18. AFFINITY CHROMATOGRAPHY

Affinity Chromatography is a highly selective separation technique based on the reversible and specific interaction between a biomolecule (e.g., enzyme, antibody, protein, nucleic acid) and an immobilized ligand. This method exploits biological recognition—such as antigen–antibody, enzyme–substrate, receptor–ligand, or lectin–carbohydrate interactions—to isolate a target molecule from complex mixtures with exceptional purity. Because of its specificity, affinity chromatography is widely used in biotechnology, pharmaceuticals, diagnostics, immunology, and protein engineering.

Classification of Affinity Chromatography

  • Bioaffinity Chromatography: Uses biological ligands such as antibodies, enzymes, or receptors.
  • Immunoaffinity Chromatography: Uses antigen–antibody specificity.
  • Lectin Affinity Chromatography: Uses lectins to purify glycoproteins.
  • Dye-Ligand Affinity Chromatography: Uses synthetic ligands like Cibacron Blue.
  • Metal Chelate Affinity Chromatography (IMAC): Uses metal ions (Ni²⁺, Co²⁺) to purify His-tagged proteins.

Common Terms in Affinity Chromatography

  • Ligand: A molecule immobilized on the matrix that binds selectively to the target.
  • Matrix/Support: Solid backbone (e.g., agarose, polyacrylamide) that holds the ligand.
  • Spacer Arm: A molecular arm used to distance the ligand from the matrix for better binding.
  • Affinity Medium: Combination of matrix, spacer, and ligand.
  • Elution: Process of releasing the bound target molecule.

Principle

The principle of affinity chromatography is based on highly specific biological interactions. When a sample passes through a column containing an immobilized ligand:

  • The target molecule binds specifically to the ligand.
  • Other non-binding molecules pass through without retention.
  • The bound molecule is then eluted using changes in pH, ionic strength, or by introducing a competitive ligand.

The binding–elution cycle is reversible, allowing recovery of purified biomolecules with minimal damage. This precision makes affinity chromatography one of the most efficient purification techniques.

Components of Affinity Medium

1. The Support (Matrix)

The matrix acts as the solid backbone on which ligands are immobilized. Ideal characteristics include:

  • Chemical and mechanical stability
  • Hydrophilicity to prevent nonspecific binding
  • Low nonspecific adsorption
  • Proper pore size to allow biomolecule entry
  • Compatibility with a wide pH range

Common matrices include agarose (Sepharose), dextran (Sephadex), polyacrylamide, and silica.

2. Spacer Arms

Spacer arms are molecular chains between the ligand and matrix. Their purpose is to:

  • Reduce steric hindrance
  • Improve accessibility of the ligand
  • Enhance binding efficiency

Spacer arm length and chemistry are chosen based on ligand size and binding requirements.

3. The Ligand

The ligand is the key functional component that binds selectively to the target molecule. Ideal ligand properties include:

  • High specificity and affinity
  • Chemical stability
  • Ability to undergo reversible binding
  • Ease of immobilization

Ligands may be biological (antibodies, enzymes, receptors) or synthetic (dyes, chelating compounds).

Steps Involved in Affinity Chromatography

1) Purification Steps

  • Column Equilibration: Buffer is passed through to prepare the column environment.
  • Sample Application: Crude mixture containing the target molecule is introduced.
  • Binding: Target selectively binds to immobilized ligand.
  • Washing: Unbound and weakly bound impurities are removed.
  • Elution: Target is released using specific conditions.
  • Regeneration: Column is cleaned to restore activity for reuse.

2) Media Selection

Media selection depends on:

  • Type of ligand
  • Nature of target biomolecule
  • pH and buffer compatibility
  • Physical stability of matrix

Choosing the right combination ensures effective binding and high recovery.

3) Media and Buffers

Buffers must maintain ligand stability, target structure, and binding conditions. Typical parameters include:

  • pH (close to physiological for protein stability)
  • Ionic strength
  • PRESENCE of cofactors (for enzyme–substrate interactions)

Special buffers like phosphate, Tris-HCl, acetate, or saline buffers are commonly used.

4) Sample Preparation and Application

Sample preparation ensures efficient binding and minimal column blockages. Requirements include:

  • Clarification by filtration or centrifugation
  • Adjusting pH and ionic strength
  • Removing interfering substances

Proper sample handling increases yield and enhances purification efficiency.

5) Elution Methods

Elution involves breaking ligand–target interactions. Methods include:

  • pH change: Alters ionization and weakens binding.
  • Ionic strength change: High salt disrupts electrostatic interactions.
  • Competitive elution: A molecule similar to the target displaces it.
  • Affinity elution: Target is released by specific cofactors or substrates.
  • Chaotropic agents: Weaken hydrophobic or hydrogen bonding.

6) Analysis of Results

Eluted fractions are analyzed using:

  • UV–Visible absorbance
  • SDS-PAGE for protein purity
  • Activity assays for enzymes

Chromatograms provide peak profiles for qualitative and quantitative interpretation.

7) Equipment for Purification

Affinity chromatography can be performed manually or on automated purification systems. Equipment includes:

  • Affinity column
  • Peristaltic or chromatography pumps
  • Fraction collector
  • UV detectors
  • Automated FPLC systems

Advantages of Affinity Chromatography

  • Highly specific and selective
  • Produces high-purity biomolecules
  • Suitable for complex mixtures
  • Low sample dilution during purification
  • Can isolate proteins in active form

Disadvantages of Affinity Chromatography

  • High cost of ligands and columns
  • Ligand leakage may contaminate product
  • Limited lifespan of affinity media
  • Requires careful optimization of binding and elution conditions

Applications of Affinity Chromatography

1. Immunoglobulin Purification (Antibody Immobilization)

Affinity chromatography is extensively used to purify antibodies, using antigens or Protein A/G/L ligands immobilized on the matrix.

2. Recombinant Tagged Proteins

His-tagged proteins are purified using nickel or cobalt IMAC columns, providing high purity in a single step.

3. Protein A, G, and L Purification

These proteins bind specifically to immunoglobulins and are widely used for antibody purification workflows.

4. Biotin and Biotinylated Molecules

Affinity methods isolate biotin-containing molecules using avidin or streptavidin ligands.

5. Affinity Purification of Albumin and Macroglobulin Contaminants

Serum protein contaminants can be selectively removed using specialized ligands.

6. Lectin Affinity Chromatography

Used to purify glycoproteins based on carbohydrate–lectin interactions.

7. Determination of Dissociation Constant

Affinity chromatography helps estimate ligand–target binding strength (Kd), important for drug discovery and protein engineering.

Detailed Notes:

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