Figure 3 illustrates the use of Cellufine Formyl coupled with an antibody for largescale antigen purification. In this application the Cellufine affinity column provides a significant concentration and purification of antigen at high yield. The subject column has been used for over 30 months to process over 3,000 liters of starting plasma with no significant degradation in performance.

To produce the gel, 45 liters of horse serum containing anti-HBs Ag antibody were first concentrated and purified by ammonium sulfate precipitation and dialysed into 0.2M phosphate (pH 7) with 0.1 M NaCl. The resulting antibody serum was added to 12 liters of Cellufine Formyl and reacted together with 80 grams of NaCNBH₃ at 4 to 8 °C for 24 hours. The antibody gel was then washed with buffer and packed into the column. The process stream consisted of human plasma positive for HBs Ag which had been previously purified by freeze-thawing, centrifugation, ammonium sulfate precipitation and gel filtration chromatography.

Cellufine Formyl can be used to immobilize lectins for glycoprotein purification, as shown in Figure 4. Con A (50mg) was immobilized on 0.5g (wet) of Cellufine Formyl by reacting at
4 °C overnight in 1ml of 0.1M acetate (pH 6.4) containing 1mM MgCl₂, 1mM MnCl₂ and 1mM CaCl₂ under the presence of methyl-alpha-D-mannoside and NaCNBH₃. After washing with water, the gel was suspended at 4 °C overnight in 2ml of 1 % glutaraldehyde with NaCNBH₃. After a second water wash the gel was suspended for one hour at room temperature in 2ml of 1M Tris/HCl (pH 7.4) and rewashed.

The availability of formyl functional groups allows great flexibility in selecting media for optimal reaction conditions (pH, temperature, activating agents, reactant concentrations, etc.). Cellufine formyl is a high-stability, functional packing optimized for an application group. Control of reaction chemistry and ligand density is straightforward.

The aldehyde active group on Cellufine Formyl packings reacts with primary amine groups on the ligand to form a Schiff’s base complex (see Figure 5). A mild reducing agent is used to convert the Schiff’s base to a highly stable linkage. Table 2 illustrates a general ligand coupling protocol.

Cellufine Formyl requires a reducing agent for formation of a highly stable linkage. A number of reducing agents are available for good results in virtually any application. The agent should be selected to produce a reasonable reaction rate and yet not be so strong as to damage the protein ligand (such as by reduction of disulfide bonds) or as to reduce the aldehyde groups. Sodium borohydride (NaBH₄), sodium cyanoborohydride (NaCNBH₃) and a newer, non-toxic reducing agent, trimethylamine borane ((CH₃)₃NBH₃) are successful agents, depending upon the particular requirements. For any agent, the quantity required is typically less than 10 milligrams per gram of wet gel.

The reaction rate of Cellufine Formyl is rapid enough to be practical, yet slow enough to be extremely gentle to most proteins. It also allows for a fine measure of control. The rate may be controlled effectively with temperature to achieve maximum protein stability. The pH of effective couplings ranges between 3 and 10.

The coupling efficiency (the ratio between amount coupled and amount offered) and total ligand density can be varied and optimized quite easily through changes in coupling ligand concentration, pH and temperature. A standard set of conditions will work well for most cases, but optimization over a broad range can be used to improve process economics for specific applications.