WHO/BS/2017.2318 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17 to 20 October 2017
Report on the WHO collaborative study to establish the 1st International Standard for antiserum to Respiratory Syncytial Virus
Jacqueline U McDonald1, Peter Rigsby2, Thomas Dougall2 , 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 18 September 2017 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 T. Zhou at email: zhout@who.int.
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Summary
A collaborative study was conducted with the aim to establish the 1st International Standard for antiserum to RSV. Two candidate standards were produced from serum samples donated by healthy adult individuals in the US. The candidate standards are intended to standardize RSV neutralization assays across multiple assay formats. These assays are particularly useful in the evaluation of immunogenicity of RSV vaccine candidates. The candidates were processed, filled and freeze-dried at NIBSC. The study consisted of 21 laboratories from 9 countries and included 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 two candidate standards,16/284 and 16/322, naturally infected adult sera, age stratified naturally infected paediatric sera, sera from RSV vaccine clinical trials in maternal and elderly subjects, a monoclonal antibody to RSV (palivizumab), two cotton rat serum samples and samples from the BEI Resources panel of human antiserum and immune globulin to RSV.
The collaborative study showed that between-laboratory variability in neutralization titres was significantly reduced when values were expressed relative to those of either of the two candidate international standards. Stability of 16/284 maintained for 6 months at different temperatures showed no significant loss of activity (relative to that at -20oC storage temperature) at temperatures of up to +20oC. Stability data are not yet available for 16/322. From these results, 16/284 is recommended as the 1st international standard for antiserum to RSV, with 16/322 as a potential replacement for 16/284 in the future, with an assigned unitage of 1,000 and 960 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 51 RSV vaccine candidates in development, 14 of which are now in human clinical trials (PATH. 2017). 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). A reference antiserum is needed to standardize clinical trials and outcomes. PATH conducted a multi-laboratory RSV neutralization assay survey study of 12 diverse assay formats and found that it was feasible to harmonize neutralization results using a standard (Hosken et al. 2017). Pooled human serum confirmed as seropositive for RSV is being proposed as the candidate material to be assessed for the international standard.
Aim of the Study
The aim of the study was to characterize two candidate anti-RSV sera in diverse RSV neutralization assays to assess their suitability to be used as the 1st international standard for anti- serum to RSV. Age stratified paediatric serum pools and vaccinee serum pools from 3 separate clinical trials were also evaluated to establish commutability of the standard. 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, as these materials are currently being used by some laboratories as working standards.
Materials and Methods Bulk materials and processing
Product summary
A donation of sixty serum samples from healthy adults was provided by PATH as source material for the proposed international standard. The samples were confirmed to be negative for HBsAg, HIV, HBV, HCV and syphilis antibodies. All samples were positive for antibodies against RSV. From the 60 samples, 12 samples with high and medium RSV antibody titre, as determined in the PATH harmonization study (Hosken et al. 2017), were selected for two candidate pools. The two candidate pools were filled and freeze-dried at NIBSC. The first fill and freeze-dry was completed in October 2016 and the second pool was filled and freeze-dried in January 2017. For both materials, 0.5ml was filled in each vial and freeze-dried. The two candidates gave yellowish, robust cakes.
Pooling, filling, freeze-drying and sealing
Serum samples were received from PATH, as frozen samples in dry ice, and then stored at - 80oC. On the day before filling, the appropriate bulk sera were thawed at room temperature, pooled in a sterile vessel and delivered to the filling plant; six samples were pooled for each candidate standard. The pooled serum was filtered prior to filling and kept at +4oC. Filling was completed for both candidate pools from homogenous stirred bulks, which were maintained at between 4oC and 8oC during fill. 3ml ampoules were filled with 0.5ml of material. Freeze- drying was carried out immediately after filling using a 4 day cycle, after which the completed product was kept at -20oC for long term storage.
Ampoules were sealed under boil-off gas from high purity liquid nitrogen (99.99%) and measurement of the mean oxygen head space after sealing served as a measure of ampoule integrity. The mean oxygen head space was measured non-invasively by frequency modulated spectroscopy (FMS 760, Lighthouse Instruments, Charlottesville, USA). Residual moisture content was measured using the colorimetric Karl Fischer method in a dry box environment (Mitsubishi CA100, A1 Envirosciences, Cramlington, UK) with total moisture expressed as a percentage of the mean dry weight of the ampoule contents.
Product summary details for each of the filled samples are shown in Table 1. Fill dates for the candidate materials are detailed below:
Anti-RSV serum Pool 1 (NIBSC 16/284) – 20th October 2016 Anti-RSV serum Pool 2 (NIBSC 16/322) – 12th January 2017 Post filling testing of freeze-dried samples
One ampoule for each of the candidate standards 16/284 and 16/322 from the beginning, middle and end of the freeze drying process were tested in two repeat assays to check the effect of the freeze drying process on RSV neutralization antibody activity. This testing was performed against RSV/A2 virus strain, using a NIBSC in-house RSV neutralization assay. The GMTs of the beginning, middle and end samples of 16/284 were 1657, 1714 and 1493 respectively (119%, 123% and 107% compared to pre-lyophilised 16/284); and for 16/322 were 2071, 1731 and 1459 respectively (160%, 134% and 113% compared to pre-lyophilised 16/322) . Overall there were no losses in activity for each of the candidates for any of the time points during the freeze drying process. The filled material was therefore fit to be used in the collaborative study as candidate standards.
Study Samples
A total of thirty-eight samples were available to participants. The samples were shipped in dry- ice and storage at ≤ -20oC was recommended. All twenty-five participating labs were provided with the core panel and the panel for commutability listed in Table 2, except for 2 labs that were unable to receive the cotton rat and paediatric samples due to internal processes. Of the 25 labs, 19 were able to receive the BEI Resources panel. Six labs were not able to receive this panel due to administrative reasons.
BEI samples were reconstituted according to instructions from BEI materials. Sample 014 (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 the majority of participating labs. Receipt of the samples was confirmed by 24 of the 25 labs.
Design of Collaborative Study Participants
Twenty six laboratories were invited to participate in the study. Twenty five laboratories from twelve countries agreed to participate. Twenty one labs returned data; two of these returned two sets of data, one lab using two distinct assay methods and the other using two virus strains, giving a total of twenty three datasets. 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 at the end of January 2017 and the coordinator requested data to be returned by the 20th March 2017. Due to issues with import of study samples for some participants, some panels were sent late and the deadline for the affected participants was extended as much as possible. The majority of participants were able to return data by mid- April 2017.
Study Plan
Participants were requested to:
- Follow the recommended protocols for storage and reconstitution of the study samples.
- Determine the neutralization titre against RSV 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 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 cases where this was not possible, participants provided the raw data and statisticians at NIBSC calculated an ED50 using Combistats. Details of participants’ in-house methods can be found in Table 3.
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 candidate standard samples. 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.
An exploratory visual assessment of sample and laboratory differences was carried out by performing a simple correspondence analysis of potencies relative to candidate standard sample 11 (16/284). 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 candidate standard samples 11 or 24 (16/284 and 16/322 respectively).
Laboratory 7, 11a & 13
These labs 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 these labs, as the assays are not optimized to calculate an ED50.
Stability Studies
Samples of the candidate standards were stored at -70°C, -20°C, +4°C, +20°C, + 37°C, +45°C and +56°C and 16/284 was tested after 6 months storage for RSV neutralization activity.
Candidate 16/322, which was filled later than 16/284, will be tested at the 6 month mark.
Samples will be tested at regular intervals to assess long term stability.
The stability of candidate standards reconstituted in liquid form was also assessed. The candidate standards were reconstituted and stored at +4°C, +22°C and + 37°C for up to four weeks then tested for RSV neutralization activity. Samples for stability were tested using a NIBSC in-house RSV neutralization assay.
Results
Study data returned
A total of 23 datasets were received from 21 out of 25 participants. Laboratory 11 returned 2 datasets using 2 different neutralization assay formats, and laboratory 14 returned 2 datasets using 2 different virus strains. The data from laboratory 6 were not included in the analysis, as the results returned did not give estimated values for either candidate standard but instead returned values of >1024.
Intra-assay and inter-assay variability in ED50s
Intra-assay variability was assessed using the coded duplicate samples included in the study.
Three pairs of coded duplicates were included in the panel of samples, these were; 1 & 3, 9 & 29, and 16 & 26. Where any of the coded duplicate ED50s within an assay differed by more than a factor of 2.5, no result for that assay was used, see Figure 1. The excluded assays accounted for
~8% of returned assays.
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. Further to the excluded assays, ~5% of samples were excluded, see Figure 2.
For each laboratory and sample, GM ED50s and relative potencies, together with inter-assay GCVs are shown in Appendix 2, following the exclusions detailed above.
Correspondence analysis outcomes
A simple correspondence analysis of log transformed potencies relative to 16/284 was carried out after exclusion of sample 32 (negative) and laboratory 15 (only 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 (less than 5%) 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 3. 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 animal and monoclonal antibody (mAb) panel samples. No particular groupings of laboratories were observed and plots of laboratory components are not shown.
Agreement between laboratories
Concordance correlation coefficients are summarised in Tables 4 and 5 for potencies relative to candidate standards 16/284 and 16/322 respectively, based on the human panel samples only.
Scatterplots of log potencies relative to 16/284 and 16/322 for all samples and all laboratory pairs are shown in Figures 4a, 4b, 5a and 5b. Concordance was noted to be poor for laboratories 02, 12, 13, 14b, 15, 17, 19, 20 and 21, all having concordance correlation coefficients <0.80 in more than 50% of cases shown in Tables 4 and 5. Levels of inter-laboratory variability have been calculated with (set A) and without (set B) these laboratories (Table 7) to illustrate the reduced level of variability that was achieved between the remaining laboratories (n=13).
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 Tables 6a and 6b and Figures 6a to 6f. A summary of the calculated GCVs can be found in Table 7 and median GCVs are summarised in Table 8. Animal samples 13 & 27, mAb samples 10 & 22, and sample 32, 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
Using the data without the removal of any laboratories (set A), with the exception of sample 7 (82%), all between-laboratory GCVs were above 100%. The GCVs ranged from 82% to 186%.
When using data excluding 9 laboratories according to the concordance analysis described above (set B), the GCVs range from 54% to 173%.
Potencies relative to 16/284
For all samples tested, the GCVs show better agreement between laboratories when the titres are expressed relative to 16/284 than when end point titres are used. When using data set A, the GCVs for relative potencies were between 26% and 86%. There was a reduction in variability for the median GCVs, from 143% to 54%. When using data set B, the GCVs for relative potencies ranged from 15% to 72%. There was a reduction in the median GCVs, from 114% to 33%.
Potencies relative to 16/322
These also show better agreement between laboratories than end point titres for all samples tested. When using data set A, the GCVs for relative potencies were between 24% and 89%.
There was a reduction in the median GCVs, from 143% to 56%. When using data set B, the GCVs for relative potencies ranged from 16% to 70%. There was a reduction in median GCVs, from 114% to 32%.
Adult Human Sera (Natural Infection)
The median GCV for this group of samples was 143% when using data set A, and 114% with data set B. When expressed relative to 16/284, the GCV was reduced to 48% with data set A, and 22% when using data set B. When expressed relative to 16/322, the GCV was reduced to 43% with data set A, and 31% when using data set B.
BEI Resources Samples
The median GCV for the BEI resources samples was 130% with data set A, and 65% with data set B. When expressed relative to 16/284, the GCV was reduced to 45% with data set A, and 25% with data set B. When expressed relative to 16/322, the GCV was reduced to 50% with data set A, and 28% with data set B. Potencies relative to samples 007, 014, and 019 were also calculated to assess the ability of these samples to perform as working standards. These samples were able to reduce the GCVs of all samples as successfully as the candidate standards (Tables 6b, 7 and 8).
Paediatric Samples
The median GCV for the paediatric samples was 158% with data set A, and 118% with data set B. When expressed relative to 16/284, the GCV was reduced to 60% with data set A, and 37%
with data set B. When expressed relative to 16/322, the GCV was reduced to 55% with dataset A, and 30% with data set B.
Vaccinee Samples
The median GCV for the vaccinee samples was 139% with data set A and 116% with data set B.
When expressed relative to 16/284, the median GCV was reduced to 65% with data set A, and
35% with data set B. When expressed relative to 16/322, the median GCV was reduced to 60%
with dataset A, and 35% with data set B.
Animal Sera
Cotton rat sera included in the study showed higher GCVs than all other sample groupings, with a median GCV of 222%, when using data set A, and a median GCV of 159% when using data set B. When expressed relative to 16/284, the GCV was reduced to 119% with data set A, and 67%
with data set B. When expressed relative to 16/322, the GCV was reduced to 94% with data set A, and 53% with data set B.
Monoclonal Antibodies
The median GCV for the monoclonal antibody sample was 154% when using data set A, and 132% with data set B. When expressed relative to 16/284, the GCV was reduced to 83% with data set A, and 71% with data set B. When expressed relative to 16/322, the GCV was reduced to 88% with data set A, and 87% with data set B.
Stability study results
At the time of this report only data up to the 6 month time point for 16/284 were available for evaluation. No accelerated stability data are yet available for 16/322. After 6 months storage at elevated temperatures, two independent assays were performed for each temperature, and potencies relative to the -20°C baseline for 16/284 were obtained. The ED50s and GMTs are shown in Table 9 and the estimated RSV neutralization activity loss per month and year are shown in Table 10. The data show that there is no loss of activity at +4°C and minimal loss at +20°C (temperatures used during laboratory manipulation for assays), relative to the -20°C baseline. The low predicted loss in activity per year (<0.01%) when stored at -20oC suggests 16/284 is sufficiently stable to serve as a WHO international standard. The stability of 16/284 and 16/322 will be monitored regularly throughout the life time of the standards.
The stability of candidate standards reconstituted in liquid form was also assessed (Table 11a and 11b). Ampoules of the three candidate materials that had been reconstituted in 0.5ml of sterile glass distilled water and maintained at different temperatures were tested. Two independent assays were performed at each temperature and time point, and potencies relative to that of an ampoule stored at -20°C and freshly reconstituted were obtained for both candidates. The data suggest that there is no loss of activity for any of the reconstituted materials that had been stored at +4°C or +22°C for both standards for up to a week. After 4 weeks at +4°C, 16/284 had lost 24% of its neutralization activity. At +37°C, 16/284 showed approximately 20% loss after 2 weeks, and 16/322 had lost >40% activity after 1 week.
Discussion
The data from this study show that both 16/284 and 16/322 are suitable as reference standards to measure RSV neutralization activity in a range of sample types, particularly human serum, due to their ability to reduce GCVs across the various assay methods included in this study. We propose 16/284 as the first International Standard for antiserum to RSV, with an assigned
potency of 1000 IU/ampoule, as stability data for this candidate are already available. Based on this proposal and the geometric mean potency of 16/322 relative to 16/284 (0.96; sample 24 in Table 6a; no laboratory results detected as outliers), 16/322 can be assigned a potency of 960 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. A deliberate decision was made that no aspect of the methods used would be controlled, to mimic the real world variation of the assays currently being used. However, once the data were expressed as potencies relative to the candidates, the GCVs were significantly decreased across all laboratories. Using data with the exclusion of 9 laboratories further reduced the GCVs.
However, there was no common aspect of the methods between the excluded laboratories, or an obvious reason why concordance correlation coefficients should be so low. There were two exceptions to this: laboratory 14b, which looked at neutralization titres against RSV B, and laboratory 17 which included guinea pig complement in their assay. Further collaborative studies will be needed to determine the usefulness of this standard against RSV B viruses.
Stability data for 16/284, based on storage at elevated temperatures up to six months, gave a low predicted loss in activity per year (<0.01%) when stored at -20oC, suggesting suitable stablilty to serve as a WHO IS. A long term programme of monitoring stability will be needed to show that 16/284 remains stable over its life time. Stability data for 16/322 is not currently available but it will also be monitored for stability over its life time. Furthermore, stability analysis showed that the candidate standards were also stable after reconstitution. Both candidates showed loss of activity at 37ºC after 2 weeks, with 16/322 showing a greater loss than 16/284.
This study has achieved its stated aims and an International Standard is proposed, with an International Unitage (IU). This study has shown that the standard is useful for multiple sample types across a wide variety of assay formats; however, the analysis suggests that the cotton rat serum samples and monoclonal antibody samples behave differently from the human serum samples, and that a more suitable standard should be considered for those sample types. This is not an issue for this International Standard, as its main role will be to look at neutralising antibody activity in human serum, mostly produced in RSV vaccine clinical trials.
The BEI Resources panel consisted of a BEI materials panel of human antisera and immunoglobulin. These were included to assess their ability to act as working standards and data from the collaborative study support their suitability, as they are able to reduce GCV when used as standards. The proposed international unitage of these samples can be found in Table 12.
Commutability for vaccine serum samples and paediatric samples was assessed in this collaborative study. Sera from both maternal and elderly clinical trials were included and both candidate standards were able to reduce GCVs for these samples. Naturally infected paediatric sera were also assessed and both candidates were able to reduce the GCVs across all laboratories for these samples. Vaccinee sera from paediatric clinical trials were not available for testing.
As well as the inter-laboratory GCVs, correspondence analysis did not suggest differences in the behaviours of the vaccinee sera and paediatric sera in comparison to the adult human naturally
infected sera, which further indicates that the candidate standards are commutable with these sample types.
Recommendations
It is proposed that the candidate 16/284 should be established as the 1st International Standard for antiserum to RSV to be used for the standardization of RSV neutralization assays. The assigned potency for this should be:
1,000 IU/ampoule
It is proposed that the candidate 16/322 should be retained as a potential secondary/replacement International Standard for antiserum to RSV to be used for the standardization of RSV neutralization assays. The assigned potency for this should be:
960 IU/ampoule
Comments from Participants
All participants were requested to comment on the draft report. Three out of the 21 did not respond to request for comments. There were no disagreements with the suitability of the candidate IS (NIBSC 16/284) to serve as the 1st international standard for antiserum to RSV.
Some responders had minor queries or suggestions for editorial changes and these have been addressed.
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 greatfully acknowledge Glaxo Smith Kline (Belgium), MedImmune (US), Novavax (US), Professor Andrew Pollard (Oxford University, UK) and Professor Pedro A. Piedra (Baylor College of Medicine, US) for donating samples included in this study. 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 3rd March 2017.
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
Table 1. Product Summary
Production Summary For Cadidate International Standards
NIBSC code 16/284 16/322
Presentation 3ml DIN
ampoules
3ml DIN ampoules
Number of containers 1703 1572
Validation of ampoule integrity Visual inspection plus Oxygen headspace on 12 random samples
Visual inspection plus Oxygen headspace on 12 random samples
Sealing gas Nitrogen from
liquid nitrogen 99.99% pure
Nitrogen from liquid nitrogen 99.99% pure Oxygen headspace measured by Near Infra-Red
spectroscopy
Near Infra-Red spectroscopy Residual moisture measured by Karl Fischer
reagent
Karl Fischer reagent
Mean fill mass 0.5239g 0.5230g
CV fill mass 0.95% 0.62%
Number of fill weights measured 83 73
Mean dry weight 0.0430g 0.0443g
CV of dry weight 0.27% 0.39%
Number of dry weights measure 6 6
Mean residual moisture 1.04% 0.32%
CV of residual moisture 25.50% 11.86%
Number of residual moisture measurements
12 12
Mean oxygen headspace 0.44% 0.44%
CV of oxygen headspace 26.02% 35.69%
Number of oxygen tests carried out 12 12
Date of fill 20/10/2016 12/01/2017
Storage temperature -20°C -20°C
Microbial contamination None detected None detected
Table 2. Samples Included in the Collaborative Study
Sample Panels Name
Sample Code
Core Human Panel
International Standard Candidate 1- NIBSC 16/284 011 International Standard Candidate 2 – NIBSC 16/322 024 International Standard Candidate 1 Pre-lyophilised 009 International Standard Candidate 1 Pre-lyophilised 029 International Standard Candidate 2 Pre-lyophilised 018 P-0015 Individual Adult Human Serum 001 P-0015 Individual Adult Human Serum 003 P-0029 Individual Adult Human Serum 031 P-0035 Individual Adult Human Serum 038 P-0041 Individual Adult Human Serum 037 P-0056 Individual Adult Human Serum 016 P-0056 Individual Adult Human Serum 026 Core Monoclonal
Antibody Panel
Palivizumab 0.1mg/ml 010
Palivizumab 1mg/ml 022
Core Animal Panel Cotton Rat Serum Pool - RSV A 027
Cotton Rat Serum Pool - RSV B 013
Panel for Commutability-
Vaccinee
Maternal sera pool from RSV F Trial - 1 006 Maternal sera pool from RSV F Trial - 2 020 Maternal sera pool from RSV F Trial - 3 017 Maternal sera pool from RSV F Trial - 4 015 Maternal sera pool from RSV F Trial - 5 004 Elderly sera pool from RSV F Trial - 1 021 Elderly sera pool from RSV F Trial - 2 025 Elderly sera pool from RSV F Trial - 3 012 Elderly sera pool from RSV F Trial - 4 034 Adult sera pool from RSV F Trial - 1 (Low) 028 Adult sera pool from RSV F Trial - 2 (Med) 035 Adult sera pool from RSV F Trial - 3 (High) 030 Panel for
Commutability - Paediatric
Pediatric Serum Pool Age < 1 year 005 Pediatric Serum Pool Age 1 - 2 years 023 Pediatric Serum Pool Age 2 - 3 years 033 Pediatric Serum Pool Age 3 - 4 years 008
BEI Resources Panel
NR-4020: Human Reference Antiserum to RSV 007 NR-4021: Human Antiserum to RSV, High Control 019 NR-4022: Human Antiserum to RSV, Medium Control 002 NR-4023: Human Antiserum to RSV, Low Control 036 NR-21973: Human Reference Immunoglobulin to RSV 014 NR-49447: Human IgG-Depleted Serum (Negative control) 032
Table 3. Details of methods used by participants
Lab Code
Plate Format
Duration of Assay
Cell Line
Complement/
% and type RSV Strain
[Virus]
added per well
Dilution Series (#dilutions /
fold dilution)
Time/temp (RT=room
temp)
Titre
Determination Read-out
01 96 well 24 - 48
hours Vero No A2 Recombinant
GFP
5,000
pfu/cell 9 / 3 fold 30 mins/ RT IC50 GFP-
fluorescence
02 96 well 2 days HEp-2 No A2 200
pfu/well 12 / 2-fold 1hr/4°C ED50 Plaque counts
03 96 well 2 days A549 No Recombinant
FFL-A2
2x106
PFU/ml 10 / 8 fold 20 - 21
hr/37°C EC50 Firefly
luciferase
04 96 well 3 days Vero No A2 1,000 pfu 10 / 3-fold
Direct to Veros at 35°C
EC50 ELISA A450
05 96 well 3 days HEp-2 No A2 75 pfu 9 / 2-fold 30 min/RT ED50 by Excel F protein EIA
06 96 well 24 hours HEp-2 No A2 100
pfu/well 8 / 2-fold 1hr/37°C ED50 by Excel Plaque counts
07 96 well 6 days vero No RSV A (long) 100
pfu/well 7 / 2-fold 1hr/33°C ED60 by Excel
Fluorescent plaques (stained with primary RSV
AB and secondary FITC) and counted with fluorescence microscope
reader
08 96 well 6-7 days HEp-2 No RSV/A/Tracy
3^3 TCID100/
.05 or 3^5.5 TCID50
12 / 2-fold 90 minutes/
36°C
The last dilution with 50% or greater intact
Hep-2 monolayer.
CPE
09 96-well 4 days Vero No rRSV/V35pX
EGFP
3,750
pfu/mL 7 / 2-fold 48 hr/37°C
2-log titer at 50% by Graphpad
showing LogEC50 as
results
Plaque counts by spots with fluorescence
Lab Code
Plate Format
Duration of Assay
Cell Line
Complement/
% and type RSV Strain
[Virus]
added per well
Dilution Series (#dilutions /
fold dilution)
Time/temp (RT=room
temp)
Titre
Determination Read-out
10 96-well 24 hours Vero No A2 Recombinat
GFP
652
ffu/well 8 / 3-fold 1hr/RT
IC50 using 2-pt interpolation of dilutions that surround 50%
virus reduction
Foci/well
11a 24 well 5 days HEp-2 No A2 25-50 pfu 6 / 4-fold 1hr/37°C 60% plaque
reduction Plaque counts 11b 96 well 4 days Vero-
81 No A2
500- 1,000 PFU/well
10/3-fold 1hr/37°C IC50 by
SoftMax Pro OD at 450nm
12 12 well 7 days Vero No A2 200
pfu/well 11 / 2-fold 1hr/37°C ED50 by Excel Plaque counts
13 96-
well 24 hours Vero No A2
500- 5,000 TCID50/
well
2-fold 1hr/37°C ND90, EXCEL RSV N gene amplicon
14a 96 well 6 days HEp-2 No RSV/A (Long) 100
TCID50 8 / 2-fold 1hr/37°C
ED50 (Reed &
Muench by excel)
CPE
14b 96 well 6 days HEp-2 No RSV/B (B1 WT) 100
TCID50 8 / 2-fold 1hr/37°C
ED50 (Reed &
Muench by excel)
CPE
15 384
well 3 days A549 No RSV A (M37) 550
ffu/well 10/2.5-fold 30 min/37°C
2-pt interpolation of
dilutions that surround 50%
virus reduction
Fluorescent foci
16 48 well 7 days Vero
E6 No A2 70 – 130
pfu/well
6 or 12 /2- fold
30 +/-5 mins /37 °C
ED50 by log- linear regression in
Excel
Plaque counts
17 96 well 2 days A549 5% guinea pig complement
A2 expressing Renilla Luciferase
250 pfu/100
ul/well
12 / 2 -fold 60mins/37°C ED50 by Excel Luciferase activity
Lab Code
Plate Format
Duration of Assay
Cell Line
Complement/
% and type RSV Strain
[Virus]
added per well
Dilution Series (#dilutions /
fold dilution)
Time/temp (RT=room
temp)
Titre
Determination Read-out
18 96 well 24 hours HEp-2 NO
RSV/A2 carrying the monomeric Katushka fluorescent protein (RSV A
mKate)
MOI=4, 5×104 cells/well
10 / 4-fold 1 hr/37°C IC50
Mean fluorescence intensity per
well
19 384
well 24 hours HEp-
2 No
Recombinant mKate-RSV
(A2)
2x104
pfu/well 3 or 4 / 9 37°C
IC50 by curve fitting with Prism software
Fluorescence Intensity
20 96 well 7 days HEp-
2 No RSV A (long) 3500
pfu/ml 12 / 2-fold 90 mins/37°C
Last dilution with > 50%
intact Hep-2 monolayer ED50 (Reed &
Muench by Excel)
CPE
21 96 well 5 days Hep-2 No RSV-A
Memphis 37b
Approx 2.75 log10
TCID50/
ml
7 / 3-fold 30 mins/37C Karber
calculation CPE
Table 4. Concordance correlation coefficients for log potencies relative to 16/284 (human panel samples only); values ≥0.8 shaded
Lab 01 02 03 04 05 07 08 09 10 11a 11b 12 13 14a 14b 15 16 17 18 19 20 21
01 02 0.78 03 0.89 0.83 04 0.73 0.69 0.89 05 0.88 0.87 0.96 0.88 07 0.81 0.75 0.90 0.81 0.85 08 0.92 0.89 0.96 0.82 0.96 0.88 09 0.78 0.80 0.95 0.87 0.90 0.87 0.90 10 0.78 0.74 0.93 0.97 0.92 0.86 0.87 0.88 11a 0.71 0.68 0.86 0.89 0.84 0.93 0.82 0.90 0.88 11b 0.80 0.72 0.93 0.94 0.94 0.87 0.89 0.91 0.94 0.94
12 0.67 0.67 0.71 0.57 0.71 0.78 0.76 0.67 0.64 0.70 0.67 13 0.49 0.51 0.69 0.82 0.64 0.66 0.59 0.70 0.83 0.72 0.71 0.44 14a 0.95 0.83 0.90 0.74 0.88 0.89 0.94 0.83 0.80 0.79 0.82 0.76 0.56 14b 0.46 0.58 0.52 0.53 0.60 0.57 0.63 0.51 0.54 0.58 0.57 0.73 0.38 0.58
15 0.34 0.28 0.39 0.51 0.36 0.35 0.34 0.37 0.51 0.36 0.38 0.19 0.62 0.31 0.14 16 0.83 0.85 0.96 0.88 0.97 0.87 0.96 0.94 0.92 0.87 0.94 0.78 0.67 0.87 0.67 0.34 17 0.65 0.66 0.83 0.87 0.80 0.70 0.73 0.80 0.89 0.73 0.81 0.41 0.84 0.66 0.33 0.55 0.77 18 0.91 0.78 0.96 0.89 0.92 0.89 0.91 0.88 0.93 0.84 0.90 0.65 0.72 0.92 0.50 0.45 0.91 0.84 19 0.87 0.83 0.75 0.58 0.78 0.64 0.80 0.64 0.64 0.53 0.62 0.54 0.40 0.82 0.34 0.28 0.69 0.58 0.75 20 0.70 0.83 0.65 0.49 0.67 0.59 0.72 0.61 0.54 0.49 0.52 0.67 0.34 0.70 0.41 0.23 0.65 0.43 0.60 0.80 21 0.78 0.76 0.79 0.75 0.90 0.59 0.82 0.72 0.76 0.62 0.80 0.51 0.46 0.69 0.44 0.32 0.81 0.67 0.74 0.74 0.62
Table 5. Concordance correlation coefficients for log potencies relative to 16/322 (human panel samples only); values ≥0.8 shaded
Lab 01 02 03 04 05 07 08 09 10 11a 11b 12 13 14a 14b 15 16 17 18 19 20 21
01 02 0.64 03 0.92 0.73 04 0.83 0.61 0.91 05 0.89 0.71 0.96 0.94 07 0.80 0.76 0.86 0.72 0.81 08 0.92 0.70 0.97 0.93 0.97 0.84 09 0.72 0.85 0.84 0.69 0.78 0.85 0.79 10 0.84 0.60 0.91 0.96 0.95 0.73 0.94 0.66 11a 0.74 0.72 0.83 0.76 0.81 0.93 0.82 0.88 0.71 11b 0.86 0.62 0.93 0.95 0.96 0.80 0.94 0.75 0.92 0.85
12 0.60 0.37 0.65 0.68 0.66 0.60 0.69 0.43 0.77 0.54 0.68 13 0.70 0.61 0.87 0.91 0.85 0.74 0.85 0.74 0.89 0.79 0.87 0.66 14a 0.92 0.77 0.90 0.76 0.85 0.92 0.89 0.84 0.77 0.85 0.82 0.58 0.74 14b 0.41 0.30 0.44 0.57 0.51 0.39 0.54 0.29 0.61 0.38 0.51 0.76 0.51 0.41
15 0.67 0.43 0.69 0.80 0.66 0.55 0.69 0.55 0.73 0.56 0.67 0.42 0.75 0.57 0.28 16 0.84 0.64 0.95 0.96 0.96 0.77 0.96 0.73 0.97 0.77 0.95 0.77 0.91 0.81 0.61 0.67 17 0.73 0.88 0.85 0.73 0.83 0.75 0.79 0.89 0.71 0.75 0.75 0.37 0.73 0.79 0.26 0.57 0.74 18 0.95 0.74 0.97 0.87 0.93 0.86 0.94 0.81 0.88 0.82 0.89 0.61 0.83 0.95 0.42 0.70 0.89 0.84 19 0.87 0.82 0.83 0.68 0.81 0.76 0.81 0.76 0.71 0.67 0.71 0.44 0.59 0.88 0.29 0.53 0.70 0.84 0.87 20 0.81 0.79 0.87 0.78 0.83 0.83 0.84 0.84 0.76 0.80 0.78 0.59 0.74 0.83 0.38 0.65 0.80 0.76 0.84 0.81 21 0.76 0.53 0.78 0.85 0.89 0.53 0.81 0.56 0.86 0.56 0.84 0.52 0.65 0.61 0.45 0.59 0.82 0.66 0.73 0.69 0.66
Table 6a. Sample geometric mean ED50 and potency estimates relative to 16/284 or 16/322
Type 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
Animal 013 183 123 213 19 0.14 28 124 19 0.14 17 106 19
027 355 257 231 18 0.25 36 114 18 0.25 18 81 18
Human
001 1392 47 156 22 1.07 4 41 22 1.11 3 31 22
003 1285 56 162 21 1.01 3 39 21 1.08 3 31 21
009 1497 17 114 21 1.12 4 42 21 1.12 3 33 21
011 1296 18 125 22 1.04 4 43 22
016 508 18 142 22 0.39 6 48 22 0.41 6 49 22
018 1293 20 122 21 1.06 4 39 21 1.07 5 38 21
024 1251 32 154 22 0.96 4 43 22
026 528 34 177 22 0.41 6 52 22 0.42 6 46 22
029 1301 25 143 21 1.06 10 54 21 1.08 4 37 21
031 669 51 150 21 0.50 4 51 21 0.52 7 57 21
037 941 15 119 20 0.74 7 54 20 0.73 6 51 20
038 187 21 134 21 0.14 17 86 21 0.14 19 77 21
mAb 010 362 55 171 22 0.28 8 90 22 0.29 14 95 22
022 3937 15 136 19 3.17 9 75 19 3.30 13 81 19
BEI Resources
002 546 19 141 18 0.50 9 60 18 0.48 4 51 18
007 801 10 82 17 0.76 3 27 17 0.76 4 40 17
014 476 17 119 16 0.46 3 31 16 0.47 7 58 16
019 2368 16 107 12 2.45 2 26 12 2.46 2 24 12
032 4 20 240 4 0.00 6 114 4 0.01 4 76 4
036 490 37 153 16 0.45 5 59 16 0.46 5 48 16
Paediatric
005 105 25 163 18 0.08 10 66 18 0.09 5 66 18
008 554 33 147 18 0.39 4 54 18 0.42 3 37 18
023 270 36 161 20 0.21 10 69 20 0.22 7 55 20
033 345 37 155 20 0.26 5 43 20 0.29 8 56 20
Vaccinee
004 382 24 123 20 0.33 8 58 20 0.35 7 57 20
006 1020 40 181 19 0.78 9 74 19 0.85 14 74 19
012 1734 27 150 20 1.37 5 54 20 1.40 7 61 20
015 1731 13 109 20 1.36 4 41 20 1.41 8 59 20
017 2589 15 134 21 1.93 9 66 21 1.94 9 71 21
020 2948 27 125 19 2.38 6 64 19 2.47 4 56 19
021 897 16 115 22 0.69 14 73 22 0.72 26 81 22
025 1328 47 139 21 1.08 11 74 21 1.10 10 70 21
028 331 72 186 19 0.27 12 71 19 0.27 6 54 19
030 1779 24 143 20 1.41 5 44 20 1.43 5 44 20
034 1635 36 162 18 1.29 19 85 18 1.33 12 89 18
035 906 31 140 22 0.70 6 50 22 0.72 5 54 22
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 6b. Sample geometric mean potency estimates relative to BEI Resources samples 7(NR-4020), 14(NR-21973) and 19(NR-4021)
Type Sample Potencies v Sample 007 Potencies v Sample 014 Potencies v Sample 019
GM Max:Min GCV N GM Max:Min GCV N GM Max:Min GCV N
Animal 013 0.23 24 153 10 0.36 17 133 10 0.07 16 151 10
027 0.41 32 166 9 0.65 22 140 9 0.12 24 145 9
Human
001 1.49 4 49 11 2.32 4 52 11 0.45 2 33 11
003 1.43 4 40 11 2.22 4 47 11 0.43 2 27 11
009 1.60 2 21 11 2.49 2 31 11 0.48 2 33 11
011 1.38 2 24 11 2.14 2 24 11 0.41 2 26 11
016 0.54 2 22 11 0.84 2 26 11 0.16 3 32 11
018 1.51 2 26 11 2.35 3 37 11 0.45 2 25 11
024 1.34 4 42 11 2.09 4 41 11 0.40 2 25 11
026 0.52 4 47 11 0.81 3 45 11 0.16 3 40 11
029 1.48 2 28 11 2.31 3 33 11 0.44 2 24 11
031 0.65 3 30 11 1.01 3 38 11 0.19 2 32 11
037 1.01 3 35 11 1.58 4 46 11 0.30 5 56 11
038 0.20 6 71 11 0.31 9 79 11 0.06 16 100 11
mAb 010 0.37 5 80 11 0.58 4 68 11 0.11 8 87 11
022 4.22 5 60 11 6.56 5 56 11 1.27 7 70 11
BEI Resources
002 0.66 2 30 11 1.03 3 37 11 0.20 3 42 11
007 1.56 2 22 11 0.30 3 30 11
014 0.64 2 22 11 0.19 2 26 11
019 3.33 3 30 11 5.19 2 26 11
032 0.00 2 48 3 0.01 3 63 3 0.00 3 83 3
036 0.59 5 55 10 0.93 6 71 10 0.18 4 48 10
Paediatric 005 0.12 5 85 9 0.19 6 90 9 0.03 7 89 9
008 0.56 4 51 9 0.88 5 68 9 0.17 4 48 9
023 0.28 5 78 10 0.44 6 88 10 0.09 7 80 10
033 0.39 2 21 10 0.61 3 31 10 0.12 4 43 10
Vaccinee
004 0.48 4 46 11 0.75 5 61 11 0.14 5 61 11
006 0.96 3 48 11 1.50 5 60 11 0.29 5 60 11
012 1.95 3 43 11 3.04 4 50 11 0.59 4 47 11
015 1.96 1 12 10 3.05 2 25 10 0.59 2 33 10
017 3.02 5 63 11 4.71 7 71 11 0.91 7 68 11
020 3.50 3 44 11 5.44 4 49 11 1.05 4 54 11
021 0.94 2 30 11 1.47 3 42 11 0.28 3 43 11
025 1.60 6 65 11 2.49 7 73 11 0.48 7 71 11
028 0.39 9 78 11 0.61 9 80 11 0.12 8 73 11
030 2.05 3 37 11 3.19 3 42 11 0.62 3 38 11
034 1.82 8 81 9 2.78 12 99 9 0.54 13 98 9
035 0.96 3 38 11 1.49 5 51 11 0.29 5 53 11
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 7. Summary of inter-laboratory GCV values
Type Sample
ED50s Potencies relative to different reference standards (All
Data)
(With Exclusions)
Set A (All Data) Set B (With Exclusions)
16/284 16/322 S7 S14 S19 16/284 16/322 S7 S14 S19
Human
1 156 133 41 31 49 52 33 22 31 21 28 29
3 160 135 39 30 40 47 27 21 31 12 29 31
9 114 103 42 33 21 31 33 18 26 16 34 32
11 125 111 43 24 24 26 18 7 19 20
16 142 113 48 49 22 26 32 23 30 20 29 31
18 122 98 39 38 26 37 25 18 18 15 19 23
24 154 120 43 42 41 25 18 24 20 25
26 177 126 52 46 47 45 40 36 41 40 39 45
29 143 115 54 37 28 33 24 15 27 16 24 25
31 150 162 51 57 30 38 32 40 37 33 35 34
37 119 100 54 51 35 46 56 32 34 28 31 30
38 134 105 86 77 71 79 100 39 31 37 24 29
BEI Resources
2 121 70 47 49 30 37 42 33 28 25 18 27
7 82 54 27 40 22 30 21 28 19 20
14 119 59 31 58 22 26 17 16 19 10
19 107 62 26 24 30 26 19 23 20 10
36 153 67 59 48 55 71 48 29 29 26 29 26
Paediatric
5 163 84 66 66 85 90 89 48 44 68 60 60
8 147 117 54 37 51 68 48 33 30 28 28 33
23 161 119 69 55 78 88 80 41 29 52 39 45
33 155 159 43 56 21 31 43 30 31 23 20 20
Vaccinee 4 123 65 58 57 46 61 61 25 24 22 29 34
6 181 166 74 74 48 60 60 35 35 32 37 41
12 150 146 54 61 43 50 47 35 42 33 28 31
15 109 100 41 59 12 25 33 26 32 15 23 26
17 134 95 66 71 63 71 68 59 56 73 69 63
20 125 110 64 56 44 49 54 38 36 36 30 34
21 115 55 73 81 30 42 43 72 70 22 32 30
25 139 89 74 70 65 73 71 35 40 35 39 42
28 186 124 71 54 78 80 73 37 36 26 26 35
30 143 122 44 44 37 42 38 22 30 27 28 31
34 162 173 85 89 81 99 98 32 35 37 35 42
35 140 136 50 54 38 51 53 28 32 31 26 23
Animal 13 213 156 124 106 153 133 151 83 63 121 95 98
27 231 162 114 81 166 140 145 52 44 74 63 71
mAb 10 171 151 90 95 80 68 87 80 95 70 68 60
22 136 113 75 81 60 56 70 61 80 50 59 50
Shading indicates level of inter-laboratory variability: darker red = increased variability
Blue shading: animal and mAb samples are shaded in blue as they behave differently to human serum samples Grey shading: empty cell
Table 8. Median inter-laboratory GCV values
Potencies relative to different reference standards
Set A (All Data) Set B (With Exclusions)
ED50 (All Data)
ED50 (With Exclusions)
16/284 16/322 007 014 019 16/284 16/322 007 014 019
All 143 114 54 56 44 50 48 33 32 28 29 31
Human 143 114 48 43 33 40 32 22 31 21 28 30
BEI Resources 120 65 39 49 30 37 42 25 28 25 19 26
Paediatric 158 118 60 55 65 78 64 37 30 40 34 39
Vaccinee 139 116 65 60 45 56 57 35 35 32 29 34
Animal 222 159 119 94 160 137 148 67 53 97 79 84
mAb 154 132 83 88 70 62 79 71 87 60 63 55
Shading indicates level of inter-laboratory variability: darker red = increased variability
Blue shading: animal and mAb samples are shaded in blue as they behave differently to human sera samples Grey shading: empty cell
Table 9. Thermal degradation assessment of 16/284; anti-RSV neutralization titres after 6 months storage
Storage
Temperature (°C) 1 2 GM % of -20°C
4 1603 1750 1675 112
20 1263 1439 1348 91
37 1069 865 962 65
45 727 710 718 49
56 81 76 78 5
Table 10. Thermal degradation assessment of 16/284; estimated percentage loss per month and year
Storage Temperature (°C) % loss per month % loss per year
-20 0.001 0.009
4 0.047 0.561
20 0.516 6.016
37 4.923 45.435
Table 11a. Thermal degradation assessment of reconstituted 16/284; percentage relative to 16/284 stored at -20°C and reconstituted on day of assay
Storage Temperature (°C) 1 week 2 weeks 4 weeks
4 166 76
22 105 123
37 93 84
Table 11b. Thermal degradation assessment of reconstituted 16/322; percentage relative to 16/322 stored at -20°C and reconstituted on day of assay
Storage Temperature (°C) 1 week 2 weeks 4 weeks
4 88 89
22 89 113
37 59 54
Table 12. Proposed IU/mL values for BEI Resources Materials
Sample Name IU/mL
NR-4020: Human Reference Antiserum to RSV 1236 NR-4021: Human Antiserum to RSV, High Control 3654 NR-4022: Human Antiserum to RSV, Medium Control 843 NR-4023: Human Antiserum to RSV, Low Control 756 NR-21973: Human Reference Immune Globulin to RSV 7346
