Plasma constitutes approximately 55% of the total blood volume. It is a clear, straw-colored, complex liquid that is 7% protein, 91% water, and 0.9% mineral salts. The majority (approximately 70%) of total plasma protein is albumin. Additional plasma proteins relevant to fractionation include immunoglobulins, coagulation factors, fibrinolytic proteins, proteases, and protease inhibitors. These constituent plasma proteins can be isolated on the basis of the different solubility characteristics of each protein when subjected to specific conditions of pH, temperature, ionic strength, and ethanol concentration. The major products derived from fractionation are listed in Table 1.

The two methods for collection of human plasma are automated apheresis (for definitions, see the Glossary preceding the appendices) and centrifugation of whole blood donations. Source Plasma collected by apheresis constitutes the majority of plasma used in the manufacture of plasma derivatives in the United States. Plasma collected for transfusion but not so used (i.e., recovered plasma) also may be used for manufacture. Flow charts delineating how apheresis and whole blood–derived plasma can be used in the manufacture of plasma derivatives in the United States and Europe are presented in Figures 1 and 2, respectively.

Regardless of the collection method, plasma for fractionation should be a clear to slightly turbid liquid without visible sign of hemolysis; it may vary in color from light yellow to green; it should be ±10% of the stated volume; and it should show no sign of clots.

Licensed Source Plasma may be manufactured only in collection centers that are approved by the FDA for the collection and distribution of Source Plasma in interstate commerce. Currently, federal regulations governing the manufacture of Source Plasma, including minimal requirements for donors, are found in 21 CFR 640, Subpart G. By definition, Source Plasma is plasma intended for further manufacture. Source Plasma donors can donate as often as twice a week and may be compensated. In addition to FDA requirements, most plasma collectors and fractionators also comply with voluntary standards established by the Plasma Protein Therapeutics Association (PPTA), a trade and standards-setting organization.2 PPTA voluntary standards address several areas of donor, plasma unit, plasma pool, and center management, and are designed to supplement existing regulatory requirements.

Plasma for transfusion is not intended for further manufacture but for direct transfusion to patients. It may be collected by either whole blood or apheresis donation. In the United States, plasma for transfusion comes from unpaid volunteer donors. Blood collecting facilities that collect plasma for transfusion typically comply with requirements of both the CFR and a voluntary trade organization, the American Association of Blood Banks (AABB). Currently, sections 640.3 and 640.31 of 21 CFR outline requirements for donors of whole blood and therefore govern most donors of plasma for transfusion, regardless of the collection method. AABB voluntary standards include information contained in FDA regulations and guidance pertaining to blood and plasma, as well as additional standards.3 Plasma for transfusion may be converted to recovered plasma, an unlicensed product that may be used for further manufacture.

Human plasma and its derivatives are used in the manufacture of other biologic products. In this role, the plasma or plasma derivative falls into the category of ancillary use. This is defined as use of a reagent or material as a processing or purification aid or a reagent that exerts an effect on the therapeutic substance but is not intended to be part of the final product formulation (see the USP general information chapter Ancillary Materials for Cell, Gene, and Tissue-Engineered Products 1043).

Human plasma is commonly used in manufacturing processes that involve primary human cells or cell lines intended for therapeutic applications. In these applications, plasma provides a source of protein and possibly other factors that enhance expansion and differentiation of cell populations. A variety of methods have been used to prepare human plasma and derivatives for ancillary use, but the practices are not standardized. Allogeneic plasma typically is obtained from either apheresis or whole blood, using citrate anticoagulation. Allogeneic plasma typically is collected from paid donors who, like Source Plasma donors, have been screened for the absence of transfusion-transmissible diseases. Preferred donors may be blood type group AB, because they lack anti-A and anti-B isohemagglutinins. Other preferred donors include untransfused males, because this group is unlikely to have human leukocyte antigen (HLA) antibodies that could react with cells in a given culture system. Serum is prepared either from nonanticoagulated whole blood that has been allowed to clot or by the addition of calcium to plasma obtained from citrated whole blood or apheresis. Heating plasma or serum to 56 inactivates heat-labile complement and other proteins.

Although there are no standardized specifications for plasma products used as ancillary materials, assays often include safety testing associated with general biologics (e.g., bacterial/fungal cultures, endotoxin, and mycoplasma). In addition, characterization of the products may include tests for irregular erythrocyte and HLA antibodies, osmolality, pH, total protein and immunoglobulin concentrations, hemoglobin concentration, and chemistries such as Na, K, Cl, Ca, and glucose.

In some instances, plasma from bovine sources has been used instead of human plasma. The differences between bovine and human plasma products include factors relevant to their efficacy as ancillary materials, as well as safety of the final manufactured product when administered to humans (see the USP general information chapter Bovine Serum 1024). Fetal bovine serum is preferred to human serum for some applications because it may be superior in promoting human cell growth in vitro. Safety considerations, especially the risk of transmissible spongiform encephalopathy and allergic reactions related to antibovine antibodies, have led to recommendations that human plasma sources be used whenever possible instead of bovine sources. However, substitution of human for bovine plasma does not completely eliminate the risk of infectious and immunologic sequelae, even when human plasma is used as an ancillary material.

Source Plasma is collected by apheresis. Recovered plasma can be obtained either by apheresis or as a by-product of whole blood collection. Collection should take place via an FDA-approved, closed, sterile, pyrogen-free collection system that contains an anticoagulant. No antibacterial or antifungal agent should be added to the plasma. Donations must be collected aseptically. The skin of the donor must be aseptically prepared. Source Plasma is collected using 4% sodium citrate as the anticoagulant. The composition of sodium citrate is given in Table 2.

Three anticoagulant solutions are licensed in the United States for collection of whole blood: citrate phosphate dextrose (CPD), citrate phosphate double dextrose (CP2D), and citrate phosphate dextrose adenine (CPDA-1). The composition of blood collection bags containing these anticoagulants is shown in Table 3. Plasma for transfusion or further manufacture can be made from a unit of whole blood collected in any of the three anticoagulant solutions. Regulations relating to plasma make no distinction among the three anticoagulant solutions. Consequently, collection, storage, and transport requirements are identical regardless of the anticoagulant solution used in the primary collection.

The labeling for Source Plasma should comply with 21 CFR 640.70 and 21 CFR 640.69(b). The labeling for whole blood should comply with 21 CFR 606.121 and 606.122, and with internal licenses.

A unique identification number is assigned so that the donation can be related to the individual donor records and test results. The origin of each donation in a plasma pool and the results of the corresponding donation and laboratory tests must be traceable while the required degree of confidentiality concerning the donor's identity is maintained. Whole blood must be labeled “This Product may transmit infectious agents” [21 CFR 121(c)(9)]. Source Plasma or recovered plasma must be labeled “Caution: For Manufacturing Use Only” if the product is intended for use in fractionation. For plasma to be used as a reagent or for in vitro use, the required labeling statement is “Caution: For Use in Manufacturing Noninjectable Products Only” [21 CFR 121(e)(5)(ii)].

Plasma for fractionation should be stored at or below 20. The plasma can still be used for fractionation if its temperature exceeds 20 on (at most) one occasion for not more than 72 hours and if the plasma has been maintained at a temperature of 5 or lower at all times. Storage temperatures must be maintained during transport.

The selection of a suitable site for blood and plasma donation activities is a first and very important step to ensure safe donations. Areas with low disease prevalence are preferred as locations for donation centers, thereby reducing the likelihood of collecting plasma from an infected donor.

One of the PPTA voluntary standards is the viral marker standard, which obliges plasma centers to report viral marker rates for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) in the donor populations. A center's rates are compared to the industry average. Alert limits are set to take into account the number of annual donations in the center. If a center exceeds the limit for any of these viruses or the aggregate of all three, the center must implement corrective actions that will bring the center into compliance with the standard.

Appropriate donor selection helps provide a safe plasma supply. A detailed donor history questionnaire in conjunction with a careful medical examination allows center personnel to recognize unsuitable donors whose behavior puts them at risk for transfusion-transmitted disease or who have underlying medical conditions that preclude donation.

An additional measure put in place by centers that collect Source Plasma is the PPTA National Donor Deferral Registry (NDDR). It lists donors throughout the United States who have been previously deferred from donation (although it provides no information about the reason for deferral). Other countries have different systems depending on their national regulations concerning personal data gathering. Any individual who tests positive for HIV, HBV, or HCV is entered into the national database (the National Donor Deferral Registry) used by all U.S. plasma centers that are certified under the International Quality Plasma Program (IQPP). All individuals who present at a U.S. plasma center for the first time are checked against the NDDR. In this manner, donors who have previously been deferred for positive test results at any participating facility can be identified and rejected quickly. This standard ensures that donors deferred for positive test results do not donate in other facilities.

A voluntary safety initiative, the Qualified Donor Standard, implemented by the plasma fractionation industry, builds on the fact that many plasmapheresis donors contribute plasma frequently. A donor who enters a plasmapheresis center for the first time is called an Applicant Donor, and the first donation is used for further manufacturing only if the donor returns a second time. Potential donors must pass two separate medical screenings and testing for HIV, HBV, and HCV on two different occasions. Only after satisfactory screenings and negative test results does that person become a Qualified Donor. If a donor does not return within 6 months, that person loses his/her Qualified Donor status and must qualify again. This standard means that plasma from a one-time-only donor (even when all test results are negative) cannot be used for further manufacture. This standard results in committed donors and eliminates the risk that plasma centers will accept so-called test seekers. The interval between permitted donations of whole blood is too long to allow a similar screening program, although quite a number of donors in whole-blood donor centers are regular and repetitive donors.

Another PPTA voluntary standard addresses donor management criteria. The Community-Based Donor Standard allows only donors who permanently reside within its defined donor recruitment area to donate at a given center. In addition, a Donor Education Standard requires new donors to engage in an educational program and follow-up assessment regarding HIV/acquired immune deficiency syndrome (AIDS) and activities that place them at risk for HIV/AIDS.

In addition to donor management strategies and standards, PPTA has issued a plasma unit management standard called Inventory Hold. This standard states that collected plasma will be held in inventory for at least 60 days from the time of collection. This allows the retrieval of units as a result of post-donation information (information that was not known at the time of donation) that would have disqualified the donor. This information could include admitting high-risk behavior; becoming reactive for HIV, HBV, or HCV; or providing incorrect information about international travel.

Testing of donations is an important safety measure both for plasma intended for transfusion and plasma intended for further manufacturing. Both enzyme-linked immunosorbent assays (ELISA) and nucleic acid amplification technologies (NAT) are used to screen donations for the presence of infectious disease. Automation provides the necessary throughput to screen every donation for a variety of potential pathogens.

Testing strategies differ depending on whether the plasma donation is intended for transfusion or further manufacture. Plasma for transfusion requires more extensive infectious disease testing, because there are no pathogen inactivation/removal technologies licensed for this product in the United States. On the other hand, plasma for further manufacture is subjected to several pathogen inactivation/removal steps during manufacture, thereby obviating the need for some disease testing. Table 4 outlines current infectious disease tests required by the FDA for plasma donations collected in the United States. Appendix 3 compares EU and U.S. disease testing and donor deferral requirements.

NAT, of which polymerase chain reaction (PCR) is the most widely used form, does not rely on the detection of antibodies produced by the infected host after exposure, but targets the nucleic acid of the infecting agent. By means of the selection of suitable priming molecules (the so-called primers), the assay is highly specific for the infecting virus (see the USP general information chapter Nucleic Acid-Based Techniques—Amplification 1127). Through several cycles of amplification, the polymerase enzyme can repetitively generate copies of the targeted fragment of the viral nucleic acid, providing an exponential amplification of a very short stretch of the viral deoxyribonucleic acid (DNA) [or ribonucleic acid (RNA)]. The exponential amplification leads to the generation of many copies of the target molecule and allows the subsequent detection of this virus-specific fragment, even if the original viral load was exceedingly low. This methodology has brought a new degree of safety.

NAT testing, because of its complexity and expense, is difficult to conduct on individual donations. Generally, aliquots from several donations are combined into a single pool, often called a minipool. Testing in pooled format remains more sensitive than serological ELISA screening of individual donations. In addition, the NAT principle circumvents several of the limitations in detecting pathogens by means of serological methods. Pooling can influence overall sensitivity, depending on the pool size and the analytical sensitivity of the NAT assay employed.

In many countries, the maximal load of a pathogen acceptable for a single donation defines the overall NAT sensitivity required. Assays of higher analytical sensitivity can use larger pools, but those of lower analytical sensitivity must test smaller pools in order to comply with regulations. The availability of commercial NAT test kits with defined analytical sensitivity has made minipools up to 512 very common, because these pool sizes, in combination with the analytical sensitivity of the assays used, comply with common regulations on overall sensitivity.

Effective NAT screening requires that the viral load of the plasma pool at the beginning of production be less than the inactivation and/or removal capacity of the process. Differences between the plasma transfusion and fractionation industries have led to different applications of NAT. For individual donations intended for transfusion, where there is no inactivation and/or removal process and where testing is the only method to interdict a contaminated donation, the safety of each individual donation must be ensured by testing with the most sensitive assays possible. Plasma intended for further manufacturing, in contrast, is pooled and serves as the starting material for a multistep process that has built-in pathogen inactivation methodologies. Therefore, NAT screening for plasma for further manufacture is focused on ensuring safe donations and limiting the viral load of the plasma pool to levels less than the known viral inactivation/removal capacity of the inactivation process.

To avoid the loss of large amounts of plasma from a reactive pool, the fractionation industry has implemented a prescreening strategy, the minipool screening concept mentioned earlier. Aliquots of plasma donations are combined to form minipools, and the minipools are tested by NAT. If a minipool is reactive for a virus tested, the individual donation that gave rise to this positive result can be identified and interdicted. The other donations demonstrated to be free of infection can be used for further manufacturing. The donations are then combined into a production pool, a sample of which is subjected to NAT testing as required by regulations.

As indicated, the NAT test portfolio is not uniform and depends on the intended use of the donation and the regulatory environment. Although screening for HCV RNA is done in most countries, screening for HIV is not universally required. NAT detection of HBV is used mainly for plasma for manufacturing. Screening for B19 virus or hepatitis A virus (HAV) is performed only on plasma for manufacturing. HBV screening using NAT detection is more widespread in European and Asian countries than in the United States.

A donor may be deferred from further donation as a result of answers provided on the donor history questionnaire, counseling of donors for reasons for deferral, a medical examination performed at the time of donation, or positive tests for infectious diseases. These processes ensure both the eligibility of the donor and the suitability of the donation. In the event that either is not acceptable, standard processes permanently remove the donor and interdict unused units previously donated. The donor registries mentioned earlier are one means of ensuring that a donor deferred at one center cannot donate elsewhere.

Each individual unit of plasma, whether for transfusion or further manufacture, is held in quarantine until all the required tests have been completed. If all required tests have been performed and found acceptable, the unit can be released; if not, the unit must be destroyed. The plasma industry has voluntarily implemented the inventory hold protocol (also discussed in the previous section Donor Screening) for plasma for further manufacture. According to inventory hold requirements, during a 60-day hold period an individual plasma donation cannot be used for further manufacture. The rationale for the hold is that donors who have been recently infected with a pathogen may not have developed levels of antibody at the time of donation, thereby donating an infectious unit despite negative disease tests. The hold provides sufficient time for an infectious donor to develop levels of antibody that will be detected during a subsequent donation if the plasma was intended for transfusion. The 60-day hold also reduces the chance of releasing an infectious unit into the manufacturing process.

The introduction of NAT may have decreased the need for inventory hold, because NAT targets the infecting virus directly and thus does not rely on the time-delayed production of antibodies. Because NAT cannot detect all viruses and because even NAT has a certain (although very low) limit of detection, inventory hold is still of value and thus remains in place in the plasma fractionation industry.

Each plasma donation must be traceable from donation to ultimate disposition in order to minimize the potential transmission of an infectious agent. Traceability encompasses all data concerning donation site, donor identifying information, test results, and data regarding transport, storage, and consignee(s).

Look-back is a process to identify and interdict (quarantine) previous donations from a donor who, at a subsequent donation event, has been found to be (1) infected with a transmissible agent or (2) unsuitable for donating plasma because of history, physical examination, or post-donation information. Although look-back strategies are similar in most countries, specific procedures may vary.