills andaccidents causing overt exposure to infectious materialsare reported promptly to the lab director/Safety Officerand appropriate treatment provided and records of theincident maintained. If warranted by the organisms at Usein the lab, baseline serum samples for all at-riskpersonnel are collected and stored. A bio-safety manual

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is developed and personnel are required to be familiarwith it and to follow its instructions.

! Bio-safety Level 3. Work done under Biosafety Level 3conditions generally occurs in clinical, diagnostic,teaching, research or production facilities and involvesorganisms that may cause serious or potentially lethaldisease following exposure through inhalation. The lab issegregated from general access areas of the building andtwo sets of self closing doors must be passed through toenter the lab from access hallways. Lab access is limitedto persons who must be present for program or supportfunctions and lab doors remain closed during experiments.All work with infectious materials is conducted in a bio-safety cabinet (Class I, II or III) or other physicalcontainment device or by personnel wearing the necessarypersonal protection clothing. Upon completing work withinfectious materials, all work surfaces aredecontaminated. Protective clothing is worn in thelaboratory and is removed before exiting the facility.All such clothing is decontaminated before laundering.Vacuum lines are protected with high efficiencyparticulate air (HEPA) filters and liquid disinfectanttraps. The HEPA-filtered exhaust air from Class I or IIbio-safety cabinets is discharged directly to the outsideor through the building exhaust system, but may berecirculated within the lab if the cabinet isappropriately certified and tested. A ducted exhaust airventilation system that draws air into the lab throughthe entry areas is in place. Walls, ceilings, and floorsare water resistant to facilitate cleaning and windowsare closed and sealed. The lab sinks are operable byfoot, elbow or automation and are located near the exitof each lab room.

! Bio-safety Level 4. This safety level is necessary forwork with organisms that present a high individual riskof life-threatening disease. These facilities are locatedin a separate building or in a completely segregated,controlled area of a building. Access to the facility iscontrolled by the use of locked doors and all personnelentering must sign a logbook. All personnel must enterand leave the facility through the clothing change andshower rooms and must shower before exiting. Any suppliesor materials that do not enter through the shower andchange rooms must enter through a double-door autoclave,fumigation chamber, or airlock that is decontaminatedbetween each use. All organisms classified as BiosafetyLevel 4 are handled in Class III bio-safety cabinets orin Class I or II bio-safety cabinets used in conjunctionwith one-piece positive pressure personnel suitsventilated by a life support system. All biological

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materials removed from a Class III cabinet or maximumcontainment lab in a viable condition are placed in annonbreakable, sealed primary container and then enclosedin a secondary container that is removed through adisinfectant dunk tank, fumigation chamber, or anairlock. All other materials must be autoclaved ordecontaminated before removal from the facility. Walls,floors, and ceilings of the facility together form asealed internal shell and any windows are resistant tobreakage. A facility is available for the quarantineisolation. and treatment of personnel with potential orknown lab-related illnesses.

5.5.2 EQUIPMENT

Studies involving bioengineered microorganisms will use many typesof equipment and instruments in the development, application, testing,transportation, and decontamination procedures that are part of abiotechnology study. While some of this equipment is common to othertypes of scientific studies, the specialization of biotechnologynecessitates the use of specific equipment. A description of many of thespecial equipment and instruments is included below to provide theinspector with the additional expertise necessary to conduct a thoroughbiotechnology inspection.

! Biosafety Cabinets. These cabinets are a commonly used primarycontainment device for work involving infectious organisms. Theirprimary function is to protect the lab worker and the immediateenvironment by containing any infectious aerosols produced duringmanipulation of organisms within the cabinet. Biosafety cabinetsare classified into three types (I, II and III) based on theirperformance characteristics. Class I and II cabinets areappropriate for use with moderate and high-risk microorganisms.These cabinets have an inward face velocity of 75 linear feet perminute and their exhaust air is filtered by HEPA filters. The ClassI cabinet can be-used with either a full-width open front, aninstalled front closure panel, or an installed front closure panelequipped with arm-length rubber gloves. The Class II cabinet is avertical laminar-flow cabinet with an open front. In addition tothe protection provided by the Class 1 cabinet, the Class IIcabinet protects materials inside the cabinet from extraneousairborne contaminants since the HEPA filtered air is recirculatedwithin the work space. The Class III cabinet is a totally enclosedventilated cabinet that is gas tight. Class III cabinets are usedfor work with infectious organisms. Work in a Class III cabinet isconducted through connected rubber gloves. The cabinet ismaintained under negative pressure with supply air drawn in throughHEPA filters and exhaust air filtered by two HEPA filters. Theexhaust air is discharged to outside the facility using an exhaustfan that is generally separate from the facility's overall exhaustfan. Each of the cabinet types is only protective if it is operatedand maintained properly by trained personnel.

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! Organism Preparation. In developing the bioengineered testsubstance, commonly used laboratory equipment include cultureplates, roller bottles, shake flasks, and seed fermenter Thesedevices are used to bring the organism from its origination inthe master cell bank through its preparation forgrowth/propagation. For work with bioengineeredmicroorganisms, the organism preparation system is generallycontained in a biosafety cabinet.

! Bioreactors. The main purpose of a bioreactor is togrow/propagate a microorganism in a controlled, asepticenvironment. Bioreactors come in various sizes and four basicdesigns (mechanical, non-mechanical, plug flow, andimmobilization). The most popular type is the mechanicalfermenter which uses mechanical stirrers to agitate theorganism. One of the most commonly used mechanical fermentersis the stirred tank fermenter (STF). Agitation is provided bycompressed gas or pumped liquid in non-mechanical fermenters.Plug flow reactors are usually tubular reactors that may Usedirect agitation. Immobilization reactors foster growpropagation on a permanent solid substrate. In order tosatisfy the metabolic requirements of the microorganism,aeration must be adequate to provide sufficient oxygen. Thosebioreactors using agitation need to be designed to maintain auniform environment within the bioreactor.

! Bioreactor Control. To control the metabolic processes withinthe bioreactor, facilities should have adequate monitoring andcontrol equipment. There are three classes of such monitoringsystems, off-line, on-line, and in-line. These systems trackparameters such as pH, temperature, and agitation and aerationrates within the bioreactor. For off-line systems, a sample istaken from the bioreactor at specified intervals andchemically analyzed using automated laboratory instruments.since off-line systems can have a lengthy turnaround time foranalytical results and do not provide a high level ofcontainment, they are not recommended for work withbioengineered microorganisms. For on-line systems, samplingand analysis are done continuously and often provideadditional secondary containment. In-line systems, through theuse of probes, sensors, and sampling devices that directlycontact the material, provide a continuous, non-invasiveindication of bioreactor conditions.

! Air Compressors and Sterilizers. Since most bioprocessing isaerobic; proper growth and production of the organism requiresthat oxygen be drawn into the bioreactor. To preventcontamination of the organism, the air needs to be compressedand sterilized before it enters the bioreactor. Compressorsgenerally used are either dynamic/centrifugal or positivedisplacement in design. To remove undesirable organisms and

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particles from the air, the compressed air is circulatedthrough filters before entering the bioreactor

! Product Recovery. To separate and concentrate the desiredfinal product from the contents of the bioreactor, a productrecovery or purification system is required. Such systemsinclude centrifugation, cell disruption, broth conditioning,filtration, extraction. chromatography, and drying/freezingtechniques. The type of equipment at a particular studyfacility depends on the type(s) of product recovery system(s)employed at the study facility. Types of centrifuges that areused to separate viable cells from liquid broth includebatch-operated solid bowl machines, semi-continuoussolids-discharging disc separators, and continuous decanters(decanter centrifuges). Types of batch centrifuges include thesolid-bowl disc centrifuge, one-chamber centrifuges (used forprotein fractionation from blood plasma), zonal centrifuges(used to separate intracellular and extracelluar products suchas virus purification or cell constituent isolation), and tubecentrifuges (used to separate liquid phases). Safety cabinetsmust be used during solids removal from batch centrifuges.Generally, semi-continuous solids-discharging machina providethe best containment and are the most widely used type forbiotechnology applications. Cell disruption is used to recoverintracellular products and can be performed using mechanicalor non-mechanical methods. Mechanical methods include ballmills and high speed homogenizers, non-mechanical methodsinclude chemical or enzymatic Iysis, heat treatment,freeze-thaw, and osmotic shock. Non mechanical methods areeasily contained and are most often used in laboratories.Broth conditioning can take place within the bioreactor (whenan STF is used) or in a separate vessel (if bioprocessing isperformed in an unstirred vessel). Filtration units are usedto separate cellular, intracellular or extracellular solidsfrom broth. Types of filtration units include continuousrotary drums, continuous rotary vacuum filters, and tangentialflow filtration systems using either microporous orultrafiltration membrane filters). The type of filtration unitused is dependent on the type of product being recovered.Affinity and gel filtration chromatography processes are usedto purify intracellular or extracellular products. Productsare purified using an eluting solvent in a packed column andare collected in a fraction collector. If adequate containmentis provided (e.g., biological safety cabinet), productrecovery using chromatography can be used to purify hazardousorganisms. Types of liquid-liquid extraction equipmentincludes centrifugal extractors (used to extract biotechnologyproducts), spray and packed towers, mechanically agitatedcolumns, pulse columns, or mixer-settler units. Eitherfreezing or drying may be used to facilitate handling andstorage of products. Organisms to be frozen are placed invials (performed in a biosafety cabinet) and frozen. The most

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common types of dryers used are freeze dryers and vacuum traydryers. since freezing provides primary containment andproduces less aerosols than dryers, it is more appropriate forproduct storage. If drying is performed, proper filtration andventilation systems must be provided.

! Waste Recovery and Decontamination. The primary method ofdecontaminating exhaust gases mixed with liquid broth, isthrough the use of filters. Before filtration, the mixturemay be passed through a condenser, a coalescing filter, and aheat exchanger. Filtration is then done using pairs of HEPA ormembrane filters. Another method of decontaminating air andgaseous waste streams is thermal destruction or incineration.Incineration may be used alone or in addition to filtration.Liquid wastes can be decontaminated through chemical or heattreatment. However, for large volumes of liquid wastes and forwastes containing genetically engineered microorganisms, heattreatment is generally preferred. Solid wastes (e.g.,microbial cultures, cell debris, glassware, protectiveclothing) is generally decontaminated by autoclaving, and ifnecessary, followed by incineration. To decontaminatelaboratory devices exposed to bioengineered substances, themost common practice is the use of pressurized steam thatcontains an appropriate chemical. For heat-sensitiveequipment, such as electronic instruments, decontamination isgenerally achieved through chemical sterilization orirradiation. Gaseous sterilants are applied by a steam ejectorthat sprays down from overhead. If decontamination by steam,liquid, or gas sterilization is not possible, ionizing orultraviolet radiation is used. However, since irradiationmethods do not always inactivate all types of microbes, steamor gaseous chemical sterilization should be used for devicescontaminated with bioengineered organisms.

! Other Common Types of Equipment. Other common types ofequipment used in laboratories performing biotechnologystudies are as follows:

- Autoclave to sterilize equipment, wastes, etc.

- Freezers, refrigerators, water baths and incubators forincubation and storage. The operating temperature of thisequipment 15 dependent on the type of organism beinggrown

- Heating blocks for conducting enzymatic reactions in testtubes or micro-centrifuge

- Geiger counter and/or scintillation counter for measuringradioactivity

- Magnetic stirrers with heaters

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- Microwave oven for agar and agarose melting

- Visible and UV spectrophotometer

- Water purification equipment

- Vacuum desiccator/lyophilizer.

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Glossary of Disciplines

Agricultural Science Discipline dealing with the selection,breeding, and management of crops and domesticanimals

Agronomy The principles and procedures of soilmanagement and of crop improvement,management, and production

Analytical Chemistry Branch of chemistry dealing with techniquesthat yield any type of information aboutchemical systems

Biochemistry The study of chemical substances occurring inliving organisms and the reactions and methodsfor identifying these substances

Bioengineering The application of engineering knowledge tothe fields of medicine and biology

Biology A division of the natural sciences concernedwith life and living organisms

Botany A branch of the biological sciences thatstudies plants and plant life

Chemical Engineering The branch of engineering dealing withchemically converting basic raw materials intoproducts

Chemistry The scientific study of the properties,composition, and structure of m a t t e r , t h echanges in structure and composition ofmatter, and accompanying energy changes

Entomology The scientific study of insects

Enzymology Branch of science dealing with the chemicalnature, biological activity, and biologicalsignificance of enzymes

Forestry The science that deals with the development,maintenance, and management of forests

Formulation Chemistry Branch of chemistry dealing with theparticular mixtures of base chemicals andadditives required for products

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Genetic Engineering The intentional production of new genes andalteration of genomes by the substitution oraddition of new genetic material

Genetics The science concerned with the study ofbiological inheritance

Histology The study of the structure and chemicalcomposition of animal and plant tissues asrelated to their function

Immunology Branch of biology concerned with the native oracquired resistance of higher animal forms andhumans to infection With microorganisms

Microbiology The science and study of microorganisms,including protozoans, algae, fungi, bacteria,viruses, and rickettsiae

Molecular Biology Part of biology that attempts to interpretbiological events in terms of thephysicochemical properties of molecules in acell

Mycology The branch of botany that deals with the studyof fungi

Organic Chemistry The study of the composition, reactions, andproperties of carbon-chain or carbon-ringcompounds and mixtures

Pathology The study of the causes, nature, and effectsof diseases and other abnormalities

Pharmacology The science dealing with the nature andproperties of drugs, particularly theiractions

Physical Chemistry The branch of chemistry that deals with theinterpretation of chemical phenomena andproperties in terms of the underlying physicalprocesses, and with the development oftechniques for their investigation

Taxonomy The study of the classification of organismsthat best reflects the totality ofsimilarities and differences

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Toxicology The study of poisons, including their nature,effects, and detection, and methods oftreatment

Virology The branch of biology dealing with the studyof viruses

Zoology The science that deals with knowledge ofanimal life

STANDARD OPERATING PROCEDURE - United States … · This standard operating procedure will be used when inspecting ... Biology of Microorganisms, Third Edition ... (May 1988). Health

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