Human plasma derived drugs separation by fractionation of plasma with polyethylene glycol

There are variety of purification techniques for separation of human plasma proteins such as salting out, ion exchange chromatography and ethanol fractionation. There are limitations for each method, for example in salting out method, the salt has to be removed in an additional step. Ion exchange chromatography is difficult for scaling up, and plasma fractionation is a time consuming method and it needs machinery and plants. In the present study the fractionation of human plasma by polyethylene glycol was investigated. The purpose of this study was to investigate the possibility of the fractionation of human plasma by polyethylene glycol. Human plasma fractionation was carried out by using polyethylene glycol with different concentrations from five to twenty percent, followed by centrifugation. After centrifugation the supernatant was used for further fractionation by addition of a higher concentration of polyethylene glycol. Suitable intermediate sources for protein purification were obtained by fractionation of human plasma by polyethylene glycol. Fibrinogen in fraction 5%, IgG and IgM in fraction 10%, IgA in fraction 20%, and finally albumin and alpha1- Antitrypsin in supernatant 20% of polyethylene glycol were achieved. By our study we could obtain four different fractions as intermediate sources for protein purification which cannot be easily obtained from plasma fractionation by cold ethanol fractionation

Pasteurization of IgM-enriched immunoglobulin

Human plasma proteins are important for therapy or prophylaxis of human diseases. Due to the preparation of human plasma proteins from human plasma pools and risk of contamination with human viruses, different viral reduction treatments such as: pasteurization, solvent/detergent, dry heat treatment, steam treatment, beta-propiolactone/UV and nanofiltration have been implemented. As pasteurization can be performed for liquid protein, this method [a 10-hour heat treatment of the aqueous solutions at 60°C] was introduced into the manufacturing procedure of IgM-enriched immunoglobulin, to improve its safety further. The efficiency of this method for inactivation of viruses was evaluated by the use of Foot-and-Mouth Disease Virus [a non-enveloped virus] and Infectious Bovine Rhinotracheitis [IBR] Virus [a lipid-enveloped virus]. Pasteurization inactivated Foot-and-Mouth Disease Virus by 7 log10 and for IBR Virus by 5log10. These findings show a significant added measure of virus safety associated with pasteurization of IgM-enriched immunoglobulin preparation

Solvent-detergent treatment of IgM-enriched immunoglobulin

Viral safety of human plasma products plays a key role in their safe uses. Solvent- detergent [SD] virus-inactivation method has gained widespread popularity in the manufacture of biological products. This treatment which inactivates lipid-enveloped viruses effectively consists of incubation of a plasma protein solution in the presence of a non-volatile organic solvent and a detergent. In this study, IgM-enriched immunoglobulin was incubated at 24 °C for 6 h under slow stirring in the presence of tri[n-butyl] phosphate [0.3% w/w] as solvent and tween 80 [1% w/w] as detergent. After completion of the inactivation process and removal of the solvent-detergent, the ability of SD-treatment to remove Infectious Bovine Rhinotracheitis [IBR] virus [a lipid-enveloped virus] and Foot-and-Mouth Disease virus [a non-enveloped virus] were evaluated by "virus spiking studies" using a scaled down process. Reduction factor of 4 log was obtained for the SD-treatment of IgM-enriched immunoglobulin spiked with IBR virus. No virus inactivation was observed in the SD-treated IgM-enriched immunoglobulin, spiked with Foot-and-Mouth Disease virus. It was concluded that treatment of IgM-enriched immunoglobulin with TNBP-TWEEN 80 may be considered as an efficient lipid-enveloped virus inactivation step in the manufacture of this product