EUROPEAN DIRECTORATE FOR THE QUALITY OF ... 2072 addition.pdf18 D. Quality control on bulk and final...

EUROPEAN DIRECTORATE FOR THEQUALITY OF MEDICINES (EDQM)

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DEPARTMENT OF BIOLOGICAL STANDARDISATION &OMCL NETWORK (DBO)

Working document, with no legally binding

status, intended exclusively for the addressees

and their associates, under the responsibility of

the addressees (listed opposite). Level 2

English /Anglais PA/PH/ISA (06) 3 DEF

This document will not be redistributed in sessions

Strasbourg, October 2006

INTERNATIONAL STANDARDS FOR ANTIBIOTICS

ISA002: Final Study Report

Collaborative Study for the Establishment of the Third International Standard for Nystatin

Authors: G. Rautmann, E. Charton, A. Daas

Administrative Assistant: L. Vignoli

Released by: J-M. Spieser, A. Artiges

DEFINITIVEDistribution

For action:

For information :

ECBS-WHO Expert Committee on Biological Standardisation - WHO

ISA002 Nystatin Study

COLLABORATIVE STUDY FOR THE ESTABLISHMENT OF THE THIRD 1

INTERNATIONAL STANDARD FOR NYSTATIN 2

Authors: G. Rautmann, E. Charton, A. Daas3

I. Introduction 4

The first International Standard (IS) for nystatin was established by the WHO in 1963 on the 5

basis of an international collaborative study (1). It was assigned with a potency of 30006

International Units per mg (IU/mg). A second IS for Nystatin was established in 1982 with an 7

assigned potency of 4855 IU/mg (2).8

As supplies of this IS were becoming exhausted, the European Directorate for the Quality of9

Medicines (EDQM) was requested by the WHO Expert Committee for Biological10

Standardization (ECBS) to undertake appropriate steps for its replacement by the11

establishment of a new batch.12

This report presents the international collaborative study in which laboratories from 17 13

countries participated. Potencies of the candidate material were estimated by microbiological14

assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the15

second IS for nystatin was used as standard.16

This report provides details about the material donated by a manufacturer, the processing to 17

generate a candidate batch and the analytical controls to assess its quality. It describes the18

statistical analysis of the results, the conclusions made thereof and the recommendation to the19

Expert Committee for Biological Standardization/ WHO.20

II. Bulk material and processing 21

A candidate bulk material was kindly donated by DSM Capua S.p.A. (Italy). Two separate 22

bottles each containing 1 kg of nystatin of the same batch of current pharmaceutical grade23

appropriate for therapeutical use were received by the EDQM in December 2005. Upon24

receipt, the bulk material was stored at +4°C before processing. The candidate material was25

claimed by the manufacturer to comply with the quality standards of both the European26

Pharmacopoeia and the United States of America Pharmacopoeia relevant monographs. A27

certificate of analysis was provided in the batch documentation.28

A. Filling 29

All powder weighting and filling were carried out in glove boxes under a controlled30

atmosphere by use of argon gas.31

The powder of one bottle was allowed to equilibrate at room temperature and was submitted 32

to extensive mixing in a Turbula. Ten weighings of 50 g were distributed in separate33

containers subsequently sealed, protected from light and stored at –20°C. The filling34

campaign was organised over 5 consecutive days from 27 to 31 March 2006. Prior to any35

further processing of the nystatin powder, containers were stored overnight unopened under a36

glove box to enable room temperature equilibration.37

B. Production of a suitable “reference standard” for monitoring purposes.38

WHO IS are primary reference materials and as such cannot be tested against higher order39

reference standards. As a consequence, real time stability studies are not usual practice and in40

many cases, stability of WHO IS were assessed by means of accelerated degradation studies.41

PA/PH/ISA (06) 3 DEF 2/22

06/10/06

In the documentation supplied with the batch, stability was confirmed by reporting data1

indicating no sign of decrease for parameters including among others, microbiological2

activities and impurity profiles by liquid chromatography after an incubation at 25 ± 2 °C3

over a period of 36 months.4

It appeared however an attractive alternative to store some of the powder at –80°C and to use 5

it, at regular intervals in the future, to assess the potency of vials stored at –20°C, the typical 6

storage temperature of the WHO IS batch. Consequently it was decided to start the filling7

process by weighing 100 mg in white glass ampoules from the first 50 g container. A total of8

190 ampoules were prepared. They were immediately sealed by fusion and tested for leaks by9

methylene blue uptake. The entire batch was stored at –80°C and is identified under the10

number 06/03-103.11

C. Production of the WHO IS for nystatin candidate batch 3.12

To increase throughput, two gloves boxes were used within which vials were filled and then13

clearly identified as batch 06/04-01A or 06/04-01B according to the glove box used. 1 671 14

vials of batch 06/04-01A and 2 148 vials of batch 06/04-01B were filled with 100 mg of15

nystatin, closed with an inert rubber stopper and sealed. Fillings were organised in morning 16

and afternoon sessions and traced by sub-batch numbering. All vials were stored at -20°C.17

D. Quality control on bulk and final batch.18

1. Conformity of bulk 19

During the first weighing session on the first morning, two lots of 5 g of nystatin powder were20

set aside to enable subsequent analysis according to the European Pharmacopoeia monograph21

since the manufacturer declared compliance with it. The results obtained by use of the22

analytical methods described under “Absorbance, Composition, Heavy Metal, Loss on Drying 23

and Sulfated Ash” were in good agreement with those of the certificate of analysis provided24

by the manufacturer. The bulk was therefore considered suitable for further processing.25

2. Homogeneity of powder fillings26

Filling was carried out to a nominal content of 100 mg with a balance connected to a27

recording device. Mean filling weights and relative standard deviations (RSD) were28

calculated for each session and for each glove box. No significant variability was observed29

between sessions and the overall mean filling weights were 103.17 mg (RSD 1.75 %) and 30

104.26 mg (RSD 2.33 %) for the two gloves boxes respectively. These results supported the 31

assumption that there was no heterogeneity in weight between vials as a result of the use of32

two glove boxes.33

3. Sampling design 34

During each half day filling session and for each glove box, 3 filled vials were sampled at the35

beginning, middle and end of the session. Each vial was individually tagged for unambiguous36

identification and stored at –20°C until subsequent analysis.37

4. Quality control 38

From the above pool of samples, the 3 vials collected at the middle of each filling session39

were tested for their water content by the loss on drying method. Samples were assayed in a 40

design that would allow elimination of a possible trend in the order of weighing and the inter-41

assay variation, while at the same time enabling to measure these effects, should there be any.42

In short this means that the samples were handled in chronological order in half of the assays43

3/22 PA/PH/ISA (06) 3 DEF

06/10/06

and in anti-chronological order in the other half and that samples from each day were1

included in each assay.2

An analysis of variance was carried out in which 4 effects were modeled:3

1. Difference between day of assay (5 classes).4

2. Difference between glove boxes (2 classes).5

3. Trend of preparation (10 sessions ordered on a chronological scale)6

4. Trend of weighing (10 weighings ordered on a chronological scale)7

The calculations were performed with the SAS-System.8

The analysis of variance (see Table 3 below) shows a highly significant difference between9

assays (p<0.0001) and a significant trend in the order of weighings (p<0.02). No trend is10

observed for the order of sampling (p=0.70) and no significant difference between the glove11

boxes can be observed (p=0.10).12

Table 3 – Analysis of variance13

Source Degrees of

Freedom

Sum of

Squares

Mean Square F-value Probability

Assay 5 0.0910 0.0182 8.87 <0.0001

Glove box 1 0.0057 0.0057 2.79 0.1022

Session 1 0.0003 0.0003 0.15 0.7011

Weighing 1 0.0142 0.0142 6.93 0.0118

Residual error 42 0.0862 0.00205

Total 50 0.2016

The mean results per group of samples are presented in Table 4 below.14

Table 4 – Mean result per group 15

Daypart Mean n Assay Mean n Weighing Mean n Hood Mean n

Mo (AM) 4.053 3 1 4.100 8 1 4.102 6 A 4.112 21

Mo (PM) 4.080 3 2 4.014 8 2 4.145 6 B 4.081 30

Tu (AM) 4.063 3 3 4.086 9 3 4.110 6

Tu (PM) 4.098 6 4 4.083 9 4 4.085 6

We (AM) 4.123 6 5 4.151 7 5 4.082 6

We (PM) 4.082 6 6 4.131 10 6 4.108 6

Th (AM) 4.092 6 7 4.075 6

Th (PM) 4.088 6 8 4.060 5

Fr (AM) 4.118 6 9 4.043 3

Fr (PM) 4.100 6 10 4.130 1

Overall mean: 4.094 (n=51)16

In conclusion, there is no significant indication for heterogeneity of the fillings between days17

and glove boxes. Two randomly chosen vials of the filling campaign are not likely to differ by18

more than 0.13% from each other. ( ts 2 = 2.020 0.00205 1.414 = 0.13). These results19

demonstrated that the two batches were to be considered as homogeneous since they did not 20

differ significantly from each other.21


06/10/06

The estimated water content is 4.09% with 95% confidence limits of 4.04% and 4.14% where1

the mean results per assay are used to estimate the uncertainty (n=6). These results are not in2

disagreement with the figure of 4.3 % stated on the certificate of analysis.3

5. Environmental control 4

During the entire filling campaign, glove boxes were continuously monitored for oxygen5

content and hygrometry. Measurements were carried out at the beginning and end of each half6

day session. Values recorded did not significantly deviate from settings.7

6. Stability studies on the product in the final container8

An accelerated stability study is being carried out. A periodical monitoring is scheduled.9

III. Collaborative study10

A. Participants.11

A total of 17 laboratories from different countries around the world volunteered to participate 12

in the study. Each participant is referred to in this report by an arbitrarily assigned number,13

not necessarily reflecting the order of listing in the Appendix.14

B. Samples15

Each participating laboratory received:16

- 7 vials of Nystatin proposed standard (EDQM code: 27979) containing approximately 17

100 mg of powder per vial (activity about 6000 IU/mg),18

- 3 vials of Nystatin 2nd International Standard (80/508), 4855 IU/mg (EDQM code: 19

28389) containing approximately 100 mg of powder per ampoule.20

C. Assay method and study design 21

The participants were asked to estimate the potency of the nystatin candidate IS by a22

microbiological diffusion assay method using the Nystatin 2nd International Standard23

(80/508) as reference preparation. It was requested that any analytical method used be in24

compliance with requirements set in regional compendia in particular with respect to method 25

validity criteria. A total of six independent assays were to be carried out by each participant.26

Prior to carrying out the study an enquiry was carried out which demonstrated that27

participants were going to use very similar testing procedures (see Annex 2). All but one28

participant used saccharomyces cerevisiae. All participants used one of the Pharmacopoeia’s29

prescribed media (medium F/ medium 19). Based on this enquiry, a pilot assay has been30

performed in the EDQM laboratory in order to develop and provide details for the study31

protocol, taking the European Pharmacopoeia as the example.32

Participating laboratories were requested to follow the study protocol as far as possible and 33

according to the prescription given in the relevant Pharmacopoeias they have as usual34

reference.35

IV. Results and statistical analysis36

Statistical methods37

The experimental data obtained in this study were analysed as parallel line assays (Finney 38

1978), using the SAS-System (GLM procedure) and CombiStats. Both programs give 39

identical outcomes but the output is somewhat easier to transform to tables with the SAS-40

system, whereas CombiStats provides a more streamlined output for individual assays.41


06/10/06

All assays were submitted to visual inspection of the plots to check for unusual features.1

Validity of the assays was assessed according to the flow chart in Figure 1. In routine2

situations where decisions are based on only one assay or only a few assays, the level of3

significance is usually taken to be P=0.05. In collaborative studies with many participants,4

however, a more conventional level of significance is often used. This is because the level of5

P=0.05 leads to about 10 per cent errors of the first kind (incorrect rejection of assays),6

whereas errors of the second kind (incorrect acceptance of assays) will not influence to global7

outcome of the study much because of the large amount of data available. Hence, the level of8

significance in this study is taken to be P=0.01 which would imply an expectation of about 29

per cent incorrect rejections. A slight but significant curvature was not considered reason for10

rejection if the mean square for quadratic regression was less then 1/100 of the mean square11

for linear regression and the difference between preparations was small (Bliss 1956, Hewitt12

1981).13

Whenever a laboratory performed several assays based on the same weighings, yielding14

several non-independent estimates of potency, a weighted mean potency of the valid sub-15

assays was calculated using weights proportional to the reciprocal of the variance. The valid16

assays per laboratory were combined using the same method of weighted combination, but a17

semi-weighted combination was used whenever the confidence intervals of the independent18

potency estimates did not satisfactorily overlap each other by means of a 2 test for19

homogeneity (P<0.10). The estimates (one for each of the participants) were then combined20

into one single estimate with a 95 per cent confidence interval using the same method of21

semi-weighted combination.22

Results 23

Seventeen (17) laboratories reported results from assays. In this report they are referred to by24

their randomly assigned code-numbers (1 to 17), not necessarily corresponding with the order25

of listing in the list of participants. Thirteen (13) laboratories used the European 26

Pharmacopoeia method, 1 used the British Pharmacopoeia (= European), 1 used the United 27

States Pharmacopoeia, 1 used the Chinese Pharmacopoeia, and 1 used the Argentinean28

Pharmacopoeia. All participants carried out at least 6 assays as requested. Laboratory 629

carried out 7 assays, one for each vial provided. Laboratory 17 also tested all 7 vials, but30

performed 2 assays on the first 2 vials and 1 assay for the remaining 5 vials. Laboratory 4 31

carried out all assays by two operators, but only the results of 2 assays from the 2nd operator32

were reported because the other results were considered invalid by the laboratory. The results33

from this 2nd operator are coded as Laboratory 4b. Laboratories 4, 7, 9 and 12 carried out34

multiple sub-assays per vial. For the calculations, all sub-assays were analyzed as individual35

assays after which they were combined into one potency estimate per vial. If all sub-assays36

are counted as individual assays a total of 192 assays were reported or 9032 zone-diameter37

readings.38

The complete computer output of the parallel line analyses as performed at the EDQM is39

available in PDF format to participants of the study (384 pages generated by CombiStats). A 40

summary of the results, as generated by the SAS-System is given in Tables 1.1 and 1.2 (See41

Annex 1 for the essential SAS-scripts used). Shown are the potency estimates and associated42

95 per cent confidence intervals, together with the relevant P-values. P-values below the43

significance level of 0.01 are printed on a grey background. The confidence intervals based on 44


06/10/06

calculations by the participants are also listed. Some laboratories reported the potency1

estimate and/or confidence intervals in terms of percentage of the estimated or assumed2

potency. In these cases the percentages were converted to IU/mg at the EDQM. Laboratory 133

did not report confidence intervals. Laboratory 14 reported confidence intervals which were4

so discrepant from the results found at EDQM that the laboratory was contacted in order to 5

find an explanation. After investigation the participant concluded having made a mistake and 6

that their recalculations concurred with the EDQM calculations. At the time of writing of this7

report the exact values were not yet available. Since participant’s calculations are only 8

included for information this should not influence the conclusions of this study.9

A graphical representation of the confidence intervals of each individual (sub)-assay is shown10

in Figure 2 (EDQM calculations) and in Figure 3 (Participants’ calculations). Potency 11

estimates ranged from 5045 IU/mg (Lab 4) to 6162 IU/mg (Lab 13) disregarding the12

extremely high results from laboratory 14 (up to 7656 IU/mg).13

Laboratory 1 14

The 6 assays were statistically valid and the potency estimates were homogeneous (P=0.890).15

The weighted combined estimate is 5829 IU/mg (±1.6%).16

Laboratory 2 17



Laboratory 3 20



Laboratory 4 23

This laboratory carried out 3 sub-assays per vial using one Latin square plate per sub-assay,24

resulting in 18 potency estimates. In addition this laboratory carried out assays by a second25

operator who also performed 3 sub-assays per vial, but only the results of 2 vials (i.e. 6 sub-26

assays) were reported because the other results were considered invalid by the laboratory. The 27

results from this 2nd operator are coded as Laboratory 4b. Of the assays from the first operator28

6 plates gave invalid results, 3 of which were part of assay 6 and therefore yielding no valid29

potency estimate from assay 6. Of the assays from the second operator 5 plates gave invalid30

results, leaving only one statistically valid result. Since this result is also of borderline quality31

with a p-value for non-parallelism of 0.015, it may be argued whether it would not be better to32

disregard all results from the second operator and only include the valid results from the first 33

operator. If this is done, the 5 resulting confidence intervals show significant heterogeneity34

(P<0.001), so a semi-weighted combination was taken. This results in a potency estimate of35

5310 IU/mg (±2.0%).36

Laboratory 5 37



40


06/10/06

Laboratory 6 1

This laboratory carried out 7 assays, 1 for each vial provided. The 7 assays were statistically2

valid and the potency estimates were homogeneous (P=0.346). The weighted combined 3

estimate is 5610 IU/mg (±1.6%).4

Laboratory 7 5

This laboratory carried out 6 sub-assays per vial using one Latin square plate per sub-assays,6

resulting in 36 potency estimates. All sub-assays were statistically valid and could be7

combined by taking the weighted mean potency, resulting in 6 potency estimates which were8

homogeneous (P=0.794). The weighted combined potency estimate is 5981 IU/mg (±0.4%).9

Laboratory 8 10

The 6 assays were statistically valid but the potency estimates were heterogeneous (P<0.001).11

The semi-weighted combined estimate is 5801 IU/mg (±1.2%).12

Laboratory 9 13

This laboratory used 6 Latin square plates per assay, but reported only the results of those 14

plates they considered valid, resulting in a total of 32 sub-assays. This laboratory used 2 dose15

levels per preparation with 2 independent weighings per dose. It was therefore not possible to 16

check for parallelism in the individual assays, but the laboratory reported that the linearity of17

the dose-response over the dose range used had previously been established. None of the sub-18

assays showed significant deviations from parallelism and visual inspection of the plots19

showed that the assays were of satisfactory quality. All assays were therefore retained as20

being valid and were combined by taking the weighted mean potency, resulting in 6 potency21

estimates which were heterogeneous (P<0.001). The semi-weighted combination is 5299 22

IU/mg (±0.7%).23

Laboratory 10 24

The 6 assays were statistically valid but had rather wide confidence limits (about ±10% on 25

average). Taken as individual assays the results should be rejected because of lack of26

precision. However, the potency estimates are remarkably homogeneous (P=1.000) and visual27

inspection of the plots did not reveal any features which could indicate fundamental28

invalidity. The weighted combined estimate is 5612 IU/mg (±3.4%). Since the combined29

potency is sufficiently precise to be acceptable, the assays are a-posterior accepted as being30

valid despite their individual lack of precision.31

Laboratory 11 32

The 6 assays were statistically valid but had rather wide confidence limits (about ±7.5% on33


precision. However, visual inspection of the plots did not reveal any features which could 35

indicate fundamental invalidity and the confidence intervals showed satisfactory homogeneity 36

(P=0.964). The weighted combined estimate is 5540 IU/mg (±2.7%). Since the combined37




06/10/06

Laboratory 12 1

This laboratory carried out 3 sub-assays per vial using one Latin square plate per sub-assays,2

resulting in 18 potency estimates. One sub-assay was statistically invalid due to significant3

deviations from parallelism. The valid sub-assays could be combined by taking the weighted4

mean potency, resulting in 6 potency estimates which were heterogeneous (P=0.006). The5

semi-weighted combined potency estimate is 5937 IU/mg (±0.5%).6

Laboratory 13 7

The 6 assays were statistically valid but had rather wide confidence limits (about ±6.0% on8


precision. However, visual inspection of the plots did not reveal any features which could 10

indicate fundamental invalidity and the confidence intervals showed satisfactory homogeneity 11

(P=0.282). The weighted combined estimate is 5940 IU/mg (±2.2%). Since the combined12



Laboratory 14 15

One (1) assay was statistically invalid due to significant deviations from parallelism. Three16

(3) other assays were statistically invalid due to significant deviations from linearity. Overall,17

all assays gave much higher responses for the test-preparation than for the standard. The18

potency was on average much higher than expected (about 7450 IU/mg). At first it was19

thought that a dilution error might have been made, or that a dilution step was overlooked in 20

the calculations but neither appeared to be the case. The participant suggested that a probable21

cause could be that the temperature excursion experienced during transit may have 22

compromised the integrity of either one of the materials. It seems therefore prudent to exclude23

the outlying results from this laboratory from the final calculation for assigning a potency24

value.25

Laboratory 15 26



Laboratory 16 29

The laboratory initially submitted results from 4 assays only because they judged assay 1 and30

assay 6 to be statistically invalid. Upon request these 2 assays were also submitted. Assay 631

was indeed found to be statistically invalid due to significant deviations from parallelism and32

linearity, but assay 1 was found to be acceptable whereas assay 4 had to be declared invalid33

because of significant deviations from linearity. The 4 valid potency estimates were34

heterogeneous (P<0.001). The semi-weighted combined potency is 5621 IU/mg (±2.4%).35

Laboratory 1736

This laboratory submitted results from 9 assays. All 7 vials were tested. The first 2 vials were37

assayed twice and the other 5 vials were assayed once. All assays were statistically valid. The38

duplicate results for the first 2 vials were first combined using a weighted combination. The39

resulting 7 potency estimates were homogeneous (P=0.501) and the weighted combination 40

was 5337 IU/mg (±1.1%).41


06/10/06

A histogram of all potency estimates per assay is shown in Figure 4 and a histogram of the1

mean results per laboratory is shown in Figure 5. The final confidence intervals per laboratory2

are summarised in Table 2 and a graphical representation is given in Figure 6.3

The χ2 value for between-laboratory homogeneity is highly significant (P<0.001) so a semi-4

weighted combination was made excluding the result from laboratory 14, which yields 57105

IU/mg with 95% confidence limits of 5673 to 5747 IU/mg (which is ±0.6%). If the result6

from laboratory 14 is included the outcome is 5780 IU/mg, but it is not easy to give7

confidence limits for this figure because the assumption of normality is violated.8

For the purpose of comparison the general unweighted geometric mean potency was9

calculated for the 104 independent assay estimates giving 5775 IU/mg if laboratory 14 is10

included, and 5684 IU/mg with 95% confidence limits of 5557 to 5814 IU/mg (which is11

±2.3%) if laboratory 14 is excluded. If all statistically invalid results are also excluded the12

unweighted geometric mean potency is 5686 IU/mg with 95% confidence limits of 5560 to13

5816 IU/mg (which is ±2.3%).14

V. Discussion15

In accordance with the precedent set for other International Standards for antibiotics the new16

replacement batch was established against the current international standard (in this case the17

2nd standard). This approach will ensure continuity of the unitage.18

All participants used their current standard method which showed no major discrepancy19

between the different laboratories (see Table 1). This was confirmed in the results submitted20

which showed a ± 9% distribution about the mean, an acceptable figure for such an21

international study.22

VI. Response from Participants23

A draft report was sent to the 17 study participants. Fourteen of them did not oppose the24

conclusions and the proposed assignment of 5710 IU per milligram of nystatin.25

One participant commented that the overall mean should be calculated using weights26

inversely proportional to the variance based on the width of the confidence limits. This would27

result in a potency estimate of 5831 IU/mg and therefore this laboratory concludes that the28

proposal of 5710 IU/mg is an underestimation.29

Answer from the organizers: If confidence intervals between laboratories do not sufficiently30

overlap, as is the case in this study, the inter-laboratory variation should also be taken into31

account to establish weights given to the potency estimates. The weighted mean of 5831 32

IU/mg does not give duly consideration to the observed inter-laboratory variation and gives33

disproportional weight to laboratories 2, 7, and 12. The semi-weighted mean of 5710 IU/mg is34

therefore considered more appropriate.35

Two participants returned editorial comments.36

VII. Proposal37

It is proposed that the WHO 3rd

IS for Nystatin (EDQM internal code ISA002) be assigned an 38

antimicrobiological activity of 5710 IU per milligram of substance.39

PA/PH/ISA (06) 3 DEF 10/22

06/10/06

VIII. Acknowledgements1

The organisers express their sincere thanks to all participants for their valuable contributions2

to this study. Special thanks go to the donator of the Nystatin drug substance, DSM CAPUA,3

Italy, for their well-appreciated contribution. The study was organised by the EDQM (project4

code ISA002) as the collaborating centre for the WHO.5

IX. References 6

(1) Lightbown et al. Bulletin of the WHO, 29, 87-94.7

(2) Thomas and Dixon, WHO/BS/82.1350 8

(3) Bliss C.I., The calculation of microbial assays, Bacteriol Rev. 1956 Dec;20(4):243-258.9

(4) Finney D.J., Statistical Method in Biological Assay, 3rd Ed., Griffin (London) 1978.10

(5) Hewitt W., Influence of curvature of response lines in antibiotic agar diffusion assays, J11

Biol Stand. 1981 Jan;9(1):1-13.12

(6) SAS Institute Inc., SAS OnlineDoc®, Version 8, Cary, NC: SAS Institute Inc., 1999.13

(7) CombiStats v3.1, EDQM – Council of Europe. www.pheur.org/combistats14

15

11/22 PA/PH/ISA (06) 3 DEF

06/10/06

LIST OF PARTICIPANTS1

By alphabetical order of contact person 2

3 N. BELIXAN 3

Instituto Nacional Medicamentos4

Buenos Aires - Argentina 5

C. BITTENCOURT/L. LAPORTA6

Farmacopéia Brasileira7

Santa Maria RS – Brazil8

L. BYSTROVA 9

Scientific Centre for Evaluation of10

Medicinal Products11

Moscow - Russia12

E. CHARTON 13

European Directorate for the Quality of14

Medicines – Council of Europe 15

Y. CORTEZ16

Agence française de sécurité sanitaire des17

produits de santé 18

Vendargues - France 19

M. CRIVELLONE 20

United States Pharmacopoeia 21

Rockville - USA 22

D. HAWKE23

Therapeutic Goods Administration 24

Woden Act - Australia 25

A. HIRECHE26

Laboratoire National de Contrôle des27

Produits Pharmaceutiques28

Hydra Alger - Algeria 29

L.M. MEIRINHOS SOARES30

Instituto Nacional de Farmacia e di31

Medicamento32

Lisbon - Portugal 33

34

B. MIRKOVIC 34

Medicines and Medical Devices Agency35

of Serbia 36

Belgrade - Serbia37

S.V. K. MOIDEEN 38

National Pharmaceutical Control Bureau 39

Petaling jaya - Malaysia40

P. NOVOTNA 41

Institute for State Control of Veterinary42

Biologicals and Medicaments43

Brno - Czech Republic 44

C. OVERBALLE PETERSEN45

Alpharma 46

Copenhagen - Denmark 47

N. RUANGRITTINON 48

Bureau of Drug and Narcotic 49

Nonthaburi - Thailand 50

M.R. SENSALE 51

DSM Food Specialities52

Capua-CE - Italy53

Y. SHANGCHEN 54

China National Institute for the Control of 55

Pharmaceutical and Biological Products56

Beijing - China57

S. TYSKI 58

Polish Institute of Public Health 59

Warsaw - Poland 60

61

PA/PH/ISA (06) 3 DEF 12/22

06/10/06

Figure 1Flow chart for assay validity check

Yes

No

Yes

No

No

Yes

No

Yes

Yes

No

Situation

No

Yes

START

Significant deviationsfrom parallelism?

Significant deviationsfrom linearity?

Significant lack of quadratic fit?

Significant quadratic curvature?

Ratio MS curvature/regression

small?

Reject the assay

Accept the assay

Reject the assay

Accept the assay

Reject the assay

Difference between preparations small? Accept the assay

Reject the assay

13/22 PA/PH/ISA (06) 3 DEF

06/10/06

Table 1.1Overview of assay results

Lab Assay95% Lower conf. limit

Estimated potency

95% upper conf. limit

95% Lower conf. limit

Estimated potency


Non-parallelism

Non-linearity

Lack of quadratic fit

Quadratic regression

Quad. regr./ Lin. regr.

1 5660 5930 6220 5659 5934 6222 0.496 0.696 0.753 0.433 0.0012 5630 5880 6140 5628 5880 6142 0.278 0.255 0.105 0.812 0.0003 5460 5720 6000 5456 5723 6001 0.875 0.311 0.322 0.243 0.0024 5500 5770 6050 5499 5767 6045 0.197 0.400 1.000 0.180 0.0025 5520 5800 6090 5519 5797 6087 0.325 0.581 0.463 0.463 0.0016 5680 5840 6010 5681 5842 6007 0.085 0.340 0.909 0.147 0.0011 5872 5943 6015 5872 5943 6015 0.097 0.400 0.923 0.179 0.0002 5901 5957 6013 5901 5957 6013 0.657 0.936 0.797 0.797 0.0003 5844 5916 5990 5844 5916 5990 0.933 0.257 0.663 0.113 0.0004 5842 5930 6020 5842 5930 6020 0.459 0.063 0.785 0.020 0.0015 5967 6026 6086 5967 6026 6086 0.289 0.785 0.623 0.623 0.0006 5902 5967 6032 5902 5967 6032 0.152 0.595 0.698 0.347 0.0001 5631 5890 6155 5632 5889 6157 0.254 0.180 0.218 0.163 0.0012 5434 5663 5905 5432 5665 5906 0.053 0.202 0.075 0.962 0.0003 5555 5814 6079 5553 5812 6082 0.910 0.115 0.425 0.054 0.0024 5561 5785 6009 5562 5783 6012 0.796 0.180 0.486 0.087 0.0015 5652 5797 5953 5650 5799 5951 0.694 0.838 0.649 0.705 0.0006 5535 5727 5918 5535 5725 5920 0.958 0.713 0.414 0.976 0.000

1.1 5627 5748 5871 5578 5748 5922 0.105 0.738 0.521 0.668 0.0001.2 5469 5547 5625 5381 5547 5715 0.015 0.380 0.170 0.914 0.0001.3 5498 5611 5726 5440 5611 5786 0.646 0.188 0.091 0.492 0.0002.1 5376 5459 5542 5295 5459 5625 0.007 0.012 0.279 0.005 0.0052.2 5280 5350 5420 5235 5350 5465 0.000 0.001 0.463 0.000 0.0052.3 5285 5366 5448 5199 5366 5535 0.078 0.012 0.751 0.003 0.0053.1 5002 5093 5185 4945 5092 5241 0.485 0.015 0.954 0.004 0.0043.2 5221 5316 5413 5179 5316 5454 0.180 0.009 0.076 0.008 0.0033.3 4989 5112 5238 4877 5112 5350 0.845 0.648 0.625 0.433 0.0014.1 5317 5417 5520 5296 5418 5541 0.168 0.070 0.030 0.418 0.0004.2 5252 5371 5493 5225 5371 5519 0.460 0.014 0.008 0.168 0.0014.3 5214 5323 5433 5114 5322 5535 0.198 0.037 0.130 0.032 0.0045.1 5036 5124 5212 4969 5123 5278 0.350 0.163 0.515 0.075 0.0025.2 4934 5022 5111 4890 5021 5154 0.908 0.367 0.740 0.173 0.0015.3 5024 5103 5183 4961 5103 5246 0.522 0.377 0.272 0.390 0.0006.1 4877 4951 5024 4818 4950 5084 0.365 0.002 0.076 0.002 0.0056.2 4969 5045 5122 4884 5045 5207 0.519 0.004 0.059 0.004 0.0056.3 5023 5083 5143 4973 5083 5194 0.347 0.001 0.023 0.002 0.0031.1 5102 5189 5279 5024 5189 5356 0.497 0.000 0.434 0.000 0.0151.2 5234 5332 5431 5180 5332 5486 0.779 0.000 0.155 0.000 0.0131.3 5186 5279 5374 5161 5279 5398 0.051 0.000 0.071 0.000 0.010032.1 5098 5182 5267 5038 5182 5327 0.000 0.001 0.511 0.000 0.0082.2 4847 4946 5046 4777 4945 5115 0.055 0.000 0.299 0.000 0.0132.3 5052 5137 5223 4995 5137 5280 0.015 0.025 0.398 0.010 0.0031 5499 5640 5781 5499 5639 5782 0.408 0.285 0.473 0.159 0.0012 5480 5697 5924 5337 5613 5899 0.197 0.080 0.528 0.032 0.0073 5643 5805 5974 5639 5806 5977 1.000 0.975 1.000 0.825 0.0004 5443 5611 5785 5444 5614 5787 0.720 0.248 0.679 0.109 0.0015 5518 5730 5948 5519 5730 5948 0.870 0.522 0.637 0.304 0.0016 5505 5687 5874 5502 5684 5870 0.092 0.082 0.234 0.054 0.0021 5277 5541 5818 5277 5542 5818 0.061 0.141 0.064 0.504 0.0012 5362 5607 5863 5362 5608 5863 0.935 0.150 0.864 0.055 0.0043 5322 5548 5785 5322 5550 5785 0.274 0.136 0.276 0.090 0.0034 5429 5671 5923 5429 5672 5923 0.093 0.681 0.968 0.387 0.0015 5133 5380 5639 5170 5422 5682 0.778 0.205 0.460 0.106 0.0036 5589 5806 6031 5589 5807 6031 0.767 0.215 0.425 0.119 0.0027 5369 5630 5903 5319 5576 5842 0.790 0.256 0.766 0.108 0.003

1.1 5754 5975 6196 5882 5978 6076 0.351 0.110 0.369 0.057 0.0001.2 5765 5901 6037 5768 5904 6043 0.085 0.388 0.606 0.206 0.0001.3 5837 5962 6087 5845 5978 6114 0.252 0.150 0.893 0.055 0.0001.4 5713 5926 6139 5713 5929 6153 0.576 0.471 0.628 0.264 0.0001.5 5751 6028 6305 5752 6031 6323 1.000 0.898 0.885 0.664 0.0001.6 5836 6016 6196 5840 6019 6203 0.653 0.643 0.604 0.438 0.0002.1 5716 5893 6070 5656 5840 6029 0.725 0.125 0.544 0.053 0.0012.2 5781 5929 6077 5797 5927 6060 0.788 0.148 0.099 0.284 0.0002.3 5863 6013 6163 5888 6037 6190 0.382 0.247 0.448 0.137 0.0002.4 5706 5925 6144 5703 5923 6151 0.323 0.136 0.445 0.065 0.0012.5 5767 5958 6149 5782 5970 6163 0.854 0.443 0.596 0.249 0.0002.6 5851 6013 6175 5850 6011 6176 1.000 0.235 0.633 0.105 0.0003.1 5822 6071 6320 5825 6069 6322 0.197 0.169 0.933 0.063 0.0013.2 5912 6089 6266 5895 6071 6253 0.833 0.152 0.399 0.080 0.0013.3 5737 5939 6141 5739 5951 6172 0.725 0.125 0.544 0.053 0.0013.4 5791 5970 6149 5785 5967 6154 0.419 0.246 0.639 0.111 0.0003.5 5825 6030 6235 5827 6028 6236 0.344 0.189 0.912 0.072 0.0013.6 5879 6061 6243 5879 6058 6243 0.398 0.236 0.806 0.096 0.0004.1 5798 5934 6070 5800 5940 6084 0.118 0.155 0.894 0.057 0.0004.2 5768 5910 6052 5770 5916 6065 0.514 0.083 0.175 0.070 0.0004.3 5806 5973 6140 5813 5979 6150 0.177 0.150 0.571 0.064 0.0014.4 5921 6067 6213 5930 6073 6218 0.637 0.109 0.282 0.067 0.0004.5 5795 5938 6081 5798 5944 6093 0.532 0.140 0.904 0.051 0.0004.6 5813 5987 6161 5820 5993 6171 0.589 0.142 0.603 0.058 0.0015.1 5788 5973 6158 5791 5976 6168 0.601 0.159 0.615 0.066 0.0015.2 5740 5985 6230 5744 5988 6243 0.667 0.179 0.934 0.067 0.0015.3 5722 5985 6248 5723 5988 6265 0.497 0.152 0.694 0.060 0.0015.4 5750 5971 6192 5755 5974 6201 0.524 0.249 0.712 0.107 0.0015.5 5744 5971 6198 5747 5974 6211 0.878 0.196 0.930 0.075 0.0015.6 5768 5959 6150 5773 5962 6157 0.478 0.589 1.000 0.310 0.0006.1 5888 6027 6166 5896 6034 6176 0.807 0.133 0.328 0.078 0.0006.2 5948 6094 6240 5948 6088 6232 0.237 0.071 0.219 0.047 0.0006.3 5807 5907 6007 5811 5914 6019 0.344 0.264 0.854 0.109 0.0006.4 5781 5972 6163 5786 5980 6180 1.000 0.144 0.839 0.053 0.0016.5 5771 5986 6201 5775 5993 6220 0.752 0.164 0.469 0.080 0.0016.6 5821 6038 6255 5856 6084 6320 0.271 0.842 0.629 0.747 0.0001 5871 5947 6025 5871 5947 6025 0.056 0.079 0.492 0.032 0.0002 5663 5909 6165 5663 5909 6165 0.804 0.822 0.668 0.652 0.0003 5471 5614 5760 5471 5614 5760 0.061 0.150 0.242 0.116 0.0014 5616 5792 5973 5616 5792 5973 0.818 0.546 0.882 0.279 0.0015 5646 5775 5907 5646 5775 5907 0.962 0.190 0.754 0.074 0.0016 5595 5712 5830 5595 5712 5830 0.796 0.105 0.661 0.039 0.001

6

7

8

Calculated by participants (IU/mg) Calculated at EDQM (IU/mg) p-Values Analysis of variance

1

2

3

4

4b

5

PA/PH/ISA (06) 3 DEF 14/22

06/10/06

Table 1.2Overview of assay results

Lab Assay95% Lower conf. limit

Estimated potency


95% Lower conf. limit

Estimated potency


Non-parallelism

Non-linearity

Lack of quadratic fit

Quadratic regression

Quad. regr./ Lin. regr.

Calculated by participants (IU/mg) Calculated at EDQM (IU/mg) p-Values Analysis of variance

1.1 5293 5364 5437 5293 5365 5437 0.179 . . . .1.2 5292 5349 5407 5294 5349 5406 0.264 . . . .1.3 5133 5260 5389 5128 5260 5395 0.756 . . . .1.4 5270 5318 5366 5270 5319 5367 0.558 . . . .1.5 5194 5258 5323 5193 5258 5324 0.348 . . . .2.1 5114 5189 5264 5115 5188 5262 0.054 . . . .2.2 5107 5181 5257 5107 5180 5255 0.157 . . . .2.3 5142 5197 5252 5143 5196 5249 0.073 . . . .2.4 5126 5173 5219 5120 5172 5225 0.091 . . . .2.5 5057 5141 5226 5054 5140 5227 0.163 . . . .2.6 5121 5219 5320 5122 5219 5317 0.157 . . . .3.1 5164 5248 5333 5167 5249 5331 0.530 . . . .3.2 5177 5265 5355 5179 5266 5355 0.856 . . . .3.3 5192 5297 5404 5190 5298 5409 0.338 . . . .3.4 5174 5240 5307 5177 5241 5307 0.076 . . . .3.5 5095 5188 5284 5098 5189 5282 0.364 . . . .3.6 5097 5220 5346 5099 5221 5345 0.831 . . . .4.1 5259 5336 5413 5261 5337 5413 0.060 . . . .4.2 5241 5327 5415 5244 5328 5414 0.206 . . . .4.3 5233 5297 5362 5236 5298 5360 0.093 . . . .4.4 5238 5321 5405 5241 5322 5404 0.082 . . . .4.5 5233 5311 5389 5238 5312 5387 0.109 . . . .5.1 5209 5284 5360 5213 5285 5358 0.071 . . . .5.2 5167 5238 5310 5170 5238 5308 0.700 . . . .5.3 5225 5293 5361 5226 5293 5362 0.327 . . . .5.4 5192 5254 5317 5192 5255 5318 0.350 . . . .5.5 5158 5210 5263 5156 5211 5265 0.597 . . . .5.6 5150 5242 5336 5152 5243 5334 0.800 . . . .6.1 5394 5462 5531 5396 5463 5532 0.807 . . . .6.2 5453 5509 5566 5456 5511 5567 0.692 . . . .6.3 5446 5526 5608 5450 5528 5608 0.246 . . . .6.4 5380 5455 5530 5384 5456 5529 0.398 . . . .1 5124 5599 6113 5126 5600 6113 0.599 0.059 0.094 0.080 0.0052 5008 5600 6251 5011 5601 6252 0.696 0.135 0.107 0.224 0.0033 5032 5607 6238 5035 5608 6238 0.708 0.132 0.175 0.130 0.0054 5151 5605 6095 5152 5606 6095 0.168 0.060 0.157 0.051 0.0055 5189 5632 6108 5190 5633 6109 0.902 0.148 0.830 0.054 0.0056 5247 5614 6005 5247 5615 6006 0.895 0.129 0.170 0.130 0.0021 5088 5573 6107 5095 5570 6092 0.450 0.197 0.754 0.079 0.0052 5268 5713 6198 5266 5710 6195 0.217 0.522 0.552 0.334 0.0013 5257 5525 5809 5259 5523 5801 0.598 0.171 0.246 0.136 0.0014 5093 5467 5870 5091 5465 5868 0.907 0.221 0.788 0.089 0.0035 5099 5471 5871 5098 5468 5869 0.912 0.178 0.655 0.073 0.0036 5162 5594 6062 5160 5591 6060 0.230 0.973 0.848 0.898 0.000

1.1 5826 5945 6067 5851 5936 6022 0.207 0.050 0.272 0.027 0.0011.2 5785 5887 5992 5797 5875 5955 0.246 0.081 0.358 0.040 0.0001.3 5725 5835 5947 5732 5823 5914 0.307 0.067 0.085 0.104 0.0002.1 5821 5969 6142 5849 5956 6064 0.268 0.038 0.273 0.020 0.0012.2 5894 5997 6101 5907 5979 6051 0.904 0.090 0.944 0.030 0.0002.3 5868 5978 6090 5898 5967 6038 0.157 0.015 0.091 0.014 0.0013.1 5719 5954 6199 5776 5939 6107 0.746 0.163 0.810 0.062 0.0023.2 5782 5943 6108 5783 5922 6065 0.316 0.234 0.559 0.112 0.0013.3 5685 5967 6262 5793 5978 6169 0.449 0.081 0.487 0.033 0.0034.1 5760 5984 6216 5770 5969 6174 0.715 0.230 0.981 0.090 0.0024.2 5729 5927 6131 5743 5903 6067 0.454 0.827 0.804 0.578 0.0004.3 5865 6030 6199 5924 6038 6155 0.118 0.094 0.555 0.037 0.0015.1 5722 5856 5992 5733 5841 5951 0.547 0.402 0.584 0.221 0.0005.2 5822 5907 5993 5823 5893 5964 0.002 0.604 0.751 0.346 0.0005.3 5733 5882 6034 5734 5867 6003 0.482 0.094 0.569 0.037 0.0016.1 5893 5983 6075 5897 5965 6034 0.714 0.049 0.944 0.015 0.0006.2 5844 6019 6199 5841 5972 6106 0.813 0.302 0.945 0.127 0.0016.3 5832 6017 6207 5894 6001 6111 0.939 0.799 0.965 0.509 0.0001 6162 6162 6162 5789 6149 6534 0.273 0.903 0.750 0.750 0.0002 5940 5940 5940 5583 5954 6348 0.064 0.787 0.827 0.513 0.0013 6078 6078 6078 5838 6093 6359 0.259 0.140 0.661 0.054 0.0044 5952 5952 5952 5538 5935 6358 0.546 0.358 0.167 0.727 0.0005 5760 5760 5760 5440 5774 6125 0.263 0.556 0.516 0.388 0.0016 5694 5694 5694 5419 5705 6002 1.000 0.067 0.147 0.065 0.0051 n.a. 7471 7656 7851 0.096 0.001 0.000 0.267 0.0002 n.a. 7093 7301 7521 0.696 0.000 0.000 0.652 0.0003 n.a. 7384 7632 7897 0.001 0.053 0.018 0.624 0.0004 n.a. 7072 7321 7588 0.734 0.011 0.003 0.844 0.0005 n.a. 7131 7349 7580 0.434 0.002 0.001 0.821 0.0006 n.a. 7246 7433 7630 0.036 0.060 0.023 0.492 0.0001 5614 5870 6136 5614 5870 6136 0.702 0.398 0.275 0.421 0.0012 5620 5857 6103 5620 5857 6103 0.887 0.227 0.171 0.291 0.0013 5516 5809 6114 5516 5809 6114 0.058 0.776 0.484 0.937 0.0004 5774 5956 6144 5774 5956 6144 0.573 0.467 0.332 0.449 0.0005 5545 5822 6111 5545 5822 6111 0.307 0.641 0.375 0.766 0.0006 5717 6008 6313 5717 6008 6313 0.407 0.631 0.731 0.375 0.0011 5812 5976 6145 5812 5976 6145 0.519 0.469 0.344 0.657 0.0002 5646 5719 5793 5646 5719 5793 0.686 0.257 0.404 0.119 0.0003 5356 5431 5506 5356 5431 5506 0.418 0.038 0.019 0.748 0.0004 5434 5534 5634 5434 5534 5634 0.398 0.007 0.005 0.220 0.0005 5361 5440 5521 5361 5440 5521 0.012 0.724 0.669 0.483 0.0006 5419 5522 5627 5419 5522 5627 0.000 0.000 0.000 0.000 0.006

1.1 5108 5410 5712 5113 5410 5717 0.680 0.454 0.932 0.213 0.0031.2 5181 5376 5572 5183 5377 5575 0.535 0.268 0.106 0.994 0.0002.1 4927 5291 5655 4984 5334 5692 0.947 0.393 0.280 0.403 0.0022.2 5065 5253 5441 5065 5252 5441 0.188 0.698 0.840 0.413 0.0003 5235 5392 5549 5235 5391 5549 0.576 0.910 0.836 0.703 0.0004 5129 5261 5394 5128 5260 5393 0.887 0.161 0.061 0.722 0.0005 4981 5234 5486 4986 5234 5492 0.681 0.201 0.113 0.402 0.0016 4989 5268 5547 4994 5268 5550 0.094 0.293 0.513 0.155 0.0037 5291 5415 5540 5292 5415 5541 0.922 0.645 0.596 0.442 0.000

10

11

16

12

13

14

15

9

17

15/22 PA/PH/ISA (06) 3 DEF

06/10/06

Table 2 - Combined potency estimates per laboratory

Lab95% Lower conf. limit

Estimated potency


1 5736 5829 59242 5937 5963 59903 5694 5776 58604 5207 5310 54155 5614 5684 57546 5523 5610 56997 5955 5981 60078 5732 5801 58709 5262 5299 5337

10 5426 5612 580511 5392 5540 569312 5906 5937 596813 5810 5940 607314 7244 7394 754715 5801 5898 599716 5485 5621 576017 5277 5337 5398

Comb. 5673 5710 5747

The combined results does not include Lab 14.

Final potency estimates (IU/mg)

PA/PH/ISA (06) 3 DEF 16/22

06/10/06

The numbers below the 95% confidence intervals are the laboratory codes. Invalid assays are shown with an empty dot.

Figure 2 - Individual estimates per assay (calculated at EDQM)

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Pote

ncy

(IU/m

g)

17/22 PA/PH/ISA (06) 3 DEF

06/10/06

The numbers below the 95% confidence intervals are the laboratory codes. Empty dots indicate assays considered to be invalid by the participants themselves.

Figure 3 - Individual estimates per assay (calculated by participants)

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ncy

(IU/m

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PA/PH/ISA (06) 3 DEF 18/22

06/10/06

11 1611 1510 15 1510 15 15 1310 15 13 1210 13 12 1210 16 8 12 1210 13 8 12 7

17 8 11 6 8 717 16 6 8 5 8 7

17 17 16 6 6 3 3 717 16 11 6 5 3 2 717 16 11 5 5 3 2 7

4 17 9 6 11 5 3 1 2 2 144 9 9 4 9 5 3 1 1 2 13 144 9 9 4 6 4 1 1 1 2 13 14 14 14 14

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Numbers in the boxes are the laboratory codes

Figure 4 - Histogram of final potency estimates per independent assay

Potency (IU/mg)

Potency (IU/mg)

Figure 5 - Histogram of final potency estimates per laboratory

19/22 PA/PH/ISA (06) 3 DEF

06/10/06

Figure 6 - 95% confidence intevals per laboratory (Combined assays)

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Pote

ncy

(IU/m

g)

PA/PH/ISA (06) 3 DEF 20/22

06/10/06

ANNEX 1 – SAS-Script used for the calculations

/* This is the essential script to perform the analysis of variance. It expects a SAS-dataset “Nystatin” with the following fields: prep: 1 for standard, 2 for test. dose: on log-scale with the primary dose set to 0. cdose: copy of dose. row: indicates the row in Latin square designs. block: the column in Latin square designs or the petri-dish in randomised block designs. block and/or row are set to 1 if not applicable for their respective designs. obs: the value of the observation (possibly transformed). */

ods select none; proc glm data=Nystatin; /* Perform the Anova by progressively relaxing model assumptions */ class prep cdose block row; model obs=block row prep dose dose(prep) dose*dose cdose*prep / ss1; ods output OverallAnova=OverallAnova ModelAnova=ModelAnova;

data Anova(keep=source df ss ms fvalue probf); /* Non-linearity has to be calculated in a separate datastep */ retain dfLin ssLin; set ModelAnova OverallAnova; if df>0 then output; if Source='dose*dose' then do; dfLin=df; ssLin=SS; end; if Source='prep*cdose' then do; dfLin=dfLin+df; ssLin=ssLin+ss; end; if Source='Error' then do; Source='Non-linearity'; FValue=(ssLin/dfLin)/ms; ProbF=1-ProbF(FValue,dfLin,df); ss=ssLin; df=dfLin; ms=ss/df; if df>0 then output; end;

ods select all; proc print data=Anova noobs; run;

/* This is the essential script to perform the potency calculations. It expects a SAS-dataset “info” with the following fields: Assigned: The assigned potency of the standard mgS: weight taken of the Standard mlS: Dilution used to prepare the primary dose of the Standard. mgT: weight taken of the Test mlT: Dilution used to prepare the primary dose of the Test. */

ods select none; proc glm data=Nystatin; /* Fit the parallel line model and output the parameter estimates and covariance matrix */ class block row; model obs=prep dose block row / inverse solution; ods output InvXPX=CovB ParameterEstimates=ParmEst;

data Estimate(keep=Low Est High); /* calculate the relative potency (m) */ set ParmEst; where Parameter='prep'; a=Estimate; set ParmEst; where Parameter='dose'; b=Estimate; m=a/b; /* Use Fieller’s theorem to compute the confidence limits */ set CovB; where Parameter='prep'; v11=prep; set CovB; where Parameter='dose'; v12=prep; v22=dose; set Anova; where source='Error'; t=tinv(0.975,df); s=sqrt(ms); g=(t*t*s*s*v22)/(b*b); root=v11-2*m*v12+m*m*v22-g*(v11-v12*v12/v22); mL=(m-g*v12/v22-t*s/b*sqrt(root))/(1-g); mU=(m-g*v12/v22+t*s/b*sqrt(root))/(1-g); /* Transform the relative potency to IU by correcting for the pre-dilutions */ set info; Correction=Assigned*mgS/mlS*mlT/mgT; Low=Correction*exp(mL); Est=Correction*exp(m); High=Correction*exp(mU); output; stop;

ods select all; proc print data=Estimate noobs; run;

21/22 PA/PH/ISA (06) 3 DEF

06/10/06

ANNEX 2: METHODS USED BY THE PARTICIPANTS

Laboratory code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Experimental

design

Randomised

block

Randomised

block

Randomised

blockLatin square

Latin square

(6X6)

Latin square

(6X6)Latin square

Randomised

block

Latin square

(8X8)

Randomised

block

Randomised

blockLatin square

Randomised

block

Randomised

block

Randomised

blockLatin square

Randomised

block

Substance

dissolved inDMF DMF DMF DMF DMF DMF DMF DMF DMF

DMF until 1000

IU/ml

DMF until 1000

IU/mlDMF DMF DMF DMF DMF DMF

Stock solution

diluted with

0.05 M

phosphate

buffer pH6.0

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

Potassium

phosphate

buffer pH6.0

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

DMF

0.05 M

phosphate

buffer pH6.0

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

0.05 M

phosphate

buffer pH6.0

5% v/v DMF

Test

microorganism

Saccharomyces

cerevisiae ATCC

2601

Saccharomyces

cerevisiae ATCC

9763

Saccharomyces

cerevisiae ATCC

9763

Saccharomyces

cerevisiae ATCC

9763

Candida

tropicalis ATCC

13803

Saccharomyces

cerevisiae ATCC

9763

Saccharomyces

cerevisiae ATCC

4098

Saccharomyces

cerevisiae CCM

8191

Saccharomyces

cerevisiae NCYC

87

Saccharomyces

cerevisiae ATCC

2601

Saccharomyces

cerevisiae ATCC

9763

Saccharomyces

cerevisiae ATCC

9763

Saccharomyce

s cerevisiae

ATCC 9763

Saccharomyces

cerevisiae ATCC

2601

Saccharomyces

cerevisiae ATCC

9763

Saccharomyces

cerevisiae ATCC

9763

Saccharomyces

cerevisiae ATCC

9763

Number of

subculture

(january-march

2006)

/ / 5th new one 4th passage 2nd passageNot more than

51

2 generations

from master

culture

/ /not more than 3

passages/ / ? /

about 8 of

subcultures

Dilution steps 1.5 1.5 2.0 1.5 1.5 1.5 2.0 1.5 3.0 2.0 2.0 1.5 1.5 1.5 1.5 1.25 1.5

Material Petri dishes Petri dishes Petri dishes Plates Plates Plates Plates Petri dishes Plates Petri dishes Petri dishes Petri dishes Plates Petri dishes Petri dishes Plates Petri dishes

Nr. of assays 6 6 6

6 (×3),

repeated by

2nd operator

6 7 6 (×6) 6

7 assays but

only 6 results

provided

6 6 6 (×3) 6 6 6 6 7 + 2

Nr. of dishes or

plates used per

assay

6 16 9 3 1 1 6 8 4 to 6 6 6 3 9 9 6 1 9 to 15

Applications per

dish or plate6 6 6 36 36 36 36 6 64 6 6 36 6 6 6 64 6

Concentrations

applied per dish

or plate

6 6 6 6 6 6 6 6 4 (×2) 6 6 6 6 6 6 8 6

Replicates of

concentrations

per dish or plate

1 1 1 6 6 6 6 1 8 1 1 6 1 1 1 8 1

Incubation

temperature31 +/- 1 °C 32°C 32°C 30°C 32°C 30°C 35-37°C 32°C 37°C 29-31°C 37°C 30 +/- 2 °C 32°C 29-31°C 32°C 28.5-32.5°C 31°C

Incubation time 18 hours 18 hours 18 hours 48 hours 18 hours 18 hours 18 hours 18 hours 18 hours 18 hours 20 hours 18 hours 18 hours 16-18 hours 24 hours 16-18 hours 18 hours

Inhibition zones

measured byPBI-ReadBiotic

Antibiotic Zone

Reader Fisher-

Lilly

Antibiotic Zone

Reader Fisher-

Lilly

naked eye, mm

ruler

Automatic Zone

Measuring

Equipment

Macro Lens

mounted on a

digital Camera

and software

"LEICA Qwin"

Zone Reader

System for

image

processing and

analysis-LUGIA

G - version 4.71

Automatic

reader

equipment

Mitutoyo digital

caliperPBI-Readbiotic

Readbiotic

viewerslide caliper

Antibiotic Zone

Reader Fisher-

Lilly

PBI-Readbiotic

Manual reading

0.1mm

precision

?

PA/PH/ISA (06) 3 DEF 22/22

06/10/06

EUROPEAN DIRECTORATE FOR THE QUALITY OF ... 2072 addition.pdf18 D. Quality control on bulk and final...

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Transcript of EUROPEAN DIRECTORATE FOR THE QUALITY OF ... 2072 addition.pdf18 D. Quality control on bulk and final...