The well-known Cohn fractionation process, originally developed in the 1940s to isolate albumin as a blood volume expander during World War II, relies primarily on a series of ethanol fractionation steps to separate the various proteins of human blood plasma from one another according to their solubility behavior in the presence of ethanol (25). As the role of other plasma components became recognized, such as factors VIII and IX for the treatment of clotting disorders and IgG for use in passive immunization, higher yield and higher purity large-scale purification methods for these components were sought, and the importance of adsorption techniques grew.
With the development of cellulose ion exchangers in the mid-1950s, followed by the introduction of agarose-based resins (Sephadex®) a few years later, new chromatographic techniques specifically designed for protein separations became available for use in purification of blood plasma components. Despite the advances made, large-scale application of these methods in the biological industry developed slowly. One reason was the aseptic processing requirements often specified for biological raw materials. Chro-matography using saline solutions and biodegradable supports could result in bacterial contamination if the appropriate precautions were not strictly followed. An inherent advantage of the Cohn fractionation process is that ethanol is bacteriostatic. But the main cause for the slow acceptance of chromatography at the large scale (i.e., fixed-bed operations rather than stirred-tank adsorption) was the poor mechanical qualities of the available macroporous supports designed for protein separations. However, these soft gels were used for some batch adsorption processes in stirred tank reactors for recovery of various blood products. Continual advances in resin technology through the 1970s provided supports that were mechanically strong and had surfaces and porosities well suited for protein chromato-graphic separations.
Although there are a number of advantages associated with column operations, packed beds of adsorptive media tended to act as depth filters for many of the biological feedstocks that were commonly used. This problem could be avoided by carrying out the adsorption in a stirred tank. This method has been used for many years for factor IX isolation from plasma, one of the first major applications of an adsorption method in plasma fractionation.
Factor IX complex, also known as prothrombin complex, can be prepared by a number of methods, but one of the most popular is to use batch adsorption with anion exchangers based on diethylaminoethyl (DEAE) groups attached to a stable support matrix such as cellulose, dex-tran, or agarose. Suomela et al. of the Finnish Red Cross Blood Transfusion Centre in Helsinki reported on a method developed to purify coagulation factor IX concentrate from human plasma (26). The production scale consisted of starting with 150 L of plasma, which was combined with pretreated and autoclaved DEAE-Sephadex A-50 and mixed for 30 minutes to complete adsorption. About 95% of the factor IX along with 5 to 6% of the other proteins were adsorbed from the plasma. The weakly bound proteins were removed by a low ionic strength wash. Adsorption was carried out in stainless steel tanks with mixing, followed by settling the gel to the bottom of the tank while the clear plasma was pumped off. The product containing DEAE-Sephadex gel was then pumped into a steel cylinder equipped with a bottom screen to retain the resin. Elution of the factor IX was achieved by increasing the buffer ionic strength. The resulting factor IX concentrate was purified about 100-fold, although it still contained relatively smaller amounts of factors II, VII, and X. The overall yield of the process was 60%.
A semicontinuous method for purification of factor IX complex from human plasma was described by Tharakan et al. (27). In the semicontinuous process, cryosupernatant plasma is pumped through a stirred column containing resin. Plasma was pumped out of the bottom of the column through the flow adapter screen at the same flow rate so that the volume in the column remained constant. After all the plasma had contacted the resin, the column was packed by gravity settling, and an upper flow adapter was installed. The column was washed with low ionic strength buffer until the absorbance of the effluent was negligible. The factor IX complex was then eluted with a higher ionic strength buffer. It was found that a residence time of 15 minutes was sufficient to capture 95% of the factor IX in the starting plasma. In the pilot plant, 550 L of plasma was passed through a 50-L column containing 8.5 L of resin, yielding a 68% recovery. Comparative studies involving batch, semicontinuous, and packed bed adsorption methods showed that although the amount of factor IX adsorbed from the plasma remained the same regardless of contacting mode, the mode of operation did affect the percentage of factor IX recovered, with increasing recoveries encountered as the resin density or ratio of resin to reactor volume increased (recoveries were 32%, 82%, and 100% batch, semicontinuous, and packed-bed operations, respectively). These results indicate that the adsorption and desorption process is not simple, and it was speculated that multipoint attachment resulting in denaturation or competitive binding of other proteins may have played a role. The semicontinuous method was developed to facilitate contacting plasma with inexpensive soft resins. It eliminates the need for handling a resin-plasma slurry, and it reduces total process time and labor requirements in comparison to batch adsorption. Furthermore, the resin is contained within one vessel (a modified column) throughout its use, thus minimizing handling and overall equipment requirements.
More recently, highly selective chromatography steps have been combined with the traditional batch adsorption step to separate factor IX from the other clotting factors (28) in human plasma. The factor IX concentrate from batch adsorption is passed through a more efficient anion exchanger, and factor VII is removed by stepwise elution. Separation of factor IX from factors II and X is carried out using a highly selective affinity column using heparin-derivatized agarose. Both factors II and X have a lower binding affinity for heparin than does factor IX. The resulting factor IX is purified more than 30-fold, resulting in an overall purification factor from plasma of about 10,000fold.
The DEAE-ion exchangers have also been applied to the preparation of IgG, which, unlike other plasma proteins, is not bound by the DEAE-group at a neutral pH and low ionic strength. Several methods have been described that use ion-exchange materials alone or in combination with ethanol fractionation to purify IgG from plasma. A DEAE-Sephadex in combination with the ethanol fractionation technique was reported for the fractionation of IgG from blood plasma (29). The plasma proteins were first separated into three main fractions by increasing ethanol frac-tionations (8%, 25%, and 40% ethanol). The y-globulin was recovered in the 25% ethanol fractionation precipitate. The paste was then dissolved and the pH adjusted to 6.5 in low ionic strength. DEAE-Sephadex (A50 coarse) was added at a level of about 1 g per gram of protein to be adsorbed, and the mixture was stirred for 30 minutes. The Sephadex gel was filtered off on a stainless steel Buchner-type funnel, and the y-globulin, which was nonbound and in the filtrate, was subsequently dispensed for direct lyophilization.
Hepatitis B Purification
Fused silica, or Aerosil, was first used in a batch adsorption mode to adsorb lipoproteins from human serum (30).
The methodology was then applied to the adsorption of hepatitis B surface antigen (HBsAg) to remove hepatitis B virus from donated plasma (31). In the mid-70s, these methods were refined with respect to optimizing HBsAg adsorption selectivity and elution conditions for the purpose of purifying HBsAg from human plasma (32,33).
The classical methods for purification of HBsAg from sera were by isopycnic banding in a CsCl density gradient, followed by rate zonal centrifugation on a sucrose density gradient. It was cumbersome, however, to process large volumes of plasma or serum containing HBsAg by ultra-centrifugation as the first step of purification. Duimel et al. (32) described a method whereby large quantities of antigen contained in serum could be partially purified by means of batch adsorption to colloidal silica (Aerosil) and then eluted with low ionic strength buffer (0.01 M borax, pH 9.3). Human positive serum was first extracted with Freon, which extracted part of the lipoids from serum, and then adsorbed with 2% Aerosil at 37 °C for 4 hours. After centrifugation for 15 min at 3,000 rpm, the Aerosil was washed four times with physiological saline. The Aerosil was eluted batchwise twice with borax, pH 9.3, for 30 min at 37 °C. Recoveries of HBsAg across this step were reported to be about 60%, providing a 15-fold purification factor. Pillot et al. (33) reported improving the recovery of the desorption step to 100% by eluting with 0.25% sodium deoxycholate in 0.01 M borax, pH 9.3, at 56 °C. Furthermore, elution at the higher temperature favored HBsAg desorption relative to some of the serum proteins, particularly albumin, which often represented the major contaminating protein eluted along with HBsAg.
The French vaccine manufacturer, Pasteur Merieux, used Aerosil adsorption with elution conditions similar to those described above for the production of a commercial plasma-derived HBsAg vaccine (34). In the mid-1980s, new second-generation hepatitis B vaccines based on recombinant DNA technology became available commercially. Many of the general purification principles that were used for the plasma-derived products were incorporated into purification schemes for HBsAg from recombinant sources (35). In the purification process for RECOMBIVAX HB® (Merck & Co., Inc.), batch adsorption of HBsAg onto colloidal silica is employed. The HBsAg is isolated from the clarified lysate by adsorption onto colloidal silica (Aerosil). The silica suspension is then collected by centrifugation, washed multiple times through resuspension and recen-trifugation, and finally eluted from the silica by treatment with warm borate buffer. Sanford et al. reported that the antigen adsorbs to bare silica by a mixed mode and is specifically eluted by a chemical interaction between the silica surface and borate ion (36). More recently, this traditional batch operation has been evaluated for application in a fixed-bed column mode using a macroporous silica packing (37). The elution uses an extended warm borate buffer recycle through the column to achieve an equilibrium distribution between the entire bed volume before product collection. Although the adsorption, washing, and elution steps all take place within a column, it is still fundamentally a batch operation because the separation is achieved in a truly single stage contactor. The column mode of operation is able to be scaled and is more efficient in terms of overall cycle time as a result of the elimination of multiple centrifugation steps, despite the more lengthy column adsorption loading time.
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