WHO/BS/09.2113 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 19 to 23 October 2009 Proposal to establish the WHO Reference Reagent for antibodies to HPV 16 (05/134) as an International Standard following stability studies

The international Reference Reagent for anti-HPV 16 (05/134) was established by ECBS in 2007. This material was not given the status of International Standard as the residual moisture was more variable than usually observed in serum fills. Two additional studies have been undertaken in which ampoules were assayed for residual moisture using non-invasive techniques so that antibody assays could be performed on ampoules of known residual moisture. The first of these studies suggested an overall significant difference of potency between the samples of high and low residual moisture (p=0.0016), but no difference in potency between samples stored at -20°C and -70°C. However, in a second study in which ampoules of high and low residual moisture stored at -20°C were tested, there were no significant differences of potency between the high and low residual moisture groups. There appears to be no consistent evidence of a problem with stability relating to the variable residual moisture content of ampoules of 05/134. In the collaborative study, within laboratory geometric coefficient of variations (GCVs) ranged from 10-80% depending on the sample and between laboratory GCVs, was greater than 40% and it is therefore unlikely that the differences in residual moisture in individual ampoules will affect the calibration of secondary reference materials. In view of the lack of consistent evidence of stability relating to residual moisture content and the inherent variability in assays in which the standard will be used, we propose that this material be upgraded to International Standard with a unitage of 5 International Units per ampoule. Background Anti-HPV 16 serum NIBSC code 05/134 05/134 is a batch of ampoules containing freeze-dried serum obtained from three women naturally infected with HPV 16 who were selected from three previously described population-based epidemiological studies of HPV infection. The serum samples were collected from the 3 individuals at intervals over a 3 year period and pooled at NIBSC. This material is therefore very valuable and almost impossible to replace. The serum was filled in ampoules and freeze-dried at NIBSC following documented procedures on 18 August 2005. This fill was 0.5g fill weight with a mean weight of 30 ampoules of 0.5086g after removal of one anomalous weight. The coefficient of variation (CV) was 0.09%. 4080 ampoules were filled and 3280 are available for issue. A high level of splashing was observed during filling. The mean dry weight was 0.0444g, CV 0.36%. Residual oxygen levels were measured on two groups of ampoules. The first group of 9 ampoules gave a mean oxygen content of 15% with CV of 30% and the second group of 6 ampoules gave a mean oxygen content of 18% with a CV of 5%, probably indicative of substantial equilibration of the products with atmospheric air prior to sealing. Although low oxygen levels are indicative of good stability, the issues relating to residual moisture described below address the issues relating to the stability of this batch. Initial routine analysis of the residual moisture content of 05/134 measured by the colorimetric Karl Fischer assay undertaken in August 2005 was higher than normally observed for plasma fills and somewhat variable (1.36% and a CV of 69.7 for the 6 determinations). Additional studies were undertaken on randomly selected samples, the ampoules used as checkweights and samples sent to FDA as part of an ongoing collaboration. For the samples used as checkweights, the position in the freeze-drier was known. All of these assays confirmed the variable and higher than normal residual moisture content of the batch. WHO/BS/09.2113 Page 3 Assays for antibody content (potency) on randomly selected ampoules stored at various temperatures were performed in two laboratories and the results were consistent. There appeared to be a loss of potency between the ampoules stored at -20°C, ie the normal storage temperature, and ampoules stored at -70°C over a period of 14 months. The reason for this apparent decrease in potency is currently not known. Further data on the potency of ampoules tested by a non-invasive infra red method which had given high and low residual moisture values indicated no loss of potency between ampoules stored at -20 and +20°C for approximately 12 months. It therefore appears that there was little difference due to the varying levels of residual moisture in the ampoules. ECBS discussions and decision 2007 Although the collaborative study report (WHO BS/07.2066) had proposed that 05/134 be established as the International Standard subject to ongoing stability studies, during the discussions at ECBS on 11 th October 2007 it was suggested that this material be established as a WHO Reference Reagent with a unitage of 5 units per ampoule. It was agreed that if the planned stability studies were undertaken, the results could be considered at ECBS, and, if at that time it is considered to meet the requirements for an International Standard with respect to stability, it could be upgraded to the status of an International Standard with a unitage assigned in International Unit (IU). Phase 1 additional studies initiated after ECBS discussions in October 2007 Non-invasive testing of samples stored at -70°C and -20°C Materials and methods An additional 200 randomly selected ampoules from the fill which had been stored at -20°C since the date of production in August 2005 were tested for residual moisture by a non-invasive near infra red (NIR) method (1, 2, 3) in January 2008, so that the range of levels in the stock could be more effectively estimated. 120 ampoules which had been set aside as archive samples at -70°C from January 2006 ie for 28 months were also tested. All ampoules were identifiable.5 ampoules selected from the lower and upper ends of the residual moisture distribution at each temperature were tested for antibodies to HPV 16 with mean residual moisture content Results Residual moisture The results for the samples stored at -20°C and -70°C are shown in the histograms in Figures 1 and 2 respectively. The comparison of the results from representative samples in the noninvasive method and Karl Fischer is shown in Tables 1 and 2 indicate good agreement which means that we can rely on the values obtained through the non-invasive testing as being indicative of moisture content. The range of residual moisture values of the samples stored at 70°C appeared to be higher than those stored at -20°C and this was reflected in the mean values of the samples of high and low residual moisture from each group which were assayed for antibodies. WHO/BS/09.2113 Page 4 Assay for antibodies to HPV 16 (potency) Five ampoules from ampoules stored at -20 and -70°C with residual moisture at the high and low ends of the ranges of residual moisture were selected for antibody testing in a laboratory with a well-established and fully validated assay (Table 3). These were designated Groups A and B for the samples stored at -20°C with low and high residual moisture respectively and , groups C and D for the samples stored at -70°C with low and high residual moisture respectively. The antibody content was calculated against an in-house standard and the values obtained shown in Table 4. A series of dilutions prepared from each ampoule was tested in triplicate on each of three plates. A total of 15 plates were used in the assay. An internal standard was included on each plate, along with validation samples. Although there was no significant difference between the assay results of samples stored at 20°C or -70°C with same level of residual moisture (group A and C, B and D), there was an overall significant difference of potency between the samples of low and high residual moisture (Groups A and C compared to B and D: p=0.0016) Phase 2 additional studies Non-invasive testing of samples stored at -20°C Materials and methods The residual moisture and oxygen content of a further 300 ampoules of 05/134 which had been stored at -20o were determined by non invasive techniques in October 2008 i.e. 38 months after production. The labels were removed by soaking overnight. The ampoules were held at ambient temperature during the assays. The number etched on the ampoules during filling were recorded so that the results from the two tests could be collated. Results Residual moisture and oxygen The results are displayed in figure 3 as a scatter plot. No samples had oxygen in headspace of <4%. The NIBSC working limit is 1.14% . The majority of ampoules (all but around 10%) have oxygen content of 20% -i.e. air. For those with an oxygen content of 20% non invasive residual moisture varies between 1 and 2 units. One ampoule had a residual moisture of 5.39% and it was realised that the glass was cracked. Assay for antibodies to HPV 16 (potency) 20 ampoules of 05/134, which had been selected to have low (10 ampoules) or high (10 ampoules) residual moisture were sent to the same laboratory that conducted the assays in phase 1 of the additional stability studies. The ampoules were randomised and coded prior to dispatch. Each ampoule was tested three times, against a common reference that was included on each plate. The results are given in Table 5. The geometric mean potency across the three tests was calculated for each ampoule. The geometric mean potency, along with 95% confidence intervals, and between ampoule % GCV, were calculated for the high and low moisture groups respectively. High: 60.19 (58.29 – 62.15) % GCV 8.98 Low: 61.76 (59.72 – 63.87) % GCV 9.41 WHO/BS/09.2113 Page 5 There were no significant differences of potency between the two groups. The % GCV of around 10% reflects the assay variability. Overall conclusions from stability studies The original stability studies, reported to ECBS previously in BS/07.2066 had found no difference of potency between samples stored at +20°C and -20°C for 14 months, indicating a stable product. However, there was an apparent drop of 10% between samples stored at -70°C and -20°C, leading to the program of further testing. These original studies did not find any evidence of differences in stability due to differences in residual moisture. Additional Phase 1 data on ampoules stored at -20°C and -70°C (5 with high and 5 with low moisture at each temperature) had shown no significant difference of potency between temperatures, but an overall significant difference in potency (p=0.0016) between the high and low moisture group. . Following these results, the further testing of the randomised coded ampoules had been recommended, to ensure that the differences were not an artefact of the assay layout. Further testing, Phase 2, has found no evidence of a difference in potencies between the high and low residual moisture ampoules. On this occasion, the ampoules were randomised and coded prior to testing. It is possible that previous differences in potency may have been the result of an assay artefact. There therefore appears to be no consistent evidence of a problem with stability relating to the variable residual moisture content of ampoules of 05/134. In the collaborative study, within laboratory GCVs ranged from 10-80% depending on the sample and between laboratory GCVs, was greater than 40% and it is therefore unlikely that the differences in residual moisture in individual ampoules will affect he calibration of secondary reference materials. Proposal to ECBS In view of the lack of consistent evidence of stability relating to residual moisture content and the inherent variability in assays in which the standard will be used, we propose that this material be upgraded to International Standard with a unitage of 5 International Units per ampoule. WHO/BS/09.2113 Page 6 Table 1: Phase 1 studies The comparison of residual moisture levels by non-invasive (NIR) and Karl Fischer (KF) methods for the first modal group with lower residual moisture Ampoule number NIR result KF result 19 0.25 0.5347 49 0.34 0.6804 70 0.31 0.5203 Mean 0.3 0.58 Standard deviation 0.05 0.09 CV (%) 15.28 15.31 Table 2: Phase 1 studies The comparison of residual moisture by non-invasive and Karl Fischer methods for the second modal group with higher residual moisture Ampoule number NIR result KF result 1 1.12 1.314 17 1.1 1.172 26 1.14 1.344 Mean 1.11 1.28 Standard deviation 0.03 0.09 CV (%) 2.74 7.18 Table 3 : Phase 1 studies Mean residual moisture by the non-invasive method of samples selected for assay for antibodies to HPV 16 by EIA Storage temperature Residual moisture group Mean residual moisture -20°C Low(Group A) 0.31 -20°C High(Group B) 1.11 -70°C Low (Group C) 0.61 -70°C High(Group D) 1.71 WHO/BS/09.2113 Page 7 Table 4: Phase 1 studies Results of anti-HPV 16 testing of samples stored at -20°C and -70°C with high and low residual moisture (RM) levels Temperature of storage and residual moisture group Mean Residual Moisture Result 1 (units/ml) Result 2 (units/ml) Result 3 (units/ml) Mean of 3 values from each ampoule Mean of 5 ampoules -20°C low RM (A) 0.31 61 67 69 65.67 65.57 61 64 * 64 62.50 66 72 70 69.33 65 67 63 65.00 64 65 67 65.33 -20°C high RM (B) 1.11 67 71 74 70.67 70.50 65 61 * 70 67.50 74 69 73 72.00 69 76 71 72.00 66 72 73 70.33 -70°C low RM (C) 0.61 62 71 65 66.00 66.00 73 57 * 73 73.00 68 74 65 69.00 57 55 61 57.67 65 65 63 64.33 -70°C high RM (D) 1.71 62 66 67 65.00 68.67 73 62 * 75 74.00 74 75 70 73.00 65 68 67 66.67 63 63 68 64.67 * One plate was re-analyzed the day after. WHO/BS/09.2113 Page 8 Table 5: Phase 2 Anti-HPV 16 results on ampoules of high and low residual moisture Sample ID Residual moisture result 1 (U/ml) result 2 (U/ml) result 3 (U/ml) Mean (U/ml) OVERALL MEANS 6 High 60 60 61 60.33 11 High 61 59 60 60.00 16 High 61 59 60 60.00 2 High 58 61 75 64.67 7 High 59 57 68 61.33 12 High 59 54 64 59.00 17 High 60 55 65 60.00 13 High 44 60 57 53.67 9 High 60 63 60 61.00 15 High 63 65 64 64.00 60.19 1 Low 60 60 61 60.33 3 Low 60 66 58 61.33 8 Low 65 62 55 60.67 18 Low 51 62 58 57.00 4 Low 65 72 61 66.00 14 Low 59 69 62 63.33 19 Low 54 56 52 54.00 5 Low 66 73 71 70.00 10 Low 63 67 64 64.67 20 Low 59 67 62 62.67 61.76 WHO/BS/09.2113 Page 9 Figure 1: Phase 1 studies – Non-invasively determined moisture values on 200 ampoules stored at -20°C, sorted by moisture content 2.8 2.4 2.0 1.6 1.2 0.8 0.4 0.0 50