METHODS OF REMOVING POLYETHYLENE GLYCOL FROM PLASMA FRACTIONS

Polyethylene glycol (PEG) is a nondenaturing, water-soluble, synthetic polymer which is widely used for purification of proteins by fractional precipitation. Although the polymer is not considered to be toxic, it is desirable to remove as much as possible from the final products. Furthermore, it may be advantageous to remove high concentrations of PEG from various fractions before further processing. Methods which have been used for this purpose include adsorption of the protein to ion exchange or affinity resins, which have no attraction for the inert polymer, or precipitation of the protein from the PEG-containing solution by addition of ethanol. More recently, mixtures of PEG-4000 and albumin were used to demonstrate the feasibility of cross-flow ultrafiltration as an alternative to conventional molecular sieve methods such as dialysis and exclusion chromatography which are of limited value. These studies have been extended to include mixtures of PEG-4000 and crude preparations of human factor VIII, the much higher apparent molecular weight of which should allow use of membranes, with greater pore size than that required to reject albumin. Most of the membranes tested exhibited drastic increases in the rejection of PEG-4000 after exposure to the factor VIII solution most likely due to irreversible protein adsorption, since the effect was not reversed by rinsing with buffer. Nuclepore membranes (0·05 /μ dia) had reasonable rejection properties but filtration rates were too low. An Amicon In-Line hollow-fiber unit (50,000 molecular weight cut-off) repeatedly gave good results but initial enthusiasm was tempered by failure to get equally good results with other units of the same type. An alternative approach involves the addition of divalent anions (sulfate or phosphate) to PEG-containing solutions to produce two aqueous phases, a lower salt-rich phase and an upper polymer-rich phase. Experiments with the 20% PEG-4000 supernate of human plasma revealed that under appropriate conditions, 90% of the protein distributes into the lower phase, whereas 98% of the PEG was recovered in the upper phase. Residual quantities of PEG could then be removed, along with the high concentration of salt, by ultrafiltration. Since phase separation occurs spontaneously, without centrifugation, such a step should tie easily integrated into large-scale processing schemes and would seem particularly attractive for use in continuous-flow systems.