CUBIST 2002
`FRESENIUS KABI v. CUBIST
`IPR2015-01572
`
`

`
`3;
`
`USP 36
`
`SIX-MONTH IMPLEMENTATION GUIDELINE
`
`The United States Pharmaco eia—National Formulary and its supplements become official six months after being released to
`the public. The USP-NF, whic 1 is released on November 1 of each year, becomes official on May 1 of the following year. This
`six—month implementation timing gives users more time to bring their methods and procedures into compliance with new
`and revised U$P—NF requirements.
`‘
`The table below describes the official dates of the U$P—NF and its supplements. The 2011 USP 35—NF 30, and its supple~
`ments, Interim Revision Anrrouncemerits (IRAs) and Revision Bulletins to that edition, will be official until May 1, 2013, at which
`time the USP 36-NF 37 becomes official.
`
`F Pub!icatj_Q_n__,_ __
`USP 36—NF 37
`
`_
`
`First Supplement to the
`bf USP 36-«NF 31
`Second Supplement to the
`USP 36~NF 37
`USP 37—NF 32
`
`_ Release Date
`November I, 2012
`
`l Official Date
`May 1, 2013
`
`_
`
`February 1, 2013
`
`August 1, 2013
`
`June 1, 2013
`
`December 1, 2013
`
`November l, 2013
`
`M3)’ l. 7014
`
`_ _,§_)_f__f_ic§9_l__|_.[;1til
`l May ‘I, 2014 (except as superseded by supplements,
`Revision Bulletins)_
`May 1, 2014 (except as superseded by Second Supplement, IR/ls,
`and Revision Bullelins1,__,___
`May 1, 2014 (except as superseded by HM; and Revision Bulletins)
`___
`May 1, 2015 (except as superseded by supplements, IR/\S, and
`Revision Bulletins)
`
`IRAS, and
`
`1
`
`The table below gives he details of the IRAs that will apply to USP 36~NF 37.
`__,_..... ._.
`N
`_r.
`l um Official Date
`IRA Postiilq Data
`comment Due Date
`V J__P_f_§fg§§i_n_¢1 D.at.:2.
`um __ ,___,
`
`U;
`1 2013
`l\/_i_;:y”3w'|“__2013
`_ March 31 2013
`__]_anuarx Z 2013
`3S_l§_1_)_
`September iL2O13
`uly 26, 2013
`May '5L_20i 3
`March 'l_,__Z013
`39(_2_)____
`Noveml'>er_1, 2013
`September 27, 2013
`MY 31. Z07 3
`. M.a.l.’._l.L..Z0l3
`39(.3.l
`|anua_ry 1, 2()__1_4 _
`Novembe_i',_2,9,,2013
`September 30 2013
`lulv 2, 2013
`39(4L__
`_ March___]_L_2()14
`1anuary_31, 2014
`November 30, 2013
`September 4,__20l3
`39 5)
`
`km 1, 2014
`| March 25, 2014
`lanuarv 31,2014
`,,_,zol3
`39_@________,__
`Revision Bulletins published on the USP website become official on the date specified in the Revision Bulletin.
`
`
`
`|
`
`NOTlCE AND WARNING
`
`Concerning U.S. Parent or Trademark Rlghts—_The inclusion in The United States Pharmacopeia or in the National Formulary of a
`monograph on any drug in respect to which patent or trademark rights may exist shall not be deemed, and is not intended
`as, a grant of, or authority to exercise, any right or privilege protected by such patent or trademark, All such rights and
`privileges are vested in the patent or trademark owner, and no other person may exercise the same without express
`permission, authority, or license secured from such patent or trademark owner.
`
`Concerning Use of USP or NF Text-—Attention is called to the fact that USP and NF text is fully copyrighted. Authors and
`others wishing to use portions of the text should request permission to do so from the Secretary of the USPC Board of
`Trustees.
`
`Copgri6
`1 2
`
`grht © 2012 The United States Pharmacopeial Convention
`winbrook Parkway, Rockville, MD 20852
`
`All rights reserved.
`ISSN: 0195-7996
`
`ISBN: 978~1-936424-12-2
`
`Printed in the United States by United Book Press, lnc., Baltimore, MD
`
`

`
`90 (81) Antibiotics——Microbial Assays / Biological Tests
`
`Table A2-1. Test for Outlier Measurements
`
`USP 36
`
`~ m les from a normal population qaps equal to or larger than the following values of Gr, 6;, and 6; occur with a probability P = 0.01, when
`5
`,;_,:i?e,r:nea5uremen:s can occur only at one end‘ or with P = 0.02, when they may occur at either end.
`N
`3
`4
`S
`6
`7
`C,
`9,937
`0.889
`0.7m
`0.698
`0.537
`
`' 7
`
`_
`
`y
`L
`
`0.597
`
`
`
`y‘
`
`6,
`
`N
`5,
`
`0.531
`
`‘I
`
`ll
`0.674
`
`0.634
`
`12
`0.543
`
`T:
`
`4.
`
`(85) BACTERIAL EN DOTOXINS
`TEST
`
`‘Portions of this general chapter have been harmonized _
`with the corresponding texts of the European Pharmacopoeia
`and/or the japanese Pharmacopoeia. Those portions that are
`not harmonized are marked with symbols (‘.) to specify this
`fdgftffe Bacterial Endotoxins Test (BET) is a test to detect or
`quantify endotoxins from Cram-negative bacteria using
`amoebocyte lysate from. the horses oe crab (timulus poly-
`phemus or Tachyplet/s tridentatus).
`There are three techniques for this test: the gel-clot tech-
`nique, which is based on
`el formation; the turbidimetric
`_
`technique, based on the
`evelopment of turbidity after
`cleavage of an endogenous substrate; and the chromogenic
`technique, based on the development of color after cleav-
`age of a synthetic peptide-chromogen complex. Proceed by
`any of the three techniques for the test.
`in the event of
`doubt or dispute, the final decision lS made based upon the
`gel-clot limit test unless otherwise indicated in the _mono— A
`graph for the product being tested. Thetest is carried out in
`a manner that avoids endotoxin contamination.
`
`APPARATUS
`
`Depyrogenate all glassware and other heat-stable materi-
`als in a hot air oven using a validated process". A com-
`monly used minimum time and temperature is 30 min at
`250”. if employing plastic apparatus, such as microplales '
`and pipet tips for automatic pipetters, use apparatus that is
`shown to be free of detectable endotoxin and does not in-
`terfere in the test. [NOTE~—ln this chapter, the term ”tube”
`includes any other receptacle such as a microtiter well.)
`
`REAGENTS AND TEST SOLUTIONS
`
`Amoebocyte Lysate—A lyophilized product obtained
`from the lysate of amoebocytes (white blood cells) from the
`horseshoe crab (Limulus polyphemus or Tcicliypleus
`triclentaius). This reagent refers onl
`to a product manufac-
`tured ln accordance with the regu ations of the com etent
`authority. [NQTE~—Amoebocyte Lysate reacts to some
`-g|u-
`cans in addition to endotoxins. Amoebocyte Lysate prepara-
`tions that do not react to glucans are availab e: they are
`prepared by removing the (3 factor reacting to glucans from
`Amoebocyte Lysote or by inhibiting the (3 factor reacting sys-
`tem of Amoebocyte Lysate and may be used for endotoxin
`testing in the presence of glucans.]
`Water for Bacterial Endotoxins Test (BET)——Use Water
`for injection or water produced by other procedures that
`‘
`‘
`f the
`rocedure for inactivating endotoxiiis, see ‘Dry-
`;.;e§,(i)r§(:e:-l;7;‘L:g;gn‘eii$r‘\cl0er 312!’/P/l'.’dl'IOIl and Sterility Assurance of comyiendial Arti-
`cles /_l2'l ii. Use Lysate 75 having a sensitivity of not less l.l'lZlil O. 5 Eridotoxin
`Unit per mL.~
`
`.__
`
`13
`lg
`0.6l7
`shows no reaction with the lysate employed, at the detec-
`tion limit of the reagent.
`Lysate TS—Dissolve Amoebocyte Lysate in Water for BET,
`or in a buffer recommended by the Iysate manufacturer, by
`, entle stirring, Store the reconstituted lysate, refrigerated or
`rozen, according to the specifications of the manufacturer.
`
`PREPARATION OF SOLUTIONS
`
`Standard Endotoxin Stock Solution——A Standard Endo-
`toxin Stock Solution is prepared from a USP Endotoxin Refer-
`ence Standard that has been calibrated to the current WHO
`international Standard for Endotoxin. Follow the specifica-
`tions in the package leaflet and on the label for preparation
`and storage of the Standard Endoloxin Stock Solution. Endo-
`toxin is expressed in Endotoxin Units (EU). [NOTE——One USP
`Endotoxin Unit (EU) is equal to one International Unit (IU)
`of endotoxin.]
`Standard Endotoxin Solutions—After mixing the Stan-
`dard Endotoxin Stock Solution vigorously, prepare appropriate
`serial dilutions of Standard Endotoxin Solution, usin Water
`for BET. Use dilutions as soon as possible to avoid (Boss of
`activity by adsorption.
`Sample Solutions—-Prepare the Sample Solutions by dis-
`solving or diluting drugs using Water for BET. Some sub-
`stances or preparations may be more appropriately dis-
`solved, or diluted in other aqueous solutions.
`if necessary,
`adjust the pH of the solution to be examined (or dilution
`thereof) so that the pH of the mixture of the lysate and
`Sample Solution falls within the pH ran e specified by the
`lysate manufacturer, usually 6.0-8.0. T e pH may be ad-
`justed b use of an acid, base, or suitable buffer as recom-
`mende
`by the lysate manufacturer. Acids and bases may
`be prepared from concentrates or solids with Water for BET
`in containers free of detectable endotoxin. Buffers must be
`validated to be free of detectable endotoxin and interfering
`factors.
`
`DETERMINATION OF MAXIMUM VALID
`DILUTION (MVD)
`The maximum valid dilution is the maximum allowable
`dilution of a specimen at which the endotoxin limit can be
`determined. Determine the MVD from the following
`equation:
`
`MVD = (endotoxin limit >< concentration of Sample Soluiion)/
`(7-)
`
`Endotoxin Limit—~The endotoxin limit for parenteral
`drugs, defined on the basis of dose, equals K/M‘-’-., where K
`W K is 5 USP-EU/kg of body wei htllor any route of administration other than
`intralthecal (for which K is
`U P-l;Ullig of body weight), For radropha.-ma.
`ceutical products not administered iritrathecally, the endotrixiii limit is calcu-
`lated as 175 EU/V, where V is the maximum recoiniriendecl dose in mt, For
`
`intramecally administered radiooharmaceiiticais, the eridotoxiri limit is ob.
`tallied by t e formula 14 EU/V. For formulations (usually zrnticzincer products)
`administered on a per square meter of body surface, the formula is K/M,
`where K = 100 EU/mi and M is the maximum dose/m7..
`
`

`
`USP 36
`
`Biological Tests‘ / (85) Bacterial Endotoxins Test 91
`
`is a threshold pyrogenic dose of ijendotoxin per kg of body
`weight, and M is equal to the maximum recommended bo-
`lus dose of product per kg of body weight. When the prod
`uct is to be injected at frequent interva s or infused continu»
`ously, M is the maximum total dose administered in a single
`hour period. The endotoxin limit for parenteral drugs is
`specified in the individual monograph in units suchas EU/
`int, EU/mg, EU/Unit of biological activity, etc.
`Concentration of Sample Solutionw
`mg/mt: in the case oi endotoxin limit specified by weight
`(EU/mg);
`Units/mt: in the case of endotoxin limit specified by unit
`of biological activity (EU/Unit);
`mL/mL: when the endotoxin limit is specified by volume
`(EU/mL).
`"i-.: the labeled sensitivity in the (iel-C/ot fechni. ue (EU/mt‘)
`or the lowest concentration used in the standar
`curve for
`the Iuroidirrmtric Technique or Chromogenic Technique.
`
`GEL-CLOT TECHNIQUE
`
`The gel-clot technique 8 used for detecting or quantiiying
`c-ndotoxins based on clotting of the lysate reagent in the
`presence of endotoxin. The minimum conceritration of en«
`dotoxin required to cause the lysate to clot under standard
`conditions is the labeled sensitivity of the lysate reagent. To
`ensure both the )FEClSl0l'i and validity of the test, perform
`the tests for con irming the labeled lysale sensitivity and for
`interfering factors as described in Preparatory Testing, imme-
`diately below.
`
`Preparatory Testing
`
`Test for Confirmation of Labeled Lysate Sensitivity—
`Confirm in tour replicates the labeled sensitivity,
`ex-
`pressed in EU/mL of the lysate prior to use in the test. The
`test for conlirrnation of lysate sensitivity is to be carried out
`when a new batch oi lysate is used or when there 35 any
`change in the test conditions that may affect the outcome
`of the test. Prepare standard solutions having at least four
`concentrations equivalent to 2).,
`"it, 0.5%, and 0.25}. by di-
`luting the USP Enclotoxin RS with Water ior BET.
`Mix a volume of the Lysote T5 with an equal volume
`(such as 0.1-mL aliquots) of one of the Staridcird Endotoxin
`Solutions in each test tube. When single test vials or ainpuls
`containinc lyophilizcd lysate are used, acid solutions directly
`to the vialjor ampul. Incubate the reaction mixture for ill
`constant period according to the directions of the lysate
`rnanuiacturer {usually at 37 .3. l" for 60 .5. 7. min‘), avoiding
`take each tube in
`vibration, To test the integrity oi the gel,
`
`turn directly from the incubator, and invert it through about
`l80'fln one smooth motion. if a firm gel has formed that
`remains in place upon inversion, record the result as posi~
`tive. A resut is negative ii an intact gel is not formed. The
`test‘ '3 considered valid when the lowest concentration of
`the[ standard solutions shows a negative result in all replicate
`ms 5.
`The endpoint is the smallest concentration in the series of
`decreasing concentrations oi standard enclotoxin that clots
`the lysate. Determine the geometric mean end oint by cal-
`culating the mean of the logarithms of the en point con
`centrations of the four replicate series and then taking the
`antilogarithm of the mean value, as indicated in the ollow—
`ing formula:
`
`geometric mean endpoint concentration = antzlog (,§;e/f)
`
`where Xe is the sum of the log endpoint concentrations of
`the dilution series used, and fis the number of replicate test
`tubes The ge-orneiric mean €‘llt.lf)C)llil <i>rirei'itrar.i0n is the
`measured sensitivity of the l)/SZ1U;’(_‘l'l EU/mt).
`If this is not
`less than O.5')t and not more than 2'/.., the Iabelec’ sensitvity
`is confirmed and is used in tests performc-d with this lysate.
`Test for interfering Factors——»-Usually‘prepare solutions
`(A—rD) as shown in Table 7, and i.ii:».ilr_:rii‘i the inhi'oitlon/en-
`hancement test on the Sample Solul/orts at a dilution less
`than the Mi/D, not containing any detectable endotoxins,
`operating as described for Test /or Corifirniotion of Lobe/ed
`Lysnte Sensitivity. The geometric mean endpoint concentra-
`tions of Solutions 8 and C are determined using the formula
`described in the Test for Confirmation of Labeled Lysate Srzrisil
`tivity. The test for interfering factors must be repeated when
`any condition changes that is likely to influence the result of
`the test.
`The test is considered valid when all replicates of Solu-
`tions A and D show no reaction and the result of Solution C
`confirms the labeied sensitivity.
`If the sensitivity of the lysate determined in the presence
`of Solution 8 is not less than 0.5‘/-. and not greater than 2/.,
`the Sample Solution does not contain factors that interfere
`under the experimental conditions used. Otherwise, the
`Sample So/iiiinn to be examined interferes with the test.
`it the szimpie under test does not comply with the tes: at
`a dilution less than the M\’D, repeat the test using a greater
`dilution, not exceeding the lvlVD. The use of a more sensi-
`tive lysate permits it greater dilution of the sainpleto be
`examined, and this may contribute to the elimination or
`interference.
`Interference may be Overcome by suitable treatment such
`as filtration, neutralization, ciiaiysis, or heatmq, To estal3lis'ri
`that the chosen treatrnc-nt effectively <1-liminates intcriereace
`without
`oss oi endotoxins, perlorm the assay i:ie.scribed
`
`nhibltion/Enhancement Test for Gel~Clo't Techniques
`._-...-_.,._.__.___..___,___,____~___,________,______,_r._~—— ~
`
`V
`
`Endocoxin
`. .C0n<s+£Ir_a_tiori..
`
`
`
`_- « ..
`
`Table 1. Preparation of Solutions for the I
`__. ._.___..__._ ,-..
`...,_.... .. ~--—n—~-4~<¢—-1..
`Endotoxin C0ncentrafl0n/
`Diiutiori
`Solution to Which Endotoxin
`
`Is Add-4.9!. .,
`
`
`
`
`
`
`hat is lie-e -tzi ti
`
`=-
`
`l‘r>'.t for ll‘.lf‘-l‘l(~’l'€‘lllI:
`:2".}’lli'i')I? B‘
`‘so/iiiimi ii": Coittrol ‘oi
`tii>el~"at: lysate sa_-nsitivity.
`' ’>-.Iuti-zii I).
`i*«li:q.-iti~/t~.-
`'..O!lll‘t'1i oi H/(TIC!
`.’o/
`iIi.'.7'.
`
`

`
`92 (85) Bacterial Endotoxins Test / Biological Tests
`
`-'
`t’ n to be examined to which Stan-
`gt::i(r)c‘l/eErLiltSjlcr>‘t%>‘Elhehgsegzgnlcadded and which has then been
`submitted to the chosen treatment.
`
`Limit Test
`
`Procedure——Prepare Solutions A, 3, C and D 35 5h°Y"” ll‘
`Table 2 and perform the test on these solutions followin
`the
`rocedure above for Preparatory Testing, Test for Con ir-
`motion of Labeled Lysate Sensitivity.
`
`,___.
`Table 2. Preparation of Solutions for the Gel-Clot Limit Test
`Endotoxin Concerrtration/
`V
`solution to Which
`Number of
`Endotoxin lsfigged
`Regllsgtes
`Solution‘
`"_‘_NonelDilutecl Sample Solution
`7.
`A
`2;LDiluted Saggigle Solution
`2
`B
`QtLWuter___[or BET
`2
`C
`Nonel Water for BET
`2
`D
`* Prepare Solution A and the positive product control Solution 3 using
`a dilution not greater than the MVD and treatments as described for
`the Test for Interfering Factors in Preparatory Testing. The positive con-
`trol Solutions B and C contain the Standard Endotoxir: Solution at a
`concentration corresponding to twice the labeled lysate sensitivity.
`The negative control Solution D consists of Water for BET.
`
`lnterpretation——The test is considered valid when both
`replicates of Solutions 3 and C are positive and those of Solu—
`tion D are negative. When a negative result is found for
`both replicates of Solution A, the preparation under test
`complies with the test. ‘When a positive result is found for
`both replicates of Solution A, the preparation under test
`does not comply with the test.
`‘
`When a positive result is found for one replicate of Solu-
`tion A and a negative result is found forthe other, repeat
`the test. in the repeat test, the preparation under test corn-_
`plies with the test if a negative result is found for both repli-
`cates of Solution A. The preparation does not comply with
`the test if a positive result is found for one or both replicates
`of Solution A. However, if the preparation does not comply
`with the test at a dilution less than the MVD, the test may
`
`USP 36
`
`be repeated using a greater dilution, not exceeding the
`MVD.
`
`Quantitative Test
`
`Procedure—-The test quantifies bacterial endotoxins in
`Sample Solutions by titration to an endpoint. Prepare Solu-
`tions A, B, C, and D as shown in Table 3, and test these
`solutions by following the procedure in Preparatory Testing,
`Test for Confirmation of Labeled Lysule Serisitiviiy.
`Calculation and lnterpreta’tion—The test is considered
`valid when the following three conditions are met: (1) Both
`replicates of negative control Solution D are negative; (2)
`Both replicates of positive product control Solution 8 are
`positive; and (3) The geometric mean endpoint concentra-
`tion of Solution C is in the range of 0.57» to 2A.
`To determine the endotoxin concentration of Solution A,
`calculate the endpoint concentration for each replicate by
`multiplying each endpoint dilution factor b '/L. The endo-
`toxin concentration in the Sample Solution IS the endpoint
`concentration of the replicates. ll the test is conducted with
`a diluted Sample Solution, calculate the concentration of en-
`dotoxin in the original Sample Solution lo multiplying by the
`dilution factor. if none of the dilutions o the Sample Solu-
`tion is positive in a valid assay, report the endotoxin concen-
`tration as less than it (if the diluted sample was tested, re-
`port as less than it times the lowest dilution factor of the
`sample). if all dilutions are positive, the endotoxin concen-
`tration is reported as equal to or greater than the greatest
`dilution factor multiplied by it (_e.g., initial dilution factor
`times eight times it in Table 3).
`The preparation under test meets the requirements of the
`test ii the concentration of endotoxin in both replicates is
`less than that specified in the individual monograph.
`
`PHOTOMETRIC QUANTITATIVE TECHNIQUES
`
`Turbidimetric Technique
`
`. This technique is a photometric assay measuring increases
`inreactant turbidity. Oh the basis of the particular assay
`principle employe , this technique may beclassified as ei»
`ther an endpoint-turbidimetric assay or a kinetic-turbidimet—
`
`Table 3. Preparation of Solutions for the Gel-Clot Assay
`Endotoxln Concentratlonl
`Solution to Which Endotoxin
`Is Added
`Solutlon
`________ None/Sample Solution
`
`
`F
`
`T
`
`i__
`
`Diluent
`Water for BET
`
`Dilution
`Factor
`I
`2
`4
`
`8
`1
`T
`
`Endotoxln
`Concentration
`_.
`-——
`—-—
`
`-~_.__....-........_
`2K
`Zit
`
`
`Number of
`
`Kgpllcates
`_m____2_
`__
`2
`2
`
`_
`
`2 _. ..
`2
`2
`
`
`
`2
`2
`2
`
`,-
`
`
`
`
`
`.
`..
`le Solution
`Zliwaier for BET
`
`C’-
`
`.
`
`_
`
`0.57;
`4
`0.252.
`8
`_‘
`I
`l
`Noni;/Water for arr
`oa
`_
`i , ——
`-‘ Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test lor Interfering Factors was coinpletcd.
`Subsequent dilution of the Sample Solution must not exceed the MVD. Use Water for BET to make a dilution series of four tubes containing the
`Sample Solution under test at concentrations of l, ‘/7,
`‘l.r, and ‘/is relative to the concentration used in the Test for interfering Factors. Other dilutions
`up to the MVD may be used as appropriate.
`0 Solution 8: Solution A containing standard endotoxin at a concentration of 2). (positive product control).
`= Solution C: Two replicates of four tubes of Water for BET containing the slaiidard enclotoxin at concentrations of Zit, it, 0.5'Jt, and 0.252s,
`respectively.
`_
`0 Solution 0: Water for BET (negative control).
`
`

`
`USP 36
`
`Biological Tests /<85) Bacterial Endotoxins Test 93
`
`rlc assay. The endpoint-turbidimetric assay is based on the
`guantitative relationship between the concentration of en-
`otoxins and the turbidity (absorbance or transmission) of
`the reaction mixture at the end of an incubation period.
`The kinetic-turbidimetric assay is a method to measure ei--
`ther the time (onset time) needed to reach a predetermined
`absorbance or transmission of the reaction mixture, or the
`rate of turbidity development. The test is carried out at the
`incubation temperature recommended by the lysate manu-
`facturer (which is usually 37 : l“).
`
`Chromogemc Techmque
`This technique is an assay to measure the chromophore
`released from a suitable chromogenic peptide b‘
`the reac-
`tion of endotoxins with I sate. On the basis of t e particular
`assay principle employe , this technique may be classified as
`either an end oint-chromogenic assay or a kinetic-chroma
`9l§““C assay‘
`.he e”dP°i”tTChr°m°9emC assay is based 0”
`t e quantitative relationship between the concentration of
`eridotoxins and the release of chromophore at the end of
`an incubation period. The kinetiochromogenic assay is a
`method to measure either the time (onset time) needed to
`reach a predetermined absorbance of the reaction mixture,
`or the rate of color development. The test is carried out at
`the
`tgttttgfralu“-* tfcggignftgded by we
`mam‘ aC“”e’ -W “C '5 “ma V
`L H '
`_
`Preparatory Testing
`To assure the precision or validity of the turbidimetric and
`gory tests are conducted to
`chromogenic techniques, prepara
`verify that the criteria for the standard curve are valid and
`that the sample solution does not interfere with the test
`Validation for the test method is required when conditions
`that are likely to influence the test result change.
`Assurance of Criteria for the Standard Curve~The test
`mu“ be Camed 0U‘ for 95”‘ '0‘ of l)’53“’- reagent U5ln9 the
`Standard Endotoxin Solution, prep_are_ at least three endotoxin
`<:onc.entrations within the range indicated by the lysate
`manufacturer to generate the standard curves Perform the
`assay using at least three replicates of each standard endo—
`toxin concentration according to the manufacturer's instruc-
`tions for the lysate (volume ratios, incubation time, temper-
`ature’ H, etc‘) if the desired range is mater than two [095
`3,, the Einem methods, additional Stan ards Shouid be m.
`cluded to bracket each leg increase in the range of the stan~
`dard curve. The absolute value of the correlation coeifl~
`cient, r, must be greater than or equal to 0.980 for the
`range of endmoxm Concentrations Set up
`Test for lnterferin Factors——Select an endotoxin con—
`centration at or near gie middle of the endotoxin standard
`curve. Prepare Solutions A, B, C, and D as shown in Table 4,
`Perform the test on Solutions A, 8, C, and D at least in dupli~
`
`cate, according to the instructions for the lysate emplo ed,
`for example, concernin volume of Sample Solution an
`Ly-
`sate TS, volume ratio o?Samp/e Solution to Lysate TS, incu-
`bation time, etc.
`The test is considered valid when the following conditions
`are met,
`l. The absolute value of the correlation coefficient of the
`standard curve generated using Solution C is greater
`than or equal to 0,980.
`2. The result with Solution 0 does not exceed the lirriit
`of the blank value required in the description of the
`lysate reagent employed, or it is less than the endo
`Ca‘CtlZJ(|3;(tl;) tcéetection limit of the lysatedgea entjemployted.
`wbtractm th: {Sean Eecdovtery. O t e d t et. 8”. Ogoxm ly
`:10“ if mg (50, [lean/fr; §,°:‘”fC°"Cet2 ‘ta ‘°”t"7 5'“? ii: U’
`added eng’jm'Ox%’n”€g’
`IIIt.O‘Z7 '5
`I 5‘ Cg” E:'“'L‘)‘§ mr?S.d_
`ered free of factor, {’h‘ t’.
`t 'f
`J: O’ 9', ° der"the'
`Conditions of the t’ St amm er 8”? ‘"21
`e a5t5a5;.”Ir1' of the
`endotoxin added t: the §(7{l’nHea/:U5r:llIIfC?rPE:LTS{ab|E?Within
`5O%_ZOO% of the known a ded pndotoxm concentration
`after Subtra t. n
`f
`d t
`. ‘d
`d .
`h
`I mm
`Without adgpfi’ egd at"y.en O 0""
`etecte
`'” t 8 5° U
`Wheq the End to .°Xm‘
`.
`f h
`.f. d
`n
`ran 8 we 5
`/0 ‘§X;“ lecoveéy '5 Out. 0 t eépecé ‘:3
`tam9h‘“erferi:£"F;,et 0 unfigj U” er tefttés Con?‘ 6.”?
`_ 0 water
`atzzzitt, irtniittset;...ite:::;i:S'iit:,i2i:ie
`of the Sam le Solution or diluted ‘Sam le Solution not to ex-
`ceed the MPVD may be eliminated by Fsuitable validated
`treatment such as filtration, neutralization, dialysis, or heat
`treatment. To establish that the chosen treatment effectively
`eliminates interference without loss of endotoxins, perform
`the assay described above, using the preparation to be ex-
`amined to which Standard Endotoxin has been added and
`which has then been submitted to the chosen treatment.
`
`Test Procedure
`Follow the procedure described tor Test for Interfering Foc-
`W5 under p,epa,mO,y rm,-mg, immediately above,
`,
`Cakulatlon
`_
`_
`, Cakulal? the €l7d0l0_Xm COHCEFIUBUOH 01‘ Bad’! 0i the WP‘
`licates of_Solution A, using the standard curve generated by
`the positive control Solution C.'The test is considered valid
`WW9” the l0”0Wln9 three FGQUIFEITIGHFS are met.
`_
`1. The {€SU|lS of the CO_f1l£TC_>l 50/W0" C C0mPl>’ Wm‘ me
`requiremeiits for validation defined for Assurance of
`Criteria for the Standard Curve under Preparatory
`_
`2 TIf5t"’9‘d
`1
`,
`_
`' Fe" f°‘°"‘”. ’eC°‘{9.’Yr Calcumed “Om. the fioncoenl?
`"‘m°".°“”‘{! '” SOIW9” 3 me’. 5“btra‘.:“”9 I. evfithm
`§e“t'at‘°” C’ e“d°t°’*‘“ f°”“d ’“ 5°’“t’°” A’
`'5
`the range of 50°/o—200%.
`
`Table 4. Preparation of Solutlons for the lnhlbltlon/Enhancement Test for Photometric Techniques
`’
`“
`—~
`Solution to Which
`W
`_ Endo_t_oxln C_oncentration___
`_S_olution
`Endotoxln Is Added
`Number of R_gpll€i!f€5
`,
`_ N909
`_____,,__
`_____
`_, /\“,,~
`__________., Salnglq SoIutio_n __
`M __>
`_ Not less than 2
`Middle concentration ol the sti§_r_i_qa_rd curv
`8"
`__
`Suirlple so/M9,,
`N0; I353 imp 2
`At least three concentrations (lowest concentration is
`- desigiizued 1.)
`_-
`__ C‘.--
`ll/uler for Bf?"
`E'd(_‘li not less than 2
`M... N005:
`...__,.
`9"’
`,__
`_‘4_/ole; lor BET
`__
`__ Not less than 2
`«~ Solution A: The Siirriple Solution may be diluted not to exceed MVD.
`tr Solulion B: The preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the
`middle of the standarcl curve.
`' Solution C: The standard endotoxin at the concentrations used in the validation of the method described for Assurance ol Criteria for the Standard
`Curve under Preparatory Testing (positive controls).
`‘I Solution D: Water for BET (negative control).
`
`"*’l
`
`,__,_
`
`S
`‘
`
`
`
`_ _
`
`
`
`

`
`94 (85) Bacterial Endotoxins Test / Biological Tests
`
`3. The result of the negative control Solution D does not
`exceed the limit of the blank value required in the
`description of the lysate employed, or it is less than
`the endotoxin detection limit of the lysate reagent
`employed.
`
`interpretation
`
`in photometric assays, the preparation under test com-
`plies with the test if the mean endotoxiri concentration of
`the replicates of Solution A, after correction for dilution and
`concentration, is less than the endotoxiri limit for the
`product.
`
`<87) BIOLOGICAL REACTlVlTY
`TESTS,
`lN VITRO
`
`tests are designed to determine the biologi-
`The followint
`cal reactivity o mammalian cell cultures following contact
`with the elastomeric plastics and other polymeric materials
`with direct or indirect patient contact or of specific extracts
`prepared from the materials under test. it is essential that
`the tests be performed on the specified surface area. When
`the surface area of the specimen cannot be determined, use
`0.1 g of elastomer or 0.2 g of plastic or other material for
`every mL of extraction fluid. Exercise care in the preparation
`of the materials to prevent contamination with microorgan-
`isms and other foreign matter.
`Three tests are described (i.e., the Agar Diffusion Test, the
`Direct Contact Test, and the Elution Test).* The decision as to
`which type of test or the number of tests to be performed
`to assess the potential biological response of a specific sam-
`ple or extract depends upon the material, the final product,
`and its intended use. Other factors that may also affect the
`suitability of sample for a specific use are the polymeric
`composition; processing and cleani_ng procedures; contact-
`ing media; inks; adhesives; absorption, adsorption, and per-
`meability of preservatives; and conditions of storage. Evalua-
`tion of such factors should be made by appropriate
`additional specific tests before determining that a product
`made from a specific material is suitable for its intended use.
`USP Reference Standards (‘I 'l‘;—USP High-Density Poly-
`ethylene RS. USP Positive Bioreaction RS.
`Cell Culture Preparation~Prepare multiple cultures of L--
`929 (ATCC cell line CCL l, NCTC clone 929) mammalian
`fibroblast cells in serurn~sup iemerized minirnum essential
`medium having a seeding ensity of about 105 cells per mL.
`incubate the cultures at 37 i l" in a humidified incubator
`for not less than 7.4 hours in a 5 Jr. 1% carbon tlioxicle at-
`mosphere until a monolayer, with greater than 80% conflu-
`ence, is obtained. Examine the prepared cultures under a
`microscope to ensure uniform, near-confluent monolayers.
`[NOTE-—The reproducibility of the in Vitro Biological Reactivity
`Tests depends upon obtaining uniform cell culture density]
`Extraction Solvents——-Sodium Chloride Injection (see mon-
`ogra h—-—use Sodium Chloride injection containing 0.9% of
`NaCf;. Alternativel , serum—free mammalian cell culture me-
`dia or serum-supp emented mamrnaliari cell culture media
`may be used. Serum su plementation is used when extrac-
`tion is done at 37" for 4 hours.
` re F iveri in the fOllO\«Vil'1g publications of the American Soci-
`ety for Testing and Materials,’ l9l_6 Race St, Philadelphia, PA_I9_l 03: "Stan-H
`dard Test Method for Agar Diffusion Cell Culture Screeriiiig lor (.ytoio><ici_ty.
`ASIM Desigriatlori F 895_-84; ”Standarcl Practice for psrect Contact Cell Cul-
`Iwe Evdiu,-moi} of Materials for Medical Dev-ces," r‘\'>fM D€$'9n‘<ll'°”
`F 8l3—83.
`
`USP 36
`
`Apparatus-—
`Autoclrive——Employ an autoclave capable of maintaining a
`temperature of TN : 2", equipped with a thermometer, a
`pressure gauge, a vent cock, a rack adequate to accommo-
`date the test containers above the water level, and a water
`cooling system that will allow for cooling of the test con-
`tainers to about 20“, but not below 20“, immediately fol-
`lowing the heating cycle.
`Oven--—-Use an oven, preferably a mechanical convection
`model, that will maintain operating temperatures in the
`range of 50° to 70" within 3; 2".
`/ncubator-Use an incubator capable of maintaining a
`temperature of 37 :r T" and a humidified atmosphere of
`5 t 1% carbon dioxide in air.
`Extraction Containers»-Use only containers, such as am-
`puls or screw-cap culture test tubes, or their equivalent, of
`Type l glass.
`if used, culture test tubes, or their equivalent,
`are closed with a screw cap having a suitable elastomeric
`liner. The exposed surface of the elastomeric liner is com-
`pletely protected with an inert solid disk 50 to 75 ),lm in
`tliii..:kriess. A suitable disk can be fabricated from polytef.
`Pre oration ofA aratus-Cleanse all
`iassware thor-
`-
`.
`‘
`'
`,
`e
`oughly) with throng? acid cleansinc mixt%re and if n ces
`sary, with hot nitric acid followed by prolonged rinsln_ with
`Sterile Water for injection. $t(:‘l'lllZ€ and dry by a suitab e
`processcontainers and devicesused for extraction, transfer,
`or administration of test material. if ethylene oxide is used
`as the sterilizing agent, allow not less than 48 hours for
`complete degassing.
`Procedure——
`
`Preparation of Sam le for Extracts-—Prepare as directed in
`the Procedure under
`iological Reactivity Tests, In Vivo (88).
`Preparation of Extracts—~Prepare as directed for Pre ara-
`tion of Extracts in Biological Reactivity Tests, In Vivo (8 > using
`either Sodium Chloride lniectiori (0.9% NaCl) or serum-free
`mammalian cell culture media as Extraction So/vents. [Nore-
`ll‘ extraction is done at 37° for 24 hours in an incubator, use
`cell culture media supplemented by serum. The extraction
`conditions should not in any instance cause physical
`changes, such as fusion or melting of