Preparation of SDTreated MiniPool Blood Products

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Preparation of SD-Treated Mini-Pool Blood
Products

• Magdy El Ekiaby, MD
• Shabrawishi Hospital BTC HTC
• Cairo, Egypt

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Definition

• Preparation of virally inactivated, SD-treated
• cryoprecipitate,
• plasma,
• and cryo-poor plasma
• by blood establishments

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Objectives

• Easy-to-use, cost/effective, reliable, and
  affordable technology
• Implementation by blood establishment with
  minimum infrastructure investment
• It should fit blood establishments that can
  prepare and test blood components following good
  practices

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Project elements

• GMP production of plasma and its components in
  blood establishments
• Development and adaptation of SD virus
  inactivation procedures into blood establishments
• Introducing principles of quality assurance and
  quality control to ensure effective uniform final
  product

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SD mini-pool virus inactivation technology

• Solvent-Detergent virus inactivation a gold
  standard and a robust technology
• Long clinical experience with SD-treated products
• Inactivation of lipid enveloped viruses (most
  pathogenic TTV)
• Less risk for transmission of non-enveloped
  viruses due to the mini-pool size

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SD mini-pool current options developed

• Viral inactivation step-
• 2 TnBP, 37C
• 1 TnBP 1 Triton X45, 31C
• Solvent-Detergent removal-
• 3 oil extractions with Castor/Soya Bean Oil
• /- SDR/C18 chromatography adsorption step
• Performed in a closed sterile disposable
  processing system with equipment that is
  routinely used in blood establishments

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Equipments

• Laminar air flow cabinet for aseptic environment
• Syringe pump for SD addition under controlled
  flow-rate, volume, and duration
• Shaker for controlled and reproducible mixing of
  SD agents
• Shaker incubator -
• Control mixing of SD plasma/cryoprecipitate
  under optimal temperature
• Control mixing of oil and plasma/cryoprecipitate
  for SD removal under optimal temperature

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Reagents

• TnBP
• Triton X45
• Soya Bean oil
• /- C18/SDR

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The Disposable

• Interconnected patented closed sterile bag system
  formed of-
• 2 bags for mixing SD with plasma, cryo-poor
  plasma or cryoprecipitate and perform virus
  inactivation
• 3 oil extraction bags for removal of solvent and
  detergent
• Inlets to the bags for addition of SD and oil
• The inlets are protected with caps and clamps
• Double clamps on the connecting lines between
  bags to segregate the different processing steps
  and control the transfer of the plasma component
  from one bag to the next

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Virus inactivation bags
1st stage

• Viral inactivation performed
• - in two stages
• And using oval bags to avoid dead-ends and poor
  mixing
• FOR OPTIMAL GUARANTEE OF VIRUS KILL

2nd stage
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SD Removal bags

• Funnel Shape bag where plasma component is mixed
  with Soya Bean oil to remove SD
• There is an inlet line for addition of the oil
• The funnel shape design of the bag allows for
  accurate separation of the plasma from the oil
• The process may be repeated for 2-3 times for
  removal of TnBP Triton after virus inactivation
  step.

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One SD technique for three plasma components
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SD-Cryoprecipitate
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VWF in SD-treated cryoprecipitate
Collaborative study with Professor Jenny
Goudemand and Dr. Claudine Caron,
CHRU, Lille, France
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No detrimental impact of SD on VWF in cryo

• No significant changes in
• VWFAg
• VWFRCo
• VWF CB
• VWF multimers including the highest MW gt 15 mers

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Residual TnBP and Triton X-45

• TnBP lt 10 ppm
• Triton X-45 lt 100 ppm

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Viral validation study of SD in the disposable
system

• Performed at Texcell/Pasteur Institute
• Spiking studies using-
• HIV1, relevant virus
• Pseudo Rabies Virus (PRV) as a model of Herpes
  Virus
• Bovine Viral Diarrhea Virus (BVDV) as a
  recognized model for HCV
• gt4 log reduction of the 3 viruses in two minutes

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Technical Steps of Mini-Pool SD Virus
Inactivation Cryoprecipitate
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Cryoprecipitate preparation

• The usual blood bank techniques
• At the final step of separation of the
  cryoprecipitate from cryo-supernatant plasma, we
  almost completely remove all the plasma (almost
  dry cryoprecipitate)
• Re-suspend the cryo in 8 ml of 5 glucose saline
  (total volume 10 ml)
• Thaw the prepared cryoprecipitate units at 31C
  and pool 40 in one disposable ( 3200 4000 IU
  FVIII in 400 ml volume)

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• Aseptic Pooling of 40 plasma depleted
  cryoprecipitate units (thawing pooling) 2
  hours
• SD treatment SD extraction 2 hours each
• Aseptic dispensing into final bag
• QC Factor VIIIc, sterility testing
• Labeling

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SD-plasma depleted cryoprecipitate
characteristics (1)

• Suspended in 5 glucose saline
• Mean 8-10 IU FVIII/ml-
• Volume of 200 IU FVIII will be 20-25 ml.
• The product will be stored frozen at lt-20C or
  colder for one year from date of preparation
• Mean clottable fibrinogen 15-20 mg/ml

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Plasma depleted SD-cryoprecipitate
characteristics (2)

• VWF data
• RCo 6 8.88 IU/ml
• CBA 5 10.6 U/ml
• Ag 9.24 11 IU/ml
• Multimers
• gt15 mers as in starting cryo
• gt10 mers as in starting cryo
• ABO iso-agglutinins
• Anti-A titer 0-1
• Anti-B titer 0 1/8
• Jenny Goudemand, Claudine Caron, CHRU, Lille,
  France

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Clinical Use

• The product can be used in the treatment of the
  following conditions-
• Hemophilia A
• vWD
• Fibrinogen deficiency
• Severe post partum hemorrhage
• DIC
• FXIII deficiency ???

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Future developments

• Mini-pool preparation of the following solvent
  detergent treated plasma products prepared from
  plasma-
• PCC
• FIX
• FVII
• FXI
• FV

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Collaborators

• Thierry Burnouf
• Miryana Radosevich
• Hadi Goubran

• Makram El Sayed
• George Gorgy
• Nermin Nabil
• Khaled Fathy
• Ahmed Abdalla