Blood plasma fractionation
Plasma fractionation is a process for the recovery of proteins from human blood plasma. Thereby, the plasma is separated and purified by various physical methods ( precipitation, filtration, adsorption, electrophoresis ) into individual fractions. Advantage is utilized in this method, the different solubilities of the different plasma proteins with varying pH, temperature and ionic strengths, as well as varying the adsorption on solid carrier substances.
Preparative plasma fractionation is, therefore, carried out in equipment which is designed for the processing of several thousands of liters in an industrial scale. Prepared from the isolated proteins products ( plasma products ) are used as drugs for the treatment of congenital and acquired bleeding disorders and immune defense, as well as shocks caused by large blood loss or severe burns and other disease states.
For diagnostic purposes, separating plasma proteins by the use of electrophoresis.
- 3.1 donor selection and control
- 3.2 Donations control
- 3.3 refurbishing process
Plasma for plasma fractionation is usually obtained by plasmapheresis. Due to the high demand for plasma from healthy donors, this method of whole blood donation is preferred. Plasmapheresis donations allowed shorter intervals and a higher volume of donations. The sample taken in a plastic bag donating plasma is frozen and stored at -25 ° C. In plasma, dissolved proteins are responsible for blood clotting, the immune system, the colloid osmotic pressure, mass transport and other vital tasks.
Several thousand plasma bags are cut open while still frozen state and thawed slowly together in a large, temperature-controlled container. The temperature in the thus formed plasma pool is about 1 ° C.
As Judith pool mid-1960s found, but not dissolve it all the proteins contained in the plasma immediately back on. It forms a cloudy precipitate ( cryoprecipitate ), which can be separated by centrifugation from the remaining plasma. The supernatant, which kryoarme plasma containing after this step, numerous proteins and is not a waste product.
In cryoprecipitate important for blood clotting proteins factor VIII, von Willebrand factor and fibrinogen accumulate. The velocity and the temperature profile during thawing decide on the quality of the cryoprecipitate. From the cryoprecipitate, said proteins can be isolated 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 ) and QAE ® adsorption of C1 inactivator be isolated using DEAE ® adsorption from kryoarmen plasma.
But even after these process steps are useful proteins in kryoarmen plasma, which are obtained by application of the Cohn fractionation.
Edwin Joseph Cohn has developed it in the 1940s. In this case, the kryoarmen plasma ethanol is added at increasingly higher concentrations and lowered the temperature below 0 ° C. At an ethanol concentration of 8%, inter alia, the factor XIII is insoluble and precipitates in the form of a precipitate ( precipitate ) out. The precipitate can be separated by centrifugation or filtration. It is a stable intermediate ( intermediate) and frozen for several weeks shelf life. For further processing, the precipitate is thawed and dissolved in a buffer. For this, the factor XIII is then purified by precipitation and chromatography. From the supernatant of the 8% ethanol precipitation is recovered by adsorption on a resin heparin antithrombin III.
By further addition of ethanol to precipitate the immunoglobulins at a concentration of 20-25%. For this intermediate, a hyperimmune globin may be obtained. However, by appropriate purification (eg affinity chromatography ), it is also possible to isolate specific antibody. So passive vaccines against many pathogens can be provided, such as hepatitis B, diphtheria, tetanus and rabies. At the end of the Cohn fractionation, the concentration of ethanol is adjusted to 40-42 %. Wherein albumin is insoluble and can be separated.
In this type of fractionation, the ionic strength is regulated in the protein solution with added salt ( ammonium sulfate, sodium sulfate, alanine ).
Donor selection and control
Blood plasma of healthy donors apparently serves as a starting material for plasma fractionation. Already by the selection of suitable donors, the risk of transmission of diseases from the beginning is to be minimized. In addition, the health of the donor is monitored from the donation for a certain period of time to unidentified risks or disease in the re-use of the donation to be considered at the time of donation. To this end, donated blood plasma is not processed immediately, but in quarantine for a defined time.
But like any material of biological origin, including blood plasma, yet the risk of disease transmission contains within itself. Every single donation for himself and later the plasma pool are equipped with highly sensitive methods for pathogens, especially hepatitis B and HIV, examined.
Through the process of plasma fractionation existing pathogens is a depletion may take place. However, a complete, safe elimination or inactivation can not afford the procedure. Therefore measures to virus reduction (physical removal of viruses by precipitation or filtration ) or virus inactivation (destruction of viruses) must be carried out in the plasma fractionation at suitable locations. Established since the 1960s, the pasteurization at 60 ° C for 10 hours, and since the 1990s in addition nanofiltration. Prerequisite for these measures are heat stable intermediates and in the case of nanofiltration adequate dilution of the substances contained whose molecular size in addition also needs to be small enough to be filtered can.
Another way of virus inactivation provides the solvent detergent method (S / D). In this case, a suitable intermediate in the process course virus-inactivating chemicals, such as tri-n- butyl phosphate ( TNBP ) and Triton X- 100 are added. After a specified exposure time, these are then extracted again and thus remain not (or only in traces ) in the product.
The regulations require at least two virus inactivation steps in the manufacturing process of each plasma product. These must in test runs, so-called virus spike experiments prove its effectiveness. For this, the production conditions in the laboratory simulated ( down -scale ) and the intermediate concerned with the virus to be inactivated offset ( spiking ). After the viral inactivation of success is checked using appropriate tests.
If a method is repeated in the laboratory and has performed reliably, it is converted to production scale (up- scale ). Here then no spike tests be performed, because this would contaminate the production equipment and lead to an incalculable risk.
The more attention is paid to a precise process control. Thus, the temperature is monitored in the pasteurization accurately, for example during pasteurization. This may not fall below the specified in the down -scale limits. Otherwise, a safe inactivation of any existing viruses is not guaranteed. The temperature must the upper limit but not exceed. Then the sensitive products could be destroyed.
According to the Working Group on Blood at the Robert Koch Institute ( RKI), is no longer a risk of becoming infected with HIV through plasma products.
In Germany, the Paul Ehrlich Institute (PEI ) in Langen monitors the manufacture of plasma products. Each batch of a product is subject to approval by the PEI. New products and methods of production and changes already approved production methods also require a manufacturing license by the PEI. In the United States, arguably the most important sales market for plasma products, this task is performed by the Food and Drug Administration (FDA).
Efficiency of the process
The objective of the plasma fractionation are pure, concentrated protein solutions whose function is to be maintained by the gentlest possible treatment and conservation (mostly freeze-drying). Parts of the process are now over 60 years old and have been changed little in the past.
By always scarce and expensive raw material it comes to the demand for a more efficient implementation of plasma fractionation. So shall from as little starting material, more and more products are produced (yield increase ). But this requirement can often be met only in a narrow frame. Thus, perceived improvements in the manufacturing process while increasing its purity and increase the yield, but not disproportionate to stabilizing auxiliary proteins may thereby arise. Otherwise, the biological activity of the active proteins can be reduced.