Overview of Chromatography in Biopurification
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Introduction
Chromatography is a method for separating molecules consisting of a stationary phase (silica, beads, membranes), a moving phase (gas or solvent or buffer) and solute molecules that separate due to different residence times in the moving and stationary phases. The different residence times occur by either a partitioning or adsorption mechanism.
Normal phase (polar interaction chromatography usually done on silica or alumina with isocratic solvent elution) and gas chromatography (GC) are two of the earliest forms of chromatography. GC is a good analytical separation method for volatile compounds and not useful for the separation on macromolecules. Normal phase is best used with small molecules that are not water soluble and is only important as a predecessor of reversed phase and to explain the names of these two techniques. A polar stationary phase (silica or alumina) and a non-polar moving phase (hexane, for example) is called normal phase. When chemists bonded a non-polar molecule, C18, to the silica, they reversed the phase, hence the term "reversed phase."
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Types of Chromatography Used for Biopurification
Ion Exchange Chromatography
This separation method is based on the electrostatic attraction between opposite charges on the stationary-phase media and the molecules. Ion-exchange columns are categorized as either strong or weak. Strong ion exchange columns are charged (ionized) at all pH levels while weak ion-exchange columns are only ionized in a certain pH range.
Cation exchange columns have negative charges on their surfaces and therefore exchange cations. Mustang® S membrane is a strong cation exchange column that has sulfonic acid groups that are ionized at all pH values. Mustang C membrane is a weak cation exchange column that has carboxyl groups that are ionized only at pH >4.
Anion exchange columns have positive charges on their surfaces and therefore exchange anions. Mustang Q membrane is a strong anion exchanger with quaternary amine groups that are ionized at all pH levels.
Typically, ion exchange columns are loaded in low ionic strength buffers. Under low ionic strength conditions, appropriately charged macromolecules will be retained on the stationary phase. After washing, the columns are eluted with either step or linear gradients to buffers of higher ionic strength. As the salt concentration of the mobile phase increases, salt molecules in the buffer exchange for the macromolecules on the stationary phase, which then elute. See below for more information.
Hydrophobic Interaction Chromatography (HIC) and Reversed Phase (RP)
These two types of chromatography are closely related. Both involve retention on the basis of hydrophobic interactions.
HIC is water-eluted hydrophobic interaction chromatography. Loading is done in a high concentration of a structure-enhancing salt (cosmotrope) such as 2M ammonium sulfate. Elution is typically achieved with a gradient down to lower ionic strength buffer.
RP is solvent-eluted hydrophobic interaction chromatography. Loading is done in water or low concentrations of a solvent such as acetonitrile or ethanol. Elution is done with a gradient to higher concentrations of solvent (solvents, urea and guanidine are chaotropes, agents that increase chaos and break up protein structure). In both cases, hydrophobic areas of the molecules bind to the hydrophobic ligands on the column. RP is a very high-resolution method that has typically one-fifth the capacity of ion exchange but is very sensitive to variations in structure. RP is a valuable separation tool for peptides and small proteins, but the method tends to be expensive (both the media and solvents), requires an explosion-proof environment, and the media is usually silica-based and cannot be cleaned with the base. Proteins over 100,000 MW are not easily eluted from RP columns because the amount of solvent needed to break up the hydrophobic interaction will usually precipitate the protein. These large proteins and proteins with subunits, such as IgG, are purified with HIC.
Neither HIC or RP are useful for hydrophobic aggregates (proteins aggregated with each other or lipid bound to proteins) because hydrophobic aggregates adsorb as aggregates and elute as aggregates. This is one area where ion exchange can be very valuable. Ion exchange will still bind proteins in the presence of chaotropes, such as urea or solvent, that break up hydrophobic complexes and allow purification of the individual molecules. Molecules do not bind to HIC or RP in the presence of chaotropes.
HIC and RP are orthogonal to ion exchange. Separation techniques are said to be orthogonal when they separate by different principle so that each technique adds a dimension to the separation process and does not merely repeat the selectivity of the other. Ion exchange separates primarily by charge; molecular structure and shape have little to do with selectivity. HIC and RP separate primarily by structure and shape; differences in charge have little to do with selectivity. HIC or RP are usually done after ion exchange because the salt in the ion exchange eluate helps the binding to HIC and RP. Hydrophobic interactions are stronger in salt solutions than in water.
Affinity Chromatography
Affinity chromatography uses a ligand attached to the media with binding affinity for either a specific molecule or a class of molecules. Researchers frequently use affinity columns with antibodies attached to bind one specific molecule or columns with antigenic proteins attached that bind a specific antibody. Protein ligands are not ideal for production, however. They must be pharmaceutically pure before being bound to the column. They are expensive. Proteins can be irreversibly denatured by cleaning solutions. Proteins will not tolerate sanitizing conditions.
Other types of affinity columns have lectins attached that will bind certain carbohydrates, but these are not used in production because most lectins are highly poisonous. The best type of ligand for affinity chromatography would be a synthetic molecule that is stable, safe, inexpensive, has good specificity, and allows gentle elution. Some dyes have been used in this way, but the problem so far has been a lack of specificity. The one form of affinity chromatography that is commonly used in production uses Protein A as a ligand for purification of immunoglobulins.
Protein A
Protein A is a cell wall protein from Staphlococcus aureas with affinity for the Fc region of IgG. For this reason it is used extensively for IgG purification. There is very little method development with Protein A; simply bind, wash and elute. Binding is usually done at pH 7-9 and elution is usually at pH 3. One of the potential problems is that pH 3 can denature or partially denature the IgG. Companies are avoiding this by selecting clones of monoclonal IgG's that are stable, and immediately raising the pH while the IgG is being collected. The other main problem is price; Protein A columns are far more expensive than conventional ion exchange columns. The other common means of IgG purification is ion exchange followed by HIC.
Metal Chelate Chromatography
This form of purification is based on an immobilized metal ion that has affinity for a chain of histidines that are added to the target protein by recombinant molecular biological techniques to create a fusion protein. The protein is eluted with imidazole (the side chain of histidine) and then the chain of histidines are removed by an enzyme. This is very common in research labs but not yet common in production.
Size Exclusion Chromatography (SEC) or Gel Filtration or Gel Permeation Chromatography (GPC)
Separation is based on the size of the molecules (hydrodynamic volume = the volume created by the movement of the molecule in water) not the molecular weight. The difference between molecular weight and hydrodynamic volume is shape. Proteins tend to be globular molecules while DNA or polysaccharides tend to be linear molecules. Linear molecules have much larger hydrodynamic volumes than globular molecules, so a 10,000 MW DNA molecule will elute much earlier than a 10,000 MW protein. The media is typically porous beads, with the size of the pore determining the exclusion limit of the column, hence size exclusion. The elution is isocratic, so no gradient pumping systems are needed. Large, excluded molecules elute first. Then molecules that are "included" (can penetrate the pores) come off, large followed by small, and salts and other small molecules elute last.
While SEC is easy to understand and perform, this separation method gives the least resolution with the lowest capacity and largest dilution of the sample with respect to all other forms of chromatography. If you are trying to separate two molecules that are both included, then loading is 2-3% of the column volume and the molecular weights should differ by a factor of 2.
This separation method is based on the electrostatic attraction between opposite charges on the stationary-phase media and the molecules. Ion-exchange columns are categorized as either strong or weak. Strong ion exchange columns are charged (ionized) at all pH levels while weak ion-exchange columns are only ionized in a certain pH range.
Cation exchange columns have negative charges on their surfaces and therefore exchange cations. Mustang® S membrane is a strong cation exchange column that has sulfonic acid groups that are ionized at all pH values. Mustang C membrane is a weak cation exchange column that has carboxyl groups that are ionized only at pH >4.
Anion exchange columns have positive charges on their surfaces and therefore exchange anions. Mustang Q membrane is a strong anion exchanger with quaternary amine groups that are ionized at all pH levels.
Typically, ion exchange columns are loaded in low ionic strength buffers. Under low ionic strength conditions, appropriately charged macromolecules will be retained on the stationary phase. After washing, the columns are eluted with either step or linear gradients to buffers of higher ionic strength. As the salt concentration of the mobile phase increases, salt molecules in the buffer exchange for the macromolecules on the stationary phase, which then elute. See below for more information.
Hydrophobic Interaction Chromatography (HIC) and Reversed Phase (RP)
These two types of chromatography are closely related. Both involve retention on the basis of hydrophobic interactions.
HIC is water-eluted hydrophobic interaction chromatography. Loading is done in a high concentration of a structure-enhancing salt (cosmotrope) such as 2M ammonium sulfate. Elution is typically achieved with a gradient down to lower ionic strength buffer.
RP is solvent-eluted hydrophobic interaction chromatography. Loading is done in water or low concentrations of a solvent such as acetonitrile or ethanol. Elution is done with a gradient to higher concentrations of solvent (solvents, urea and guanidine are chaotropes, agents that increase chaos and break up protein structure). In both cases, hydrophobic areas of the molecules bind to the hydrophobic ligands on the column. RP is a very high-resolution method that has typically one-fifth the capacity of ion exchange but is very sensitive to variations in structure. RP is a valuable separation tool for peptides and small proteins, but the method tends to be expensive (both the media and solvents), requires an explosion-proof environment, and the media is usually silica-based and cannot be cleaned with the base. Proteins over 100,000 MW are not easily eluted from RP columns because the amount of solvent needed to break up the hydrophobic interaction will usually precipitate the protein. These large proteins and proteins with subunits, such as IgG, are purified with HIC.
Neither HIC or RP are useful for hydrophobic aggregates (proteins aggregated with each other or lipid bound to proteins) because hydrophobic aggregates adsorb as aggregates and elute as aggregates. This is one area where ion exchange can be very valuable. Ion exchange will still bind proteins in the presence of chaotropes, such as urea or solvent, that break up hydrophobic complexes and allow purification of the individual molecules. Molecules do not bind to HIC or RP in the presence of chaotropes.
HIC and RP are orthogonal to ion exchange. Separation techniques are said to be orthogonal when they separate by different principle so that each technique adds a dimension to the separation process and does not merely repeat the selectivity of the other. Ion exchange separates primarily by charge; molecular structure and shape have little to do with selectivity. HIC and RP separate primarily by structure and shape; differences in charge have little to do with selectivity. HIC or RP are usually done after ion exchange because the salt in the ion exchange eluate helps the binding to HIC and RP. Hydrophobic interactions are stronger in salt solutions than in water.
Affinity Chromatography
Affinity chromatography uses a ligand attached to the media with binding affinity for either a specific molecule or a class of molecules. Researchers frequently use affinity columns with antibodies attached to bind one specific molecule or columns with antigenic proteins attached that bind a specific antibody. Protein ligands are not ideal for production, however. They must be pharmaceutically pure before being bound to the column. They are expensive. Proteins can be irreversibly denatured by cleaning solutions. Proteins will not tolerate sanitizing conditions.
Other types of affinity columns have lectins attached that will bind certain carbohydrates, but these are not used in production because most lectins are highly poisonous. The best type of ligand for affinity chromatography would be a synthetic molecule that is stable, safe, inexpensive, has good specificity, and allows gentle elution. Some dyes have been used in this way, but the problem so far has been a lack of specificity. The one form of affinity chromatography that is commonly used in production uses Protein A as a ligand for purification of immunoglobulins.
Protein A
Protein A is a cell wall protein from Staphlococcus aureas with affinity for the Fc region of IgG. For this reason it is used extensively for IgG purification. There is very little method development with Protein A; simply bind, wash and elute. Binding is usually done at pH 7-9 and elution is usually at pH 3. One of the potential problems is that pH 3 can denature or partially denature the IgG. Companies are avoiding this by selecting clones of monoclonal IgG's that are stable, and immediately raising the pH while the IgG is being collected. The other main problem is price; Protein A columns are far more expensive than conventional ion exchange columns. The other common means of IgG purification is ion exchange followed by HIC.
Metal Chelate Chromatography
This form of purification is based on an immobilized metal ion that has affinity for a chain of histidines that are added to the target protein by recombinant molecular biological techniques to create a fusion protein. The protein is eluted with imidazole (the side chain of histidine) and then the chain of histidines are removed by an enzyme. This is very common in research labs but not yet common in production.
Size Exclusion Chromatography (SEC) or Gel Filtration or Gel Permeation Chromatography (GPC)
Separation is based on the size of the molecules (hydrodynamic volume = the volume created by the movement of the molecule in water) not the molecular weight. The difference between molecular weight and hydrodynamic volume is shape. Proteins tend to be globular molecules while DNA or polysaccharides tend to be linear molecules. Linear molecules have much larger hydrodynamic volumes than globular molecules, so a 10,000 MW DNA molecule will elute much earlier than a 10,000 MW protein. The media is typically porous beads, with the size of the pore determining the exclusion limit of the column, hence size exclusion. The elution is isocratic, so no gradient pumping systems are needed. Large, excluded molecules elute first. Then molecules that are "included" (can penetrate the pores) come off, large followed by small, and salts and other small molecules elute last.
While SEC is easy to understand and perform, this separation method gives the least resolution with the lowest capacity and largest dilution of the sample with respect to all other forms of chromatography. If you are trying to separate two molecules that are both included, then loading is 2-3% of the column volume and the molecular weights should differ by a factor of 2.
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