The international standard for kanamycin

Join'halof Biologica!St.'ndardlzation(1986) I~, 35-44

The International Standard for Kanamycin* A. H. Thomas,t M. V. Mussett~ and R. A. Broadbridget

Tile International Referencc Preparatiotl of Kanamycin has been replaced by the International Standard f~r Kanamycin. The potency of the standard is based upon a collaborativestudy in ten laboratories in ten countries. Eacb ampoule of :be International Sta~ldard of Kanamycin is defined as containing tbe activity of 10345 I~tema~ionalUnits of Kanamycin.

INTRODUCTION As stocks of the International Reference Preparation of Kanamycin established by the W H O Expert Committee on Biologica! Standardization in I959 t were becoming depleted, suitable material was obtai.'Jed to serve a~ a replacement standard and a collaborative study of this material, in comparison with the International Reference Preparation, was arranged at the National Institute fi~r Biological Standards and Control on behalf of the World Health Organization. 2 This report describes the results of the international colIaborative study. MATERIALS AND METHODS

The ProporedInternational Standard for Kanamyd~J (PIS ) Approximately 50 g of a sample of kanamycin sulphate, Lot No. KM WS- 11(5), was made available through the generosity of Meiji Seika Kaisha Ltd, Tokyo, Japan and * Received for p~bilcation 14 June 1985. t Division of Antibiotics and Chemistry, :~ Statistics Section, WHO International ~boratory for BiologicalStandards, National Institute for BiologicalStandards and Control, Holly Hilk Han~psteao, London NW3 6RB, U.K. 0092 1157i86/010035+lO S03.00/O ~ 1986The InternationalA~sociationof Bit*logicalStandardizatit~n 35

A. H. THOMAS ET AL, through the good offices of Dr T. Mayama. The sample was received in a single container at NIBSC, London, in September 1982, The following analytical data were supplied by the manufacturer: 'Lot No. Potency Loss on drying Form

KMWS 11(51 790 "mcg"/mg (Dry) 0% Crystalline monosulphate-monohydrate

[0~]1~~ Sulphate content Sulphated ash Kanarnycin A content

+ 1 lg ~ ~c, 1'0, water) 16" 1% 0% 79"0%

In addition to sulphate, the sample contains water of crystallization (theoretical: 3%), which could not be detected on "Loss on drying". In March 1983, the sample was d issolved at a concentration of 12'66 mg of hydrated kanamycin sulphate per 1' I g. in double-distilled water prepared in a glass still. The solution was filtered through a Millipore HA membrane {,t.45 ~*m APD and distributed into approximately 4000 non-actinic glass ampoules. In accordance with the recommendation of the W H O Expert Committee on Biological Standardization 3 the material has been distributed into ampoules in precise equal volumes of an aqueous solution and the solution freeze-dried so that each ampoule is expected to contain 10 000 IU ofkanamycit~. Cheek weighings at frequent intervals during the fill showed that the volumes dispensed were within a range of-+ 0* 16% with a mean of I" 10582 g. After freeze-drying a vented polythene plug was inserted into each of tile ampoules which were then further dried in vacuum over phosphorus pentoxide for six days. The ampoules were then filled with pure dry nitrogen, sealed by fi*aion of the glass, tested for leaks and stored at -20~ in the dark. Four ampoules, chosen at random, were opened and the weight of the contents were determined as described for the Third International Standard tot Streptomycin ~ (Table 1). The determined content by weight was 100.8% of target. Tile moisture content of the material in the ampoules was found to be less than 0"2% by the method of Biichler e! a/.r" and by Karl Fischer determination. Tile material irl the ampoules was extremely hygroscopic when exposed to the atmosphere even at a relative humidity of 20% as is shown in Table 2, TattLE 1,

Kanamydn sulphate content of ampoules of the Proposed International Standard for Kanamycin

Ampoule no.

Weight of plug remowd (rag)

Weight of residual kanamycin determined biologically ling)

Total weight of contents (rag)

1 2 3 4

t2.1386 12.2210 12.04411 12.1600

).22:;9 0"3983 {1-3363 0.2556

12.3625 12.6193 12"381}3 12"4156 Mean I2"4444

Theoretical weight per ampoule (rag) 12"66 • 1"10582 X 97t1.1 • I00 36

12'3452

INTERNATIONAL STANDARD FOR KANAMYCIN TABLE 2.

Percentage moisture uptake of the Proposed International Standard for Kanamycln RH

Min

1

2

5

10

15

20 65

2.6 7.0

4.3 10.8

6.6 15'3

7'8 18.8

8"3 21.3

10.1 23'0

Accelerated degradation studies showed no loss of potency after storage at 56~ for 20 months.

The International Referemv Preparation of Kanamycin (IRP ) This preparation has a defined potency of 812 IU per mg; each ampoule containing approximately 50 mg. v The material in the ampoules increased 0' 1% in weight in 5 min when exposed to the atmosphere at a relative humidity of 65% and equilibrium was obtained at 0"2%. DESIGN OF STUDY Six ampouJes of the International Reference Preparation (IRP) and six ampooles of the Proposed International Standard (PIS) were supplied to each laboratory. Workers in the participating laboratories were asked to carry out six independent assays of the PIS against the IRP of Kanamycin. For this purpose an independent assay was defined as one based on a separate ampoule olthe IRP and the PIS, Weighinga of the IRP were to he made with a minimum precision of t-2%, taking suitable precautions to prevent the excessive uptake of moisture and recorded to at least three significant figures. The PIS was not to be weighed but the total contents were to be washed out with a suirabie diluent. Participants were permitted to use any fissay method they chose, provided that each assay contained sufficient information to provide an estimate of potency for the PIS in terms oflRP with associated fiducial limits. A minimum of three dilutions of each preparation was required to allow assessment of the Iinearity and parallelism of the log dose-response lines. There was to be adequate replication (at least five responses for each dilution of each preparation) and each response was to be reported to three significant figures. It was suggested that a potency of 10 000 IU per ampoule should initially be assumed when making test dilutions of PIS. RESULTS Assay results were received from workers in ten laboratories in ten countries and the names of those participating in the study are given in the Appendix. Throughout this report code numbers are used to refer to laboratories and these numbers are nor related to the order of listing in the Appendix. In Laboratory 5 a turbidimetic assay method was used and in all other laboratories diffusion methods with some variations of technique and design (Table 3) were used. Each dilution of each preparation (i.e. all treatments) was tested in Laboratories 1, 2, 6, 7 and 8 six or eight times on large square plates; in the other laboratories the various treatments were applied singly ro each of a number of Petri dizSes. Workers from all laboratories provided results of six assays, as requested. In 37

A. H. THOMAS ET AL. TAgt.E 3. Lab no. Test organism

Assay raenlods used by participarlng laboratories

IRP doses Assay Medium (IU/ml) medium * pH

1 B. subtilis 1'5, 3, 6 A NCTC 8236 2 B. pumilus 5, 10, 20 A NCTC 82zi l 3 S. aureus 5, 10, 20 B 209p 4 B. sub/ills l'6, 2'0, 2"5 " C ATCC 6633 5 St. ~m~,us 8, lO, 12.5 D ATCC 29737 6 B. subtilis I, 2, 4 A ATCC 6633 7 B. subtihs 5, I0, 20 E ATCC 6633 8 B. puv,'ilus 10, I7, 29, 50 A NCTC 8241 9 B. sub#l# 5, 20 G ATCC 6633 10 f3, subtilis 5, 8'5, 15 A NCTC 8236

7'8 -

-

7'8 8'0 7'8-8'(/ 6'95-7'05 8"0 7.4 7'8 7'g-8"0 7,8

Assay Design F~ur square plates 6 • 6 Latin Square One square plate 6 • 6 Latin Square Three operators using 6 Petri dishes Two sets of 9 Petri dishes Turbidimetrie 5 robes per dose One square plate 6 • 6 Latin Square Two square plates 6 • 6 Latin Square Four square plates 8 • 8 Latin Square I5 Petri dishes Six Petr/dishes

Responses per assay 144 36 1(18 108 30 36 72 256 90 36

* A, 0.6~ pt,proge. 0.4Vc pancre.aric dJgesr of caesin, 0']5~ beef extract, 0'3~,~ yeast extracr, (I.lc~ D-glucose. 1"5%agar; B, Hottinger's broth, 0"3% NazHPOI. l'Sr?kagar; C, 0"5% peptone, 0'3~ beef extract, 0"3% KzHPOa, 2"0@agar; D, U'5% peptone, 0" 15% beefextract, 0' 15% yt ~t ex:ract, 0.35% NaCI, (J' I~ I>gJucose,()'3fi8~/cK_~HPOj, 0" 132~'~KH2PO,; E, 1.0rT~peptone, 15~7, L~':fextract, 0. ~'7, NaCl, 0"2~ Na2HPOa, 0"9',~ agar; G, 0"5% peptone, 0'3% beef extract, 1.5~ agar. Laboratories I to 6 and Laboratory 10 there were two assay sessions on each of three days while in L~boratories 8 and 9 comparisons of PIS with 1RP were made on six different days. The resuks from Laboratory 7 were not dated. Participants in six laboratories used f3acil/us subtiliJ as the test organism, two used Bacilus pumi/us and two Stapky/ocoaws aureuJ. In Laboratories 4 and 9 stainless steel cylinders were used to apply the solutions to the plates. In Laboratory l, paper discs were used; in Laboratories 2, 3, 6 and 8, wells were made in the agar; and in Laboratory 1() solutions were pipetted on to the surface of the agar. The standard preparations were diluted in phosphate buffer at pH 7 " 8 - 8 ' 0 for diffusion assays and in distilled water for the turbidimetric method. Pre-incubation diffhsion periods of between 0.75 and 2.0 hours at room temperature were used in Laboratories 1, 2, 6, 8, 9 and 10 (4~ for Laboratory 3). Incubation temperatures were about 37~ (35~ for Laboratory 3) and the duration of incubation was 18 hours ( 16 hours for Laboratory 4). In Laboratory 5 the tubes were incubated for 4 hours at 37~ Participants in most laboratories tested three dilutions of each preparation, but the design in Laboratory 8 was (4 + 4) and in Laboratory 9 it was (2 + 2). in seven laboratories responses in diffusion assay were measured as the diameters of the zones of inhibition, but in Laboratories 1 and 10 zone area was recorded. For the turbidimetric assay, optical densities were used as responses. Participants in some laboratories 38

INTERNATIONAL STANDARD FOR KANAMYCIN repeated their basic assay design for each ampoule comparison, i.e. in Laboratories 1,7 and 8 the contents of each pair of ampoules were tested on more than one large plate, in Laboratory 4 two sets of Petri dishes were used and in Laboratory 3 each comparison was made by tbme operators.

Statistical nl~,lhods The results were analysed by the usual methods for parallel line assay, 8 i.e. responses were related to the logarithms of dilutions and analyses of variance were carried out in order to assess the significance of the regressions of response on log dose and the llnearity and parallelism of the log dose-response lines. Zone diameters recorded in Laboratories 6 and 8 were squared before analysis as the participants in these laboratories reported improved linearity by use of this procedure. When isolating error terms in analyses of variance, account was taken of experlmental design, e.g. data from Labnrarnries 3, 4 and 9 were of a randomized block design, since all treatments were tested on each Petri dish. Results obtained by each operator in Laboratory 3 were initially analysed separately and, after consideration of possible difft-rences in potencies estimated by different operators and interactions between treatments and operators, all data relating to a single weighing/ampoule comparison were pooled for analysis. Similarly, results derived from each pair ofampoules used in Laboratory 4 were pooled after initial examination of responses on each set of dishes. In Laboratory 10 all treatments were applied to a Petri dish simultaneously and a set of six dishes was used for each assay; solutions were rotated between pipettes so that each solution was dispensed by each pipette within the set of dishes. Thus, pipette variation could be isolated as a variable, in addition to dish variation, anti ~he design was Latin Square. Participants in laboratories using large plate diffusion methods arranged the six (or eight) treatments in Latin Square designs and the results from each plate were analysed separately. For Laboratories l, 7 and 8 all information constituting one assay was then included in a single analysis. The turbidimerric assays from Laboratory 5 were assumed to be of random design. When tests of validity for the parallel line model had been examined, relative potencies were estimated as the anrilogs of the horizontal distances between log dose-response lines for IRP and P1S. The Z 2 test for homogeneity was applied to sets of potency estimates within and between laboratories and, for homogeneous sets, weighred mean potencies were calculated using the reciprocals of the variances of log potencies as weighting factors. For heterogeneous sets, unweighted 8eometric means were calculated together with weights or limits of error obtained directly from disrributions of log potencies. Laboratory comparisom of PIS with IRP Potencies of the P1S relative to the IRP which were estimated from each set of assay data are expressed in international units per ampoule and are given in Table 4. Although responses in diffusion assays were variously expressed as zone area, zone diameter and squared zone diameter there were no deviations of the log dose-response lines from linearity that were significant at the I% level. There were significant curvatures in the first two turbidimetric assays but this was probably due to small error variances, as shown by the high weights. One potency obtained in Laboratory 9 was rejected from the combined values on grounds of non-parallelism but, in general, there 39

A. H. THOMAS ET AL.

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INTERNATIONAL STANDARD FOR KANAMYCIN

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Fig. 1. Frequencydistribution of log potency estimatesfor the ProposedInternational Standard for Kanamycln in terms of the international ReferencePreparation of Kanamycinobtained in laboratories 1-10. was good slope agreement in the study with about a half of the total number of assays showing a steeper slope for the IRP than for PIS and 1dee versa. There were no significant differences between the potencies obtained by different operators in Laboratory 3 (or between any of the assay sub-sets introduced by other laboratories) and thus nothing to prevent estimation of a single potency for each assay as defined in the protocol.

Combinedpotenciesfor IRP in terms of PIS Individual potencies accepted for inclusion in mean values are presented in logarithmic form in Fig. 1. Sets of log potencies were homogeneous for six laboratories but heterogenous within Laboratories 2, 4, 7 and 8. The laboratory means potencies are given in Table 5 together with associated 95% confidence limits and total weights. The ten laboratory means were heterogeneous so the overall mean of 10 414 IU/amp gave equal weight to the contribution from each laboratory and was unduly affected by the outlying value from Laboratory 7. Removal of this value reduced the overall value to 10 338. Combination of individual potencies led to similar conclusions, the 59 values in the figure being very heterogeneous (X2 = 1302) with a geometric mean of 10 422 (10 264-10 583) IU/amp. Exclusion of results from Laboratory 7 reduced X2 to t86 and the heterogeneous set still remaining combined to a value of 10 345 (10 289-10 402). DISCUSSION Both intra- and inter-laboratory heterogeneity of the potency estimates were observed in the collaborative study, but the intra-labotatory variations were no greater titan those usually encountered with this type of high precision assay of antibiotics. 41

A. H. THOMAS E T A L . TABLE 5. Laboratory number

Laboratory mean potencies of the Proposed International Standard for Kauamycin (IU/ampoule) Number of assays

Mean potency

Confidence limits (P = 0"95)

Weight

10 395-10 510

674 483

1

6

10 '452

2

6

I0 lOO'

3 4 5 6 7

6 6 6 6 6

10 076 10 375' 10 400 I0 298 11 125'

8

6

5 6

9 10 Labs 1-10 Omhting Lab 7

9772-10 440

9998-10 l0 196-10 10 28|-10 10 212-10 9270-13

~2 006

155 558 533 384 352

335 45l I I4 863 139 ~,85 290 330 1052

10 472'

lO 338-10 607

210 869

I0 335 IO 537 10 414 ~ 10 338*

10 In,8- 10 5~6 10 444-10 631 10 212-I0 621 lO 215-10 462

54 8,ll 258 162 70 468 198 8~0

Unweighted mean from heterogeneousset Although there was inter-laboratory heterogeneity even after excluding the results of Laboratory 7, the variation about the remaining laboratory means was limited to • 1.19% and the variation about the geometric mean of tl~e individual potencies was reduced from _+ 1'53~/~ to-+0"55~. The ampoules were prepared with an intended nnminal content of 10 000 International Units of activity per ampoule. The weight of solution delivered would be expected to yield 10 054 International Units of activity per ampoule; they were found to contain l0 345 International Units per ampoule with confidence limits of +0'55')~, i.e. approximately 3"5% more than the target. The quantity of PIS distributed into the ampoules was caleu}ated on the supplier's estimation of potency and water content. The weight of material found in the ampoules was very close to tile target weight confirming that the PIS did contain 3% water of crystallization. The potency of the PIS, 831 IU/mg, calculated from the potency and weight content per ampoule is the potency expected for theoretically pure anhydrous kanamycin monosulphate. Based on the manufacturer's estimate of potency the purity of the PIS was calculated as 97"904 assuming it to be kanamycin monosulphate monohydrate. In originally assigning the international unit of kanamyein the manufacturer's estimate of purity was accepted so as to equate one international unit to I p g of theoretically pure material.9 The IRP was established in 1959 without a collaborative study, the potency was calculated on an anhydrous basis. Recent Karl Fischer determinations on the ampouled material showed less than 0'04~. of water confirming the anhydrous nature of the 1RP. High-performance liquid chromatographic analysis showed the relative base composition of the IRP and P|S were 96"9~. and 97.5cX, kanamycin A, respectively. The PIS has a potency about 39~ greater than expected and this could indicate that either the estimate of the chemical purity of the IRP was incorrect or possibly that the IRP had lost activity. No evidence for such a loss is available and ampoules oflRP stored at 20 and 37~ have shown no significant loss of activity after 23 years relative to material stored at -20~ 42

INTERNATIONAL STANDARD FOR KANAMYCIN In accordance with the authorization given in the thirty-fifth report 2 of the WHO Expert Committee on Biological Standardization the National Institute for Biological Standards and Control established the preparation studied as the International Standard for Kanamycio and, with the agreement of the participants, defined the activity of the contents of each ampoule as 10 345 IU of Kanamycin.

Acknowledgements Grateful acknowlegement is made to the participants in this collaborative study and to Gillian Darbyshire for considerable assistance with numerical analysis.

REFERENCES 1. WHO Expert Committee (m Biological Standardization, WHO Tech ReD Ser 1960; 187: 6, 2. WHO Expert Committee on Biological Standardlzatian. WHO Tech Rep Set 1985; 725: 15. 3. WHO Expert Committee on Biological Standardizati
APPENDIX

List of participantJ in the cdlabora#t,e study Austraha Mr R. K. Howard and Mrs J. Andres National BiologicalStandards Laboratory Canberra City A.C.T

Eire Dr D Hayes State Laboratory Castleknock Co. Dublin

Hu~gar) Professor I. Bayer, Dr G. Keseru and Mr L. Gampe National Instituteof Pharmacy Budapest

Japan Dr A. Otsubo Production Planningand Control Department Pharmaceutical Division Meiji Seika Kaisha Lrd Tokyo

The Netherlands Dr 13. van Klingeren Laboratory for Chemotherapy Rijks lnstituut voor Volksgezondheid en Milieuhygiene l~ilthoven 43

A, H. THOMAS ET AL, Ne~eZealand Miss H. Heffi?rnan and MissJ. Cadeton National Health Institute Porirua Wellington

People~ Repleblieof Cbipta Dr Bi Ruren Division of Antibiotics National Institute for the Control of Pharmaceutical and Biological Products Temple of Heaven Beijing Umon of ,%etet S,~'taltrl Repulfll,s ProfessorJ, F. Krilov State Research Institute for Standardization and Control of Drugs Nauchni Proyezd Moscow

44

United Kingdov~ of Great Britain a~ld Northern ~re/and Mr R. A. Broadbridge and Mr L. J. Bates National Institute for Biologic,~lStandards and Control Hampstead London United States of America DrJ. H. Grallam Antimkrobial Drugs Branch National Center for Drugs and Bioh)gics Food and Drug Administatinn Washington D,C.