The scope of this chapter is limited to the virus testing performed on human plasma for the further manufacture of pharmaceuticals, which are referred to as plasma-derived products (see Virology Test Methods 1237 for virus testing of other therapeutic products). These types of plasma include either source plasma collected by apheresis or recovered plasma obtained from whole blood collection or as a byproduct in the production of blood components. In all cases, the source material is obtained through voluntary donations. The following topics are specifically excluded from the scope of this chapter:

This chapter introduces the virus testing that is performed on plasma used for the production of therapeutic proteins. Topics that are addressed include:

The chapter also includes an Appendix that contains pertinent regulatory guidances and supporting references.

Human-plasma-derived products are used to treat coagulation disorders, primary immune deficiency, and congenital emphysema as well as other diseases (see Human Plasma 1180). Because these products are manufactured from pooled human plasma donations, the presence of blood-borne viral pathogens from individual plasma donations can potentially contaminate the resulting final products manufactured from a large pool of donations and thus transmit the virus to many recipients. Sufficient measures must be taken to ensure that these products are as safe as possible. In order to minimize the risk of transmission of viruses by these products, manufacturers use several strategies, which include:

Plasma used for further manufacture can be either source plasma or recovered plasma. In the United States, licensed human plasma products are derived mainly from source plasma. Because plasma for further manufacture is obtained by pooling a large number of donations, there is a risk of viral contamination of the pool, thus resulting in a much higher potential risk of virus transmission to multiple recipients than is the case for blood for transfusion. The manufacturing process, which is used to purify and concentrate the desired protein, is not capable of completely removing the viral load, and therefore validated virus-reduction steps capable of effectively reducing transfusion-transmissible viruses in the starting material are included in the manufacturing process. A detailed discussion of virus inactivation and removal procedures for pathogen reduction can be found in the 2004 WHO Technical Report Series 924 cited in the Appendix.

Approaches for screening plasma for further manufacture can be categorized into two groups: donor-screening and in-process testing methods. The donor-screening method takes into account not only the plasma-derived end product but also the plasma donor. This category of testing typically is required for blood-transmissible viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV). Virus transmission is a major public safety concern, because infections with these highly pathogenic viruses typically progress to chronicity. During donor screening the objectives of the test laboratory or manufacturer are not only to identify positive units for destruction before production pooling but also to identify and notify infected donors. Donors who test positive for HCV or HIV are permanently deferred from donating both blood and plasma. Although fewer than 5% of HBV-infected adults develop persistent asymptomatic infection (i.e., a carrier state), HBV-positive donors are deferred permanently. Collection of source plasma from donors who are convalescing from HBV is sometimes permitted for further manufacturing into plasma-derived products such as Hepatitis B Immune Globulin (Human) [21 CFR 610.41(3)].

In contrast to viruses that are associated with donor screening, viruses such as hepatitis A virus (HAV) and parvovirus B19 (B19V) usually cause self-limiting infections in immunocompetent individuals, and thus manufacturers use in-process nucleic acid amplification technology (NAT) testing that results in only the removal of plasma units with high levels of virus before pooling for production. In these cases, there is no donor-management procedure and hence no requirement to inform the donor of the result. This approach focuses primarily on the product, not the donor, because some recipients of these products are susceptible to an infection that may occur if the pathogens were present in the plasma-derived product. Donor and donation-management procedures are well developed in the blood and plasma industry, and more details on these specific topics can be found in 1180. Other permanent or temporary donor-deferral criteria are in place to avoid donations from potentially infected donors based on the epidemiological surveillance of a country or region or donor population for transfusion-transmissible infections relevant to the safety of blood components.

Viruses that greatly affect public health, such as HIV, HBV, and HCV, are detected by serological assays that measure either a viral antigen, such as hepatitis B surface antigen (HBsAg), or an antibody, such as anti-HCV or anti-HIV-1/2 antibodies, in infected donors and associated donations. These immunoassays for detecting viral markers in plasma donations must be sensitive and able to detect a viral infection as early as possible following infection in order to identify and exclude potentially infectious donations.

There is a finite time period, known as the window period, between the infection of a donor and the time at which the test method can detect the antibody response to the virus, the viral antigens, or the viral nucleic acid. This window period varies from disease to disease as well as from person to person. The window period can be effectively “shortened” by changing from a test based on detecting antibodies to one based on detecting the virus directly, namely the viral antigen or, especially, the viral nucleic acid (see Figure 1), thereby interdicting donations that contain transfusion-transmissible viruses. Tests for detecting the viral nucleic acids (i.e., NAT tests) were introduced in the 1990s. NAT tests are sensitive and can considerably shorten the window period (see Figure 1). In a later development after B19V transmission incidents involving plasma-derived products, NAT tests were initiated to interdict high-titer donations, thereby decreasing the B19V virus load in manufacturing pools.

Both source plasma and recovered plasma are tested by serological and NAT tests that are approved by competent regulatory authorities or, in the case of in-process testing, are validated to manufacturers' requirements. Plasma units found acceptable by these tests are combined into large pools, called fractionation pools, for manufacturing of plasma-derived products. The fractionation pool size can vary from several hundred donations (typically used for the production of specific immunoglobulins) to several thousand donations (used, for example, for the manufacture of albumin). Finally, the fractionation pools are retested for the target viruses. Testing of the plasma donations, at the individual or minipool level, and the fractionation pools are two of the elements that manufacturers put into place to maintain the safety margins of these products. Serology testing is performed on the individual donations. In contrast to serology tests, current NAT tests are highly sensitive and specific; therefore, manufacturers, in addition to testing individual donations, test minipools made up of equal volumes of each donation. Currently, the minipool size used for NAT testing varies from 6 to 512 donations. The high sensitivity of NAT tests also allows earlier virus detection compared to an antigen- or antibody-based test, thereby reducing the average length of the window period.

Plasma-derived products are produced from tested fractionation pools and are further manufactured by using a combination of fractionation and purification steps. These steps may have some inherent potential to remove or inactivate viruses and thus reduce viral contaminants that may have been present in the starting plasma. Nevertheless, manufacturing of plasma-derived products also includes dedicated steps designed solely to inactivate (e.g., by solvent-detergent treatment) or remove (e.g., by virus filtration) potential viral contaminants.

Historically, virological test methods have been used for detecting viral antigen or antibodies in clinical settings for disease diagnosis, intervention, and containment. Subsequently, these methods were adapted to screen blood and plasma donations with high sensitivity and specificity for transfusion-transmissible viruses. In order to develop a new virus screening test, scientists must know the biochemical properties of a new emerging pathogen (e.g., the nucleic acid sequence) for the development of an NAT test or the protein (e.g., antigen) for immunological tests. When implementing such a test for a given pathogen, the public health implications of positive test results and the potential for early intervention and treatment of the disease have to be considered. In addition, the availability of plasma for further manufacture and the effects of the virus on the safety of the finished product should be taken into consideration.

The emergence of a viral pathogen in the donor population could result in a considerable virus load in the plasma donations and in the resulting fractionation pools. For viruses such as HBV, HCV, HIV-1, and HIV-2 that can cause chronic diseases with potential public health effects, all donations positive for one or more of these viruses, irrespective of the virus titer, must be removed, and the donor must be informed. Some viruses such as B19V are prevalent in the population (as many as 1 in 5000 individuals may be infected during an epidemic period) and can be present at high virus titers in infected individuals. Thus, the removal of all B19V-positive donations could lead to a shortage of plasma. Instead, in-process NAT testing is done to interdict high-titer donations and thereby limit the B19V load in the manufacturing pool. The rationale for such screening is that B19V causes a self-limiting infection in most immunocompetent individuals. Following recovery, such individuals have neutralizing antibodies to B19V. Seroconversion occurs early in life, because B19V infection is common in childhood, and approximately 50% of 15-year-old adolescents have B19V antibodies. Infection of susceptible individuals continues throughout adult life, and B19V seroprevalence increases with age. The B19V neutralizing antibodies present in a plasma pool and the validated virus reduction steps included in the manufacturing process ensure that the inclusion of such donations does not compromise either the safety of the plasma-derived products or the availability of plasma for further manufacture.

In some instances it may not be necessary or feasible to test for a blood-borne virus. For example, testing of plasma for cell-associated viruses such as Human T Lymphotropic Virus (HTLV) types I and II, which present with no or with only limited virus load in plasma (but with a considerable virus load in whole blood donations) is not required. Similarly, testing for West Nile Virus (WNV), a member of the Flaviviridae family, is unnecessary because the virus load is low during the asymptomatic window period, the prevalence in the donor population is low (resulting in a low virus load in a plasma pool for fractionation), and WNV can be effectively inactivated by the manufacturing process as demonstrated by validation studies using relevant Flaviviridae model viruses. Therefore, WNV NAT testing for plasma (source and recovered) for further manufacture is not required. However, in the United States, the Food and Drug Administration (FDA) recommends WNV NAT testing for blood and blood components for transfusion because of the epidemiological situation and the risk of WNV transmission by blood components (see the FDA Guidance for Industry cited in the Appendix).

Other pathogenic viruses such as influenza viruses and severe acute respiratory syndrome coronavirus (SARS-CoV) are associated with clinical disease after a short incubation period and have a low or no virus load during the asymptomatic window period. No transmission by blood transfusion or plasma-derived products has been reported for these viruses. Furthermore, the manufacturing process for plasma-derived products has been shown to effectively inactivate influenza viruses. Therefore, NAT testing of plasma is not required for these viruses.

For a virus with a high prevalence in the donor population but without known clinical implications, no screening program, neither NAT nor serology, is required because the majority of donors would no longer be eligible to donate, thereby threatening the supply of blood and plasma and of plasma-derived products. Viruses that fall in this category are Torque Teno Virus (TTV), which is present in greater than 80% of the general population, and GB virus C (GBV-C, previously known as hepatitis G virus, HGV).

Newly emerging pathogens such as hepatitis E virus (HEV) can potentially enter the blood and plasma donor population, resulting in viral infections in recipients of blood and plasma-derived products. Monitoring the emergence of such agents is a continuous effort that involves academia, public organizations that monitor health and develop early warning systems, regulatory agencies, and industry. Currently, several epidemiological surveillance systems are in place and include hemovigilance or biovigilance to address the potential risk of emerging pathogens to the recipients of blood and plasma-derived products. This risk can be mitigated by appropriate measures, such as donor deferral because of geographic risk and risk behaviors, the testing of donations if appropriate, and the inclusion of virus-reduction steps for a wide range of enveloped and nonenveloped viruses during the manufacturing process.

Virological screening assays are designed to detect antibodies, antigens, or nucleic acid sequences of the infectious virus via serological and NAT testing. Sensitive virological test methods are a prerequisite for the quality control of fractionation pools in order to interdict and discard infected donations before manufacturers process these donations into pools to produce plasma-derived products.

All assays used to screen blood or plasma donations should be designed for their intended use and should meet the performance requirements specified by the Clinical and Laboratory Standards Institute (CLSI) guidelines for qualitative and quantitative tests. Assays also should comply with guidance from regulators, such as the European Common technical specifications for in vitro diagnostic assays (see Appendix). Associated calibrators or control materials must be traceable to reference material of a higher order or to reference measurement procedures. The Appendix includes FDA guidance documents pertaining to the manufacture and clinical evaluation of these assays and the use of controls. U.S. and EU requirements or recommendations for tests for screening plasma for further manufacture are described in the Regulatory Environment section.

Regulatory agencies have the goal of ensuring that plasma-derived products are safe with respect to risk from blood-borne pathogens. In addition to regulating the final products, regulators also oversee the assays used to test for infectious agents and set policies about how those tests will be used. Although regulatory agencies in the United States and Europe share the common goal of safety, they use different legal structures and strategies. In general, the hierarchy of regulatory documents is similar. Both start with laws that set the definitive requirements for donor selection and plasma screening.

In the United States, the primary laws that regulate plasma-derived products and the assays that test their safety are the Public Health Service (PHS) Act and the Federal Food, Drug, and Cosmetic (FD&C) Act. The PHS Act addresses biologics and communicable disease controls, and the FD&C Act addresses drugs and medical devices. Donor-screening tests are licensed under the PHS Act rather than being cleared or approved under the medical device provisions of the FD&C Act. Testing requirements for communicable disease agents, including viral pathogens such as HBV, HCV, and HIV, are required under 21 CFR, including not only test requirements (21 CFR 610.40) but also donor deferral (21 CFR 610.41) and look-back requirements (21 CFR 610.46 through 610.48). If a plasma or blood donation is reactive in one of the screening tests, especially in the donor-screening tests for HBV, HCV, or HIV, supplementary or confirmation tests should be conducted to clarify whether the donor is infected [21 CFR 610(b)]. The donor must be informed (21 CFR 630.6) and should be excluded from donating blood or plasma (temporarily or permanently according to 21 CFR 630.6), and manufacturers should initiate a look-back procedure (21 CFR 610.40–48). For HIV and HCV, the procedure includes not only the quarantine and destruction of unused, previously donated units from an infected donor, but also the further testing of the donor and notification of the recipients of the blood and blood components (21 CFR 610.47–48). The look-back period can be as long as 1 year (21 CFR 610.46–48).

The responsibility for legislation of the European Union (EU) is shared between the EU and the European Member States. The European Commission (EC) is responsible for the regulation of the common European market and thus is responsible for medicinal products for human use, whereas the Member States are responsible for health care, which includes the supply of hospitals with blood components such as plasma and cellular components for transfusion. Because the EC is responsible for the regulation of medicinal products derived from human blood or plasma, it is, as a consequence, also responsible for the regulation of plasma for further manufacture. The laws of the EU are found in regulations and directives from the EC and in the binding monographs of the European Pharmacopoeia (Ph. Eur.).

Additional tests and specifications for plasma for further manufacture have been developed as part of the Plasma Product Therapeutics Association (PPTA) Voluntary Standards Program. The PPTA Quality Standard for Excellence, Assurance, and Leadership (QSEAL) includes additional routine testing of blood and plasma donations or plasma pools for HCV RNA, HIV RNA, HBV DNA, HAV RNA, and B19V DNA. Companies certified by PPTA under the QSEAL program have implemented this testing.

All the measures discussed in this chapter, along with virus-reduction steps included during the manufacturing process, ensure the safety of plasma-derived products. However, testing of plasma for further manufacture is only one of the steps taken to ensure the safety of the final plasma-derived products. Both manufacturers and regulators face continuing challenges because of the emergence of new blood-borne viruses, mutants, and variants of existing viruses not detected by current serological/NAT technology. The development of new screening tests and regulatory guidance documents depends on whether the emerging virus is a risk to the safety of plasma-derived products.