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N IBSC Investigation of the efficacy of novel pathogen removal/inactivation methods for plasma products using B19 as a model Cécile El Hana Department of Virology, NIBSC N IBSC B19 DNA quantification • LightCycler real-time PCR (Roche) • Standard curve established with the B19 international standard (NIBSC) N IBSC B19 infectivity assay •A clone of UT-7 cells: UT-7/EPO-S1, dependent on the presence of erythropoietin in the culture medium •2x105 cells were infected by 30ml of a virus dilution during the S phase •Cells were harvested 48 hours post-infection •mRNA was extracted and amplified by one step multiplex RT-PCR •One set of primers detect B19-specific transcripts and the other set detect a housekeeping gene: Actin N I BSC Inactivation by dry heat treatment •BioProducts Laboratory, UK •Factor VIII (8Y) from non-B19 screened plasma was spiked with high titre (1011 IU/ml) human parvovirus B19 •Spiked freeze-dried product was treated for 24 and 72 hours at 80ºC N I BSC Inactivation bybydry heat treatment Inactivation dry heat treatment •Bioproducts Laboratory, UK •Although quantification showed that the •Factor VIII B19 (8Y)DNA was spiked with high titre human unspiked factor parvovirus B19 VIII contained 104 IU/ml, no infectious particles could be detected thetreated infectivity •Spiked freeze-dried productby was for 24assay and 72 hours at 80ºC • •There was a 2.5 log10 reduction in the number of B19 infectious units after 24 hours at 80ºC, compared to the untreated control •After 72 hours of treatment, no infectious units could be detected by the infectivity assay N IBSC Inactivation bybydry heat critical treatmentfluids Inactivation super •Bioproducts Laboratory, UK • Aphios Corporation, USA •Factor VIII (8Y) was spiked with high titre human parvovirus B19 •SuperFluids™ are normally gases which, when •Spiked freeze-dried product was treated for 24 and 72 compressed, show enhanced penetration and expansion hours at 80ºC properties. • • SuperFluids™ penetrate the virus particles and inflate them until the decompression causes their expansion. The overfilled particles rupture at their weakest point. N IBSC Inactivation bybydry heat critical treatmentfluids Inactivation super •Bioproducts Laboratory, UK •Serum was spiked with high titre human parvovirus •Factor VIII (8Y) was spiked with high titre human B19 parvovirus B19 •Three supercritical fluids were tested: Freon-22, Freon •Spiked freeze-dried -23 and N2O/CO2 product was treated for 24 and 72 hours at 80ºC • Pressure was 206 bars and temperature was either 25 • or 50°C •Although the DNA titre was unchanged by the treatment (1010 IU/ml), treatment with N2O/CO2 at 206 bars and 50°C resulted in more than 5 log10 reduction of the number of infectious units (undetectable by our infectivity assay). Inactivation heat treatment byby thedry INACTINE™ system N IBSC Inactivation •Bioproducts Laboratory, UK • VI technologies or Vitex, USA •Factor VIII (8Y) was spiked with high titre human parvovirus B19 • INACTINE™ system includes small molecules that, due •Spiked freeze-dried product was treated for 24 and 72 to their size and stability in blood, are able to penetrate hours at 80ºC through the protective walls of resistant pathogens. • • These molecules are triggered only when they bind with their target, the DNA or RNA of the pathogen. The loss of DNA or RNA replication is a fatal event for viruses or bacteria whereas the red blood cells do not need DNA or RNA to function. Inactivation heat treatment byby thedry INACTINE™ system N IBSC Inactivation •Bioproducts Laboratory,inUK • Red cells resuspended medium AS1 were spiked with •Factor VIII (8Y) parvovirus was spikedB19 with high titre human high titre human parvovirus B19 •Spiked freeze-dried treated forhours 24 and • Samples were takenproduct after 3,was 6, 18 and 22 of 72 hours at 80ºC treatment • • B19 DNA titre decreased with time (7 log10 reduction) • The number of infectious units in the treated sample was reduced from 105 to undetectable level after only 3 hours of INACTINEä treatment B19 inactivation by INACTINE 350000 300000 "infectious units"/per 250000 200000 150000 100000 50000 0 0 5 10 15 Time (hours) 20 25 N IBSC Inactivationby bythe dryHelinxâ heat treatment Inactivation technology •Bioproducts Laboratory, UK • Cerus Corporation •Factor VIII (8Y) was spiked with high titre human • Helinx® B19 compounds are able to cross cell walls or viral parvovirus membranes and bind to and crosslink nucleic acids (DNA •Spiked freeze-dried product was treated for 24 and 72 and RNA), thereby preventing their replication hours at 80ºC • The psoralen compound S-59 requires UV light for • activity •Although the reduction in B19 DNA titre was only 1 log10, the number of infectious units per ml was reduced by 4.5 log10. N IBSC Removalby bydry nanofiltration Inactivation heat treatment •Bioproducts UK • Asahi KaseiLaboratory, Pharma, Japan •Factor VIII (8Y)solution was spiked with high titretitre human • 0.5% albumin spiked with high human parvovirus parvovirus B19 B19 •Spiked freeze-dried product was treated for 24 andPCR 72 • B19 DNA was titrated by real-time LightCycler hours at 80ºC • Prefiltration through 35N PlanovaÒ filter removed 1 •log of human parvovirus B19 10 • Then filtration through 15N or 20N PlanovaÒ filters removed up to 6 log10 reduction in the B19 DNA titre • Results correlated with study on haemoglobin solutions by Abe et al., 2000. Artif Cells Blood Substit Immobil Biotechnol 28(5): 375-383.