Coupling Small Ligands Through Carboxyl Groups via a Spacer Arm

EAH Sepharose 4B

The partial structure of EAH Sepharose 4B is shown in Figure 4.10.

Figure 4.10. Partial structure of EAH Sepharose 4B.

Ligands are coupled in a simple one-step procedure in the presence of a coupling reagent, carbodiimide. The carbodiimides may be regarded as anhydrides of urea. The N,N’ di-substituted carbodiimides promote condensation between a free amino and a free carboxyl group to form a peptide link by acid-catalyzed removal of water. Thus EAH Sepharose 4B can be coupled with carboxyl-containing ligands. The carbodiimide yields an isourea upon hydration. The coupling reaction is shown in Figure 4.11.

Figure 4.11. Carbodiimide coupling reaction.

Chromatography medium characteristics

Characteristics of EAH Sepharose 4B chromatography medium are shown in Table 4.7.

Table 4.7. Characteristics of EAH 4B chromatography medium

Product	Compositionh	pH stability1	Average particle size (µM)
EAH Sepharose 4B	Covalent linkage of 1,6-diamino-hexane by epoxy coupling creates a stable, uncharged ether link between a 10-atom spacer arm and Sepharose 4B.	Short term: 3 to 14 Long term: 3 to 14	90

¹ Stability data refers to the coupled medium provided that the ligand can withstand the pH. Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

Purification options

The purification options for EAH Sepharose 4B are shown in Table 4.8

Table 4.8. Purification options for EAH Sepharose 4B

Product	Spacer arm	Substitution (µmol/mL of medium)	Coupling conditions	Maximum operating flow velocity (cm/h)1	Comments
EAH Sepharose 4B	11-atom	7 to 11 amino groups	pH 4.5, 1.5 to 24 h, 4 °C to room temp.	75	Couple ligands containing free carboxyl groups. Supplied as a suspension ready for use.

¹ See Appendix 4 to convert flow velocity (cm/h) to volumetric flow rate (mL/min). Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Preparation of coupling reagent

Use a water-soluble carbodiimide such as N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-cyclohexyl-N’-2-(4’-methyl-morpholinium) ethyl carbodiimide p-toluene sulfonate (CMC). These two carbodiimides have been used in a variety of experimental conditions and at a wide range of concentrations (Table 4.9). EDC often gives better coupling yields than CMC.

Table 4.9. Examples of conditions used during coupling via carbodiimides

Coupled ligand	Carbodiimide	Conc. of carbodiimide (mg/mL)	pH	Reaction time (h)
Methotrexate	EDC	18	6.4	1.5
UDP-glucuronic acid	EDC	32	4.8	24
p-amino-benzamidine	CMC	2	4.75	5
Folic acid	EDC	5	6.0	2
Mannosylamine	EDC	19	4.5 to 6.0	24

Use a concentration of carbodiimide greater than the stoichiometric concentration, usually 10- to 100-fold greater than the concentration of spacer groups.

The coupling reaction is normally performed in distilled water adjusted to pH 4.5 to 6.0 to promote the acid-catalyzed condensation reaction. Blocking agents are not usually required after the coupling reaction if excess ligand has been used.

Always use freshly prepared carbodiimides.

Coupling buffer:	Dissolve the carbodiimide in water and adjust to pH 4.5
Wash buffer:	100 mM acetate, 500 mM NaCl, pH 4.0

Avoid the presence of amino, phosphate, or carboxyl groups as these will compete with the coupling reaction.

Preparation of EAH Sepharose 4B

Wash the required amount of matrix on a sintered glass filter (porosity G3) with distilled water adjusted to pH 4.5 with HCl, followed by 500 mM NaCl (80 mL in aliquots/mL sedimented matrix).

Ligand preparation

Dissolve the ligand and adjust to pH 4.5. The optimal concentration depends on the ligand. Organic solvents can be used to dissolve the ligand, if necessary. If using a mixture of organic solvent and water, adjust the pH of the water to pH 4.5 before mixing it with the organic solvent. Solvents such as dioxane (up to 50%), ethylene glycol (up to 50%), ethanol, methanol, and acetone have been used.

If organic solvents have been used, use pH paper to measure pH since solvents can damage pH electrodes.

Ligand coupling

1. Add the ligand solution followed by the carbodiimide solution to the matrix suspension and leave on an end-over-end or similar mixer. Use a matrix: ligand solution ratio of 1:2 to produce a suspension that is suitable for coupling. Typically the reaction takes place overnight either at 4 °C or room temperature.
2. Adjust the pH of the reaction mixture during the first hour (pH will decrease) by adding 100 mM sodium hydroxide.
3. Wash at pH 8.0 and pH 4.0 to remove excess reagents and reaction by-products.

If a mixture of aqueous solution and organic solvent has been used, use this mixture to wash the final product as in Step 3. After Step 3 wash in distilled water, followed by the binding buffer to be used for the affinity purification.

Do not use magnetic stirrers as they can disrupt the Sepharose matrix.

Storage

Store preactivated matrices 4 °C to 8 °C in 20% ethanol.

Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, see Appendix 8, or 20% ethanol in a suitable buffer.

The pH stability of the chromatography medium when coupled to a ligand will depend upon the stability of the ligand.

Performing a separation

See Binding and elution conditions earlier in this chapter for a preliminary separation protocol and Chapter 2 for general guidelines.