Plasma fractionation

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Plasma fractionation is a process for extracting proteins from human blood plasma . The plasma is separated into individual fractions and purified with the aid of various physical methods ( precipitation , filtration , adsorption , electrophoresis ). This process makes use of the different solubilities of the various plasma proteins with changing pH values , temperatures and ionic strengths, as well as the varying adsorption behavior on solid carrier substances.

Preparative plasma fractionation is carried out in plants that are designed to process several thousand liters, i.e. on an industrial scale. Products made from the isolated proteins (plasma products) are used as drugs for the treatment of congenital and acquired disorders of blood coagulation and immune defense , as well as for shocks caused by large blood loss or severe burns and other illnesses.

For diagnostic purposes, plasma proteins are separated using electrophoresis.

Plasma extraction

Plasma for plasma fractionation is mostly obtained by plasmapheresis . This method is preferred to whole blood donation due to the high demand for plasma from healthy donors . Plasmapheresis allows shorter donation intervals and a higher donation volume. The plasma donation removed in a plastic bag is frozen at −25 ° C and stored. Proteins dissolved in the plasma are responsible for blood coagulation, immune defense, colloid osmotic pressure , substance transport and other vital tasks.

Fractionation

Cryoprecipitation

Schematic representation of plasma fractionation

Several thousand plasma bags are cut open while still frozen and slowly thawed together in a large, temperature-controlled container. The temperature in the resulting plasma pool is around +1 ° C.

As Judith Pool found in the mid-1960s, however, not all proteins contained in the plasma dissolve immediately. A cloudy precipitate (cryoprecipitate) forms, which can be separated from the rest of the plasma by centrifugation . The supernatant, the cryogenic plasma, still contains numerous proteins after this step and is not a waste product.

The proteins factor VIII , Von Willebrand factor and fibrinogen, which are important for blood coagulation, accumulate in the cryoprecipitate . The speed and the temperature profile during thawing determine the quality of the cryoprecipitate. The proteins mentioned are isolated from the cryoprecipitate by further precipitations and chromatographic purification steps and concentrated by ultrafiltration .

The factors of the prothrombin complex ( factor II , factor VII , factor IX and factor X ) are isolated from the cryo-poor plasma with the help of DEAE ® adsorption and the C1 inactivator is isolated by QAE ® adsorption .

Ethanol fractionation

Even after these process steps, however, there are usable proteins in the cryogenic plasma, which are obtained using Cohn extraction .

Edwin Joseph Cohn developed this process in the 1940s. Ethanol is added to the cryogenic plasma in ever higher concentrations and the temperature is lowered below 0 ° C. At an ethanol concentration of 8%, inter alia, is the factor XIII insoluble and precipitates in the form of a precipitate ( precipitate ) out. The precipitate can be separated off by centrifugation or filtration. It is a stable intermediate product and can be stored frozen for a few weeks. For further processing, the precipitate is thawed and dissolved in a buffer. The factor XIII is then purified from this by precipitation and chromatography . Antithrombin III is obtained from the supernatant of the 8% ethanol precipitation by adsorption on a heparin resin .

By adding further ethanol, the immunoglobulins precipitate at a concentration of 20 to 25% . A hyperimmune globin can be obtained from this intermediate. However, by means of suitable purification (e.g. affinity chromatography ) it is also possible to isolate specific antibodies. Passive vaccines against many pathogens, such as hepatitis B , diphtheria , tetanus and rabies, can be provided . At the end of the Cohn fractionation, the concentration of the ethanol is adjusted to 40-42%. This makes albumin insoluble and can be separated off.

Salt fractionation

With this type of fractionation, the ionic strength in the protein solution is regulated by adding salt ( ammonium sulfate precipitation , sodium sulfate , alanine ).

safety

Donor selection and control

Blood plasma from apparently healthy donors is used as the starting material for plasma fractionation. By selecting suitable donors, the risk of disease transmission should be minimized from the start. In addition, the state of health of the donor is monitored for a certain period of time from the donation in order to take into account risks or diseases that were not recognized at the time of the donation when the donation is further used. For this purpose, donated blood plasma is not processed immediately, but rather quarantined for a defined period of time .

Donation control

But like any material of biological origin, blood plasma also carries the risk of disease transmission. Each individual donation for itself and later the plasma pool are examined for pathogens, especially hepatitis B and HIV , using highly sensitive methods .

Work-up process

The process of plasma fractionation depletes any pathogens that may be present. However, the process cannot achieve complete, safe elimination or inactivation. For this reason, measures for virus reduction (physical separation of viruses by precipitation or filtration) or virus inactivation steps (destruction of viruses) must be carried out in the plasma fractionation at suitable points . Pasteurization at +60 ° C for 10 hours has been established since the 1960s, and nanofiltration since the 1990s . The prerequisites for these measures are heat-stable intermediates and, in the case of nanofiltration, sufficient dilution of the substances contained, whose molecular size must also be small enough to be able to be filtered.

The solvent detergent process (S / D) offers another possibility of virus inactivation . In this case, a suitable intermediate in the process history virus-inactivating chemicals, such as are tri- n -butyl phosphate (TNBP) and Triton X-100 added. After a specified exposure time, these are then extracted again and therefore do not remain in the product (or only in traces).

The regulations require at least two virus inactivation steps in the manufacturing process of each plasma product. These have to prove their effectiveness in test runs, so-called virus spike tests. For this purpose, the production conditions in the laboratory are simulated ( down-scale ) and the relevant intermediate is mixed with the virus to be inactivated (spiking). After the virus inactivation has been carried out, the success is checked using appropriate tests.

If a method has been carried out repeatedly and reliably in the laboratory, it is implemented on the production scale ( up-scale ). Spike tests are then no longer carried out here, as these would contaminate the production equipment and lead to an incalculable risk.

All the more attention is paid to precise process control. During pasteurization, for example, the temperature in the pasteurization container is precisely monitored. This must not fall below the limits set in the down-scale. Otherwise, safe inactivation of any viruses that may be present cannot be guaranteed. However, the temperature must not exceed the upper limit. Then the sensitive products could be destroyed.

According to the Blood Working Group at the Robert Koch Institute (RKI), there is no longer any risk of contracting HIV through plasma products.

In Germany, the Paul Ehrlich Institute (PEI) in Langen monitors the manufacture of plasma products. Every single batch of a product is subject to approval by the PEI. New products and manufacturing methods, as well as changes to manufacturing methods that have already been approved, also require manufacturing authorization from the PEI. In the USA, arguably the most important sales market for plasma products, this task is taken over by the Food and Drug Administration (FDA).

Process efficiency

The goal of plasma fractionation is pure, concentrated protein solutions, the function of which is to be preserved through the gentlest possible treatment and preservation (usually freeze-drying). Parts of the process are now over 60 years old and have not been changed much in the past.

The increasingly scarce and expensive raw material means that the plasma fractionation must be carried out more efficiently. More and more product should be produced from as little starting material as possible (increase in yield). But this requirement can often only be met within a narrow framework. So supposed improvements in the manufacturing process can indeed increase its purity and increase the yield, but there must be no disproportion to stabilizing auxiliary proteins. Otherwise the biological effectiveness of the active proteins can be reduced.

literature

  • A. Gröner, SC Martin, Virus Elimination by Purification Steps During the Production of Plasma Preparations. The yellow booklets 38: 81 (1998), http://www.diegelbenhefte.de/
  • PFW Strengers, WG Van Aken u. a., blood - from magic to science. Spektrum Akademischer Verlag (1996), ISBN 3-86025-364-6
  • Human immunodeficiency virus (HIV) ; Statement by the Blood Working Group of the Federal Ministry for Health and Social Security, PDF file

Individual evidence

  1. Commemorative publication for Judith Pool (English) ( Archived copy ( memento of the original from March 28, 2012 in the Internet Archive ) Info: The archive link has been inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice. PDF 15 kB). @1@ 2Template: Webachiv / IABot / histsoc.stanford.edu

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