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Replacement of the 1 st Parvovirus B19 DNA International Standard SoGAT XXI, Brussels, 28th-29th May 2009 Sally Baylis.

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Presentation on theme: "Replacement of the 1 st Parvovirus B19 DNA International Standard SoGAT XXI, Brussels, 28th-29th May 2009 Sally Baylis."— Presentation transcript:

1 Replacement of the 1 st Parvovirus B19 DNA International Standard SoGAT XXI, Brussels, 28th-29th May 2009 Sally Baylis

2 Replacement of the Parvovirus B19 DNA IS 99/800 The 1 st International Standard for B19V DNA was established by the WHO ECBS in October 2000 The 1 st International Standard for B19V DNA was established by the WHO ECBS in October 2000 Estimated date of exhaustion of the IS will be ~2009 at current rate of usage Estimated date of exhaustion of the IS will be ~2009 at current rate of usage Materials coded AA (99/800) & BB (99/802) showed no significant difference in potency in the collaborative study, i.e IU/ml Materials coded AA (99/800) & BB (99/802) showed no significant difference in potency in the collaborative study, i.e IU/ml BB (made from the same bulk as AA) could be reserved for potential future use as a replacement standard BB (made from the same bulk as AA) could be reserved for potential future use as a replacement standard

3 Collaborative Study to Establish 2 nd International Standard for B19V DNA Current study designed to demonstrate the equivalence of the candidate replacement (BB) to AA Current study designed to demonstrate the equivalence of the candidate replacement (BB) to AA Real-time data on samples AA & BB Real-time data on samples AA & BB Accelerated degradation data for samples AA & BB Accelerated degradation data for samples AA & BB 5 laboratories participated in study 5 laboratories participated in study The 1 st International Standard and candidate BB were recoded as samples 1 and 2 respectively The 1 st International Standard and candidate BB were recoded as samples 1 and 2 respectively Participants requested to test samples in four independent assays Participants requested to test samples in four independent assays 6 sets of data were received, all from quantitative real-time PCR assays (NS1, VP1; in-house and commercial) 6 sets of data were received, all from quantitative real-time PCR assays (NS1, VP1; in-house and commercial)

4 Estimated IU/ml log 10 for 99/800 & 99/802 Laboratory99/800 (Sample 1)99/802 (Sample 2) Overall mean

5 Analysis of Accelerated Thermal Degradation Studies of 99/800 & 99/802 SampleIncubation temperature Mean IU/ml log 10 95% CI 99/8004°C /80020°C /8024°C /80220°C Samples incubated at -20ºC, +4 ºC and +20 ºC for ~7.5 years Estimated IU/ml (log 10 )

6 Establishment of the 2 nd International Standard for B19V DNA Real-time & accelerated degradation data indicate 99/800 & 99/802 are very stable & suitable for long term use, with no degradation observed after ~7.5 years at +20°C Real-time & accelerated degradation data indicate 99/800 & 99/802 are very stable & suitable for long term use, with no degradation observed after ~7.5 years at +20°C No significant differences found in estimated IU/ml or PCR-detectable units/ml for 99/800 & 99/802 No significant differences found in estimated IU/ml or PCR-detectable units/ml for 99/800 & 99/802 At the WHO ECBS Meeting in October 2008, 99/802 was established as the 2 nd International Standard for B19V DNA with a unitage of 10 6 IU/ml At the WHO ECBS Meeting in October 2008, 99/802 was established as the 2 nd International Standard for B19V DNA with a unitage of 10 6 IU/ml

7 Investigation of B19V DNA of 99/802 Following Nuclease Digestion Is B19V DNA present in lyophilised preparation 99/802 susceptible to nuclease degradation? Is B19V DNA present in lyophilised preparation 99/802 susceptible to nuclease degradation? Investigation involved treatment of re-constituted preparations of 99/802 with Benzonase ® at 37ºC, for 1 hr in the presence of Mg 2+, followed by immediate extraction and analysis Investigation involved treatment of re-constituted preparations of 99/802 with Benzonase ® at 37ºC, for 1 hr in the presence of Mg 2+, followed by immediate extraction and analysis Benzonase ® Benzonase ® used to remove DNA impurities in biopharmaceuticals used to remove DNA impurities in biopharmaceuticals has a very high specific activity attacking and degrading all forms of DNA and RNA has a very high specific activity attacking and degrading all forms of DNA and RNA enzyme is effective under a wide range of operating conditions enzyme is effective under a wide range of operating conditions

8 Investigation of B19V DNA of 99/802 Following Nuclease Digestion Controls included preparations of 99/802 which were not treated with nuclease; B19V DNA negative plasma spiked with high titres of previously extracted B19V DNA (with and without nuclease treatment) Controls included preparations of 99/802 which were not treated with nuclease; B19V DNA negative plasma spiked with high titres of previously extracted B19V DNA (with and without nuclease treatment) All samples were analysed by real-time PCR for levels of B19V DNA All samples were analysed by real-time PCR for levels of B19V DNA

9 Nuclease Treatment of 99/802 +Nuc. -Nuc. 99/802 NTC Control DNA spiked in neg. plasma -Nuc. +Nuc.

10 Conclusions > 7 log 10 IU/ml of spiked/extracted B19V DNA was effectively removed from plasma by the nuclease treatment > 7 log 10 IU/ml of spiked/extracted B19V DNA was effectively removed from plasma by the nuclease treatment No loss of titre for 99/802 DNA was observed following to nuclease digestion, which suggests the viral DNA remains intact in the B19V capsid No loss of titre for 99/802 DNA was observed following to nuclease digestion, which suggests the viral DNA remains intact in the B19V capsid The accelerated thermal degradation samples further confirm the stability of the lyophilised B19V preparations The accelerated thermal degradation samples further confirm the stability of the lyophilised B19V preparations

11 B19V DNA Collaborative Study Participants Sally BaylisSouth Mimms, UK Sally BaylisSouth Mimms, UK Daniel CandottiCambridge, UK Daniel CandottiCambridge, UK Michael Chudy/Johannes BlümelLangen, Germany Michael Chudy/Johannes BlümelLangen, Germany Giulio PisaniRome, Italy Giulio PisaniRome, Italy Marta JoséBarcelona, Spain Marta JoséBarcelona, Spain


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