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Plasma standards and protein yield

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Plasma standards and protein yield

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    1. Plasma standards and protein yield Albert Farrugia Head, Blood & Tissues Unit Australian Therapeutic Goods Administration Fourth World Federation of Hemophilia Global Forum on the safety and supply of treatment products for bleeding disorders Montreal, Canada - September 26-27 2005

    2. Available standards

    3. Available standards

    4. The regulatory requirements underpinning blood and plasma storage, freezing and frozen storage are predicated on the needs of Factor VIII Is this justified?

    5. European plasma standards FVIII levels Council of Europe (for transfusion) Requirement for > 70% of the “average normal value” controlled through measurement of FVIIIc every two months on a pool of six units of mixed blood groups during the first and last months of storage European Pharmacopeia (for fractionation) On a pool of not fewer than ten units, measurement of factor VIII, using the EP reference method and a reference plasma calibrated against the International Standard for blood coagulation factor VIII in plasma. The activity is not less than 0.7 I.U. per millilitre.

    6. Factors claimed to affect FVIII yield in fractionated concentrates Anticoagulant Collection method Time/Temperature to separation/freezing Freezing rate Storage conditions of frozen plasma Thawing conditions Purification chemistry Viral inactivation

    8. Factors claimed to affect FVIII yield in fractionated concentrates Anticoagulant Collection method Time/Temperature to separation/freezing Freezing rate Storage conditions of frozen plasma Thawing conditions Purification chemistry Viral inactivation

    9. Source vs recovered plasma FVIII yield in low purity concentrates

    10. Factors claimed to affect FVIII yield in fractionated concentrates Anticoagulant Collection method Time/Temperature to separation/freezing Freezing rate Storage conditions of frozen plasma Thawing conditions Purification chemistry Viral inactivation

    11. Factor VIII content – Freezing rates and pack types Smith et al (1985) Dev Hem & Imm 13 :15-23

    12. DOES IT MATTER? There is no doubt that delayed blood processing to frozen plasma decreases FVIII levels in plasma for fractionation Does this affect the yields and quality of fractionated products?

    13. Distribution of FVIII in manufacture to LP concentrates Effect of overnight storage

    14. DOES IT MATTER? There is no doubt that delayed blood processing to frozen plasma decreases FVIII levels in plasma for fractionation Does this affect the yields and quality of fractionated products? IT DEPENDS Cryo yield affected LP & IP sometimes affected No data for current generation of FVIII concs

    15. Factors claimed to affect FVIII yield in fractionated concentrates Anticoagulant Collection method Time/Temperature to separation/freezing Freezing rate Storage conditions of frozen plasma Thawing conditions Purification chemistry Viral inactivation

    16. “Plasma should be frozen at……. …….-18OC, -20OC, -23OC,-30OC?” Remarkably ambiguous language in standards “cooling rapidly at -30OC, frozen at -20OC” (EP) “shall be stored at a temperature not warmer than -20OC” (CFR) Little recognition of the important - obvious - parameter THE FREEZING RATE

    17. Plasma freezing time to -25oC with different equipment Carlebjork et al (1986) Transfusion 26:159-162

    18. Plasma freezing What is important? Rapid freezing - ca -30oC in 30 minutes - results in better FVIII yields in cryo relative to slower freezing - - ca -30oC in 3-4 hours The ice crystal structure and the physical nature of cryoprecipitate are affected by the plasma freezing rate. Slower freezing also increases fibrinogen in cryo; this has its pro’s and con’s The effect of freezing rates on FVIII yields in current concentrates is not well recorded

    19. Factors claimed to affect FVIII yield in fractionated concentrates Anticoagulant Collection method Time/Temperature to separation/freezing Freezing rate Storage conditions of frozen plasma Thawing conditions Purification chemistry Viral inactivation

    20. Plasma Quality Effect of poor storage conditions “…….Source Plasma intended for manufacture into injectable products that is inadvertently exposed (i.e., an unforeseen occurrence in spite of compliance with good manufacturing practice) to a storage temperature warmer than -20 deg.C and colder than +10 deg.C may be issued only if labeled as ``Source Plasma Salvaged.'’” CFR 21- 640

    22. The alternative methods result in equivalent FVIII yields, but cold softening results in a product with significantly lower fibrinogen levels with the attendant benefits. Again, control of this process is crucial, and analysis of the material is of little use in assessing its suitability at this stage.The alternative methods result in equivalent FVIII yields, but cold softening results in a product with significantly lower fibrinogen levels with the attendant benefits. Again, control of this process is crucial, and analysis of the material is of little use in assessing its suitability at this stage.

    23. Plasma Storage What is important? As long as freezing is optimised, storage requirements appear to be flexible in the range -20oC to -40oC Maintaining a steady storage temperature is more important than the absolute storage temperature, within this range While temperature changes can affect the quality of cryoprecipitate, this can be exploited to improve both blood bank and industrial cryo

    24. Eutectic point of plasma?

    25. Freezing of 700 ml plasma Energy consumption at different stages of freezing Carlebjork et al (1986) Transfusion 26:159-162

    26. Plasma freezing and storage Conventional eutectics offer no guidance Freezing so that phase change is as rapid as possible Storage so that this is maintained - -20oC is adequate AND WHY SHOULD THIS BE AN ISSUE FOR REGLATORS ANYWAY? IS THERE ANY EVIDENCE THAT BLOOD/PLASMA PROCESSING AFFECTS SAFETY AND QUALITY (AS OPPOSED TO YIELD)?

    27. FVIII and activation of coagulation in plasma freezing Split 300 ml pairs from 600 ml source plasma units, n=12

    28. Inhibitor outbreaks caused by manufacturing problem?

    29. FVIII from concentrates made from plasma pools with evidence of coagulation activation

    30. And of course, there are other things one can get out of plasma………...

    31. Plasma Quality Effect on IMIG fragmentation during storage “……. When obtained from whole blood, plasma intended solely for the recovery of proteins that are not labile in plasma is separated from cellular elements and frozen at – 20 °C or below as soon as possible and at the latest within 72 h of collection..” EP Monograph

    32. …………are these issues mainly of historical interest……or can other plasma proteins be affected by poor storage conditions?……… …..is this part of the great unknown…..and therefore subject to regulatory precautionism?…..

    33. How can plasma be assured to a high level of ……...

    34. Tentative conclusions and possible approaches There is a need for clear and unambiguous standards for plasma freezing and storage A process which results in a consistent product, irrespective of scale and location, should form the basis of any standard Empirical observations appear to support greater flexibility than some current requirements There is little evidence that any of these requirements have a bearing on product safety Basic conditions for minimising microbial contamination and preserving product integrity should be defined Other requirements reflecting product yield eg FVIII levels should be left to be negotiated between the manufacturer and plasma supplier

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