Hydrophobic Interaction Chromatography (HIC)

Hydrophobic Interaction Chromatography (HIC) is a chromatographic technique used for separation and purification of proteins, peptides, enzymes and monoclonal antibodies. It separates molecules according to their surface hydrophobic nature.

In this method the stationary phase has hydrophobic groups attached to it. These groups may be phenyl, butyl, octyl or other alkyl groups. These groups bind with hydrophobic regions present on the surface of protein.

The sample is added to the column in high salt condition. High salt removes some water layer around the protein and increases hydrophobic interaction. So the protein binds with the hydrophobic matrix.

After binding, the proteins are eluted by decreasing the salt concentration. When salt becomes low, the hydrophobic interaction becomes weak. The proteins then come out from the column.

Less hydrophobic proteins come out first. More hydrophobic proteins remain bound for longer time and elute later. In this way different proteins are separated from each other.

This method is done in mild aqueous condition. So the natural structure and activity of proteins are mostly maintained. It is used in protein purification, enzyme purification and antibody purification.

The basic idea of this method is related to salting out. Arne Tiselius first observed that proteins bind at high salt concentration below their precipitation point. Later Stellan Hjertén developed this method as Hydrophobic Interaction Chromatography.

Principle of Hydrophobic Interaction Chromatography (HIC)

Principle of Hydrophobic Interaction Chromatography (HIC) is based on hydrophobic interaction between protein and hydrophobic group present on the stationary phase. The separation takes place according to the surface hydrophobicity of protein.

In this method the column matrix contains hydrophobic ligands. These ligands may be phenyl, butyl, octyl or other alkyl groups. These groups are non-polar in nature.

The protein sample is applied in high salt buffer. Generally ammonium sulfate is used. High salt condition reduces the water covering around the hydrophobic part of protein.

So the hydrophobic regions of protein become exposed. These exposed regions bind with hydrophobic ligands of the column matrix. The protein having more hydrophobic surface binds more strongly.

The elution is done by lowering the salt concentration. When salt concentration is decreased, water again surrounds the hydrophobic regions of protein. So the binding becomes weak.

The proteins then leave the column. Less hydrophobic proteins come out first. More hydrophobic proteins come out later.

Thus Hydrophobic Interaction Chromatography separates proteins without denaturing them. The reaction is done in aqueous condition, so the natural structure and activity of protein is maintained.

Parts of Hydrophobic Interaction Chromatography (HIC)

The following are the main parts of Hydrophobic Interaction Chromatography (HIC)–

1. Stationary phase
   It is a porous matrix present inside the column. It may be made up of cross-linked agarose or synthetic polymer. Hydrophobic groups are attached to this matrix.
2. Hydrophobic ligand
   These are the non-polar groups fixed on the stationary phase. Common ligands are butyl, octyl and phenyl groups. These ligands bind with hydrophobic patches of protein.
3. Mobile phase
   It is the buffer system which passes through the column. It contains high salt buffer for binding and low salt buffer for elution.
4. Binding buffer
   It is a high salt buffer used during sample loading. Generally ammonium sulfate is used. This buffer helps the protein to bind with hydrophobic ligand.
5. Elution buffer
   It is a low salt buffer used for removing the bound protein from the column. When salt concentration is decreased, the protein leaves the stationary phase.
6. Sample
   The sample contains the molecule which is to be separated. It may contain proteins, peptides, enzymes or monoclonal antibodies.
7. Chromatography column
   It is the main part where separation takes place. The column contains the stationary phase media. It may be pre-packed or manually packed.
8. Pump
   Pump is used to pass the buffer through the column. It maintains constant flow rate and helps in mixing of buffers during elution.
9. Sample injector
   It is used for loading the sample into the system. It may contain sample loop, injection valve or loading valve.
10. Detector
    Detector is used to detect the protein coming out from the column. UV-Visible detector is commonly used because proteins absorb at 280 nm.
11. pH monitor
    It is used to check the pH of buffer during the run. Change in pH may affect protein binding and elution.
12. Conductivity monitor
    It is used to measure salt concentration in the running buffer. It helps to follow the decrease of salt during elution.
13. Fraction collector
    It is used to collect the separated protein fractions. The eluted sample is collected in tubes or microplates for further analysis.

Types of Hydrophobic Interaction Chromatography (HIC)

The following are the types of Hydrophobic Interaction Chromatography (HIC)–

A. Based on ligand chemistry

1. Straight-chain alkyl HIC
   In this type alkyl ligands are attached with the stationary phase. The ligands may be methyl, ethyl, propyl, butyl and octyl groups. The separation is mainly due to hydrophobic interaction.
2. Aromatic aryl HIC
   In this type aromatic ligands are attached with the matrix. Common ligands are phenyl and benzyl groups. It gives hydrophobic interaction and also π-π interaction with some molecules.

B. Based on operational mode

1. Bind and elute mode
   In this type the protein is first bound with the resin in high salt condition. Then it is eluted by decreasing the salt concentration.
2. Flow-through mode
   In this type the target protein does not bind with the resin. It comes out in flow-through. The more hydrophobic impurities and aggregates remain attached with the resin.

C. Based on elution strategy

1. Gradient elution HIC
   In this type the salt concentration is decreased slowly and continuously. It is used for better separation of complex protein mixture.
2. Step elution HIC
   In this type the salt concentration is decreased in fixed steps. It is faster and also uses less buffer than gradient method.

D. Based on base matrix material

1. Carbohydrate-based HIC
   In this type the base matrix is made up of carbohydrate material. Cross-linked agarose is commonly used. It has open porous structure and is suitable for protein separation.
2. Synthetic polymer-based HIC
   In this type synthetic polymer matrix is used. Polystyrene-divinylbenzene (PS-DVB) is one example. It is rigid and can be used at high flow rate.

Protocol of Hydrophobic Interaction Chromatography (HIC)

The following are the steps of Hydrophobic Interaction Chromatography (HIC)–

1. The work is done mostly at 4°C. This temperature helps to protect the protein from degradation.
2. At first high salt binding buffer is prepared. Generally ammonium sulfate containing buffer is used for binding of protein with column matrix.
3. Low salt elution buffer is also prepared. This buffer is used later for elution of bound protein from the column.
4. The protein sample is mixed with high salt buffer. This makes the sample suitable for binding with hydrophobic ligand of the column.
5. The sample is filtered through 0.45 μm filter. This removes dust, precipitate and other particulate matter from the sample.
6. The chromatography system is made ready before the run. The pump, tubing and buffer lines are filled properly with buffer.
7. The HIC column is first washed with low salt buffer. Then the column is equilibrated with high salt start buffer.
8. Equilibration is continued until pH, conductivity and UV baseline become stable. This shows that the column is ready for sample loading.
9. The prepared protein sample is loaded into the column. The loading may be done by sample loop or loading valve.
10. In high salt condition, hydrophobic regions of protein bind with hydrophobic ligands present on the stationary phase.
11. After sample loading, elution is started by decreasing the salt concentration. This may be done by descending salt gradient.
12. As the salt concentration becomes low, the hydrophobic interaction becomes weak. The bound proteins then start to leave the column.
13. The eluting proteins are monitored by detector. UV absorbance at 280 nm is commonly used for protein detection.
14. The separated proteins are collected in different tubes by fraction collector. Each fraction may contain different protein.
15. The collected fractions are tested by SDS-PAGE, ELISA or western blotting. This is done to check the purity and identity of target protein.
16. After the run, the column and system lines are washed with deionized water. This removes remaining salt from the column and tubing.
17. The column is then stored in suitable storage buffer. This prevents drying of column and keeps the column ready for next use.

Factors that Affect Hydrophobic Interaction Chromatography (HIC)

Factors that Affect Hydrophobic Interaction Chromatography (HIC)

The following are the factors that affect Hydrophobic Interaction Chromatography (HIC)–

• Ligand type
  The type of ligand present on the matrix affects the separation. Butyl, octyl and phenyl groups give different hydrophobic interaction with protein.
• Alkyl ligand
  Straight chain alkyl ligands like butyl and octyl mainly show hydrophobic binding. Longer chain ligand usually gives stronger binding.
• Aryl ligand
  Aromatic ligand like phenyl gives hydrophobic interaction and also some π-π interaction. So it may separate some closely related proteins in different way.
• Ligand density
  The amount of ligand attached on the matrix affects binding capacity. More ligand density increases binding of protein.
• Excess ligand density
  Too much ligand on the matrix may bind protein very strongly. The protein may attach at many points and then elution becomes difficult.
• Base matrix
  The base matrix affects porosity and stability of the column. Cross-linked agarose and synthetic polymer are commonly used matrix.
• Matrix porosity
  Good porous matrix allows protein to enter and bind properly. Poor porosity may reduce binding and separation.
• Salt type
  The type of salt is very important in HIC. Ammonium sulfate is commonly used because it strongly promotes hydrophobic interaction.
• Salt concentration
  High salt concentration increases binding of protein with hydrophobic ligand. But very high salt may precipitate the protein.
• Protein solubility
  Some proteins are not stable in high salt condition. If precipitation occurs, the salt concentration should be reduced.
• Temperature
  Temperature affects hydrophobic interaction. Higher temperature usually increases binding of protein with stationary phase.
• pH
  pH changes the surface charge of protein. It can affect binding near the hydrophobic regions and may change separation pattern.
• Organic modifier
  Mild organic solvent like isopropanol may be added in low amount. It weakens hydrophobic interaction and helps in elution of strongly bound protein.
• Gradient slope
  Salt gradient affects peak shape and separation. Shallow gradient gives better separation, but peak may become broad.
• Flow rate
  Flow rate affects binding and elution. Very fast flow may reduce binding and poor separation may occur.
• Sample load
  Too much sample may overload the column. This gives broad peak and poor resolution.

Uses of Hydrophobic interaction chromatography

The following are the uses of Hydrophobic Interaction Chromatography (HIC)–

• It is used for purification of proteins, peptides and other biomolecules. The separation is done according to surface hydrophobicity.
• It is used for purification of enzymes. The native structure and activity of enzyme is mostly maintained because mild aqueous condition is used.
• It is used in monoclonal antibody production. It helps in polishing step after main purification.
• It is used for removal of process impurities. Host cell proteins, leached Protein A and some viral impurities can be removed by this method.
• It is used for removal of protein aggregates. Aggregated proteins have more exposed hydrophobic regions, so they bind differently from normal protein monomers.
• It is used for separation of monomer and high molecular weight aggregates. This is important in therapeutic protein preparation.
• It is used for study of antibody-drug conjugates (ADCs). Different ADC species are separated according to number of hydrophobic drug molecules attached with antibody.
• It is used for determination of drug-to-antibody ratio (DAR). The different peaks show different drug loaded antibody forms.
• It is used for studying protein folding. Folded and partially unfolded proteins show different hydrophobic surface and bind differently with HIC resin.
• It is used for studying protein stability and aggregation. It helps to know whether the protein has hydrophobic region exposed or not.
• It is used for studying protein-ligand interaction. Change in hydrophobic behaviour can show binding of ligand with protein.
• It is used in protein engineering. Engineered proteins are screened for desired hydrophobic property and unwanted hydrophobic nature.
• It is used in proteomics study. With suitable volatile salt and mass spectrometry, intact proteins and modified proteins can be studied.
• It is used for biomarker discovery. Native-like proteins and their post-translational changes can be separated and analysed.

Advantages of Hydrophobic interaction chromatography

The following are the advantages of Hydrophobic Interaction Chromatography (HIC)–

• It is carried out in mild aqueous condition. So the native structure and biological activity of protein is maintained.
• It does not require strong organic solvent. So sensitive proteins and enzymes are not denatured easily.
• It gives good separation of biomolecules. The separation is based on surface hydrophobicity of the molecule.
• It is useful for separating target protein from complex protein mixture. Proteins having different hydrophobic surface bind differently with the resin.
• It removes impurities from protein preparation. Misfolded proteins, aggregates, host cell proteins and product variants can be removed.
• It is useful as polishing step in protein purification. It works by hydrophobicity, so it is different from ion exchange and affinity chromatography.
• It can be used in small laboratory scale and also in industrial scale. So it is useful in research work and biopharmaceutical production.
• It can be done by bind and elute mode. In this method protein first binds with the resin and then elutes by lowering salt concentration.
• It can also be done by flow-through mode. In this method target protein comes out and more hydrophobic impurities remain bound with the column.
• It is simple to perform. The sample preparation is not very complicated and the method can be adjusted easily.
• It is useful for purification of proteins which do not have affinity tag. Such proteins can be separated by their hydrophobic nature.
• It can also concentrate protein during purification. The bound protein is eluted in smaller volume.
• The selectivity of separation can be changed. Salt type, salt concentration and pH can be adjusted according to protein behaviour.
• It gives good recovery when proper condition is used. The protein remains active and useful for further study.

Limitations of Hydrophobic interaction chromatography

The following are the limitations of Hydrophobic Interaction Chromatography (HIC)–

• Proteins having very low hydrophobicity may not bind properly with the hydrophobic matrix. So separation may become poor.
• High salt concentration is needed for binding of protein. This may affect solubility of some proteins.
• Some proteins may precipitate in high salt condition. So salt concentration should be controlled carefully.
• It is less specific than affinity chromatography. It separates proteins by hydrophobic nature, not by a particular binding site.
• Non-specific binding may occur in the column. Some unwanted proteins may also bind with the hydrophobic ligand.
• Column fouling may occur due to strong binding of impurities. This reduces column performance and column life.
• Method development is not always simple. Salt type, salt concentration, pH and flow rate must be selected properly.
• Temperature affects the separation. A method working at room temperature may not give same result in cold room.
• It is not fully suitable with mass spectrometry (MS) in traditional method. High amount of non-volatile salt creates problem in MS analysis.
• Large volume of high salt waste is produced after the run. This waste needs proper disposal.
• High salt buffer may cause corrosion of some metal parts. So the instrument and tubing should be washed properly after use.
• Some sensitive proteins may lose stability during high salt exposure. This can decrease recovery of active protein.

Precautions of Hydrophobic Interaction Chromatography

The following are the precautions of Hydrophobic Interaction Chromatography (HIC)–

• The temperature should be maintained constant during the run. Usually the work is done at 4°C or on ice to prevent degradation of protein.
• Sudden change in temperature should be avoided. It may change the hydrophobic interaction and affect the separation pattern.
• Salt concentration should be selected carefully. High salt is needed for binding, but too much salt may precipitate the target protein.
• The protein sample should be checked after adding salt. If turbidity or precipitate appears, the salt concentration should be reduced.
• Organic modifier should be used in low amount only. Solvents like isopropanol may denature protein if used in high concentration.
• The concentration of organic solvent should not be very high. Otherwise the separation may become like reversed phase chromatography.
• Air bubbles should not enter into the column. Bubbles disturb the flow and may give irregular peak.
• All tubing and fittings should be connected properly. Loose connection may cause leakage and air entry into the system.
• The column and buffer lines should be flushed before use. This removes trapped air and makes the system ready for running.
• Back pressure should be observed during the run. Sudden increase in pressure may show column blockage or air block.
• Conductivity, pH and UV tracing should be monitored. These parameters help to know whether binding and elution are going properly or not.
• The column should be washed after use. Remaining salt and bound impurities should be removed from the column.
• Regular cleaning of column should be done. This prevents non-specific binding, column fouling and loss of column performance.
• The column should be stored in proper storage buffer. Drying of resin should be avoided because it damages the column bed.

Troubleshoot of Hydrophobic Interaction Chromatography

The following are the troubleshoot of Hydrophobic Interaction Chromatography (HIC)–

• If the protein does not bind with the column, the hydrophobic interaction is weak. In this case more hydrophobic medium can be used.
• If the protein comes out in flow-through, the salt concentration may be low. The binding buffer may need higher salt concentration.
• If the protein binds very strongly and does not elute, the starting salt concentration should be decreased. This reduces very strong binding with the resin.
• If the protein is not eluted even after lowering salt, less hydrophobic medium can be used. This helps the protein to leave the column more easily.
• If elution is difficult, small amount of organic modifier may be added. Isopropanol is sometimes used in low concentration to weaken hydrophobic binding.
• If peak resolution is poor, the salt gradient should be adjusted. A shallow gradient gives better separation of close peaks.
• If the peak becomes broad, the gradient may be too shallow. A steeper gradient can make the peak sharper and improve sensitivity.
• If retention time changes in different run, temperature may not be constant. HIC is very sensitive to temperature.
• If the run at room temperature and cold room gives different result, the method should be repeated at same temperature. Same temperature gives more reproducible result.
• If protein precipitates during sample loading, salt concentration may be too high. Lowest salt concentration should be used which can still allow binding.
• If protein solubility is poor in high salt, a dual salt system may be tried. Sodium citrate and sodium phosphate can be combined to maintain solubility.
• If closely related variants are not separated, pH of mobile phase should be changed. Change in pH can change surface charge near hydrophobic regions of protein.
• If variant separation is still poor, different salt type can be tested. Different salts may give different selectivity in HIC.
• If column pressure becomes high, the sample or buffer may contain particles. The sample and buffer should be filtered before loading.
• If baseline becomes unstable, air bubbles may be present in the system. The column and tubing should be flushed properly before running.

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