© World Health Organization WHO Technical Report, Series No. 924, 2004 Annex 4 Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products List of abbreviations and definitions used in this Annex 151 1. Introduction and scope 154 2. General considerations 155 3. Infectious agents 157 3.1 Viruses, viral burden and screening methods 157 3.2 Other infectious agents 161 3.3 Validation of viral inactivation and removal procedures 161 4. Review of well-recognized methods for viral inactivation and removal 166 4.1 Methods of inactivation 167 4.2 Methods of virus removal 179 4.3 Protein issues 186 4.4 Clinical trials to assess safety 193 4.5 Implementation in a manufacturing setting 193 5. Virally inactivated plasma for transfusion 198 5.1 Quarantine or donor-retested plasma 199 5.2 Solvent/detergent-treated plasma 200 5.3 Methylene blue and visible light 201 6. Review of newer viral inactivation methods under development 203 6.1 Psoralen-treated fresh frozen plasma 203 6.2 UVC light irradiation 204 6.3 Gamma-irradiation 205 6.4 Iodine 208 6.5 Pasteurized fresh frozen plasma 208 7. Summary 209 8. Authors 211 Acknowledgements 212 WHO Secretariat 213 References 213 Appendix Example of a study on the inactivation of human immuodeficiency virus-1 by treating a therapeutic plasma protein preparation with tri(n-butyl)phosphate and Tween 80 219 G 150 ECB text 150 19/11/2004, 09:49 AM Black List of abbreviations and definitions used in this Annex The definitions given below apply to the terms used in these guidelines. They may have different meanings in other contexts. AHF Antihaemophilic factor. Blood coagulation factor VIII, missing in patients with classic haemophilia. Blood components These typically refer to red blood cell concentrates, platelet concentrates and plasma. BEV Bovine enterovirus. A non-enveloped, single- stranded RNA virus used as a model for hepatitis A virus. BVDV Bovine viral diarrhoea virus. An enveloped, single-stranded RNA virus used as a model for hepatitis C virus. CMV Cytomegalovirus. An enveloped, double- stranded DNA virus, typically cell-associated. Coxsackie virus A non-enveloped, single-stranded RNA virus. CPV Canine parvovirus. A non-enveloped, single- stranded DNA virus. Donor retested plasma A process for reducing window period transmissions whereby fresh frozen plasma is held in the inventory for a designated period of time until the donor returns and tests negative for virus exposure. The initial unit is then released for use. Also called quarantine plasma. Dry heat A process of heating protein following lyophilization, typically at 80 °C or higher. EBV Epstein–Barr virus. An enveloped, double- stranded DNA virus, typically cell-associated. EMCV Encephalomyocarditis virus. A non-enveloped, single-stranded, RNA virus. Factor IX Blood coagulation factor IX, missing in patients with haemophilia B. Factor VIII Blood coagulation factor VIII, missing in patients with haemophilia A. Also called antihaemophilic factor. FFP Fresh frozen plasma. Fluence The total quantity of light delivered. Expressed in J/cm2. Gamma-irradiation A process of virus inactivation or bacterial sterilization using gamma-irradiation of liquid, frozen or lyophilized product. G 151 ECB text 151 19/11/2004, 09:49 AM Black GE Genome equivalents. The amount of nucleic acid of a particular virus assessed using nucleic acid testing. GMPs Good manufacturing practices. Sometimes referred to as current good manufacturing practices. HAV Hepatitis A virus. A non-enveloped, single- stranded RNA virus. HBsAg Hepatitis B surface antigen. The antigen on the periphery of hepatitis B virus. HBV Hepatitis B virus. An enveloped, double- stranded DNA virus. HCV Hepatitis C virus. An enveloped, single- stranded, RNA virus. HDV Hepatitis delta virus. A defective virus which requires co-infection by hepatitis B virus. High purity factor VIII Factor VIII concentrate with a specific activity typically greater than 100 IU/mg. HIV Human immunodeficiency virus. An enveloped, single-stranded RNA virus. HSV Herpes simplex virus. An enveloped, double- stranded DNA virus, typically cell-associated. HTLV 1 and 2 Human T-cell lymphotropic virus, types 1 and 2. Enveloped, single-stranded RNA viruses, typically cell-associated. ID50 The quantity of virus or other infectious agent that will infect 50% of subjects or tissue cultures. Frequently expressed on a log scale; thus, 6 log10 ID50 represents 1 million infectious units. Immunogenic Causing the formation of antibody. Harsh processing conditions may modify the structure of a protein so as to make it immunogenic. Intermediate purity factor VIII Factor VIII concentrate with a specific activity between 1 and 50 IU/mg. IVIG Intravenous immunoglobulin. Limiting dilution A way of determining titre by diluting the sample continually until the positive signal is lost. LRF Log reduction factor. The quantity of virus, expressed on a log 10 scale, inactivated or G removed. 152 ECB text 152 19/11/2004, 09:49 AM Black MB-plasma Methylene blue-treated plasma intended as a substitute for fresh frozen plasma. Nanofilters Filters that usually have effective pore sizes of 50 nm or less, designed to remove viruses from protein solutions. NAT Nucleic acid testing, using amplification techniques such as polymerase chain reaction. Pasteurization A process of heating protein in solution, typically at 60 °C. Polio virus A non-enveloped, single-stranded, RNA virus. PPRV Porcine pseudorabies virus. An enveloped, double-stranded DNA virus. PPV Porcine parvovirus. A non-enveloped, single- stranded DNA virus. Prion The infectious particle associated with transmissible spongiform encephalopathies. It is believed to consist only of protein and to contain no nucleic acid. PRV Pseudorabies virus. An enveloped, double- stranded DNA virus. Psoralen A furocoumarin ring structure, which when exposed to light, cross-links nucleic acid. Quarantine plasma See donor retested plasma. RT3 Reovirus type 3. A non-enveloped, double- stranded RNA virus. Rutin A flavonoid used as an antioxidant that reduces the action of reactive oxygen species. Solvent/detergent treatment A process of treating protein in solution, usually with the organic solvent, tri(n- butyl)phosphate, and a detergent such as Tween 80 or Triton X-100. SD-Plasma Solvent/detergent-treated plasma intended as a substitute for FFP. Sindbis virus An enveloped, single-stranded RNA virus. SLFV Semliki forest virus. An enveloped, single- stranded, RNA virus. Titre The quantity of virus, typically expressed on a log10 scale. Six logs of virus are equal to 1 million infectious units. TNBP Tri(n-butyl)phosphate. The organic solvent used with solvent/detergent treatment. Triton X-100 A non-ionic detergent frequently used as part of solvent/detergent treatment. G 153 ECB text 153 19/11/2004, 09:49 AM Black Tween 80 A non-ionic detergent frequently used as part of solvent/detergent treatment. UVC Ultraviolet irradiation, usually at a wavelength of 254 nm. Vaccinia virus An enveloped, double-stranded DNA virus. Vapour heating A process of heating protein following lyophilization and then reintroducing moisture normally at 60 °C and in some cases at 80 °C. Viral inactivation A process of enhancing viral safety in which virus is intentionally “killed”. Viral removal A process of enhancing viral safety by removing or separating the virus from the protein(s) of interest. VSV Vesicular stomatitis virus. An enveloped, single-stranded RNA virus. West Nile virus An enveloped, single-stranded RNA virus. 1. Introduction and scope Human blood is the source of a wide range of medicinal products used for the prevention and treatment of a variety of often life-threatening injuries and diseases. Despite measures such as donor selection, test- ing of donations and of plasma pools, the transmission of blood-borne viruses by plasma and purified plasma products is still considered to constitute a risk to patients. Over the past 15–20 years, the transmis- sion of the principal viral threats historically associated with these products — hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) — has been greatly reduced or eliminated in many areas of the world. This is a consequence of the more sensitive methods being used to screen donated blood and plasma pools, and of the establishment of manufacturing practices that lead to significant virus inactivation and removal. Several proce- dures for virus inactivation and removal have proven to be robust and to contribute substantially to blood product safety. Viral inactiva- tion methods should be applied to all blood plasma-derived protein solutions. Continuing concerns about the quality and safety of plasma-derived medicinal products have resulted in a number of urgent requests from Member States for support and advice from WHO. Moreover, the World Health Assembly Resolution No 50.20, of 13 May 1997 on the “Quality of biological products moving in international commerce”, G requested WHO to extend the assistance offered to Member States to 154 ECB text 154 19/11/2004, 09:49 AM Black develop and to strengthen their national regulatory authorities and control laboratories to increase competence in the area, and to extend efforts to upgrade the quality and safety of all biological products worldwide. The present WHO Guidelines on viral inactivation and removal pro- cedures intended to assure the viral safety of human blood plasma products were developed to complement the WHO Requirements for the collection, processing and quality control of blood, blood compo- nents and plasma derivatives”(1), in response to the above requests. These Guidelines pertain to the validation and assessment of the steps for viral inactivation and removal employed in the manufacture of human blood plasma derivatives and virally inactivated plasma for transfusion, prepared either from plasma pools or from individual donations.. It is hoped that this document, by summarizing current experience with well recognized methods, will help set expectations, serve as a guide to speed implementation, and ensure that implemen- tation is appropriate.