Matrices

By definition matrices must be inert and play no part in the separation. In practice most play a (usually) negative role in the separation process. To minimize these disadvantages matrices have to be selected with great care. There is a theoretically perfect matrix, defined as consisting of monodispersed perfectly shaped spheres ranging from 5 to 500 |im in diameter, of high mechanical strength, zero nonspecific adsorption and with a range of selectable pore sizes from 10-500 nm, a very narrow pore size distribution and low cost. This idealized matrix would then provide the most efficient separation under all experimental conditions. As always, a compromise has to be reached, the usual approach being to accentuate the most attractive characteristics while minimizing the limitations, usually by manipulating the experimental conditions most likely to provide the optimum result.

The relative molecular masses of proteins vary from the low thousands to tens of millions, making pore size the most important single characteristic of the selected matrix. Very large molecules need very open and highly porous networks to allow rapid and easy penetration into the core of the particle. Structures of this type must therefore have very large pores, but this in turn indicates low surface areas per unit volume, suggesting relatively low numbers of surface groups to which ligands can be covalently attached. The matrix must also be biologically and chemically inert. A special characteristic demanded from biological macromolecular separations media is an ability to be sanitized on a routine basis without damage. This requires resistance to attack by cleansing reagents such as molar concentrations of strong alkali, acids and chaotropes. In contrast to analytical separations, where silica-based supports are inevitably used, silica cannot meet these requirements and is generally not favoured for protein separations. Table 2 contains examples of support matrices used in affinity separations.

The beaded agaroses have captured over 85% of the total market for biological macromolecule separations, and are regarded as the industry standard to which all other supports are compared. They have achieved this position by providing many of the desirable characteristics needed, and are also relatively inexpensive. Beaded agaroses do have one severe limitation - poor mechanical stability. For analytical applications speed and sensitivity are essential, demanding mechanically strong, very small particles. Beaded agaroses are thus of limited use analytically, a gap filled by high performance liquid chromatography (HPLC) using silica matrices. For preparative and large scale operations other factors are more important than speed and sensitivity. For example, mass transfer between stationary phase and mobile phase is much less important when compared to the contribution from the chemical kinetics of the binding reaction between stationary phase and protein. Band spreading is also not a serious problem. When combined with the highly selective nature of the affinity mechanism, these factors favour the common use of large sized, low mechanical strength particles.

In recent years synthetic polymeric matrices have been marketed as alternatives. Although nonbiodeg-radable, physically and chemically stable, with good permeabilities up to molecular weights greater than 107 Da, the advantages provided are generally offset by other quite serious disadvantages, exemplified by high nonspecific adsorption. Inorganic matrices have also been used for large scale protein separations, notably reversed-phase silica for large scale recombinant human insulin manufacture (molecular weight approximately 6000 Da), but are generally not preferred for larger molecular weight products. A very slow adoption of synthetic matrices is

Table 2 Support matrices

Support matrix

Operational

pH range

Agarose

2-14

Cellulose

1-14

Dextran

2-14

Silica

<8

Glass

<8

Polyacrylamides

3-10

Polyhydroxymethacrylates

2-12

Oxirane-acrylic copolymers

0-12

Styrene-divinylbenzene copolymers

1-13

Polyvinyl alcohols

1-14

A/-Acryloyl-2-amino-2-hydroxy-1, 2-propane

1-11

PTFE

Unaffected

PTFE, polytetrafluoroethylene.

PTFE, polytetrafluoroethylene.

indicated as improvements are made to current materials and the prices of synthetics begin to approach those of agarose beads. Other factors resist any significant movement towards synthetic matrices. Most installed processing units are designed for low performance applications. Higher performance matrices would need reinstallation of new, much higher cost high performance plant; plant operators would need retraining; operating manuals would need rewriting; and plant and factory would need reregistration with the FDA. In combination, the implication is that penetration of high performance systems for large scale applications will be slow, and agarose beads will continue to dominate the market for protein separations.

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