WHO/BS/2019.2372 ENGLISH ONLY EXPERT COMMITTEE ON

WHO/BS/2019.2372

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 21 to 25 October 2019

Update on the WHO collaborative study to establish the 1st International Standard for

antiserum to Respiratory Syncytial Virus

Jacqueline U McDonald1, Peter Rigsby2, Eleanor Atkinson2 , Othmar G Engelhardt1 and Study

Participants

1Division of Virology and 2Biostatistics

National Institute for Biological Standards and Control (NIBSC),

South Mimms, Potters Bar, Herts, EN6 3QG, UK

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 27 September 2019 and

should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to

the Responsible Officer: Dr I. Knezevic at email: [email protected].

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WHO/BS/2019.2372

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SUMMARY

A collaborative study was conducted with the aim to expand the use of the 1st International

Standard for antiserum to RSV (16/284) to include RSV neutralisation titres against RSV/B. The

study included 11 laboratories from 6 countries, representing university laboratories,

manufacturers/developers of RSV vaccines and public health laboratories. All participants used

their own in-house virus neutralization assay and their own virus stocks. The study samples

comprised the current international standard (16/284) and its potential replacement (16/322),

individual sera from naturally infected humans, a monoclonal antibody to RSV (palivizumab) and

samples from the BEI Resources panel of human antiserum and immune globulin to RSV. Of the

11 laboratories that took part in the study, 5 returned data from neutralisation assays with and

without the inclusion of serum complement.

The collaborative study showed that between-laboratory variability in neutralisation titres was

significantly reduced when values were expressed relative to 16/284 and 16/322. Complement did

not affect the ability of the international standard to decrease between-laboratory variability and

the international standard was able to decrease the variability between titres from assays with and

without complement. Based on these results, it is recommended that 16/284 and 16/322 be

expanded to include titres against RSV/B with assigned unitages of 1,000 and 690 International

Units (IU) of anti-RSV neutralising antibodies per vial, respectively.

INTRODUCTION

Development of an RSV vaccine is recognised as a global priority by national governments, the

World Health Organization, the pharmaceutical industry and not for profit health organisations.

Activity in this area has increased significantly in recent years, with at least 39 RSV vaccine

candidates in development, 19 of which are now in human clinical trials (PATH. 2018). RSV

neutralising activity in serum has been reported to correlate with protection against RSV acute

lower respiratory infection in both rodent models and human infants (Graham. 2016). Quantifying

this neutralising activity is vital in the development of future RSV vaccines.

RSV neutralization assays come in multiple formats and one of the challenges in RSV vaccine

research is accurately comparing the neutralization titres in sera from multiple clinical trials, each

using a different neutralization assay format (Hosken et al. 2017). The WHO endorsed the

production of a reference antiserum standard to enable standardization of clinical trial data and

outcomes. In 2017 the WHO first International Standard for Antiserum to RSV was established by

the WHO ECBS (WHO ECBS, TRS 1011. 2018). This standard, now publically available from

NIBSC, is recommended for use as a reference standard in assessing RSV neutralization titres of

human serum against RSV subtype A. To expand the use of this standard to assess RSV

neutralization titres against RSV subtype B (RSV/B), a new collaborative study was conducted.

It has been shown that the use of guinea pig complement increases the RSV neutralization titres

of human sera (Yoder. 2004). In the previous collaborative study used to establish the first RSV

WHO/BS/2019.2372

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International Standard only a single lab included the use of guinea pig complement in their assay,

and this lab produced poor concordance correlation coefficients with the other study labs. As a

result of this, it was decided that assessing the impact of the use of guinea pig complement on

assay titres and the effectiveness of the standard was necessary and was therefore included as a

secondary objective in this study.

Aim of the Study

The aim of this study was to expand the use of the first WHO International Standard for Antiserum

to RSV to look at titres against RSV/B and to determine the effect, if any, of the use of guinea pig

complement on the effectiveness of the international standard. A set of coded samples including

the international standard (16/284) and its potential replacement (16/322) were to be tested using

in house RSV neutralisation assays, against RSV/B, with and/or without guinea pig complement.

The BEI Resources panel of human antiserum and immune globulin to RSV (NR-32832) was also

included to allow comparison with and potential calibration against the proposed international

standard.

MATERIALS AND METHODS

Product summary

Full details on the production of 16/284 and 16/322 can be found in the report to establish the 1st

International Standard for antiserum to RSV (WHO ECBS, TRS 1011. 2018)(Mcdonald et al.

2018).

Study Samples

A total of 20 blinded samples were available to participants. The samples were shipped in dry-ice

and storage at ≤ -20oC was recommended. All 11 participating labs received the full panel of 20

samples (Table 1).

BEI samples were reconstituted according to instructions from BEI materials. Sample 1 (NR-

21973) was diluted 1/10 before being sent to participants. All titres shown for this sample are for

the 1/10 diluted product. The international unit assigned to this sample has been converted to take

this dilution into account.

A sample information sheet explaining how to store and handle the samples was sent with each

package. There were no issues with the shipment and the receipt of samples for all participating

labs.

DESIGN OF COLLABORATIVE STUDY

Participants

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Twenty-eight laboratories were invited to participate in the study. Eleven laboratories from six

countries agreed to participate, with five agreeing to perform their assays both with and without

guinea pig complement. All eleven labs returned data. They are referred to by a code number,

allocated at random, and not reflecting the order of listing in Appendix 1.

Study time-frame

The study samples were sent to participants in May 2018 and the coordinator requested data to be

returned by October 2018. All data was received by January 2019.

Study Plan

Participants were requested to:

- Follow the recommended protocols for storage and reconstitution of the study samples.

- Determine the neutralization titre against RSV/B, with or without complement where

applicable, of each of the samples in the panel by performing four independent assays using

their in-house method and using in-house reagents, including their own virus stocks.

- Avoid multiple freeze-thaw cycles of the study samples.

Laboratory methods

Participants used their own in-house assays for determining RSV/B neutralization antibody titres.

Each participant used their own virus stocks. Participants provided calculated titres based on their

own in-house method of titre calculation. In most cases, an ED50 (or equivalent) was provided.

In a single case where this was not possible, participants provided the raw data and statisticians at

NIBSC calculated an ED50 using Combistats.

Documentation of Study results

Participants were requested to report their results electronically using standard forms provided by

the study coordinator. The forms requested both raw data and calculated endpoint titres.

Statistical Analysis

Analysis was performed using ED50s reported by the participants or calculated at NIBSC and also

using relative potencies i.e. ED50s expressed relative to IS 16/284 or 16/322. All ED50s and

relative potency estimates were combined as Geometric Means (GM) and variability within

laboratories (between assays) and between laboratories was expressed using Geometric

Coefficients of Variation (%GCV) i.e. (10s-1)x100%, where s is the standard deviation of the log10

ED50s or potency estimates. In some cases, the endpoint ED50 was not covered by the range of

dilutions used by the participant and results were reported as “less than” or “greater than” and all

estimates for that sample in that laboratory were excluded from further analysis. Any exclusions

due to high intra-assay or inter-assay variability within a laboratory are described in the results

section of this report.

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Further assessment of agreement in geometric mean potencies for each pair of laboratories was

performed by calculating Lin’s concordance correlation coefficient with log transformed potencies

relative to IS 16/284 or 16/322.

Laboratory 3

This lab reported their data in a format other than ED50 or equivalent. To allow for like for like

comparisons ED50 values for these datasets were calculated using Combistats. The re-calculation

could affect the within assay variations for this lab, as the assays may not be optimized to calculate

an ED50.

RESULTS

Study data returned

A total of 16 datasets were received from 11 participants (results from one laboratory from assays

with and without complement are counted as two datasets). All laboratories submitted datasets

excluding guinea pig complement from their assays. Laboratories 1, 3, 7, 8 and 9 submitted

additional datasets including complement in their assy.

Intra-assay and inter-assay variability in ED50s

Intra-assay variability was assessed using the coded duplicate samples included in the study. One

pair of coded duplicates was included in the panel of samples, these were 9 & 15. As none of the

coded duplicate ED50s within an assay differed by more than a factor of 2.5, no assays were

excluded in this case, see Figure 1.

Inter-assay variability was assessed for each sample from the ratio of the maximum and minimum

ED50 results across all assays within a laboratory. Where the ratio for a sample exceeded 3.5, all

results for that sample for that laboratory were excluded, leading to ~3% of samples being

excluded from all further analysis, see Figure 2.

Agreement between laboratories

Concordance correlation coefficients are summarised in Tables 2, 3 and 4 for log 10 ED50s and

potencies relative to standards 16/284 and 16/322 respectively. Concordance was noted to be poor

for all laboratories when expressed as ED50, with almost all concordance correlation values being

<0.8. When potencies were expressed relative to 16/284 or 16/322, concordance correlation values

improved for most laboratories. Lab 11b was noted to have particularly poor concordance that did

not improve when potencies were expressed relative to 16/284 or 16/322.

Inter-laboratory variability in ED50s and relative potencies

Variability between laboratories for ED50s and potencies relative to different candidate standards

was assessed using the inter-laboratory GCV values and ratios of maximum and minimum

estimates shown in Table 5 and Figures 3 to 5. A summary of the calculated GCVs can be found

WHO/BS/2019.2372

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in Table 6. Sample 2, an IgG deficient serum sample included as a negative control, will be

excluded from all further discussions of data unless specifically related to these samples.

ED50 Results

The inter-lab variability for all samples was high with GCVs ranging from 120% to 215%.

Potencies relative to 16/284

When titres were expressed relative to RSV International Standard 16/284, inter-lab variability

noticeably decreased with much smaller GCVs ranging from 19% to 191%. Except for samples 3

and 14 (palivizumab samples), the GCVs for all samples fell below 70%.

Potencies relative to 16/322

When titres were expressed relative to International Standard replacement 16/322, inter-lab

variability decreased, with smaller GCVs ranging from 20% to 199%. All samples, except for

sample 3 and 14 palivizumab samples), had GCVs that fell below 70%.

BEI Resources NR-4020, NR4021 and NR-21973

The BEI resources human reference antiserum to RSV (NR-4020), human antiserum to RSV high

control (NR-4021) and human reference immunoglobulin to RSV (NR-21973) were also tested for

their ability to improve inter-laboratory agreement. Except for samples 3 and 14, when expressed

relative to NR-4020 and NR-40-21, inter-laboratory GCVs improved. While NR-4020 and NR-

4021 minimally decreased the the GCVs for sample 3 and 14 (palivizumab samples) by up to 22%,

expressing relative to NR-21973 increased GCVs by up to 21%.

Complement

The data was separated by the exclusion/inclusion of complement in the methods (Tables 7 and 8,

and Figures 6 - 11). Both NIBSC standards and the BEI Resources samples NR-4020, NR-4012

and NR-21973 were able to reduce inter-lab variability and bring down GCVs. The only

exceptions were samples 3 and 14, where GCVs increased after normalisation to a standard, in the

presence of complement (Table 9).

Principal component analysis

Principal component analysis of log transformed potencies relative to 16/284 was performed

after the exclusion of laboratory 11 (limited data available). This multivariate statistical

technique allows an exploratory visual assessment of the results profiles observed for samples

(across different laboratories) or laboratories (across different samples). In the small number of

cases where no result was reported, the median result for that sample from all other laboratories

was included in place of the missing value for the purposes of this analysis.

A scatterplot of the first three sample components is shown in Figure 12. Samples exhibiting

similar results profiles across different laboratories would be expected to be in close proximity on

this plot. In this case, there is evidence that results profiles across laboratories are different for the

monoclonal antibody (mAb) samples. No particular groupings of laboratories were observed

(Figure 13).

WHO/BS/2019.2372

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Complement effects

The inclusion of complement in the assay showed significantly increased end point titres

(P=0.0345, Figure 14). This difference in titres decreased when expressed as international units

until significance was lost. The percentage difference between the titres of the samples when

tested with and without complement was calculated (Figure 15). Expressing the titres as

international units significantly decreased this percentage difference (16/824: P=0.0111, 16/322:

P=0.0354). Concordance correlation coefficients, comparing assays with and without

complement, are summarised in Table 10 for log 10 ED50s and log 10 international units (16/284

and 16/322). When expressed as international units using either 16/284 or 16/322 as standards, all

labs that returned data showed CCC values of at ≥ 0.8.

DISCUSSION

The data from this study shows that both 16/284 and 16/322 are suitable as reference standards to

measure RSV neutralization activity against RSV/B in human serum samples, due to their ability

to reduce GCVs across the various assay methods included in this study. We propose extending

the use of 16/284 as the first International Standard for antiserum to RSV to include RSV/B

neutralisation titres, with an assigned potency of 1000 IU/ampoule. Based on this proposal and

the geometric mean potency of 16/322 relative to 16/284 (0.69; sample 12 in Table 5), 16/322 can

be assigned a potency of 690 IU/ampoule and may be used as a replacement standard, once 16/284

has been depleted.

The data generated showed high GCVs across the laboratories for all sample types. This can be

attributed to the varying formats and methods used to assess RSV neutralizing antibody titres.

Once the data were expressed as potencies relative to the candidate standards, the GCVs were

significantly decreased across all laboratories. Samples 3 and 14, palivizumab at 0.1mg/ml and

1mg/ml respectively, are notable exceptions to the above. This observation of differing behaviour

of samples 3 and 14 is supported by the principal component analysis and by a previous study

(McDonald et al. 2018).

This study has achieved its stated aims and an International Standard is proposed for titres against

RSV/B, with an International Unitage (IU). This study has shown that the standard is useful for

human serum samples across a wide variety of assay formats but unsuitable for monoclonal

antibody samples. A more suitable standard should be considered for titres against non-human

serum sample types including monoclonal antibodies.

The samples within the BEI Resources panel, consisting of a panel of human antisera and

immunoglobulin, were also shown to reduce inter-lab variability of RSV/B titres. The proposed

international unitage of these samples can be found in Table 11

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RECOMMENDATIONS

It is proposed that the International Standard 16/284 should be expanded to include assays looking

at RSV/B neutralisation titres. The assigned potency for this should be:

1,000 IU/ampoule

It is proposed that the sample 16/322 should be retained as a potential secondary/replacement

International Standard for antiserum to RSV/B. The assigned potency for this should be:

690 IU/ampoule

ACKNOWLEDGEMENTS We gratefully acknowledge the important contributions of the collaborative study participants. We

would also like to thank NIBSC Standards Production and Dispatch for the filling, freeze-drying and

distribution of the candidate material. We also acknowledge PATH for arranging blood collection

from those who gave consent to the use of their sera in preparing the candidate material, donating

said material to NIBSC and funding this study.

REFERENCES

- Graham B. Vaccines against respiratory syncytial virus: The time has finally come.

Vaccine. 2016 Jun 24;34(30):3535-41. DOI: 10.1016/j.vaccine.2016.04.083

- PATH. RSV Vaccine and mAb Snapshot 10th December 2018.

http://www.path.org/vaccineresources/details.php?i=1562

- Hosken N, Plikaytis B, Trujillo C, Mahmood K, Higgins D, Participating Laboratories

Working Group. A multi-laboratory study of diverse RSV neutralization assays indicates

feasibility for harmonization with an international standard. Vaccine. 2017 May

25;35(23):3082-3088. DOI: 10.1016/j.vaccine.2017.04.053

- Yoder SM, Zhu Y, Ikizler MR, Wright PF. Role of complement in neutralization of

respiratory syncytial virus. J Med Virol. 2004 Apr;72(4):688-94. DOI: 10.1002/jmv.20046

- WHO Expert Committee on Biological Standardisation, Page 73. TRS 1011. 2018.

https://apps.who.int/iris/bitstream/handle/10665/272807/9789241210201-

eng.pdf?ua=1&ua=1

- McDonald JU, Rigsby P, Dougall T, Engelhardt OG; Study Participants. Establishment of

the first WHO International Standard for antiserum to Respiratory Syncytial Virus: Report

of an international collaborative study. Vaccine. 2018 Nov 29;36(50):7641-7649. doi:

10.1016/j.vaccine.2018.10.087

WHO/BS/2019.2372

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Table 1. Samples Included in the Collaborative Study

Panel Sample Name Sample

Code

International Standard Panel

RSV IS NIBSC 16/284 7

IS Potential Replacement NIBSC 16/322 12

Individual Human Serum Panel

P-0007 Individual Adult Human Serum 18










Monoclonal Antibody Panel

Palivizumab 0.1mg/ml 3

Palivizumab 1mg/ml 14

BEI Resources Panel

NR-4020: Human Reference Antiserum to RSV 6

NR-4021: Human Antiserum to RSV, High Control 19

NR-4022: Human Antiserum to RSV, Medium Control 4

NR-4023: Human Antiserum to RSV, Low Control 8

NR-21973: Human Reference Immunoglobulin to RSV 1

NR-49447: Human IgG-Depleted Serum (Negative control) 2

WHO/BS/2019.2372

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Table 2. Concordance correlation coefficients for log ED50s; values ≥0.8 shaded

Lab 01a 03a 07a 08a 09a 01b 02b 03b 04b 05b 06b 07b 08b 09b 10b 11b

01a

03a 0.55

07a 0.38 0.79

08a 0.51 0.27 0.09

09a 0.47 0.63 0.54 0.31

01b 0.96 0.39 0.27 0.59 0.35

02b 0.73 0.58 0.28 0.42 0.77 0.52

03b 0.3 0.76 0.87 0.1 0.31 0.22 0.28

04b 0.46 0.89 0.46 0.24 0.51 0.34 0.61 0.49

05b 0.25 0.74 0.54 0.19 0.47 0.19 0.46 0.68 0.55

06b 0.93 0.54 0.32 0.55 0.53 0.85 0.77 0.27 0.52 0.29

07b 0.46 0.9 0.9 0.17 0.71 0.35 0.49 0.66 0.64 0.69 0.45

08b 0.51 0.23 0.1 0.95 0.25 0.61 0.35 0.1 0.2 0.17 0.51 0.17

09b 0.57 0.42 0.44 0.44 0.8 0.5 0.78 0.2 0.36 0.27 0.71 0.6 0.37

10b 0.82 0.28 0.21 0.4 0.22 0.88 0.33 0.17 0.23 0.1 0.63 0.24 0.49 0.32

11b -0.2 0.02 -0 0.17 -0 -0.1 0.04 0.03 0.06 0 0.03 -0 0.11 0.11 0.12

Table 3. Concordance correlation coefficients for log potencies relative to 16/284; values

≥0.8 shaded

Lab 01a 03a 07a 08a 09a 01b 02b 03b 04b 05b 06b 07b 08b 09b 10b 11b

01a

03a 0.89

07a 0.92 0.8

08a 0.55 0.71 0.33

09a 0.59 0.76 0.64 0.73

01b 0.99 0.86 0.89 0.51 0.57

02b 0.81 0.84 0.64 0.83 0.79 0.72

03b 0.84 0.94 0.87 0.63 0.78 0.83 0.89

04b 0.79 0.9 0.63 0.7 0.54 0.8 0.77 0.82

05b 0.68 0.81 0.52 0.84 0.84 0.66 0.88 0.75 0.66

06b 0.93 0.94 0.8 0.66 0.67 0.94 0.84 0.87 0.9 0.78

07b 0.89 0.83 0.95 0.48 0.68 0.91 0.7 0.82 0.64 0.74 0.85

08b 0.67 0.82 0.47 0.97 0.8 0.62 0.88 0.76 0.75 0.93 0.77 0.61

09b 0.54 0.74 0.69 0.72 0.95 0.55 0.84 0.79 0.62 0.81 0.68 0.73 0.79

10b 0.81 0.9 0.84 0.4 0.56 0.78 0.69 0.88 0.77 0.52 0.78 0.75 0.51 0.55

11b -0.2 0.59 -0.3 0.26 -0 -0.1 0.09 0.3 0.2 0.01 0.03 -0.2 0.21 0.21 0.12

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Table 4. Concordance correlation coefficients for log potencies relative to 16/322; values

≥0.8 shade

Lab 01a 07a 08a 09a 01b 02b 03b 04b 05b 06b 07b 08b 09b 10b 11b

01a

07a 0.94

08a 0.58 0.32

09a 0.59 0.6 0.74

01b 0.99 0.9 0.56 0.6

02b 0.84 0.65 0.78 0.68 0.79

03b 0.89 0.87 0.62 0.76 0.89 0.84

04b 0.78 0.59 0.73 0.56 0.81 0.73 0.84

05b 0.7 0.49 0.85 0.82 0.71 0.87 0.75 0.65

06b 0.94 0.8 0.66 0.63 0.96 0.87 0.88 0.86 0.78

07b 0.83 0.9 0.56 0.74 0.86 0.66 0.83 0.65 0.79 0.8

08b 0.72 0.47 0.94 0.77 0.69 0.87 0.76 0.75 0.94 0.79 0.69

09b 0.59 0.68 0.72 0.93 0.63 0.79 0.79 0.64 0.82 0.69 0.8 0.78

10b 0.9 0.87 0.38 0.52 0.9 0.68 0.89 0.84 0.52 0.83 0.82 0.51 0.54

11b -0.24 -0.41 0.17 -0.02 -0.1 0.12 0.37 0.22 0.02 0.04 -0.18 0.27 0.16 0.05

Table 5. Sample geometric mean ED50 and potency estimates relative to 16/284 or 16/322

Sample

ED50 Potencies v 16/284 Potencies v 16/322

GM Max:Min GCV N GM Max:Min GCV N GM Max:Min GCV N

1 1098 31 168 16 0.60 2 31 16 0.85 4 48 15

3 312 82 215 13 0.16 31 191 13 0.26 34 199 12

4 915 27 174 14 0.52 2 19 14 0.72 3 31 13

5 249 23 168 15 0.13 4 48 15 0.19 3 42 14

6 1360 26 162 16 0.75 2 25 16 1.08 3 28 15

7 1821 19 165 16 1 1 0 16 1.44 3 29 15

8 609 18 156 14 0.38 6 63 14 0.55 3 45 14

9 1574 17 145 15 0.80 3 30 15 1.18 2 20 14

10 681 26 211 14 0.36 4 45 14 0.55 6 54 14

11 974 28 197 15 0.49 4 40 15 0.75 3 40 14

12 1204 18 149 15 0.69 3 29 15 1.00 1 0 15

13 4564 27 173 15 2.31 2 21 15 3.52 2 29 14

14 3867 63 163 13 2.12 29 152 13 3.23 32 155 12

15 1743 22 171 14 0.86 4 47 14 1.26 2 34 13

16 961 28 183 14 0.51 3 33 14 0.77 3 34 14

17 1178 26 146 16 0.65 7 69 16 0.91 9 63 15

18 390 24 186 14 0.22 6 70 14 0.34 5 65 13

19 4014 24 161 14 2.20 2 28 14 3.29 3 31 13

20 1007 12 120 15 0.61 4 46 15 0.87 5 44 14

GM: Geometric Mean

Max:Min: Ratio of maximum and minimum laboratory geometric means

GCV: Geometric Coefficient of Variation (%)

N: Number of laboratories used in calculation of GM and GCV

Table 6. Summary of inter-laboratory GCV values

Type Sample ED50 16/284 16/322 NR4020 NR4021 NR21973

International

Standard

7 165 29 25 28 31

12 149 29 28 31 48

Individual Human

Serum

5 168 48 42 38 64 70

9 145 30 20 32 32 42

10 211 45 54 50 44 46

11 197 40 40 44 49 68

13 173 21 29 31 35 35

15 171 47 34 43 50 63

16 183 33 34 39 56 54

17 146 69 63 73 80 95

18 186 70 65 57 91 87

20 120 46 44 52 50 56

BEI Resources

1 168 31 48 43 34

4 174 19 31 30 36 27

6 162 25 28 40 43

8 156 63 45 56 54 70

19 161 28 31 40 34

Monoclonal

Antibody

3 215 191 199 203 193 226

14 163 152 155 154 155 184

% GCV

0 70 140+

Grey shading: Reference, GCV not applicable

Table 7. Sample geometric mean ED50 and potency estimates relative to 16/284 or 16/322 (Data without complement)

GM: Geometric Mean




Sample ED50 Potencies v 16/284 Potencies v 16/322


1 927 16 160 11 0.54 2 30 11 0.76 4 50 11

3 291 80 295 8 0.16 13 160 8 0.25 14 150 8

4 821 15 160 10 0.52 2 19 10 0.71 3 37 10

5 254 23 182 10 0.13 4 48 10 0.20 3 35 10

6 1315 26 176 11 0.77 2 32 11 1.08 3 34 11

7 1715 19 180 11 1 1 0 11 1.41 3 35 11

8 575 15 146 10 0.38 6 73 10 0.53 3 46 10

9 1443 17 153 10 0.75 3 31 10 1.13 2 18 10

10 713 26 231 10 0.37 4 54 10 0.56 6 68 10

11 995 28 222 10 0.52 3 39 10 0.78 2 39 10

12 1220 18 146 11 0.71 3 35 11 1 1 0 11

13 4389 27 189 10 2.28 2 22 10 3.44 2 31 10

14 3369 63 225 9 2.04 10 127 9 3.11 10 110 9

15 1510 22 179 10 0.79 4 53 10 1.18 2 36 10

16 1044 26 178 10 0.54 2 37 10 0.82 3 39 10

17 1234 26 168 11 0.72 5 67 11 1.01 5 57 11

18 356 21 196 9 0.22 6 75 9 0.33 5 64 9

19 3511 24 170 9 2.06 2 27 9 3.11 3 34 9

20 842 10 108 10 0.57 3 46 10 0.80 3 34 10

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Table 8. Sample geometric mean ED50 and potency estimates relative to 16/284 or 16/322 (Data with complement)

GM: Geometric Mean




Sample ED50 Potencies v 16/284 Potencies v 16/322


1 1593 23 221 5 0.77 1 13 5 1.18 1 13 4

3 352 11 161 5 0.17 31 326 5 0.27 34 437 4

4 1202 23 289 4 0.53 2 26 4 0.76 1 18 3

5 239 11 202 5 0.12 3 58 5 0.17 3 70 4

6 1463 17 189 5 0.70 1 3 5 1.08 1 13 4

7 2079 16 187 5 1 1 0 5 1.54 1 10 4

8 707 16 259 4 0.40 3 58 4 0.61 3 55 4

9 1871 11 175 5 0.90 2 28 5 1.32 2 26 4

10 609 20 265 4 0.34 2 32 4 0.52 2 27 4

11 934 15 222 5 0.45 3 49 5 0.67 3 54 4

12 1161 18 231 4 0.65 1 10 4 1 1 0 4

13 4933 20 203 5 2.37 2 24 5 3.73 2 33 4

14 5276 2 46 4 2.32 29 297 4 3.62 32 470 3

15 2495 12 206 4 1.10 2 20 4 1.54 2 27 3

16 782 26 286 4 0.44 2 22 4 0.67 2 22 4

17 1065 9 144 5 0.51 4 81 5 0.67 4 79 4

18 461 17 234 5 0.22 5 79 5 0.37 5 92 4

19 5110 15 191 5 2.46 2 31 5 3.74 2 26 4

20 1440 8 158 5 0.69 3 53 5 1.05 4 72 4

WHO/BS/2019.2372

Page 17

Table 9. Summary of inter-laboratory GCV values with and without complement

Type Sample ED50 16/284 16/322 NR4020 NR4021 NR21973

With

C'

Without

C'

With

C'

Without

C'

With

C'

Without

C'

With

C'

Without

C'

With

C'

Without

C'

With

C'

Without

C'

International

Standard

7 187 180 10 35 2 30 27 26 12 28

12 231 146 10 35 11 32 23 32 11 47

Individual

Human

Serum

5 202 182 58 48 70 35 50 31 61 59 60 62

9 175 153 28 31 26 18 26 29 19 38 33 45

10 265 231 32 54 27 68 28 58 38 45 28 42

11 222 222 49 39 54 39 42 44 50 43 48 65

13 203 189 24 22 33 31 21 34 42 29 19 35

15 206 179 20 53 27 36 16 41 35 55 30 74

16 286 178 22 37 22 39 18 44 43 52 10 51

17 144 168 81 67 79 57 70 71 64 75 83 78

18 234 196 79 75 92 64 68 51 92 88 74 89

20 158 108 53 46 72 34 45 51 68 37 60 53

BEI

Resources

1 221 160 13 30 13 50 12 42 29 34

4 289 160 26 19 18 37 23 31 22 40 13 25

6 189 176 3 32 13 34 30 41 12 42

8 259 146 58 73 55 46 48 59 60 51 54 71

19 191 170 31 27 26 34 30 41 29 34

Monoclonal

Antibody

3 161 295 326 160 437 150 266 164 256 148 302 176

14 46 225 297 127 470 110 231 118 234 115 273 143

% GCV

0 70 140+

Grey shading: Reference, GCV not applicable

Table 10. Concordance correlation coefficients for log 10 potencies comparing assays with

and without guinea pig complement; values ≥0.8 shade

Grey shading: Not calculated as no value for 16/322 provided.

Table 11. Proposed RSV/B without complement IU/mL values for BEI Resources Materials

Sample Name IU/mL

NR-4020: Human Reference Antiserum to RSV 1494

NR-4021: Human Antiserum to RSV, High Control 4409

NR-4022: Human Antiserum to RSV, Medium Control 1005

NR-4023: Human Antiserum to RSV, Low Control 669

NR-21973: Human Reference Immune Globulin to RSV 12059

Lab ED50 16/284 16/322

1 0.96 0.99 0.99

3 0.76 0.94 7 0.78 0.88 0.80

8 0.95 0.97 0.94

9 0.72 0.92 0.90

WHO/BS/2019.2372

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Figure 1. Ratios of ED50s for coded duplicate samples in each assay and the cut-off for

acceptability

WHO/BS/2019.2372

Page 20

Figure 2. Ratios of the maximum and minimum ED50s for each sample in each laboratory

and the cut-off for acceptability

WHO/BS/2019.2372

Page 21

Figure 3. Laboratory geometric mean ED50s

Sample

Geo

mean

1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2010

100

1000

10000

50000

Figure 4. Laboratory mean potencies relative to 16/284

Sample

Geo

mean

1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200.01

0.1

1

10

50

WHO/BS/2019.2372

Page 22

Figure 5. Laboratory mean potencies relative to 16/322

Sample

Geo

mean

1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200.01

0.1

1

10

50

WHO/BS/2019.2372

Page 23

Figure 6. Laboratory geometric mean ED50s (Without Complement)

Figure 7. Laboratory mean potencies relative to 16/284 (Without Complement)

201918171615141312111098765431

50000

10000

1000

100

10

Sample

Geo

mean

201918171615141312111098765431

50

10

1

0.1

0.01

Sample

Geo

mean

WHO/BS/2019.2372

Page 24

Figure 8. Laboratory mean potencies relative to 16/322 (Without Complement)

201918171615141312111098765431

50

10

1

0.1

0.01

Sample

Geo

mean

WHO/BS/2019.2372

Page 25

Figure 9. Laboratory geometric mean ED50s (With Complement)

Figure 10. Laboratory geometric mean potencies relative to 16/284 (With Complement)

201918171615141312111098765431

50000

10000

1000

100

10

Sample

Geo

mean

201918171615141312111098765431

50

10

1

0.1

0.01

Sample

Geo

mean

WHO/BS/2019.2372

Page 26

Figure 11. Laboratory geometric mean potencies relative to 16/322 (With Complement)

201918171615141312111098765431

50

10

1

0.1

0.01

Sample

Geo

mean

WHO/BS/2019.2372

Page 27

Figure 12. Scatterplot of sample coordinates from PCA.

Figure 13. Scatter plot of laboratory coordinates from PCA.

WHO/BS/2019.2372

Page 28

Figure 14. RSV/B Neutralisation titres (ED50 or IU/mL) with and without complement

ED50

C'

ED50

16/2

84 C

'

16/2

84

16/3

22 C

'

16/3

22

0

500

1000

1500

2000

Tit

res

( E

D5

0 o

r IU

/mL

)

*

Figure 15. Percentage difference between titres from assays with and without complement

ED

50

16

/ 28

4

16

/ 32

2

0

2 0

4 0

6 0

8 0

1 0 0

% D

iffe

re

nc

e *

*

WHO/BS/2019.2372

Page 29

APPENDIX 1

List of Collaborative Study Participants

Marina Boukhvalova

Sigmovir Biosystems

Rockville, MD, US

Hans Layman

AstraZeneca

South San Francisco, CA,

USA

Joyce Nyiro/ James Nokes

Kemri-Wellcome Trust

Kilifi, Kenya

Pedro A Piedra

Baylor College of Medicine

Houston, TX, US

David Cooper

Pfizer

Pearl River, NY, US

Lien Anh Ha Do/ Jeremy

Anderson/ Paul Licciardi/

Kim Mulholland

Murdoch Children’s research

Institute

Parkville, Australia

Surender Khurana

FDA

Silver Spring, MD, US

Carolin Schmittwolf

Bavarian Nordic

Munich, Germany

Zi-Zheng Zheng/ Yong-Peng

Sun

Xiamen University

Xiamen, China

Carel van Baalen

Viroclinics

Rotterdam, The Netherlands

Natalie Thornbug

Centers for Disease Control

and Prevention (CDC)

Atlanta, GA, US

WHO/BS/2019.2372

Page 30

APPENDIX 2

Laboratory geometric mean ED50 and relative potency estimates

Lab Sample ED50 Potencies v 16/284 Potencies v 16/322

GM GCV N GM GCV N GM GCV N

01a 1 1117 68 4 0.77 39 4 1.30 68 4

01a 3 70 19 4 0.05 19 4 0.08 19 4

01a 5 83 55 4 0.06 19 4 0.10 40 4

01a 6 1024 33 4 0.71 0 4 1.19 22 4

01a 7 1448 33 4 1.00 0 4 1.68 22 4

01a 8 304 56 4 0.21 22 4 0.35 49 4

01a 9 939 55 4 0.65 19 4 1.09 39 4

01a 10 332 39 4 0.23 19 4 0.39 19 4

01a 11 332 39 4 0.23 19 4 0.39 39 4

01a 12 861 22 4 0.59 22 4 1.00 0 4

01a 13 4871 56 4 3.36 22 4 5.66 49 4

01a 16 609 56 4 0.42 22 4 0.71 49 4

01a 17 724 49 4 0.50 33 4 0.84 56 4

01a 18 181 33 4 0.13 0 4 0.21 22 4

01a 19 2896 49 4 2.00 33 4 3.36 56 4

01a 20 724 33 4 0.50 0 4 0.84 22 4

01b 1 724 33 4 0.65 19 4 1.19 22 4

01b 3 70 19 4 0.06 33 4 0.11 40 4

01b 4 558 19 4 0.50 33 4 0.92 39 4

01b 5 70 19 4 0.06 0 4 0.11 19 4

01b 6 664 19 4 0.59 22 4 1.09 39 4

01b 7 1117 19 4 1.00 0 4 1.83 19 4

01b 8 197 39 4 0.18 49 4 0.32 68 4

01b 9 609 22 4 0.55 19 4 1.00 33 4

01b 10 304 22 4 0.27 39 4 0.50 49 4

01b 11 304 22 4 0.27 19 4 0.50 0 4

01b 12 609 22 4 0.55 19 4 1.00 0 4

01b 13 2896 49 4 2.59 39 4 4.76 56 4

01b 14 939 19 4 0.84 22 4 1.54 19 4

01b 15 512 63 4 0.46 39 4 0.84 41 4

01b 16 512 33 4 0.46 39 4 0.84 41 4

01b 17 609 22 4 0.55 39 4 1.00 49 4

01b 18 128 0 4 0.11 19 4 0.21 22 4

01b 19 2435 22 4 2.18 19 4 4.00 33 4

01b 20 558 19 4 0.50 33 4 0.92 39 4

02b 1 760 44 4 0.42 43 4 0.49 88 4

02b 4 1044 26 4 0.58 28 4 0.68 10 4

02b 5 310 37 4 0.17 40 4 0.20 6 4

02b 6 2272 71 4 1.26 77 4 1.47 32 4

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02b 7 1808 5 4 1.00 0 4 1.17 37 4

02b 8 670 13 4 0.37 9 4 0.43 37 4

02b 9 1448 12 4 0.80 15 4 0.94 41 4

02b 10 443 27 4 0.25 22 4 0.29 54 4

02b 11 851 20 4 0.47 20 4 0.55 20 4

02b 12 1547 34 4 0.86 37 4 1.00 0 4

02b 13 4963 19 4 2.74 19 4 3.21 25 4

02b 14 6644 22 4 3.67 23 4 4.29 26 4

02b 15 1739 22 4 0.96 21 4 1.12 27 4

02b 16 1112 13 4 0.61 12 4 0.72 23 4

02b 17 1078 30 4 0.60 32 4 0.70 7 4

02b 18 680 29 4 0.38 33 4 0.44 13 4

02b 19 2682 26 4 1.48 26 4 1.73 24 4

02b 20 679 67 4 0.38 70 4 0.44 31 4

03a 1 2919 23 3 0.77 6 3 - - -

03a 3 528 35 3 0.14 16 3 - - -

03a 4 2264 42 3 0.59 42 3 - - -

03a 5 494 5 3 0.13 13 3 - - -

03a 6 2749 50 3 0.72 35 3 - - -

03a 7 3812 17 3 1.00 0 3 - - -

03a 9 4103 38 3 1.08 58 3 - - -

03a 11 1975 30 3 0.52 24 3 - - -

03a 13 8343 31 3 2.19 53 3 - - -

03a 14 7735 35 3 2.03 57 3 - - -

03a 15 5035 30 3 1.32 51 3 - - -

03a 17 3634 45 3 0.95 67 3 - - -

03a 18 598 20 3 0.16 26 3 - - -

03a 19 9812 35 3 2.57 57 3 - - -

03a 20 2763 71 3 0.72 91 3 - - -

03b 1 3027 30 3 0.40 39 3 0.55 39 3

03b 3 777 26 3 0.10 29 3 0.14 49 3

03b 4 2768 35 3 0.36 21 3 0.50 36 3

03b 5 1127 24 3 0.15 32 3 0.20 45 3

03b 6 7331 34 3 0.96 33 3 1.33 33 3

03b 7 7660 62 3 1.00 0 3 1.38 30 3

03b 8 2617 51 3 0.34 43 3 0.47 26 3

03b 9 6673 79 3 0.87 37 3 1.21 6 3

03b 10 3558 73 3 0.46 34 3 0.64 6 3

03b 11 6369 52 3 0.83 19 3 1.15 18 3

03b 12 5532 78 3 0.72 30 3 1.00 0 3

03b 13 21359 67 3 2.79 18 3 3.86 10 3

03b 15 10302 84 3 1.34 21 3 1.86 14 3

03b 16 4270 103 3 0.56 31 3 0.77 20 3

03b 17 5648 79 3 0.74 64 3 1.02 32 3

03b 19 23759 48 3 3.10 32 3 4.29 22 3

04b 1 1422 26 4 0.39 27 4 0.68 75 4

WHO/BS/2019.2372

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04b 5 516 46 4 0.14 42 4 0.25 55 4

04b 6 2013 42 4 0.55 36 4 0.97 36 4

04b 7 3650 23 4 1.00 0 4 1.75 58 4

04b 8 778 56 4 0.21 46 4 0.37 45 4

04b 9 3191 37 4 0.87 18 4 1.53 79 4

04b 10 981 17 4 0.27 29 4 0.47 35 4

04b 11 2013 18 4 0.55 43 4 0.97 49 4

04b 12 2086 42 4 0.57 58 4 1.00 0 4

04b 13 6060 11 4 1.66 24 4 2.91 58 4

04b 14 4541 21 4 1.24 15 4 2.18 57 4

04b 15 3929 3 4 1.08 22 4 1.88 41 4

04b 16 3169 49 4 0.87 52 4 1.52 109 4

04b 17 6671 37 4 1.83 58 4 3.20 81 4

04b 18 637 36 4 0.17 39 4 0.31 80 4

04b 19 5216 26 4 1.43 36 4 2.50 44 4

04b 20 2080 32 4 0.57 44 4 1.00 80 4

05b 1 3662 18 4 0.63 35 4 0.96 20 4

05b 3 2370 16 4 0.41 8 4 0.62 20 4

05b 4 2626 10 4 0.45 15 4 0.68 10 4

05b 5 746 20 4 0.13 22 4 0.19 12 4

05b 6 4059 21 4 0.70 10 4 1.06 16 4

05b 7 5822 15 4 1.00 0 4 1.52 15 4

05b 9 3926 9 4 0.67 14 4 1.02 11 4

05b 10 3162 31 4 0.54 33 4 0.82 20 4

05b 11 3598 16 4 0.62 28 4 0.94 11 4

05b 12 3834 9 4 0.66 15 4 1.00 0 4

05b 13 12801 12 4 2.20 5 4 3.34 12 4

05b 14 29635 6 4 5.09 10 4 7.73 11 4

05b 15 3734 9 4 0.64 24 4 0.97 10 4

05b 16 2198 21 4 0.38 34 4 0.57 27 4

05b 17 2474 3 4 0.42 15 4 0.65 12 4

05b 18 1427 13 4 0.25 22 4 0.37 24 4

05b 20 2527 15 4 0.43 25 4 0.66 16 4

06b 1 777 22 4 0.53 19 4 0.78 22 4

06b 3 209 37 4 0.14 44 4 0.21 11 4

06b 4 858 18 4 0.59 31 4 0.87 7 4

06b 5 145 9 4 0.10 18 4 0.15 22 4

06b 6 883 33 4 0.60 49 4 0.89 14 4

06b 7 1467 20 4 1.00 0 4 1.48 32 4

06b 8 323 38 4 0.22 55 4 0.33 23 4

06b 9 1017 19 4 0.69 28 4 1.03 14 4

06b 10 306 22 4 0.21 43 4 0.31 22 4

06b 11 604 46 4 0.41 66 4 0.61 28 4

06b 12 991 23 4 0.68 32 4 1.00 0 4

06b 13 4190 16 4 2.86 9 4 4.23 22 4

06b 14 2352 22 4 1.60 32 4 2.37 23 4

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06b 15 833 16 4 0.57 6 4 0.84 29 4

06b 16 1072 11 4 0.73 10 4 1.08 30 4

06b 17 1036 18 4 0.71 25 4 1.04 6 4

06b 18 172 19 4 0.12 39 4 0.17 19 4

06b 19 2998 16 4 2.04 28 4 3.02 25 4

06b 20 799 28 4 0.54 38 4 0.81 14 4

07a 1 6958 34 3 0.93 27 3 1.26 19 3

07a 2 32 29 3 0.00 27 3 0.01 51 3

07a 3 315 35 3 0.04 36 3 0.06 64 3

07a 4 5074 14 3 0.68 9 3 0.92 27 3

07a 5 735 40 3 0.10 34 3 0.13 51 3

07a 6 5156 41 3 0.69 49 3 0.93 51 3

07a 7 7488 6 3 1.00 0 3 1.36 21 3

07a 8 3169 9 3 0.42 7 3 0.57 29 3

07a 9 6395 25 3 0.85 18 3 1.16 25 3

07a 10 3116 18 3 0.42 24 3 0.56 31 3

07a 11 3478 17 3 0.46 21 3 0.63 44 3

07a 12 5518 23 3 0.74 21 3 1.00 0 3

07a 13 18580 77 3 2.48 67 3 3.37 77 3

07a 14 3130 59 3 0.42 54 3 0.57 29 3

07a 15 6451 39 3 0.86 38 3 1.17 67 3

07a 16 4066 40 3 0.54 32 3 0.74 26 3

07a 17 1955 11 3 0.26 17 3 0.35 25 3

07a 18 1632 14 3 0.22 10 3 0.30 10 3

07a 19 22609 54 3 3.02 48 3 4.10 69 3

07a 20 3171 74 3 0.42 64 3 0.57 52 3

07b 1 3326 41 3 0.65 45 3 1.45 42 3

07b 2 26 21 3 0.01 32 3 0.01 30 3

07b 3 454 42 3 0.09 76 3 0.20 73 3

07b 4 2556 18 3 0.50 14 3 1.11 11 3

07b 5 497 24 3 0.10 43 3 0.22 41 3

07b 6 3209 10 3 0.62 37 3 1.40 36 3

07b 7 5147 25 3 1.00 0 3 2.24 2 3

07b 8 1924 16 3 0.37 14 3 0.84 15 3

07b 9 2386 22 3 0.46 32 3 1.04 30 3

07b 10 3909 15 3 0.76 30 3 1.70 27 3

07b 11 2356 14 3 0.46 35 3 1.03 33 3

07b 12 2293 24 3 0.45 2 3 1.00 0 3

07b 13 12619 37 3 2.45 54 3 5.50 51 3

07b 14 5364 44 3 1.04 79 3 2.34 78 3

07b 15 1971 51 3 0.38 78 3 0.86 74 3

07b 16 2160 25 3 0.42 37 3 0.94 34 3

07b 17 1767 22 3 0.34 35 3 0.77 33 3

07b 18 851 8 3 0.17 23 3 0.37 22 3

07b 19 10198 7 3 1.98 16 3 4.45 16 3

07b 20 1595 20 3 0.31 47 3 0.70 47 3

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08a 1 302 26 4 0.66 26 4 0.99 30 4

08a 3 598 11 4 1.31 12 4 1.96 6 4

08a 4 218 17 4 0.48 12 4 0.71 9 4

08a 5 65 27 4 0.14 28 4 0.21 35 4

08a 6 311 17 4 0.68 7 4 1.02 24 4

08a 7 455 11 4 1.00 0 4 1.49 18 4

08a 8 201 18 4 0.44 19 4 0.66 11 4

08a 9 557 16 4 1.22 15 4 1.82 28 4

08a 10 155 15 4 0.34 9 4 0.51 12 4

08a 11 228 10 4 0.50 18 4 0.75 16 4

08a 12 305 16 4 0.67 18 4 1.00 0 4

08a 13 908 7 4 2.00 9 4 2.97 18 4

08a 14 5478 15 4 12.04 16 4 17.93 26 4

08a 15 542 4 4 1.19 8 4 1.77 13 4

08a 16 154 19 4 0.34 20 4 0.50 37 4

08a 17 411 8 4 0.90 16 4 1.35 17 4

08a 18 98 31 4 0.22 22 4 0.32 24 4

08a 19 1505 13 4 3.31 24 4 4.93 20 4

08a 20 384 17 4 0.84 13 4 1.26 11 4

08b 1 226 24 4 0.56 33 4 0.73 21 4

08b 3 287 28 4 0.71 7 4 0.93 29 4

08b 4 188 26 4 0.46 25 4 0.61 16 4

08b 5 50 19 4 0.12 10 4 0.16 20 4

08b 6 287 9 4 0.71 17 4 0.93 14 4

08b 7 406 26 4 1.00 0 4 1.32 31 4

08b 8 177 46 4 0.44 33 4 0.58 34 4

08b 9 386 19 4 0.95 8 4 1.25 26 4

08b 10 149 48 4 0.37 25 4 0.48 40 4

08b 11 227 22 4 0.56 11 4 0.74 26 4

08b 12 308 15 4 0.76 31 4 1.00 0 4

08b 13 789 18 4 1.94 20 4 2.56 24 4

08b 14 3304 16 4 8.14 28 4 10.73 6 4

08b 15 462 28 4 1.14 14 4 1.50 24 4

08b 16 165 17 4 0.41 30 4 0.54 11 4

08b 17 258 24 4 0.64 23 4 0.84 17 4

08b 18 67 35 4 0.17 22 4 0.22 26 4

08b 19 995 26 4 2.45 27 4 3.23 19 4

08b 20 260 39 4 0.64 44 4 0.84 29 4

09a 1 1494 4 4 0.72 6 4 1.19 10 4

09a 2 152 22 4 0.07 17 4 0.12 26 4

09a 3 775 10 4 0.38 6 4 0.62 16 4

09a 4 836 10 4 0.40 15 4 0.67 10 4

09a 5 403 2 4 0.20 6 4 0.32 6 4

09a 6 1489 7 4 0.72 9 4 1.19 10 4

09a 7 2065 5 4 1.00 0 4 1.65 11 4

09a 8 1290 22 4 0.62 24 4 1.03 27 4

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09a 9 1673 2 4 0.81 7 4 1.34 7 4

09a 10 857 9 4 0.42 5 4 0.68 15 4

09a 11 1361 3 4 0.66 8 4 1.09 6 4

09a 12 1252 7 4 0.61 11 4 1.00 0 4

09a 13 4261 3 4 2.06 4 4 3.40 8 4

09a 14 5841 4 4 2.83 6 4 4.66 9 4

09a 15 2200 17 4 1.07 16 4 1.76 25 4

09a 16 979 8 4 0.47 10 4 0.78 14 4

09a 17 647 6 4 0.31 9 4 0.52 4 4

09a 18 1193 5 4 0.58 8 4 0.95 4 4

09a 19 3604 8 4 1.75 11 4 2.88 13 4

09a 20 2539 5 4 1.23 6 4 2.03 6 4

09b 1 590 22 4 0.46 23 4 0.65 19 4

09b 2 53 6 4 0.04 9 4 0.06 15 4

09b 3 491 18 4 0.38 14 4 0.54 10 4

09b 4 749 11 4 0.58 10 4 0.83 15 4

09b 5 299 17 4 0.23 19 4 0.33 23 4

09b 6 1049 4 4 0.81 8 4 1.16 11 4

09b 7 1292 5 4 1.00 0 4 1.43 7 4

09b 8 778 11 4 0.60 12 4 0.86 19 4

09b 9 908 11 4 0.70 11 4 1.01 17 4

09b 10 393 9 4 0.30 5 4 0.44 8 4

09b 11 1049 4 4 0.81 5 4 1.16 8 4

09b 12 903 10 4 0.70 7 4 1.00 0 4

09b 13 2168 8 4 1.68 7 4 2.40 6 4

09b 14 3390 4 4 2.62 5 4 3.75 11 4

09b 15 1047 2 4 0.81 4 4 1.16 9 4

09b 16 530 12 4 0.41 11 4 0.59 11 4

09b 17 695 6 4 0.54 4 4 0.77 4 4

09b 18 835 19 4 0.65 14 4 0.92 13 4

09b 19 2563 18 4 1.98 18 4 2.84 14 4

09b 20 1189 12 4 0.92 16 4 1.32 20 4

10b 1 494 44 4 0.93 68 4 1.14 60 4

10b 3 29 16 4 0.06 24 4 0.07 22 4

10b 4 358 43 4 0.67 58 4 0.82 59 4

10b 5 112 5 4 0.21 20 4 0.26 19 4

10b 6 630 35 4 1.19 55 4 1.45 47 4

10b 7 532 16 4 1.00 0 4 1.22 12 4

10b 8 289 12 4 0.54 11 4 0.66 17 4

10b 9 624 13 4 1.17 12 4 1.44 18 4

10b 10 325 24 4 0.61 43 4 0.75 38 4

10b 11 235 28 4 0.44 24 4 0.54 36 4

10b 12 434 14 4 0.82 12 4 1.00 0 4

10b 13 1233 14 4 2.32 24 4 2.84 14 4

10b 14 471 50 4 0.89 49 4 1.08 42 4

10b 15 576 28 4 1.08 34 4 1.33 19 4

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10b 16 448 23 4 0.84 22 4 1.03 10 4

10b 17 690 23 4 1.30 36 4 1.59 29 4

10b 18 141 25 4 0.26 30 4 0.32 26 4

10b 19 1283 14 4 2.41 27 4 2.95 19 4

10b 20 419 28 4 0.79 19 4 0.97 16 4

11b 1 294 83 3 0.54 109 3 0.38 185 3

11b 4 295 94 3 0.54 195 3 0.38 93 3

11b 6 420 59 3 0.77 77 3 0.54 49 3

11b 7 545 54 3 1.00 0 3 0.70 132 3

11b 8 588 49 3 1.08 129 3 0.75 54 3

11b 12 780 65 3 1.43 132 3 1.00 0 3

11b 17 730 7 3 1.34 64 3 0.94 64 3

11b 20 545 80 3 1.00 175 3 0.70 74 3

GM: Geometric Mean


N: Number of assays used in calculation of GM and GCV

WHO/BS/2019.2372

Page 37

Appendix 3

Proposed Instructions for use

1st WHO International standard for antiserum to Respiratory Syncytial Virus

NIBSC code 16/284

Version 03, 11/03/2019

“This material is not for in vitro diagnostic use”

1. INTENDED USE

This preparation was established by the WHO Expert Committee on Biological Standardization

in 2017 as the 1st International Standard for antiserum to respiratory syncytial virus. It has been

shown to be suitable for the standardization of virus neutralization methods to measure antibody

levels against RSV subtypes A and B in human sera.

2. UNITAGE

1,000 International Units (IU) of anti-RSV/A and anti-RSV/B neutralizing antibodies per ampoule.

3. CAUTION

This preparation is not for administration to humans or animals in the human food chain.

The preparation contains material of human origin, and either the final product or the source

materials, from which it is derived, have been tested and found negative for HBsAg, anti-HIV and

HCV RNA. As with all materials of biological origin, this preparation should be regarded as

potentially hazardous to health. It should be used and discarded according to your own laboratory's

safety procedures. Such safety procedures should include the wearing of protective gloves and

avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to

avoid cuts.

4. CONTENTS

Country of origin of biological material: United States. Each ampoule contains the freeze dried

residue of 0.5 ml human serum. The candidate standard has been prepared from a bulk of human

sera. 0.5 ml aliquots of this bulk were filled in DIN ampoules and freeze dried at NIBSC following

documented procedures. This fill was 0.5g fill weight with a mean dry weight of 0.043g. The

coefficient of variation (CV) was 0.27%. Residual moisture measured on 12 samples gave a mean

of 1.04% with a CV of 25.50% and oxygen imeasured in the headspace of 12 ampoules, gave a

mean of 0.44% with a CV of 26.02%.

5. STORAGE

The ampoules should be stored at -20°C or below until use.

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Please note: because of the inherent stability of lyophilized material, NIBSC may ship these

materials at ambient temperature.

6. DIRECTIONS FOR OPENING

DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow ampoule stem joins

the wider ampoule body. Tap the ampoule gently to collect the material at the bottom (labelled)

end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of

the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one

hand and the disposable ampoule breaker covering the ampoule stem between the thumb and first

finger of the other hand. Apply a bending force to open the ampoule at the coloured stress point,

primarily using the hand holding the plastic collar.

Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for

example, by the use of suitable gloves and an eye shield. Take care that no material is lost from

the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly

less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN

ampoule.

7. USE OF MATERIAL

No attempt should be made to weigh out any portion of the freeze-dried material prior to

reconstitution.

Ampoules should be reconstituted on the day of the assay by adding exactly 0.5 ml of pure sterile

distilled water to give 2000 IU/mL. Shake gently without the formation of foam to ensure that all

contents are reconstituted. To remove the reagent from the ampoule it is necessary to use some

form of transfer pipette rather than a volumetric pipette. The contents of the ampoules should not

be assumed to be sterile.

International Unit Conversion:

8. STABILITY

It is the policy of WHO not to assign an expiry date to their international reference materials. They

remain valid with the assigned potency and status until withdrawn or amended.

Reference materials are held at NIBSC within assured, temperature-controlled storage facilities.

Reference Materials should be stored on receipt as indicated on the label. Users should determine

(GMT antiserum to RSV IS/2000)

GMT SampleIU/mL =

GMT=Geometric Mean Titre

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the stability of the material according to their own method of preparation, storage and use. Multiple

freeze/thaw cycles should be avoided.

NIBSC follows the policy of WHO with respect to its reference materials.

9. CITATION

In all publications, including data sheets, in which this material is referenced, it is important that

the title of the preparation, the NIBSC code number, and the name and address of NIBSC are

cited and cited correctly.

10. LIABILITY AND LOSS

In the event that this document is translated into another language, the English language version

shall prevail in the event of any inconsistencies between the documents. Unless expressly stated

otherwise by NIBSC, NIBSC‟s Standard Terms and Conditions for the Supply of Materials

(available at http://www.nibsc.org/About_Us/Terms_and_Conditions.aspx or upon request by the

Recipient) (“Conditions”) apply to the exclusion of all other terms and are hereby incorporated

into this document by reference. The Recipient's attention is drawn in particular to the provisions

of clause 11 of the Conditions.

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MATERIAL SAFETY SHEET

Physical properties (at room temperature)

Physical appearance

Freeze dried and has a small white/yellowish cake

Fire hazard None

Chemical properties

Stable Yes Corrosive: No

Hygroscopic Yes Oxidising: No

Flammable No Irritant: No

Other (specify) Contains material of human origin(RSV antibodies)

Handling: See caution, section 1

Toxicological properties

Effects of inhalation: Not established, avoid inhalation

Effects of ingestion: Not established, avoid ingestion

Effects of skin absorption: Not established, avoid contact with skin

Suggested First Aid

Inhalation: Seek medical advice

Ingestion: Seek medical advice

Contact with eyes: Wash with copious amounts of water. Seek medical advice.

Contact with skin: Wash thoroughly with water.

Action on Spillage and Method of Disposal

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Spillage of ampoule contents should be taken up with absorbent material wetted with a virucidal

agent. Rinse area with a virucidal agent followed by water.

Absorbent materials used to treat spillage should be treated as biologically hazardous waste.

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Appendix 4

Summary of Participants comments on the report

Laboratory 1, 2, 5, 6, 7 and 11

• No comments received. This indicates that participants agreed with the report

recommendations.

Laboratory 3

• Replied but had no comments.

Laboratory 4

• Corrected information for Appendix 1. Recommended assigning international unitage to

palivizumab. Final draft not revised as standard has been shown to not work at

harmonising data from this sample type.

Laboratory 8


Laboratory 9


Laboratory 10

• Corrected information for Appendix 1. Minor editorial changes. Comment on improving

clarification on excluded data. Final draft revised.

Documents

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