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logical molecules and cause strong, often irreversible binding of proteins. For most separation applications, surface modification of CPG with organosilanes is necessary, although native CPG has been used for the purification of a number of biological molecules, such as factor VIII (24). Surface modification also enhances the stability and durability of CPG.

Surface Modification

Siliceous materials such as CPG can be reacted with the organosilane compounds in a number of different ways to obtain the desired bonding through hydrolyzable groups of the organosilane. Depending on the type of organosilane, the reaction can be performed in organic, aqueous, or va pour phase, resulting in matrices suitable for different applications. The majority of the work cited on this subject relates to the surface modification of silica material (25), which is not always applicable to CPG because of the difference in the surface characteristics of the two matrices.

Surface modification of CPG is carried out by treating the material with an organosilane containing an organic functional group at one end and a silylalkoxy group at the other. Attachment of the silane compound to the matrix is through the surface silanol or oxide groups to the silyl-akoxy groups with polymerization between the adjacent silanes. The result is an inorganic matrix with available organic functional groups that can be used in the preparation of chromatography adsorbents. A large number of different organosilanes are commercially available with functional groups such as epoxy, amine, and sulfhydryl (Table 4).

The first step in the surface modification of porous glass is to clean the surface by washing the matrix in nitric acid at elevated temperature. The matrix is then washed with water and dried in the oven at temperatures not exceeding 180 °C. The dry matrix is reacted with the silane compound dissolved in water or an organic solvent at temperatures ranging from 40 °C to 120 °C, depending on the solvent. After removing the excess silane by washing the matrix with suitable solvents or water, the functional groups on the matrix can be further modified to obtain various adsorbents. The exact conditions used in the surface modification of CPG will depend on the nature of the silane compound and the application of the resulting adsorbent.

Extensive optimization of the surface modification procedure is usually required to obtain a suitable adsorbent for a specific application. At Bioprocessing, special tech-

niques for preparing surface modified CPG (PROSEP®) have been developed for applications in affinity and other types of separation processes. PROSEP® has been designed for effective immobilization of both large and small ligands, resulting in affinity adsorbents with a high capacity for the target molecules, minimum nonspecific binding of contaminating proteins, and low ligand leakage (26).

Applications

CPG has been effective in the separation of a range of biological molecules, using a variety of chromatography techniques that include adsorption, size exclusion, affinity, and ion exchange. It has also been used extensively in the field of enzyme immobilization for the modification of biological molecules. The rigidity and incompressibility of the matrix allows it to be used in large columns at very high flow rates generating low backpressure (Fig. 5), which makes this matrix especially useful for processing large volumes of feedstock in short cycle times.

Application of native CPG in adsorption chromatogra-phy has been generally limited because of its denaturing surface characteristics and irreversible binding of proteins. A number of proteins, such as factor VIII (24), have been purified from plasma by first blocking the surface of CPG with albumin. The use of CPG in ion exchange chro-matography applications has also been very limited because of the instability of the matrix in sodium hydroxide solution, which is the standard cleaning and sanitization solution for ion exchange adsorbents. Furthermore, ion exchange adsorbents prepared with CPG as the matrix have generally shown low functional capacity when compared to the polymetric matrices. Nevertheless, some applications of surface-modified CPG in ion exchange chromatography have been reported (27-29).

The use of CPG in size-exclusion chromatography has generally been for the separation of macromolecules such

Table 4. Surface Modification of CPG

Organosilane

Reactant

Activated CPG

3-Aminopropyltriethoxysilane

CPG-amine

3-Aminopropyltriethoxysilane

Glutaraldehyde

CPG-aldehyde

3-Aminopropyltriethoxysilane

Succinic anhydride

CPG-carboxyl

3-Glycidoxypropyltrimethoxysilane

CPG-epoxy

3-Glycidoxypropyltrimethoxysilane

Acid solution

CPG-diol

3-Glycidoxypropyltrimethoxysilane

Sodium periodate

CPG-aldehyde

Mercaptopropyltrimethoxysilane

CPG-sulfyhyryl

Figure 5. Backpressure generated from a column (11.3 X 10.0 cm) packed with PROSEP®.

Linear velocity (cm/h)

Figure 5. Backpressure generated from a column (11.3 X 10.0 cm) packed with PROSEP®.

Figure 6. Principle of fluidized-bed adsorption. From left to right: PROSEP® adsorbent is packed into a column specially adopted for fluidized-bed operation. The bed is gradually expanded by an upward flow of solution through the column until full expansion is reached.

as viruses, cell components, and polymers (30,31). Separation of these species from low molecular weight contaminants has been achieved by taking advantage of the very sharp exclusion limits and narrow pore size distribution of CPG. Generally, surface-modified CPG has been used for size-exclusion chromatography to eliminate the problem of nonspecific and irreversible binding of biological molecules to native CPG. The surface of the matrix is modified with glycidoxypropyltrimethoxysilane, converting the solid phase to diol-CPG (Table 4). This is a nonionic, hydrophilic coating compatible with aqueous and most solvent systems (32).

Surface-modified CPG has been widely applied in the area of affinity chromatography for the purification of biological molecules. Biologically active molecules will often bind to other molecules in a reversible and highly specific manner. The formation of these stable, specific, and dissociable complexes forms the basis of this powerful separation technique.

Affinity adsorbents prepared using PROSEP® and other surface-modified CPG have been used in many applications for the purification of biological molecules, such as antibodies (33,34) and enzymes (35). PROSEP® adsorbents have been used in packed-bed chromatography columns and in fluidized or expanded bed mode (Fig. 6) for the direct capture of biological molecules from feedstocks containing particulate or colloidal solids (36,37). Table 5 shows the application of PROSEP® adsorbents in the field of affinity purification.

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