Human blood as a unique tissue (1), contains many important proteins with therapeutic uses (2). As a biological drugs, virus inactivation is one of the crucial steps in their preparations of these plasma derived proteins (3-6). The most important proteins in human plasma are: albumin, immunoglobulin, fibrinogen, and coagulation factors VII, VIII and IX (7). There are varieties of purification techniques for separation of these proteins (8-11). One of the methods is salting out (12, 13). There are not many salts which can be used in this method and most of the researchers use ammonium sulphate for precipitation of plasma protein (14, 15). But the problem is fractionation of plasma by ammonium sulphate needs to be repeated several times. The other compound which can be implemented for protein precipitation is the lactate of 2-ethoxy-6, 9-diamino-acridine (Rivanol) (16). After plasma fractionation by this substance, it has to be removed from the plasma, which sometimes its complete removal could be difficult. The other method for protein separation is ion exchange chromatography (17, 18). So far many commercially available gels have been produced for ion exchange chromatography for which scaling up for industrial scale is not so easy. The quality of plasma play an important role in the quality of final product of plasma fractionation (19). Ethanol fractionation of plasma is a very suitable method for the preparation of albumin, and immunoglobulin in large scale (20, 21). However, for separation of protein from human plasma by ethanol fractionation, different equipments are needed that to be installed in a plasma fractionation factory. In our study we used polyethylene glycol as an alternative method to ethanol plasma fractionation, to investigate protein separation by addition of polyethylene glycol.
The purpose of this study was to investigate the possibility of the fractionation of human plasma by polyethylene glycol.
3. Materials and Methods
Human fresh frozen plasma (22) was thawed overnight at 4ºC in cold room. After centrifugation at 2500 ´g of fresh frozen plasma and the removal of the paste, the supernatant was used as starting material. In order to minimize the protein denaturation all procedures were carried out at 4ºC in the cold room. Solid PEG-4000 which is a water soluble and non-toxic synthetic polymer from Merck-Germany at the concentration of 5% (50 g/L) was added to the cryosupernatant and stirred for 1 h. After centrifugation a 5% paste was obtained. After removal of the 5% paste, addition of 50 g/L of polyethylene glycol was added to the supernatant to obtain 10% paste by centrifugation. The 20% fraction was achieved by addition of 100 g/L of PEG to the supernatant and by adding an additional 100 g/L of PEG to the supernatant resulted in obtaining of a 30% paste.
It should be mentioned after each step of PEG addition, the solution was stirred for 1h, and it was followed by centrifugation at 2500 ´g for removal of related paste. By this method we could obtain four fractions of 5%, 10%, 20%, and 30% of PEG in addition to fraction of cryosupernatant as starting material which could be compared to those fractions from cold ethanol fractionation.
Determination of fibrinogen was carried out by clotting assay method. Total protein determination was measured by a colorimetric test with biuret reagent from Merck, Germany by using a Philips PU 8750 UV/Vis Scanninig Spectrophotometer.
Albumin measurement was carried out by a colorimetric method using bromcresol green (BCG), and determined by reading the absorbance by Philips PU 8750 UV/Vis Scanning Spectrophotometer.
The concentrations of IgG, IgA, and IgM were measured by immunoturbidimetric test using Immunoglobulin IgG Kit, Immunoglobulin IgA Kit, and Immunoglobulin IgM Kit from Human Diagnostics, Germany by Cobas Mira Photometer from Roche, USA.
As it has been shown in table 1, albumin is the main contaminant in fraction with 5% polyethylene glycol, while fibrinogen is the dominant protein in this fraction. Also albumin as contaminant could be observed in fractions 10% and 20% of polyethylene glycol. The highest concentration of immunoglobulin G and immunoglobulin M could be found in 10% polyethylene glycol fraction. The fraction 20% PEG is rich in immunoglobulin A with less amount of IgG and IgM. Albumin and a1-Antitrypsin because of their similarity in physicochemical properties are dominant in final supernatant with 20% PEG. This fraction is a suitable source for albumin and a1-antitrypsin preparation.
Human plasma proteins showed different solubility in variations of polyethylene glycol concentrations. In our study plasma fractionation by different concentration of polyethylene glycol resulted in four fractions which is shown in Table 1.
By cold ethanol fractionation of human plasma, five fractions can be achieved (23,24). Table 2 shows these fractions with approximately recovery of protein. Human plasma fractionation by cold ethanol is a time consuming process and for adjustments of different parameters such as pH, ionic strength, temperature, ethanol concentration, and protein concentration it needs hours for conditioning. It has to be noticed that ethanol may influence protein-protein interactions and there are growing indications that exposure to ethanol affects some of the plasma proteins.
One of the advantages of polyethylene glycol in comparison with ethanol is the shorter period of time of processing, which is required for the precipitation process. Using polyethylene glycol instead of alcohol makes it possible to work at below 0?C without the need for extreme temperature control to avoid freezing and it is due to its low heat of PEG solution in water. Different distribution of proteins in fractions with 5% to 20% PEG is shown in the Table 1.
Fraction with 5% PEG is a good source for further purification of fibrinogen, while IgG is dominant in fraction with 10% PEG. Most of the IgA could be obtained in fraction with 20% PEG, and at the end final supernatant is a very good source for purification of albumin.
By our study we could obtain different fractions as intermediate sources for protein purification which cannot be easily obtained from plasma fractionation by cold ethanol fractionation.
In compare with ethanol, polyethylene glycol is nonflammable and is not toxic. Using PEG allowed us working above zero temperature without denaturation of protein, and permitted direct centrifugation without the time period for conditioning which is time consuming in the case of using cold ethanol.
This study was supported by Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
KMH: Designed and carried out the study and prepared the manuscript. MP: Participated in the experimental work of the fractionation and drafting the manuscript. MHR: Was involved in the experimental work of the fractionation. MS: Carried out the analytical methods.
There was no conflict of interest.
The study has been supported by Iranian Blood Transfusion Organization.