Plasma standards and protein yield

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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

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Available standards
Plasma for manufacture
Plasma for transfusion
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Available standards
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• FVIII in 2005
• Plasma-derived FVIII production is becoming
  increasingly marginal in the developed blood
  economies
• Fractionators still ship plasma for FVIII
  manufacture in the hope of supplying the
  emerging markets
• Factor VIII is the most labile plasma therapeutic
  protein
• Conditions affecting FVIII may affect other
  proteins in ways which are still unknown
• Tailoring conditions to optimising FVIII
  preservation is therefore still a valid goal

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

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European plasma standardsFVIII levels

• 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.

• Council of Europe
• (for transfusion)
• Requirement for gt 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

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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

Blood/plasma centre
Fractionator
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Over et al (1990) Dev Hem Imm 2411-24
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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

Blood/plasma centre
Fractionator
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Source vs recovered plasma FVIII yield in low
purity concentrates
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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

Blood/plasma centre
Fractionator
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Factor VIII content Freezing rates and pack
typesSmith et al (1985) Dev Hem Imm 13 15-23
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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?

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Distribution of FVIII in manufacture to LP
concentratesEffect of overnight storage
Significant
Significant
Not significant
LP
Hughes et al 1989
Hellings et al (1982)
. plasma intended for the recovery of proteins
that are labile in plasma is frozen by cooling
rapidly at 30 C or below as soon as possible
and at the latest within 24 h of collection. EP
Monograph
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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

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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

Blood/plasma centre
Fractionator
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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

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Plasma freezing time to -25oC with different
equipmentCarlebjork et al (1986) Transfusion
26159-162
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Plasma freezingWhat 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 pros and cons
• The effect of freezing rates on FVIII yields in
  current concentrates is not well recorded

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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

Blood/plasma centre
Fractionator
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Plasma QualityEffect of poor storage conditions
Plasma temperature cycling

• .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

Cryoprecipitate quality
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Foster et al (1985) Dev Hem Imm 13 15-23
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Plasma conditioningEffect on FVIII concentrate
CRYO
CRYO ELUATE
ELUATE
FVIII FIBRINOGEN
Farrugia et al (1992) Transfusion 32755-759
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Plasma StorageWhat 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

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Eutectic point of plasma?
Phase transitions in frozen plasma
Resistivity of normal plasma saline measurements
on slow thawing after fast freezing McIntosh 1990
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Freezing of 700 ml plasmaEnergy consumption at
different stages of freezing Carlebjork et al
(1986) Transfusion 26159-162
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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)?

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FVIII and activation of coagulation in plasma
freezingSplit 300 ml pairs from 600 ml source
plasma units, n12
NB - FAST FREEZING RESULTS IN HIGHER ACTIVATION
Hellstern et al (2001) Trans 411601-1605
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Inhibitor outbreaks caused by manufacturing
problem?

• Octavi/Bisinact - high purity pdFVIII (SD and
  pasteurisation steps), marketed in Europe
  (1993-95) by OctaPharma, Bisinact in Belgium
• 12/109 PTPs developed inhibitors on Octavi SDPlus
• Anti-C2 specificity
• Cold-chain failure 40 kDa degradation product
  found in some batches manufactured from plasma
  from some countries but not all.

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FVIII from concentrates made from plasma pools
with evidence of coagulation activation
Saeneko et al 2001Thrombosis Research 101501-511
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And of course, there are other things one can get
out of plasma...
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Plasma QualityEffect 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

McIver 1980
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are these issues mainly of historical
interestor can other plasma proteins be
affected by poor storage conditions?..is
this part of the great unknown..and therefore
subject to regulatory precautionism?..
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How can plasma be assured to a high level of ...

• A defined manufacturing process
• Specified freezing and storage conditions
• Robustness to volume and temperature changes

• Reliability
• Consistency
• Ability to continue performance in stress or
  volume situations

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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