Chromatography and immobilization materials including monolith versions rely on a suitable matrix which is robust enough to withstand the rigours of regular use, buffers, extreme temperatures etc.
A number of matrices have been routinely exploited to this effect. A great many of these matrices are naturally derived and include agarose, cellulose, dextrans and chitosan. One of the earliest were the dextran-based matrices such as Sephadex G-25 and G-50.
One feature in common with all natural and synthetic matrices is the unfortunate property of non-specific adsorption because of their hydrophilic character although this is manageable and generally low-level. It does however mean that undesirable componentry can be eluted with the protein or nucleic acid of choice
Agarose
As in all forms of chromatography, the raw material for a matrix is commonly agarose. Originally, the agarose gels were developed for gel filtration but have also been repurposed for other applications.
It is especially popular it seems in affinity adsorption applications although ion-exchange chromatography appears to exploit its properties considerably. Agarose is a polysaccharide derived from agar. It has free hydroxyl groups that can be modified using various activation chemistries. There is a very low non-specific binding. It is resistant to microbial degradation which means that it has a long shelf-life in columns if treated carefully. There are some sulphate and carboxyl groups but these do not contribute to any type of non-specific adsorption unless the ionic strength of the solutions used in chromatography are above 0.02. As cited later, non-specific adsorption is an issue with agarose in immunoaffinity chromatography which cannot be resolved readily.
Agarose gels form spontaneously when a hot saturated solution of agarose is cooled. The agarose concentration is available between 2 and 6 % w/v.
The main suppliers are Bio-Rad, Cytiva and Pharmacia.
The gel is porous and readily compresses with high-pressure gradients that occur in packed bed columns. It is one of its limiting factors in scaling-up chromatography or adsorption.
Cross-linking improves the robustness and rigidity of the agarose matrix. It also appears to make it more chemically stable and less prone to damage or degradation especially with the chemical binding processes used.
Suppliers such as Merck through Cytiva will supply 4% cross-linked agarose which is supplied in an antimicrobial solution of pure isopropanol. The isopropanol is always washed away before use. The issue with cross-linking is the reduction in the number of hydroxyl groups available for activation.
The particle size is often between 45 and 165 micron diameter with an average of 90 microns, which means it is suitable for laboratory and process-scale chromatography.
Agarose can be cleaned in place but it is ligand dependent.
Agarose gels have found their greatest application in gel chromatography because they possess greater resistance to mass transfer than those supports specifically designed for affinity chromatography. One of the issues confronted in any chromatography application is that the greater the mass transfer resistance, the longer the loading and elution steps. As these steps become longer, productivity reduces and lowers the economic viability of the process.
Glass and Silica
Support materials using controlled pore silica and glass are popular. Most chromatographic suppliers offer these. These supports are derivatized using their hydroxyl groups. The porous inorganic supports have great rigidity and cost less than agarose but they suffer with non-specific adsorption and will degrade in alkaline solutions unlike agarose.
Silanes are used to modify the surfaces of glasses and presumably silica to severely reduce non-specific adsorption. They reduce the charge on the surface but retain the hydrophilic character needed (Regnier & Noel, 1976). The paper referenced refers to the use of glycerolpropyl silane bonding which not only helps reduce non-specific adsorption but also minimises protein and nucleic acid denaturation.
Polyacrylamide
Organic polymers such as polyacrylamide, polymethacrylamide and hydroxyethyl methacrylate beads head a large group of these materials used in all forms of adsorption. These matrices are stable in a variety of process conditions and hydrophilic. They are also prone to non-specific adsorption too.
A well-known brand still in use is Eupergit® C that is made by Rohm Pharma. It it is available as a macroporous bead and routinely touted as a carrier for immobilizing enzymes on an industrial scale (Katchalski-Katzir & Kraemer, 2000). Another variant of note is Eupergit C 250 L which has the same chemical structure as the ‘C’ form but with larger pores.
Eupergit C suffers from non-specific adsorption of proteins and it is problematic in some forms of chromatography such as immunoaffinity. It can be so bad that the specifically eluted antigens are heavily contaminated. The same effect occurs with agarose in immunoaffinity chromatography.
Unfortunately, all these materials have poor mechanical stability at the level of porosity needed for both adsorption and immobilization.
N-hydroxysuccinamide Activation
To bind ligands to a matrix such as agarose requires activation of chemical groups with a ligand that is suitable for most coupling chemistries. One of the best is to have N-hydroxysuccinimide activated groups (NHS-activated groups). This NHS group is ideal for binding amino-containing smaller proteins and peptides. NHS (N-hydroxysuccinimide) coupling forms a chemically stable amide bond with ligands containing primary amino groups. The resin offers a spacer arm to the coupled ligand and is thus most suitable for immobilising smaller proteins and peptide ligands.
The ideal commercial situation is to exploit the NHS pre-activated medium where the coupling method produces a chemically stable ligand attachment. There should also be a high degree of substitution of the selected ligand for best optimization.
References
Katchalski-Katzir, E., & Kraemer, D. M. (2000). Eupergit® C, a carrier for immobilization of enzymes of industrial potential. Journal of molecular catalysis B: enzymatic, 10(1-3), pp. 157-176.
Regnier, F.E., Noel, R. (1976) Glycerolpropylsilane bonded phases in the steric exclusion chromatography of biological macromolecules. J Chromatogr. Sci. Jul;14(7):316-20. doi: 10.1093/chromsci/14.7.316. PMID: 956322.
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