Administering Files, Records, and Grants

6.1 Methods used to obtain plasma for fractionation

Human plasma for fractionation may be obtained by separation of plasma from whole blood, or by apheresis.

6.1.1 Recovered plasma

Recovered plasma is plasma recovered by centrifugal separation from the cells and cellular debris of whole blood under the conditions described below.

6.1.2 Apheresis plasma (source plasma)

Apheresis plasma is obtained by a procedure in which anticoagulant-treated blood is removed from the donor, the plasma is separated from the formed elements, and at a minimum the red cells are returned to the donor. The separation of cellular elements and plasma may be achieved either by centrifugation or fi ltration. The equipment used for the collection of plasma by automated methods is designed for separating cellular elements and plasma by centrifugation or fi ltration. The manufacturers of the equipment provide operating manuals that include instructions for installation validation, routine preventive maintenance, periodic performance checks (e.g. weight scale checks), alert mechanisms (e.g. haemoglobin detector) and troubleshooting. Annual preventive maintenance should be performed by a qualifi ed fi eld service engineer. It includes e.g. visual inspection, initial operational integrity, inspection of equipment integrity, inspection of fi lter and/or centrifuge, calibration testing and safety testing. In addition, the manufacturers of the equipment usually provide support for the installation and train on-site technicians to maintain the equipment. Apheresis collection potentially increases the availability of plasma for fractionation, enabling higher donation frequency and a larger volume per donation, and is the preferred approach for the regular collection of plasma from hyperimmune donors who have high antibody titres against specifi c disorders.

In principle, the method of preparation should remove cells and cell debris as completely as possible and should be designed to prevent the introduction of microorganisms. No antibacterial or antifungal agent is added to the plasma. The residual blood cell content of the plasma, in the absence of dedicated leukoreduction fi ltration, may vary with the collection method.

6.2 Characteristics of plasma for fractionation

6.2.1 Plasma frozen within 24 hours of collection

Subject to appropriate handling (storage and transport), plasma frozen, at –20 °C or –30 °C, within 24 hours of blood collection or apheresis (see section 6.6.2.1) will normally be suitable for optimal recovery of both labile factors (factor VIII and other coagulation factors and inhibitors) and stable plasma proteins (usually albumin and immunoglobulins). Plasma meeting these quality specifi cations is also used for direct clinical applications; it is then referred to as fresh frozen plasma (FFP), clinical plasma or plasma for transfusion. Table 4 sets out the main characteristics of plasma prepared either from whole blood (recovered plasma) or by apheresis.

Both sources of plasma have been found by experience to be appropriate for the manufacture of the whole range of plasma products. That said, the method of collection and preparation has some impact on the characteristics and/or yield of the proteins fractionated from the plasma. Apheresis plasma collected from donors undergoing frequent plasmapheresis contains lower levels of IgG than plasma units produced by moderate serial plasmapheresis or from whole blood (23, 24). The content of various coagulation factors is usually higher in apheresis plasma than in recovered plasma (24, 25), for various reasons that include rapid separation of blood cells and plasma, differing ratios of anticoagulant added, and the possibility of freezing the apheresis plasma soon after completion of collection.

Table 4

Characteristics of plasma for fractionation used in the manufacture of labile plasma products

a Based on a standard donation size of 450 ml, with blood:anticoagulant ratio of 7:1. The maximum volume of blood to be collected during one donation procedure is determined by national authorities.

b With anticoagulant. The maximum volume of plasma to be removed during one plasmapheresis procedure is determined by national authorities.

Preservation of factor VIII and other labile factors depends on the collection procedure and on the subsequent handling of the blood and plasma. With good practice, an average of 0.7 IU/ml factor VIII can usually be achieved both with apheresis and recovered plasma. Units of plasma for fractionation with a lower activity may still be suitable for use in the production of coagulation factor concentrates, although the fi nal product yield may be reduced.

The implementation of GMP in the preparation of plasma for fractionation should ensure that the plasma bioburden is controlled, labile proteins are conserved as far as possible, and minimal proteolytic activity is generated.

6.2.2 Plasma frozen after 24 hours of collection

Plasma may be available that does not fulfi l the above-defi ned criteria but still has value as a source of some plasma proteins. This would include:

· plasma separated from whole blood and frozen more than 24 h but usually less than 72 h after collection;

· plasma, separated from whole blood stored at 4 °C, and frozen within

72 h of separation but within the assigned shelf-life of the blood);

• Plasma frozen within 24 h but stored under conditions that preclude its use for the manufacture of coagulation factors.

Provided the circumstances of manufacture and storage of such plasma does not result in increased bioburden, the plasma may be considered suitable for the manufacture of stable plasma proteins, but not coagulation factors.

Plasma which is not frozen within 72 h of collection or separation from whole blood should not be used for fractionation.

6.2.3 Plasma not meeting the requirement for fractionation

Plasma obtained by therapeutic plasma exchange does not meet the criteria for fractionation into plasma products. Indeed, plasma from individuals subjected to therapeutic plasma exchange for the treatment of a disease state may present an enhanced risk of transmitting blood-borne diseases (due to infectious risks associated with plasma) and a high risk of irregular antibodies, and should not be offered for fractionation. In addition, such plasma cannot be classifi ed as being obtained from a voluntary donor.

Plasma from autologous blood donations is excluded from use as plasma for fractionation and may have higher prevalence of viral markers (27).

6.2.4 Hyper-immune (antibody-specifi c) plasma

Detailed information regarding immunization of donors for the preparation of hyperimmune plasma is provided in Appendix 3. The following are the three approaches for the preparation of plasma for the manufacture of specifi c immunoglobulins (antibody-specifi c immunoglobulins):

· Individuals selected from the normal population by screening of plasma units for antibody titres (Screening may be random, or may be informed by knowledge of history of recovery from an infectious disease — for example varicella).

· Individuals with a high titre of a specifi c antibody resulting from prophylactic immunization.

· Volunteers recruited to a panel for a targeted immunization programme. The clinical and ethical requirements for such a programme are considered in Appendix 3.

Clinically relevant specifi c immunoglobulins include anti-D (anti-Rho), and HAV, HBs, tetanus, varicella/herpes zoster and rabies immunoglobulins. Hyperimmune globulins are prepared for intramuscular administration, but products for intravenous use are also available. The typical derivation of hyperimmune plasma of each specifi city is summarized in Table 5.

Table 5

Types of hyperimmune plasma

Acceptable minimum antibody potencies in individual plasma donations for fractionation should be agreed to by the fractionator. Those will usually depend upon:

· the size and composition of the fractionation pool (which may include high-titre donations to increase the mean titre of the fractionation pool);

· the characteristics of the immunoglobulin fractionation process; and

· the minimum approved potency of the fi nal IgG product.

The following general guidance may be useful for each specifi city.

6.2.4.1 Anti-D (anti-Rho)

• Antibody potency should be estimated in international units, using an appropriate quantitative assay (e.g. auto analyser-based assay or fl ow cytometry method) agreed by the fractionator.

6.2.4.2 Anti-hepatitis A

· Antibody potency should be estimated in international units, using a quantitative assay agreed by the fractionator.

· The minimum acceptable potency in an individual donation is unlikely to be less than 50 IU/ml.

6.2.4.3 Anti-hepatitis B

· Antibody potency should be estimated in international units, using a quantitative assay that detects antibody to hepatitis B surface antigen (typically radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA)) agreed to by the fractionator.

· The minimum acceptable potency in an individual donation is unlikely to be less than 10 IU/ml.

6.2.4.4 Anti-tetanus

• Antibody potency should be estimated using either a neutralization assay or a quantitative assay with established correlation to the neutralization assay, agreed by the fractionator.

6.2.4.5 Anti-varicella/zoster

· Antibody potency should be estimated using a quantitative assay (typically ELISA, immunofl uorescence or complement fi xation) agreed by the fractionator.

· The minimum potency should be shown to be equal to or greater than that of a control sample provided by the fractionator.

6.2.4.6 Anti-cytomegalovirus

· Antibody potency should be estimated using a quantitative assay (typically ELISA, immunofl uorescence or complement fi xation) agreed by the fractionator.

· The minimum potency should be shown to be equal to or greater than that of a control sample provided by the fractionator.

6.2.4.7 Anti-rabies

• Assessing plasma for rabies antibody is rarely done. A donor may be considered to have acceptable antibody titres between 1 and 3 months after a second (or booster) dose of vaccine. Plasma should not be collected from persons immunized after exposure to infection with rabies virus.

6.3 Premises and devices for collection of plasma for fractionation

6.3.1 Premises

The collection of blood or plasma for fractionation should be performed in licensed, or regulated, permanent premises or mobile sites which are compliant with the intended activity and comply with the GMP standards approved by the national regulatory authority. The area for blood donors should be separated from all processing and storage areas. The area for donor selection should allow confi dential personal interviews with due regard for the safety of donors and personnel. Before premises are accepted for mobile donor sessions, their suitability should be assessed against the following criteria:

— the size (to allow proper operation and ensure donor privacy);

— safety for staff and donors; and

— adequate ventilation, electrical supply, lighting, hand washing facilities, blood storage and transport equipment, and reliable communication capabilities.

6.3.2 Containers

Because plasma is a complex and variable mix of proteins in aqueous solution, the way in which it is handled will have consequences for its safety, quality and quantity. Furthermore, the effects of mishandling will not always be as simple (or as obvious) as a reduction in the content of recoverable factor VIII — they are just as likely to affect the behaviour of the plasma when it is thawed (this is very important to the fractionator, who requires consistency from this particularly important process step).

The containers used for the collection and storage of plasma for fractionation should comply with the appropriate regulatory provisions and should be under the control of the regulatory authority. Containers should also comply with the regulatory and technical requirements of the plasma fractionator. Containers should be labelled with batch numbers traceable to individual donations. The quality of containers has a direct impact on the quality of the plasma produced and it is therefore part of GMP to control the suitability of this starting material before use.

Containers for whole blood collections are the same as for donations of whole blood from which plasma is used for fractionation. They should be plastic, and should have been manufactured in such a way as to assure internal sterility; they should be hermetically sealed to exclude contamination. If the container is not manufactured as an integral part of a blood collection set, there should be a mechanism for docking with the collection set that minimizes the risk of adventitious microbial contamination.

Validation studies will be required to confi rm the suitability of the container material (and the material of any tubing or harness through which plasma should pass) during storage in contact with the plasma. Specifi cally, it will be necessary to establish that the plastic is physically compatible with the proposed methods for freezing and opening (or thawing) the packs and to establish the quantities of extractable materials (for example, plasticizers) during the claimed periods of storage in the liquid and frozen forms. These studies are carried out by the manufacturer of the containers. When using collection sets and containers previously established by a manufacturer as being suitable, a cross-reference to such a study may be suffi cient. Validated collection and storage containers for blood/plasma are available from several manufacturers worldwide.

The choice of the containers (e.g. type of plastic bags for recovered plasma or plastic bags or bottles for apheresis collection) has a direct impact on the design of the container opening machine that is used at the plasma fractionation plant at the plasma pooling stage.

6.3.3 Anticoagulants

Most anticoagulant solutions developed and introduced for the collection of blood cellular components and plasma for transfusion are compatible with the preparation of plasma for fractionation and with the manufacture of plasma products (although some infl uence on factor VIII content in plasma has been described (28–32)). One exception is when heparin is added to the anticoagulant solution. The main anticoagulant solutions currently in use for collection of either whole blood or apheresis plasma are listed in Table 6.

Anticoagulant solutions should comply with the appropriate regulatory provisions. They can be already present in the collection container (e.g. plastic containers used for whole blood collection) or added to the blood fl ow during apheresis procedures. In both cases, information on the device and the anticoagulant should be provided to the regulatory authorities. The fractionator will need to know what anticoagulant has been used, and its concentration as these may have an impact on the fractionation process.

6.4 Blood/plasma collection process

6.4.1 Procedure

A standardized and validated procedure for the preparation of the phlebotomy site should be followed using a suitable antiseptic solution, and should be allowed to dry (depending on the type of disinfectant). The prepared area should not be touched before insertion of the needle. Prior to venipuncture the containers should be inspected for defects. Any abnormal moisture or discoloration suggests a defect. A careful check of the identity of the donor should be performed immediately before venipuncture.

The collection of a whole blood unit used to prepare plasma for fractionation should be performed following already established recommendations (for instance as described in the Council of Europe Guide (13)). In particular, good mixing of the blood with the anticoagulant solution should be ensured as soon as the collection process starts to avoid risks of activation of the coagulation cascade. The mixing can be done manually, every 30 to 45 seconds, and at least every 90 seconds. Collection of one standard unit of blood should be achieved within 15 minutes, as longer collection periods may result in activation of the coagulation factors and cellular components.

In automated apheresis procedures, whole blood is collected from the donor, mixed with anticoagulant, and passed through an automated cell separator. The plasma for fractionation is separated from the cellular components of the blood, which are returned to the donor in a series of collection/ separation and return cycles. The plasma is separated from the red blood cells by centrifugation or fi ltration, or a combination of both (33, 34). The operational parameters of the plasmapheresis equipment are defi ned by the manufacturers of the equipment and by the requirements of national regulatory authorities. In general, the anticoagulant (often 4% sodium citrate) is delivered at a rate to yield a specifi ed ratio of anticoagulant to blood. The volume of plasma collected from the donor during one procedure and over a period of time is regulated. The number of collection/separation and return cycles for each donor depends on the total volume of plasma that is to be collected. For determining the number of cycles employed, the equipment requires programming by input of data. These data elements may include such parameters as donor weight and haematocrit values. The amount of time required for the donation procedure depends on the number of cycles (and hence the volume of plasma collected) but is generally between 35 and 70 minutes.

Table 6

Examples of anticoagulant solutions commonly used in the preparation of plasma for fractionation

(×), seldom used; ×, commonly used.

6.4.2 Labelling of collection bags

There should be a secure system for procurement, printing and storing of the bar code labels used to identify the main collection bags and the satellite bags, associated samples and documentation to ensure full traceability at each stage of plasma production. There should be a defi ned procedure for labelling collection bags and samples — in particular to ensure that the labels correctly identify the association between samples and donations. Labelling should be performed in a secure manner, e.g. at the donor couch, prior to collection, or immediately after the start of collection, to avoid mislabelling. Duplicate number sets of bar code donation numbers should not be used. Information on the label of the donation should include: offi cial name of the product; volume or weight; unique donor identifi cation; name of the blood establishment; shelf-life or shelf term; shelf temperature; and name, content and volume of anticoagulant.

6.4.3 Equipment

Equipment used for the collection and further separation of blood should be maintained and calibrated regularly, and the collection and separation process needs to be validated. When validating the quality of the recovered plasma, a set of quality control tests, including measurement of total proteins, residual blood cells, haemoglobin, and relevant coagulation factors, such as Factor VIII, should be included. In addition, markers of activation of the coagulation and fi brinolytic systems may, if necessary, be performed with the support of the plasma fractionator based on the specifi cations of the plasma for fractionation set out by the fractionator and/or the national regulatory authority.

Likewise, apheresis equipment and apheresis procedures should be validated, maintained and serviced. Validation criteria for assessing the quality of plasma for fractionation also include protein recovery, residual content of blood cell and haemoglobin, and relevant coagulation factors. Validation studies of new apheresis procedures should also evaluate possible risks of activation of the coagulation, fi brinolysis, and complement systems potentially induced by the material in contact with blood (25, 35, 36); such studies are usually performed by the manufacturer of the apheresis machines.

6.4.4 Laboratory samples

Laboratory samples should be taken at the time of blood/plasma collection. Procedures should be designed to avoid any mix-up of samples and samples awaiting testing should be stored at an appropriate temperature, as specifi ed in the operating instructions of the test kits.

6.4.5 Volume of plasma per unit

The volume of recovered plasma per container varies depending upon the volume of whole blood collected, the respective haematocrit of the donor, and the volume of the anticoagulant solution. The volume of apheresis plasma per container depends directly upon the volume collected during the apheresis session and the volume of anticoagulant. The range of volume of blood and plasma collected per donor is usually defi ned in national regulations taking into consideration criteria such as the weight of the donor.

Although in most countries the volume of whole blood collected is close to 400–450 ml per donor, in some it may be as low as 200 ml (under those circumstances, the volume of anticoagulant solution is reduced so that the plasma:anticoagulant ratio is constant). As a result the volume of recovered plasma per unit (including anticoagulant) may vary from about 100 to 260 ml per container. In the case of plasmapheresis plasma, the volume may range from about 450 to 880 ml per container, depending upon the regulations in the country of collection.

The volume of plasma per container has direct practical impact on the fractionation process and manufacture of plasma products. Small-volume donations (e.g. 100 ml) will require more handling by the plasma fractionation operators at the stage of plasma preparation, at the container opening step, and during plasma thawing. The overall container opening process will take longer, requiring additional care to control bacterial contamination. Another consequence is that the number of donations contributing to a plasma pool will be higher (for instance, 20 000 plasma donations for a pool size of 2000 litres).

6.4.6 Secure holding and reconciliation

When the collection process is fi nished, it should be ensured that blood/ plasma donations are held at the donation site using a secure system to avoid mishandling.

Prior to dispatching the collected donations to the blood/plasma processing site, reconciliation of the collected donations should be performed according to a standardized procedure. The procedure should also specify the actions to be taken if there are found to be missing numbers or leaking containers. Documentation should accompany the donations to the plasma processing site, to account for all donations in the consignment.

6.4.7 Donor call-back system

A system should be in place in the blood establishment which allows recall of a donor if further analysis or investigation is necessary.

6.5 Separation of plasma

6.5.1 Premises

Blood processing should be carried out in adequate facilities suitable for the needs of the intended activity. The donor area and plasma processing areas should be separated whenever possible. Each area used for processing and storage should be secured against the entry or intervention of unauthorized persons and should be used only for the intended purpose. Laboratory areas and plasma storage areas should be separate from the donor and processing areas.

6.5.2 Intermediate storage and transport

Transport of the donations and samples to the processing site should be done according to procedures that ensure both constant approved temperature and secure confi nement. This is especially important when blood/plasma is transported from distant blood drive sessions.

Temperature monitoring is important to ensure optimal compliance and quality. One way is to use packaging methods that can keep the blood/ plasma within the required temperature limits. Portable temperature loggers can be used to monitor and record temperatures during the transportation of blood/plasma to the processing site.

6.5.3 Impact of whole-blood holding period

It has been shown that whole blood anticoagulated with CPD, transported and stored at 22 °C for up to 8 h prior to separation of plasma is suitable for the production of plasma for fractionation, but factor VIII activity is reduced by an additional 15–20% if blood is stored for 24 h (37). Rapid cooling of whole blood to 22 °C +/– 2 °C immediately after collection (e.g. using cooling units with butane-1,4-diol) (38) protects factor VIII and may allow storage of blood for 24 h (39). A temperature of 4 °C during transportation or storage of blood collected with either ACD, ACD-adenine, or CPD anticoagulants consistently appears to reduce the factor VIII content, but not necessarily that of other proteins, especially after 8 hrs of holding time (40–43). Holding blood at 4 °C for longer than 8 h is therefore not recommended when plasma is used for fractionation in the manufacture of factor VIII products.

6.5.4 Centrifugation of whole blood

Documentation on blood and plasma collection should be checked at the processing laboratory on receipt of the donations; reconciliation between consignment and documentation received should be performed. Blood separation procedures should be performed using a closed system and should be validated, documented and proven to ensure that containers are correctly identifi ed.

Reproducible production characteristics of the plasma for fractionation, following a validated procedure, should ensure consistency in the residual blood cell count and protein content and quality to meet the specifi cations set out by the blood establishment or the national regulatory authority and the plasma fractionator.

Comparisons have shown that CPD whole blood units that were centrifuged under conditions of low g force for a long time and those subjected to high g force for a short time yielded blood components of similar quality (44). Blood separation classically starts with the isolation of the platelet-rich plasma (PRP) fraction from whole blood by low-speed centrifugation. Subsequent high-speed centrifugation of PRP in turn yields the corresponding platelet concentrate and the plasma.

Fully automated systems for blood processing including removal of the buffy-coat layer have replaced manual extraction procedures. This allows standardized extraction and contributes to compliance with GMP in the preparation of blood components including plasma for fractionation (45). Blood component separation systems may be based on buffy coat extraction by the “top and bottom” technique (46). Its effi cacy in terms of yield, purity, and standardization of blood components has been well established.

Several technical approaches have been developed to separate blood components. The process may involve normal centrifugation to separate the blood components, which are subsequently squeezed out from the top and bottom simultaneously under control of a photocell. This primary separation step results in three components: a leukocyte-poor red-cell suspension, plasma, and a buffy-coat preparation (46). A multiple-bag system with top and bottom drainage of the primary bag allows automatic separation of blood components; plasma containing 14.6 ± 5.6 × 103 platelets/µl and 0.04 ± 0.035.6 × 103 leukocytes/µl is obtained (47). Blood components may be separated by initial high-speed centrifugation (4158 g, 14 min, 22 ºC) of whole blood in sealed triple or quadruple bag systems, followed by simultaneous extraction of fresh plasma at the top, and the red blood cell concentrate at the bottom, of the respective satellite bags that constitute the blood extraction bag system — keeping the leukocyte-platelet buffy coat layer stable throughout the process within the original extraction bag. The buffy coat component yields the platelet concentrate after low-speed centrifugation and removal of the plasma from the PRP. Automatic separators that subsequently express the various components into their respective satellite bags in top and bottom systems yield plasma containing 3 ± 3 × 106 leukocytes and 4 ± 3 × 109 platelets per unit (48). The “top and bottom” approach allows a marked reduction in leukocyte contamination of the different blood components (38, 49), and may yield optimal plasma volume (38).

6.5.5 Impact of leukoreduction

Recently, several countries have implemented universal leukoreduction of the blood supply (50, 51) to avoid cell-mediated adverse events or improve viral safety of blood components. It has also been considered as a precautionary measure against the risk of transmission of variant Creutzfeldt-Jakob disease (vCJD). A recent study in an endogenous animal infectivity model reports that leukoreduction of whole blood removes 42% of the vCJD infectivity associated with plasma (52), whereas further investigation by the same group found a ~70% removal of infectivity (R. Rohwer, unpublished data). The impact of leukoreduction on plasma protein recovery and activation markers appears to depend upon the chemical nature of the fi lters (53, 54). Some loss of coagulation factors and sometimes an increase in the markers of coagulation and complement activation has been found, although the impact on the quality of fractionated plasma derivatives is not known (54, 55).

Therefore, until more scientifi c data are gathered, the benefi ts of leukoreduction for the quality and safety of plasma products remains debated. The decision to leukoreduce plasma for fractionation should be assessed with the plasma fractionator and the national regulatory authority.

6.6 Freezing of plasma

Freezing is an important processing step that has an impact on some aspects of the quality of plasma for fractionation, in particular with regard to the content of factor VIII.

Several aspects of the freezing conditions of plasma for fractionation have been evaluated.

6.6.1 Holding time of plasma

Holding plasma, freshly harvested from CPD-whole blood, at ~ 4 °C for up to 24 h before freezing at –20 °C for 4 months was shown to induce almost 25% loss of factor VIII activity compared to that in plasma frozen immediately, whereas other coagulation factors were not affected (56). Storing plasma at 22 °C for 2–4 h does not seem to induce a signifi cant loss of factor VIII activity; however, after 4 h, some loss of activity takes place (41, 57).

Therefore, placing recovered plasma in a freezer as soon as possible, or at least within 4 h, after separation from cellular elements, would be favourable to the recovery of factor VIII. Similarly apheresis plasma should be frozen as soon as possible after completion of the collection procedure.

6.6.2 Freezing rate and freezing temperature

6.6.2.1 Freezing conditions

The regulatory requirements for the temperature at which plasma should be frozen vary (58), and depend upon the type of proteins fractionated.

The fractionator may also wish to specify freezing conditions depending on the intended use of the plasma.

The European pharmacopoeia currently states that recovered or apheresis plasma for fractionation to be used for the manufacturing of labile proteins (e.g. production of factor VIII concentrate) should be frozen rapidly, within 24 hours of collection, at –30 °C or colder (26), as this temperature has long been claimed to ensure complete solidifi cation (59), and to be needed for optimal freezing (60). However, freezing conditions are currently under debate and the wording used in the European pharmacopoeia monograph may be revised. Recovered plasma used to manufacture only stable plasma proteins (e.g. albumin and immunoglobulins) should be frozen within 72 h of collection at –20 °C or colder.

The US Code of Federal Regulations specify that plasma collected by apheresis and intended as source material for further manufacturing should be stored at –20 °C or colder immediately after collection.

The rate at which freezing proceeds is considered to be an important quality factor, at least when coagulation factors are intended to be produced (61, 62). Rapid freezing of plasma prevents or reduces loss of factor VIII in frozen plasma either recovered or obtained by apheresis (23, 63, 64), whereas slow freezing of plasma has been shown to infl uence the purity and recovery of factor VIII in cryoprecipitate (61, 64–66). An ice front velocity of 26 mm/h during freezing was recently shown to preserve factor VIII:C in plasma better than 9 mm/h or less (57).

Therefore, freezing plasma rapidly (typically in less than 2 h, so as to ensure a high ice front velocity) down to a core temperature of at least –20 °C, and preferably colder, appears to be the best approach for the preservation of labile proteins.

6.6.2.2 Impact of containers and equipment

To ensure optimal and consistent freezing and storage conditions, it is important to use standardized plasma containers as freezing time is infl uenced by container shape, volume and thickness (57, 64, 65).

Optimum conditions used by some plasma collectors to ensure reproducible freezing are achieved by freezing “well separated” plasma packs in a stream of moving cold air at the lowest temperature tolerable to the plastic of the pack (a so-called “blast freezer”), and then to store the frozen packs “closepacked” in a storage freezer at the agreed upon storage temperature. The worst case would be to place a large number of unfrozen plasma bags, close together, in a domestic (–18 °C to –22 °C) freezer, adding more plasma bags for freezing each day, and storing the plasma under these conditions for several months. With good practice at the time of loading (i.e. not putting too many packs in at the same time and keeping them separated), a walk-in freezer at a suitable temperature offers a workable compromise.

The plasma fractionator should specify precisely to the plasma collector, with the approval of the national regulatory authority, which precise freezing parameters to use.

6.6.2.3 Validation of the freezing process

Recovered plasma and apheresis plasma should be shown to be frozen in a consistent manner at the required temperature. A system should be in place for ensuring that plasma is frozen to the correct core temperature within the time limit agreed upon with the plasma fractionator, keeping in mind that the speed of freezing will be infl uenced by the type of plasma container as well as by the volume of plasma (64). Validation of the freezing process by recording the temperature of plasma donations during a freezing process allows evaluating the freezing capacity of the equipment to be evaluated. Validation studies should be available, and should demonstrate that the temperature of a frozen pack reaches the proposed storage temperature following the specifi cations agreed upon with the manufacturer.

As indicated above, the aim should be to achieve rapid freezing, and thereafter to minimize temperature changes to the frozen plasma.

6.7 Storage of plasma

6.7.1 Storage conditions and validation

Plasma for fractionation should be stored at –20 °C or colder.

A multicentre study showed no detectable storage-related changes in three pools of plasma (2 recovered CPD plasma and 1 apheresis plasma) that had been quick-frozen at –30 °C, or colder, and stored over a period of 36 months at –20 °C, -25 °C, -30 °C, or –40 °C. An 11% reduction in factor IX was found in one of the recovered plasma pools during storage at –20 °C for 2 years (67). The authors concluded that plasma may be stored at –20 °C for 2 years, or at –25 °C, –30 °C, or –40 °C for 3 years.

By keeping the average storage temperature of the frozen plasma as constant as possible, at or below –20 °C, the original quality of the plasma is maintained, without having any impact on the fractionation process, in particular the cryoprecipitation step (60, 61, 66).

The European pharmacopoeia has a provision stating that if the temperature of the plasma is between –20 °C and –15 °C for a maximum of 72 h, or if it is above –15 °C (but colder than –5 °C) in no more than one occurrence, the plasma can still be used for fractionation. Therefore, maintaining a constant storage temperature of –20 °C or colder is a recommended approach to ensure a consistent and optimal plasma quality.

6.7.2 Premises and equipment

Storage conditions should be controlled, monitored and checked. Temperature records should be available to prove that the full plasma containment is stored at the temperature agreed upon with the plasma fractionator throughout the storage area. Appropriate alarms should be present and regularly checked; the checks should be recorded. Appropriate actions on alarms should be defi ned. Areas for storage should be secured against the entry of unauthorised persons and should be used only for the intended purpose. Storage areas should provide effective segregation of quarantined and released materials or components. There should be a separate area for rejected components and material.

If a temporary breakdown of the freezing machine or failure of the electricity supply occurs (e.g. electricity used for the stored plasma), examination of the temperature records should be made together with the plasma fractionator to evaluate the impact on plasma quality.

6.7.3 Segregation procedures

The following should be taken into account in the storage and boxing of plasma for fractionation.

· Untested plasma and released plasma should be stored in separate freezers, or if both types of plasma are stored in a single freezer a secure segregation system should be used.

· Initially reactive plasma donations should be stored in a separate quarantine freezer or a secure system (e.g. validated computer hold system) should be used to prevent boxing of non-released plasma.

· Donations that are found to be unacceptable for fractionation should be retrieved, disinfected and discarded using a secure system.

· Plasma donations for shipment to the plasma fractionator should be boxed in a secure manner and an effective procedure (such as a computerized system) should exist to make sure that only fully tested and released plasma donations are boxed.

· Prior to shipment, plasma boxes should be reconciled appropriately.

· Prior to release of the plasma shipment to the fractionator, there should be a formal review of the documentation to ensure that the plasma shipped complies fully with the specifi cations agreed upon with the plasma fractionator.

The goal of the above-mentioned measures is to make sure that donations that do not comply with the specifi cations agreed upon with the fractionator will not be released and shipped, and that traceability of donations is ensured.

6.8 Compliance with plasma fractionator requirements

Any plasma collected and prepared for fractionation should meet the plasma product manufacturer requirements as the specifi cations of plasma for fractionation are part of the marketing authorization granted by the national regulatory authority for a specifi c plasma derivative. In addition, to the regulatory criteria related to donor selection and screening of donations, the quality specifi cations agreed upon with the fractionator may encompass:

— compliance with GMP during production and control;

— residual level of blood cells (platelets, leukocytes) that should be below a certain level that may vary depending upon the requirements of different countries or fractionators;

— protein content possibly including a minimal mean level of Factor VIII coagulation activity if this product is manufactured;

— guarantee of an appropriate ratio of plasma:anticoagulant solution (see Table 6) and evidence of appropriate mixing with the anticoagulant

during the collection process (for instance, clots should be absent);

— acceptable maximum titre of ABO blood group antibodies (risks of haemolytic reactions due to the presence of ABO antibodies, or antibodies to other blood group systems, in intravenous IgG and lowpurity factor VIII preparations have been described (68)). The European pharmacopoeia requires an ABO titre of less than 1:64 for the release of plasma products for intravenous use.

— maximum haemoglobin content;

— absence of haemolysis;

— colour;

— absence of opalescence (due to lipids);

— citrate (anticoagulant) range content (usually between 15 and 25 mM); and

— minimum titre of a specifi c antibody when the donation is used for the production of hyperimmune IgG such as anti-Rho, anti-HBs, anti-tetanus or anti-rabies.

6.9 Release of plasma for fractionation

Each blood establishment should be able to demonstrate that each unit of plasma has been formally approved for release by an authorized person preferably assisted by validated information technology (IT)-systems. The specifi cations for release of plasma for fractionation should be defi ned, validated, documented and approved by quality assurance and the fractionator.

There should be a system of administrative and physical quarantine for plasma units to ensure that they cannot be released until all mandatory requirements have been satisfi ed. In the absence of a computerized system for control of product status, the label of the plasma unit should identify the product status and should clearly distinguish released from non-released (quarantined) plasma. Records should demonstrate that before a plasma unit is released, all current declaration forms, relevant medical records and test results have been verifi ed by an authorized person.

Before fi nal product release, if plasma has been prepared from a donor who has donated on previous occasions, a comparison with previous records should be made to ensure that current records accurately refl ect the donor history.

In the event that the fi nal product is not released due to potential impact on plasma quality or safety, all other implicated components from the same donation should be identifi ed. A check should be made to ensure that (if relevant) other components from the same donation(s) and plasma units or other components prepared from previous donations from the same donor(s) are identifi ed. There should be an immediate update of the donor record(s) to ensure that the donor(s) cannot make a further donation, if appropriate.

6.9.1 Plasma release using electronic information systems

Special documented evidence is needed if release of plasma is subject to use of electronic information systems (EIS) to ensure that the system correctly releases plasma units only if all requirements are met. The following points should be checked:

· The EIS should be validated to be fully secure against the possibility of plasma which does not fulfi l all test or donor selection criteria, being released.

· The manual entry of critical data, such as laboratory test results, should require independent verifi cation by a second authorized person.

· There should be a hierarchy of personnel permitted access to enter, amend, read or print data. Methods of preventing unauthorized entry should be in place, such as personal identity codes or passwords which are changed regularly.

· The EIS should block the release of plasma or other blood components considered not to be acceptable for release. There should also be a means to block the release of any future donation from a donor.

6.10 Packaging of plasma

The packaging requirements should be specifi ed by the fractionator. The specifi cation should include the following information:

— how the plasma containers are to be packed to prevent damage during shipment;

— that plasma of different types should be kept discrete and packaged into separate cartons; and

— that each carton should have a unique identifi cation number or a bar code which should be clearly displayed on the carton and recorded in the shipping documentation.

6.11 Transportation of plasma

Although it is possible to think of transport as an extension of storage, some additional qualifi cation is appropriate. This need arises because of the additional requirements for risk management during transport. Plasma is at increased risk when:

· Responsibilities for storage and transportation conditions change (especially when handling is the responsibility of individuals with little understanding of the consequences of temperature elevation, as will often be the case with contract shippers).

· Plasma is moved from one freezer or container to another (especially if this involves even temporary exposure to ambient temperatures, as on the loading dock of a blood establishment or a fractionation facility).

· The usual provisions for backup in the event of failure of the refrigeration system are not available (as during sea-transportation of several weeks duration).

The recommendations for cold chain maintenance, as mentioned for plasma storage, should also apply during transportation of plasma. The arrangements for temperature control and monitoring during shipping should be clearly defi ned and documented. The requirements for number and location of temperature logging devices during shipping should be based on a documented assessment of risk throughout the process. The temperature to be maintained during transportation should be defi ned by the fractionator in accordance with relevant regulations.

The responsibilities of organizations and individuals during shipping should be identifi ed; in particular any requirements for documented handover checks should be specifi ed. The fi nal responsibility for acceptance of quality as compliant with specifi cation lies with the quality department of the fractionation facility.

Table 7 summarizes some recommendations on the handling of blood and plasma to optimize the recovery of labile proteins such as factor VIII in plasma. These recommendations should be examined keeping in mind that the relationship between the content of factor VIII in the starting plasma and its recovery in factor VIII concentrates is unclear (40, 69), possibly in part due to the loss of factor VIII that takes place during industrial cryoprecipitation (70) as well as during purifi cation and virus reduction procedures.

Table 7

Processing of plasma for fractionation to optimize factor VIII stability

a Collection of plasma by apheresis makes it possible to freeze plasma immediately after the end of the collection procedure by contrast to whole blood processing.

6.12 Recall system

In the case of known or suspected quality defects of a plasma unit that has already been shipped, a person within the blood establishment should be nominated to assess the need for product recall and to initiate and coordinate the necessary actions. An effective recall procedure should be in place, including a description of the responsibilities and actions to be taken. Actions should be taken within predefi ned periods of time and should include tracing all relevant components of the donation and, where applicable, should include look-back procedures.

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