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Chromatographic separation technologies for product purification

Protein Purification

Protein purification is the process of extracting the purest form of protein or concentrating it from a crude mixture. The extent of purification is determined by its purpose in an industrial setting. A relatively impure sample is sufficient, for an enzyme that is to be used in a washing powder, provided it does not contain any inhibiting activities. If the protein is aimed for therapeutic use it must be extremely pure and purification must then be done in several subsequent steps.

The earlier method of purification was based on their relative solubility. Fractional precipitation is still used for separation of gross impurities, membrane proteins and nucleic acids. Calcium phosphate gels are used to specifically absorb proteins from heterogeneous mixtures. Owing to their high resolving power, different chromatographic techniques are now being used for protein purification.

Chromatography

Chromatography refers to the separation techniques that involve a deceleration of molecules with respect to the solvent front that progresses through the material. The different characteristics of a number of proteins, such as size, charge, hydrophobicity and biospecific interaction are used to purify proteins from one another.  

The first step in a typical chromatography process is a capturing step, where the product binds to the adsorbent while the impurities do not. Then the weakly bound proteins are washed off before the conditions are changed so that the target protein is eluted. Different types of liquid chromatography, differing primarily in types of stationary phase are used.

Typical Paper Chromatography

Ion Exchange Chromatography

The basis of Ion Exchange Chromatography is ionic interactions and the separation of proteins takes place as a result of the interactions between proteins with different surface charges and the oppositely charged groups on an ion exchanger adsorbent. One of the first processes of ion exchange purification is credited to Moses, who purified acrid water with the help of a special type of wood. Primitive ion exchangers were made of hydrophobic polymer matrices, with substituted ionic groups.

Proteins are Zwitter ions that carry both positive and negative charges. The Isoelectric point (pI) of a protein is a measure of the ionizable amino acids present within. Free N- terminal amines contribute to a positive charge below a pH 8 And C-terminal carboxyl Groups contribute to a negative charge above ph6. Usually the charged groups are present on the protein surface. The charge of the protein is the overall influence of  the charges of the individual amino acids. Net charge and ionic strength are the factors that influence the interaction between a protein and ion exchange. Structural changes affect the separation by Ion exchange chromatography because the chromatographic behaviour also depends on protein conformation.

The strength of Ion exchangers also influence the separation and they are usually classified as weak or strong. The strength is calculated on the basis of the pKa values and does not say anything about the strength of the interaction. The functional groups of strong Ion exchangers are sulphonates quaternary ammonium salts etcetera. The changes in the pH does not affect the change in the charge of Ion exchanger. Weak Ion exchangers have functional ionic groups such as carboxylate and diethyl ammonium. Thus the separation of weakly ionizable proteins requiring a very high pH or very low pH for ionization can only be done by a strong exchanger. The extent of adsorption depends on the pH and the conductivity of the running buffer. pH and conductivity is directly proportional to optimal charge of the protein to be chosen. Separation is also influenced by the choice of buffering ions.

Ion Exchange

Chromatographic techniques based on hydrophobicity

Depending on the hydrophobicity of proteins, they can be separated by using two different methods: Hydrophobic Interaction Chromatography and Reverse Phase Chromatography.

Hydrophobic Interaction Chromatography

The  basis of this technique is the reversible interaction between a protein and the chromatographic adsorbent which are of hydrophobic nature. In Hydrophobic Interaction Chromatography separation of proteins is according to differences in the number of exposed hydrophobic amino acids. The crude protein mixture is loaded onto a chromatographic column with a buffer that contains high concentration of salt in order to facilitate hydrophobic interactions. High concentrations of neutral salts were observed to enhance the binding of proteins onto the adsorbent. The elution of the bound proteins was achieved by washing the column with a salt free buffer. The washing can also be done by decreasing the polarity of the eluent.

The most extensively used stationary phase for Hydrophobic Interaction chromatography is Agarose. Silica and organic polymer resins are also used. The elution is performed at low ionic strength and hence the adsorbent must preferably be free of charge in order to inhibit ionic interactions between protein and column. The amount of immobilized ligands will increase the protein binding capacity. The most widely used ligands for this technique are linear chain alkanes with or without a terminal amino group. This technique requires certain salt ions which take up the ordered water molecules and hence promote hydrophobic interactions. Adsorption of proteins to the adsorbent is also dependent on the pH of the buffer used in this technique. An increase in pH value has an inhibiting effect on the hydrophobic interaction between proteins and hydrophobic ligands. The reason for this is the hydrophilicity which is promoted by the change in the charge of protein. The temperature of the column is directly proportional to the adsorbing nature of the proteins, hence, labile proteins must be eluted at lower temperatures.

Reversed Phase Chromatography

This technique is also based on the interactions between hydrophobic ligands which are covalently bound to the adsorbent and the hydrophobic molecules in the mobile phase. Adsorbent reversed phase chromatography in an order of magnitude is more highly substituted with hydrophobic ligand than Reverse Phase Chromatography. This technique is efficient enough to adsorb proteins even in pure water. The interactions are very strong and hence requires the use of organic solvents and other addictive chemicals in order to obtain the protein from the column. Hence the risk of denaturation of protein is an added disadvantage to this technique. The basic molecular interactions of this technique is very similar to Hydrophobic Interaction Chromatography.

Silica beads with modified hydroxyl groups attached to the ligand is the most common base matrix for Reverse Phase Chromatography gels. The mechanical strength and chemical stability of silica beads in organic solvents is the reason for its use in Reverse Phase Chromatography. The silica particles should be covered with chemically bonded hydrocarbon chains in order to obtain strong hydrophobic interaction. This represents the hydrophobic phase. Proteins and peptides adsorb on the stationary phase and are much less sensitive. Short chain hydrocarbons are used for longer polypeptide chains and long aliphatic hydrocarbons are used for smaller peptides polypeptides. The mobile phases used in this technique are aqueous with a high degree of organised water structure surrounding the column. The column is hydrophobic in nature. This results in protein binding that is strong and requires the use of organic solvents in the mobile phase. Organic solvents lowers the polarity of the mobile phase. Polarity of the mobile phase is inversely proportional to the extent of elution.

Hydrophobic repulsion in a peptide

Affinity Chromatography

A protein has binding sites with complementary surfaces to the ligands. This binding may be due to electrostatic or hydrophobic interactions or other molecular interactions like vander Waals forces and hydrogen bonds. Affinity chromatography derives its name from the biological affinity of proteins which is used for its purification. A ligand with a particular specificity is covalently bonded to an inert chromatographic column under the favourable conditions. Only the intended protein is then adsorbed onto the column and the other substances are washed away. To elute the target molecule the experimental condition is reversed such that the protein ligand interaction does not take place.

Affinity Chromatography

The gel material to be used in the stationary phase must have certain characteristics such as a suitable chemical group to which the ligand is covalently bonded. The chemicals used during the derivation of the protein must be chemically and mechanically stable. Inert solvent and buffer should be employed in this process especially during the elution of the protein. The Matrix should be macroporous, hydrophilic and neutral. Affinity purification requires a certain biospecific ligand that can be attached to the matrix. The ligands must be selectively chosen to bind only to a single or a very small number of proteins. To prepare the affinity adsorbent the ligand should be compatible with the solvents that is used during the procedure and should possess at least one functional group by which it can be immobilized on the matrix. The most common functional groups used for this are amines, thiols, carbohydroxides and hydroxyl groups.

Immobilized Metal Affinity chromatography

This Technique depends on the formation of weak coordinate bonds between the metal ions that are immobilized and some amino acids which are present on proteins and mainly on the histidine residues. Immobilized Metal Affinity chromatography can be considered as pseudo-affinity technique since it does not operate through a biospecific interaction. The interaction used in Immobilized Metal Affinity chromatography depends upon the formation of coordinated complexes between metal Ions and electron donor groups on the surface of the protein. However the residues must be present on the protein surface for efficient coordination and the extent of interaction depends on the number of such coordinations.

Certain spacer arm and chelator is attached to the matrix. Some commonly used chelators include imino diacetate (IDA), nitro triacetic acid (NTA) and tris ethylenediamine (TED). The predominantly used stationary phase is a beaded Agarose. The extent of affinity of a protein to a metal chelate depends on the metal Ion that is involved during coordination. The most commonly used ions are the divalent ions of Iron, Cobalt, Nickel, Copper, and Zinc and the trivalent ions of metals like Iron and aluminium are rarely used. The metal column process and overall charge as the chelates are negative and the metals have a positive; they do not always cancel out. Hence buffers of higher ionic strength are used in order to reduce the non specificity of absorption.

Size Exclusion Chromatography

Size Exclusion Chromatography

Size Exclusion Chromatography is slightly different from the other chromatographic techniques. Most of the chromatographic techniques used in protein purification involve techniques that separate proteins according to some property, that allows the interaction to the matrix. In Size Exclusion Chromatography the matrix is made up of porous particles and the separation is achieved according to the shape and size of the molecules. This technique is also known as gel filtration, Molecular Sieve Chromatography and Gel Permeation Chromatography.

For separation of molecules in Size Exclusion Chromatography porous materials with molecular sieve properties are used. The matrices of this technique have a range of Beads with different sizes of pores. The Separation depends on the ability of various proteins to enter a few or none of the channels in the beads. The molecules run through the Size Exclusion Chromatographic column according to molecular weight and it is more accurate. The extent of diffusion of the molecules depends on the hydrodynamic volume, which is the volume that is created by the movement of the molecules in water. Hydrodynamic volume is different from molecular weight by shape of the proteins. Proteins are generally globular molecules while DNA and other polysaccharides tend to be linear.

The Matrix used in Size Exclusion Chromatography are composed of polymers like agarose or Dextrin and may also be composed of synthetic Polymers like polyacrylamide gels. These gels are generally formed by the cross linking of polymers to form a three dimensional network. The surface of these gels contain predominantly hydroxyl groups and provide good conditions for hydrophilic proteins. The hydrophilicity is reduced by the cross linking reagents. The selection of gel with a suitable separation range must be considered. Rapid desalting procedures require smaller pores for peptide purification. The pores should have a carefully controlled range of sizes for reproducible separation of proteins. In contrast to other techniques the mobile phase of Size Exclusion Chromatography is not adjustable by changing its composition. Ideally there is no adsorption involved and the mobile phase is considered as a carrier phase. The sample may require a buffer solution with a defined pH and ionic composition to preserve its structure and biological activity.

See Also

• Elution
• Mobile Phase
• Molecular sieve
• Immunoaffinity
• Glowmatography
• Purnell equation
• Van Deemter equation
• Weak affinity chromatography