`
`CENTER FOR DRUG EVALUATION AND RESEARCH
`
`Guidance for Industry
`
`The FDA published Good Guidance Practices in February 1997.
`This guidance was developed and issued prior to that date.
`
`Additional copies are available from:
`Office of Training and Communications
`Division of Communications Management
`Drug Information Branch, HFD-210
`5600 Fishers Lane
`
`Rockville, MD 20857
`
`(Tel) 301 —82 7-45 73
`(Internet) http://www.fda. gov/cder/guidance/index. htm
`
`US. DEPARTMENT OF HEALTH AND HUMAN SERVICES, FOOD AND DRUG ADMINISTRATION
`
`Page 1
`
`CUBIST 2203
`
`AGILA V. CUBIST
`IPR2015-00143
`
`CUBIST 2203
`AGILA v. CUBIST
`IPR2015-00143
`
`

`

`GUIDELINE ON
`
`VALIDATION OF THE LIMULUS AM EBOCYTE LYSATE TEST.
`AS AN END-PRODUCT ENDOTOXIN TEST FOR HUMAN
`AND ANIMAL PARENTERAL DRUGS, BIOLOGICAL PRODUCTS, AND
`MEDICAL DEVICES
`
`US. DEPARTMENT OF HEALTH AND HUMAN SERVICES
`PUBLIC HEALTH SERVICE
`FOOD AND DRUG ADMINISTRATION
`
`Page 2
`
`Page 2
`
`

`

`GUIDELINE ON
`VALIDATION OF THE LIMULUS AMEBOCYTE LYSATE TEST
`AS AN END—PRODUCT ENDOTOXIN TEST FOR HUMAN
`
`AND ANIMAL PARENTERAL DRUGS, BIOLOGICAL PRODUCTS, AND
`MEDICAL DEVICES
`
`December 1987
`
`Prepared by: Center for Drug Evaluation and Research
`Center for Biologic Evaluation and Research
`Center for Devices and Radiological Health
`Center for Veterinary Medicine
`
`Maintained by: Division of Manufacturing and Product Quality (HFN—BZO)
`Office of Compliance
`Center for Drug Evaluation and Research
`Food and Drug Administration
`5600 Fishers Lane
`
`Rockville, MD 20857
`
`Page3
`
`Page 3
`
`

`

`CONTENTS
`
`INTRODUCTION
`
`I.
`
`Background ...... ..... ..................... . ................ .....1
`
`II.
`
`Legal Effect ........................... . ........................ 3
`
`III. Regulatory Requirements ......................................... 4
`
`IV.
`
`Human and Animal Drugs and Biological Products .................. 6
`
`A. Validation of the LAL Test ................................... 6
`
`B. Testing of Drugs by the LAL Test ............................. 8
`
`V.
`
`Medical Devices...... .......................................... 11
`
`A. Validation of the LAL Test.... .......................... ....11
`
`B. Testing of Devices by the LAL Lest .......................... 14
`
`VI.
`
`Appendices ................... . ......... . ....... . ......... . ..... l6
`
`A. Initial Quality Control ..................................... 17
`
`B. USP XXI/NF XIV Bacterial Endotoxins Test .................. ..18
`
`C. Determination of the Relationship Between the Control
`
`Standard Endotoxin and the Reference Standard Endotoxin ..... 19
`
`D. Maximum Valid Dilution Calculation .......................... 22
`
`E. Maximum Dose and Endotoxin Limit Table ...................... 25
`
`Page4
`
`Page 4
`
`

`

`INTRODUCTION
`
`This guideline sets
`
`forth acceptable conditions
`
`for use of
`
`the
`
`Limulus Amebocyte Lysate
`
`test.
`
`It also describes procedures
`
`for
`
`using this methodology as an end—product endotoxin test
`
`for human
`
`injectable drugs (including biological products),
`
`animal
`
`injectable
`
`drugs,
`
`and medical devices.
`
`The procedures may be used in lieu of
`
`the rabbit pyrogen test.
`
`For
`
`the purpose of
`
`this guideline,
`
`the terms "lysate" or "lysate
`
`reagent"
`
`refer only to Limulus Amebocyte Lysate licensed by
`
`the
`
`Center
`
`for Biologic Evaluation and Research.
`
`The
`
`term "official
`
`test" means
`
`that
`
`a
`
`test
`
`is
`
`referenced
`
`in
`
`a United States
`
`Pharmacopeia drug monograph, a New Drug Application, New Animal Drug
`
`Application or a Biological License.
`
`PageS
`
`Page 5
`
`

`

`i.
`
`BACKGROUND
`
`FDA announced that Limulus
`In a notice of January 12, 1973 (38 FR 1404),
`circulating
`blood
`cells
`Amebocyte
`Lysate
`(LAL),
`derived
`from
`(amebocytes) of the horseshoe crab,
`(Limulus polyphemus),
`is a biological
`product.
`As such,
`it
`is subject
`to licensing requirements as provided in
`section 351 of
`the Public Health Service Act
`(42 U.S.C. 262).
`Since
`1973,
`LAL has proved to be
`a sensitive indicator of
`the presence of
`bacterial
`endotoxins
`(pyrogens).
`Because
`of
`this
`demonstrated
`sensitivity, LAL can be of value in preventing the administration or use
`of products which may produce fever, shock, and death if administered to
`or used in humans or animals when bacterial endotoxins are present.
`
`available data and
`1973 notice was published,
`the January 12,
`When
`its adoption. as
`the
`experience with IJU. were not adequate to support
`final pyrogen test
`in place of
`the rabbit pyrogen test, which had been
`accepted and recognized for many years.
`In order
`to establish.
`a data
`base and gain experience with the use of LAL,
`that notice permitted the
`introduction of LAL into the marketplace without
`a
`license.
`This was
`upon the condition that its use be limited to the in—process testing of
`drugs
`and other products,
`that
`the decision to
`use
`it
`be
`reached
`voluntarily by affected firms,
`and the labeling on. LAL state that
`the
`test was not suitable as a replacement for the rabbit pyrogen test.
`
`time, production techniques have been. greatly improved and
`Since that
`standardized so
`that
`they consistently yield LAL with an
`endotoxin
`sensitivity over 100 times greater
`than originally obtained. Moreover,
`it is widely recognized that
`the LAL test is faster, more economical, and
`requires a smaller volume of product
`than does
`the rabbit pyrogen test.
`In addition,
`the procedure is less labor intensive than the rabbit test,
`making it possible to perform many tests in a single day.
`
`In a notice published in the Federal Register of November 4, 1977 (42 FR
`57749),
`FDA described conditions for
`the use of LAL as an end—product
`test
`for endotoxins
`in. human biological products
`and medical devices.
`The notice stated further that
`the application of LAL testing to human
`drug products would
`be
`the
`subject
`of
`a
`future Federal Register
`publication.
`
`for Devices and
`now FDA's Center
`then Bureau of Medical Devices,
`The
`for
`the use of
`Radiologic Health (CDRH),
`issued recommended procedures
`LAL testing as an end—product endotoxin test on March 26, 1979.
`These
`procedures were
`revised as
`a
`result
`of
`the
`comments
`received
`from
`interested parties .
`
`result of CDRH'S experience in approving petitions for the
`a direct
`As
`LAL
`test
`in place of
`the
`rabbit pyrogen
`test,
`several
`use of
`the
`procedures for using the LAL test have evolved and have been adopted for
`devices.
`
`FDA announced
`In the FEDERAL REGISTER of January 18, 1980 (45 FR 3668),
`the availability of
`a draft guideline that set
`forth procedures for use
`of the LAL test as an end~product
`testing method for endotoxins in human
`and animal
`injectable drug products. This draft guideline was made
`_ l ,
`
`Page6
`
`Page 6
`
`

`

`available to interested parties to permit manufacturers, especially those
`who had used the LAL test
`in parallel with the rabbit pyrogen test,
`to
`submit data that could be considered in the preparation, of
`any final
`guideline.
`
`FDA
`received on the January 18 draft guideline,
`In response to comments
`and
`changes
`(i.e. Endotoxin
`limits
`changed
`made
`several
`significant
`deletion of section on. Absence of Non—endotoxin Pyrogenic Substances),
`and many minor editorial changes.
`The agency also determined that
`a
`single document
`should be made available 'covering all
`FDA regulated
`products that may be subject
`to LAL testing.
`Primarily because of
`the
`addition of biological products and medical devices to the guideline,
`the
`the agency made,
`in the FEDERAL REGISTER of March 29, 1983 (43 FR 13096),
`another draft of the guideline available for public comment.
`
`FDA has
`Based on the comments received on the March 29 draft guideline,
`made
`several
`changes
`in this final guideline.
`The
`comments used in
`support of
`these
`changes may be viewed at
`FDA's Dockets Management
`Branch, Room 4—62, 5600 Fishers Lane, Rockville, MD between 9 am and 4 pm
`Monday through Friday. Briefly,
`the significant changes made are:
`
`A.
`
`chromogenic,
`the
`for
`criteria
`validation
`of
`Inclusion
`endpoint—turbidimetric and kinetic-turbidimetric LAL techniques.
`
`can be
`turbidimetric)
`chromogenic or
`B. Any technique (gel—clot,
`for endotoxin. However, if a gel—clot
`used in testing a product
`lysate is used in.
`a different
`technique the results must be
`interpreted using the criteria for the technique being used.
`
`requirement
`the
`C. Elimination of
`rabbit pyrogen testing colony.
`
`to test
`
`the sensitivity of
`
`a
`
`D.
`
`has
`(CDRH)
`and Radiological Health
`for Devices
`The Center
`adopted the USP Endotoxin Reference Standard and revised the
`limit
`expressions
`from ng/mL to EU/mL.
`The
`new limit
`for
`medical devices is 0.5 EU/mL except for devices in contact with
`cerebrospinal
`fluid for which the limit
`is 0.06 EU/mL.
`These
`limits for devices are equivalent
`to those for drugs for a 70 Kg
`man when consideration is given to the following:
`
`1.
`
`in
`In the worst case situation, all endotoxin present
`the combined rinsings of 10 devices could have come from
`just
`one device.
`A wide variation in bioburden
`is
`common to some devices.
`
`2. Published FDA studies
`
`indicate that
`
`less than half of
`
`recovered
`is
`endotoxin
`added
`non—pyrogenic water rinse.
`
`from devices
`
`using
`
`a
`
`E.
`
`for Drug Evaluation and Research (CDER) has added a
`The Center
`the maximum doses
`per
`Kg
`per
`hour
`and
`the
`listing of
`endotoxin
`limits
`for most
`the
`aqueous
`corresponding
`of
`injectable drugs
`and biologics currently on.
`the market.
`This
`listing was added to promote uniformity among
`companies making
`the same product.
`
`Page7
`
`Page 7
`
`

`

`II.
`
`LEGAL EFFECT OF THE GUIDELINE
`
`(21 CFR lO.90(b)) of
`section lO.90(b)
`issued under
`This guideline is
`for use of guidelines
`FDA's administrative regulations, which provides
`to outline procedures or standards of general applicability that are
`acceptable to FDA for
`a subject matter within its statutory authority.
`Although guidelines are not
`legal
`requirements,
`a person who follows an
`agency guideline may be assured that
`the procedures or standards will be
`acceptable to FDA.
`The following guideline has been developed to inform
`manufacturers of human drugs
`(including biologicals), animal drugs,
`and
`medical devices of procedures FDA considers necessary to validate the use
`of LAL as an end—product endotoxin test.
`A manufacturer who adheres
`to
`the guideline would be considered in compliance with relevant provisions
`of
`the applicable FDA current good manufacturing practice regulations
`(CGMP)
`for drugs
`and devices
`and other applicable
`requirements.
`As
`provided in 21 CFR 10.90(b), persons who use methods and techniques not
`provided in the guideline should be able to adequately assure,
`through
`validation,
`that
`the method or
`technique they use is adequate to detect
`the endotoxin limit for the product.
`i
`
`Page 8
`
`Page 8
`
`

`

`III.
`
`REGULATORY PROVISIONS THAT PERMIT INITIATION
`OF END—PRODUCT TESTING WITH LAL
`
`firm must meet before using the LAL test
`The regulatory provisions that
`the same
`for all categories of products
`as an end-product
`test are not
`because
`applicable
`statutory
`provisions
`and
`of
`the
`different
`regulations. These provisions are as follows:
`
`a
`
`A.
`
`Human Drugs
`Abbreviated
`Applications,
`Applications
`AWE.-
`
`or
`gNDAs)
`New Drug Applications
`to
`subject
`New Drug, Applications
`gANDAs), Antibiotic Drug
`and
`animal
`drugs
`subject
`to New Animal Drug
`(NADAs),
`and Abbreviated
`New
`Animal
`Drpg
`
`a
`to submit
`are
`these classes of drugs, manufacturers
`For
`supplemental application to provide for LAL testing.
`However,
`under
`21 CFR 314.70(c)
`for drugs
`for
`human use
`and 21
`CFR
`514.8(d)(3)
`for drugs for animal use various changes may be made
`before FDA approval. Under these sections changes in testing of
`a human or animal drug that give increased assurance that
`the
`drug will have the characteristics of purity it purports or
`is
`represented to possess
`should be placed into effect at
`the
`earliest possible time. Therefore,
`if a firm validates the LAL
`test
`for
`a particular
`drug product
`covered by
`a
`new drug
`application by the procedures
`in this guideline using a
`LAL
`reagent
`licensed by
`the Center
`for Biologic Evaluation and
`Research (OBER)
`for the technique being used,
`the change may be
`made
`concurrently with
`the
`submission
`of
`the
`supplement
`providing for it.
`The supplement should contain initial quality
`control data,
`inhibition/enhancement data
`and
`the
`endotoxin
`limit for the drug product.
`
`Biological products for human use.
`
`in the manufacturing
`changes
`21 CFR 601.12 significant
`Under
`methods of biological products are required to be reported to
`the agency and may not become effective until approved by the
`Director,
`OBER.
`Therefore,
`a manufacturer
`of
`a biological
`product
`shall obtain an
`approved
`amendment
`to
`its
`product
`license before changing to the use of LAL
`in an end—product
`test,
`irrespective of the validation procedure used.
`
`C.
`
`Drugs not subject
`
`to premarket approval.
`
`A manufacturer of an injectable drug for human or animal use
`that
`is not subject
`to premarket approval would be able to use
`the LAL
`test
`as
`an
`end—product
`test
`for
`endotoxins without
`submitting any
`information to the agency.
`CGMPs
`require the
`manufacturer to have data on file to validate the use of
`the LAL
`test for each product for which it is being used.
`
`flaw.
`
`the basis of extensive experience in review of LAL data on
`On
`devices since November 1977, CDRH believes that
`the LAL test,
`_ 4
`_
`
`Page9
`
`Page 9
`
`

`

`least
`at
`is
`guideline,
`this
`to
`according
`validated
`when
`test
`for
`equivalent
`to the rabbit pyrogen test as an end—product
`medical
`devices.
`A manufacturer
`labeling
`a
`device
`as
`non—pyrogenic must validate the LAL test
`for
`that device in the
`test
`laboratory to be used for end—product
`testing before using
`the LAL test as an end—product endotoxin teSt for any device.
`
`this guideline may be
`The data discussed under Section V of
`expressed graphically or
`in tabular
`form and should be on file
`at
`the manufacturing site; no preclearance prior
`to use of
`the
`LAL
`test
`as
`an
`end—product
`test
`is
`required if
`it
`is used
`according to this FDA guideline. Voluntary submission of
`LAL
`validation and inhibition data obtained following issuance of
`this guideline will be accepted for CDRH review and comment.
`
`that
`test procedures
`LAL
`to use
`a manufacturer plans
`When
`deviate
`significantly from the
`LAL guideline,
`a premarket
`notification under section 510(k) of the Federal Food, Drug,
`and
`Cosmetic Act (the Act) or a Premarket Approval Application (PMA)
`supplement under section 515 of
`the Act
`should be
`submitted.
`Significant deviations would 'include—— but not necessarily’ be
`limited to—— higher endotoxin concentration release criteria,
`sampling from fewer
`than three lots for
`inhibition/enhancement
`testing,
`lesser sensitivity to endotoxin,
`rabbit retest when the
`LAL method
`shows
`endotoxin
`above
`the
`recommended
`allowable
`endotoxin dose,
`and
`a device
`rinsing protocol
`resulting in
`greater dilution of
`endotoxin than that
`recommended
`in this
`guideline.
`
`for another deviation from this
`PMA supplement
`notification or
`draft guideline; process control of endotoxin contamination with
`reduced end—product
`testing,
`i.e.,
`a decrease in the number of
`devices
`per
`lot
`undergoing
`end—product
`testing.
`The
`manufacturer must demonstrate adequate control of the production
`process by the use of routine checks for endotoxin at key stages
`of production except where it has been shown that no possibility
`of contamination exists.
`
`of
`providers
`review,
`PMA
`subsequent
`facilitate
`To
`to 21 CFR part 812 or 813 are
`investigational devices subject
`encouraged to use this guideline when a non—pyrogenic device is
`to be manufactured.
`
`PagelO
`
`Page 10
`
`

`

`IV. HUMAN AND ANIMAL DRUGS AND BIOLOGICAL PRODUCTS
`
`GENERAL REQUIREMENT
`
`licensed
`reagent
`LAL
`an
`use
`shall
`Manufacturers
`validation,
`in—process, and end—product LAL tests.
`
`by
`
`CBER
`
`in all
`
`A.
`
`VALIDATION OF THE LAL TEST
`
`for the release of
`the LAL test as an endotoxin test
`Validation of
`human
`and
`animal
`drugs
`includes
`the
`following:
`(1)
`initial
`qualification of
`the laboratory, and (2)
`inhibition and enhancement
`tests.
`
`INITIAL QUALIEICATION OF THE LABORATORY
`
`the detection of
`Various methodologies have been described for
`endotoxin,
`using
`limulus
`amebocyte
`lysate.
`Currently,
`commercially available
`licensed
`lysates
`use
`the
`gel
`clot,
`chromogenic,
`endpoint—turbidimetric
`or
`kinetic—turbidimetric
`techniques.
`Other methods which
`have
`been
`reported
`show
`potential
`increasing further
`the
`sensitivity of
`the LAL
`method.
`
`for
`
`testing
`the
`the variability of
`assess
`should
`Manufacturers
`Each analyst
`tests are performed.
`laboratory before any offical
`using a single lot of LAL and a single lot of endotoxin should
`perform the
`test
`for
`confirmation
`of
`labeled LAL
`reagent
`sensitivity or of performance criteria.
`Appendix A gives
`the
`procedures and test criteria for the current
`licensed techniques.
`
`INHIBITION AND ENHANCEMENT TESTING
`
`the LAL
`enhancement of
`inhibition or
`The degree of product
`procedure should be determined for each drug formulation before
`the LAL
`test
`is used to assess
`the endotoxin content of any
`drug. All validation tests should be performed on undiluted
`drug product or on an appropriate
`dilution. Dilutions should
`not exceed the Maximum Valid Dilution (MVD)
`(see Appendix D).
`At
`least
`three production batches of
`each finished product
`should be tested for inhibition and enhancement.
`
`8) GEL—CLOT TECHNIQUE
`
`testing should be conducted according
`Inhibition/enhancement
`to
`the directions
`in the preparatory section of
`the USP
`Bacterial Endotoxins Test
`(see Appendix B). Briefly,
`the
`a
`method
`involves
`taking
`drug
`concentration containing
`varying concentrations of a standard endotoxin that bracket
`the sensitivity of
`the lysate and comparing it
`to a series
`of
`the same endotoxin concentrations
`in water alone.
`The
`
`then diluted
`"spiked” with endotoxin and
`is
`drug product
`with additional drug product
`(so that
`the drug concentration
`remains constant)
`to
`the
`same
`endotoxin concentrations
`in
`6
`
`_
`
`_
`
`Pagell
`
`Page 11
`
`

`

`water. Results of endotoxin determination in water and the
`
`twofold
`a
`plus/minus
`fall within
`should
`product
`drug
`If the undiluted drug
`dilution of
`the labeled sensitivity.
`product
`shows
`inhibition,
`the drug product
`can be diluted,
`not
`to exceed the MVD, with the same diluent
`that will be
`used
`in
`the
`release
`testing
`and
`the
`above
`procedure
`repeated. Negative controls (diluent plus lysate) should be
`included in all inhibition/enhancement testing.
`
`b)
`
`CHROMOGENIC AND ENDPOINT—TURBIQIMETRIC TECHNIQUES
`
`a
`techniques,
`these
`testing by
`In inhibition/enhancement
`the
`drug concentration containing 4 lambda concentration of
`RSE
`or
`CSE
`(lambda
`is
`equal
`to
`the
`lowest
`endotoxin
`concentration used to generate the standard curve)
`is tested
`in
`duplicate
`according
`to
`the
`lysate manufacturer's
`methodology.
`The standard curve for
`these techniques shall
`consist of at
`least
`four RSE or CSE concentrations in water
`
`If the desired range is
`that extend over the desired range.
`standards concentrations
`greater
`than one
`log, additional
`should
`be
`included.
`The
`standard curve must meet
`the
`
`The
`linearity' as outlined in Appendix A(2).
`criteria for
`be
`detected amount of endotoxin in the spiked drug must
`within plus or minus
`25% of
`the 4 lambda concentration for
`the drug concentration to be considered to neither enhance
`nor
`inhibit
`the assay.
`If the undiluted drug product shows
`inhibition,
`the drug product can be diluted, not
`to exceed
`the MVD, and ti
`test repeated.
`
`An alternate procedure may be performed as described above
`except
`the RSE/CSE
`standard
`curve
`is
`prepared
`in LAL
`negative drug product,
`i.e. no detectable endotoxin,
`instead
`of LAL negative water.
`The standard curve must meet
`the
`test
`for
`linearity,
`i.e.
`r equal
`to or greater than 0.980,
`and in addition the difference between the 0.D.
`readings for
`the
`lowest
`and highest
`endotoxin concentrations must
`be
`greater
`than 0.4 and less than 1.5 0.D. units.
`If
`the
`standard curve
`does not meet
`these criteria,
`the
`drug
`product cannot be tested by the alternate procedure.
`
`c) KINETIC-TURBIDIMETRIC TECHNIQUE
`
`a drug
`testing by this technique,
`In inhibition/enhancement
`the RSE
`concentration containing 4
`lambda concentration of
`endotoxin
`or
`CSE
`(lambda
`is
`equal
`to
`the
`lowest
`concentration used to generate the standard curve) is tested
`in
`duplicate
`according
`to
`the
`lysate manufacturer's
`methodology.
`The standard curve shall consist of at
`least
`four RSE or CSE concentrations.
`If
`the desired range
`is
`greater
`than one
`log,
`additional
`standard concentrations
`should
`be
`included.
`The
`standard curve must meet
`the
`criteria
`outlined in Appendix A(3).
`The calculated Inean
`of
`amount
`endotoxin in
`the
`spiked
`drug
`product,
`when
`referenced to the standard curve, must be within plus or
`minus
`25% to be considered to neither enhance nor
`inhibit
`
`the
`
`assay.
`
`If
`
`the
`
`7,
`
`__
`
`undiluted
`
`drug
`
`product
`
`shows
`
`PagelZ
`
`Page 12
`
`

`

`the drug product can be diluted,
`inhibition or enhancement,
`not
`to exceed the MVD, and the test repeated.
`
`An alternate procedure may be performed whereby the RSE/CSE
`standard
`curve
`is
`prepared
`in drug
`product
`or
`product
`dilution instead of water.
`The drug product cannot have a
`background
`endotoxin
`concentration
`of more
`than
`10%
`(estimated by extrapolation of
`the regression line) of
`the
`lambda concentration (lambda equals the lowest concentration
`used to generate the standard curve).
`The standard curve
`must meet
`the test
`for
`linearity,
`i.e.
`r equal
`to or
`less
`than. —O.980,
`and in addition the slope of
`the regression
`must
`be
`less
`than —O.1
`and greater
`than —l.0.
`If
`the
`standard curve
`does not meet
`these criteria,
`the
`drug
`product cannot be tested by the alternate procedure.
`
`various
`in
`is manufactured
`drug
`the
`instances when
`those
`In
`concentrations of active ingredient while the other components of the
`formulation remain constant, only the highest and lowest concentration
`need be
`tested.
`If there is a significant difference,
`i.e. greater
`than
`twofold,
`between
`the
`inhibition endpoints
`or
`if
`the
`drug
`concentration, per mL,
`in the test solutions is different,
`then each
`remaining concentrations should be tested.
`If the drug product
`shows
`inhibition or enhancement at
`the MVD, when tested by the procedures in
`the above sections, and is amenable to rabbit
`testing,
`then the rabbit
`test will still
`be
`the appropriate test
`for
`that drug.
`If
`the
`inhibiting
`or
`enhancing
`substances
`can
`be
`neutralized without
`affecting the sensitivity of
`the test or
`if the LAL
`test
`is more
`sensitive than the rabbit pyrogen test
`the LAL test can be used.
`For
`those drugs not amenable to rabbit pyrogen testing,
`the manufacturer
`should determine
`the
`smallest quantity of
`endotoxin
`that
`can
`be
`detected. This data should be submitted to the appropriate FDA Office
`for review.
`
`the
`tests must be repeated on one unit of
`The inhibition/enhancement
`product
`if
`the
`lysate manufacturer
`is
`changed.
`If
`the
`lysate
`technique is changed,
`the inhibition and enhancement
`tests must be
`repeated using three batches. When
`the manufacturing process,
`the
`product formulation,
`the source of a particular ingredient of the drug
`formulation, or
`lysate lot
`is changed,
`the positive product control
`can be used to reverify the validity of the LAL test
`for the product.
`Firms
`that are obtaining an ingredient
`from a new manufacturer are
`encourged to include as part of their vendor qualification the rabbit
`pyrogen
`test
`to
`determine
`that
`the
`ingredient
`does
`not
`contain
`non—endotoxin pyrogens.
`
`Routine Testing qwarugs b
`
`the LAL Test.
`
`the
`from
`data
`on
`based
`be
`to
`is
`testing
`End—product
`Samples,
`testing as outlined in Section A(2).
`inhibition/enhancement
`standards, positive product controls and negative controls should be
`tested at least
`in duplicate.
`
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`
`Page 13
`
`

`

`an endotoxin standard series does not
`technique,
`the gel—clot
`For
`to be
`run with each set of
`tests if consistency of
`standard
`have
`endpoints has been demonstrated in the test
`laboratory.
`It should
`be run at least once a day with the first set of
`tests and repeated
`if
`there
`is
`any
`change
`in lysate lot,
`endotoxin lot or
`test
`conditions during the day.
`An endotoxin standard series should be
`run when
`confirming end—product
`contamination.
`Positive product
`controls (
`two lamda concentration of standard endotoxin in product)
`must be positive.
`If your
`test protocols state that you are using
`the USP Bacterial Endotoxin Test,
`remember
`that
`it
`requires
`a
`standard series to be run with each test.
`The above deviation must
`be noted in your test protocol.
`
`an
`techniques,
`endpoint—turbidimetric
`and
`chromogenic
`the
`For
`endotoxin standard series does not have to be run with each set of
`tests if consistency of standard curves has been demonstrated in the
`test
`laboratory.
`It
`should be
`run at
`least once a day with the
`first set of
`tests and repeated if there is any change in lysate
`lot,
`endotoxin lot or
`test conditions during the day. However, at
`least duplicates of a 4 lambda standard concentration in water and
`in each. product
`(positive product control) must be
`included with
`each run of
`samples.
`The mean
`endotoxin concentration of
`the
`standard must be within plus/minus 25% of
`the actual concentration
`and the positive product control must meet
`the same criteria after
`subtraction of
`any
`endogenous
`endotoxin.
`An
`endotoxin standard
`series should be run when confirming end—product contamination.
`If
`the alternate procedure is used,
`a standard in product series must
`be conducted each time the product is tested.
`
`is not necessary to run a
`it
`the kinetic—turbidimetric test,
`For
`standard curve each day or when confirming end product contamination
`if consistency of standard curves has been demonstrated in the test
`laboratory..
`However, at
`least duplicates of a 4
`lambda standard
`concentration in water
`and
`in
`each
`product
`(positive
`product
`control) must
`be
`included with each run of
`samples.
`The mean
`endotoxin concentration of
`the standard when calculated using an
`archived standard curve (See Appendix C), must be within plus/minus
`25% of
`the actual concentration and the positive product control
`must meet
`the
`same criteria after subtraction of
`any endogenous
`endotoxin.
`If
`the alternate procedure
`is used,
`a
`standard
`in
`product series must be conducted each time the product
`is tested.
`
`the labeled sensitivity of
`Before a new lot of lysate is used,
`lysate or
`the performance criteria should be
`confirmed by
`laboratory, using the procedures in Appendix A.
`
`the
`the
`
`The sampling technique selected and the number of units to be tested
`should be based on the manufacturing procedures and the batch size.
`A minimum of
`three units,
`representing the beginning, middle,
`and
`end,
`should
`be
`tested from a
`lot.
`These
`units
`can
`be
`run
`individually or pooled.
`If the units are pooled and any endotoxin
`is detected,
`repeat
`testing can be performed.
`The
`LAL
`test may be
`repeated no more than twice.
`The first repeat consists of twice the
`initial number of replicates of
`the sample in question to examine
`the possibility that extrinsic contamination occurred in the initial
`
`, g _
`
`Pagel4
`
`Page 14
`
`

`

`if any endotoxin is detected in
`On pooled samples,
`assay procedure.
`the
`first
`repeat,
`proceed to second
`repeat.
`The
`second repeat
`consists of an additional 10 units tested individually.
`None of
`the
`10 units tested in the second repeat may contain endotoxin in excess
`of the limit concentration for the drug product.
`
`following should be
`The
`parenteral drugs
`to meet
`end—product endotoxin test:
`
`for all
`endotoxin limit
`considered the
`if
`the
`LAL
`test
`is
`to be used as
`an
`
`1. K/M:
`
`administered
`those
`except
`drug
`any parenteral
`For
`endotoxin is
`for
`endotoxin limit
`intrathecally,
`the
`defined as K/M, which equals
`the amount of
`endotoxin
`(EU) allowed per ng or mL of product.
`K is equal
`to 5.0
`EU/Kg.
`(SEE appendix D for definition of M).
`
`an
`have
`that
`drugs
`For parenteral
`administration, K is equal
`to 0.2 EU/Kg.
`
`intrathecal
`
`route
`
`of
`
`Drugs exempted from the above endotoxin limits are:
`
`l. Compendial drugs
`established.
`
`for which other
`
`endotoxin limits have been
`
`applications,
`drug
`new
`by
`covered
`drugs
`2. Non—compendial
`antibiotic drug applications, new animal drug applications, and
`biological product
`licenses where different
`limits have been
`approved by the agency.
`
`3.
`
`Investigational drugs or biologicals for which an IND or
`exemption has been filed and approved.
`
`INAD
`
`4. Drugs or biologicals which cannot be tested by the LAL method.
`
`A batch which fails a validated LAL release test should not be retested
`
`the high
`to
`Due
`it passes.
`released if
`and
`test
`rabbit
`the
`by
`test
`as
`an
`rabbit
`reproducibility of
`the
`lack of
`variability and
`endotoxin assay procedure, we do not consider
`it an appropriate retest
`procedure for LAL failures.
`
`PagelS
`
`Page 15
`
`

`

`V. MEDICAL DEVICES
`
`General Reguirements
`
`the "HIMA Collaborative Study for
`reviewed the results of
`The CDRH has
`the Pyrogenicity Evaluation of
`a Reference Endotoxin by the USP Rabbit
`Test."
`This study' recommends 0.1 ng/mL (10 mL/kg) of E; coli 055:B5
`endotoxin from Difco Laboratories as the level of endotoxin which should
`
`testing of
`end—product
`test when used for
`be detectable in the LAL
`sensitivity (0.1
`ng/mL given 10 mL/kg)
`is
`medical
`devices.
`This
`LAL
`sufficient
`for
`testing and
`for
`retest
`of devices
`in rabbits.
`According to recent collaborative studies in the rabbit pyrogen and LAL
`tests, one nanogram of E. coli 055:85 endotoxin is similar in potency to
`5 EU of
`the USP Endotoxin Reference Standard.
`The endotoxin limit
`for
`medical devices has been converted to EU and is now 0.5 EU/mL using the
`rinse volume recommended in Section 2 below.
`Liquid devices should be
`more appropriately validated and tested according to the requirements for
`drugs by taking the maximum human dose per kilogram of body weight per
`hour into consideration (See Section IV,B).
`
`a USP
`failures with the LAL test or
`Manufacturers may retest LAL test
`rabbit pyrogen test.
`If the endotoxin level
`in a device eluate has been
`quantitated by LAL at 0.5 EU/mL endotoxin or greater,
`then retest
`in
`rabbits is not appropriate. Medical devices that contact cerebrospinal
`fluid should have
`less
`than 0.06 EU/mL of
`endotoxin.
`These values
`
`correspond to those set by the CDER for intrathecal drugs.
`
`licensed by
`reagent
`LAL
`an
`use
`shall
`Manufacturers
`validation,
`in—process, and end—product LAL tests.
`
`OBRR
`
`in all
`
`A. Validation of the LAL Test
`
`demonstrating
`Data
`1. Sensitivity:
`reproducibility of the LAL test.
`
`the
`
`sensitivity
`
`and
`
`2.
`
`line of devices
`Each product
`Inhibition/Enhancement Testing:
`utilizing different materials or methods of manufacture should
`be checked for inhibition or enhancement of the LAL test.
`
`Further explanation of the above points is given as follows:
`
`1.
`
`SENSITIVITY
`
`A manufacturer must be able to demonstrate a sensitivity of at
`least
`0.5
`EU/mL.
`The
`level
`of
`endotoxin selected as
`the
`
`for evaluating pyrogenicity of products using
`pass/fail point
`the
`LAL
`test must
`be
`equivalent
`to
`or
`below this
`level.
`Manufacturers may
`use
`another
`endotoxin
`if
`a
`reproducible
`correlation between it and the USP Reference Endotoxin Standard
`
`has been demonstrated in their laboratory (see appendix C).
`
`Pagel6
`
`Page 16
`
`

`

`the LAL technique used should be determined
`The sensitivity of
`and
`criteria
`in Appendix A.
`Routine
`by
`the
`procedures
`performance of
`the LAL
`test
`should include standards
`(run in
`duplicate) and a negative control.
`An endotoxin standard series
`is useful
`for checking lysate sensitivity and the competence of
`the technician,
`and for
`identifying other problems such as the
`contamination of glassware.
`
`The stability of the endotoxin standards and appropriate storage
`conditions should also be considered; dilute endotoxin solutions
`are not as stable as more concentrated solutions under certain
`conditions.
`
`INHIBITION AND ENHANCEMENT TESTING
`
`inhibition or enhancement of the LAL test should
`Lack of product
`be shown for each type of device before use of
`the LAL
`test.
`Possible inhibition of different chemical components of similar
`devices
`should be
`considered.
`A manufacturer may ilogically
`divide its device products into groups of products according to
`common
`chemical
`formulation;
`and may
`then qualify only
`a
`representative product
`from each such group.
`Ideally,
`the
`product chosen from each group would be the one with the largest
`surface area contacting body or
`fluid for administration to a
`patient.
`
`type should be
`three production lots of each product
`least
`At
`the
`sampling
`for
`inhibition.
`In general,
`use
`of
`tested
`technique selected should result
`in a
`random sampling of
`a
`finished production lot.
`CDRH recommends
`testing 2 devices for
`lot sizes under 30,
`3 devices
`for
`lot
`sizes
`30—100,
`and
`3
`percent of
`lots above size 100, up to a maximum of 10 devices
`per lot.
`
`or
`pyrogen
`for
`of preparing an eluate/extract
`The process
`Some
`inhibition/enhancement
`testing may vary for each device.
`medical devices can be flushed,
`some may have to be immersed in
`the non—pyrogenic rinse solution, while others may be tested by
`disassembling or by cutting the device into pieces prior
`to
`extraction by immersion.
`In general,
`for device