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Tutorials : Apr 15, 2008 ( )
Development of an Ion-Exchange Process
Guidelines for Dealing with Complications that Arise with Workhorse Purification Procedure!--h2>
Cation- and anion-exchange chromatography columns are used routinely for the downstream processing of therapeutic antibodies and proteins. Despite their frequent use, they are considered less glamorous than the high-profile affinity adsorbents such as protein A sepharose that enjoy celebrity status in the world of industrial antibody purification.
While the protein A adsorbents may be compared to formula one racing cars—continually nurtured, tweaked, redesigned, or redeveloped to improve performance with due care and attention—ion exchangers are often considered the workhorses of the purification process—well-characterized adsorbents with well-defined, charged chemical ligands that have been used for decades and typically perform in a predictable manner.
Ion exchangers are often used as a second chromatography step, following primary capture of an antibody or therapeutic protein to remove potential impurities such as DNA, host cell proteins, endotoxins, and viruses. Therefore, the performance and robustness of the process step may be critical.
As the name suggests, ion exchangers were initially developed for the purification of ions and small molecules including ammonium, radioactive isotopes, and amines. As such, the results were often consistent and predictable. The seemingly simple application of ion exchangers for what perhaps should be described as protein-exchange or ion:protein-exchange, however, often presents a multitude of unexpected technical challenges that have no doubt been the focus of many purification scientists’ woes.
The apparent simplicity of the ion-exchange (IEX) process for the purification of proteins may hoodwink even the most experienced of scientists. The pI, or isoelectric point (the pH at which the molecule has a neutral net charge), may be accurately predicted or determined experimentally. Therefore, a theoretical ion-exchange step for the purification of a protein of interest may be developed with the minimum of effort.
The use of buffers one pH unit above or below the pI will ensure that the protein of interest binds or flows through the column. If the buffer pH is maintained, then the purification step will perform consistently and reproducibly.
The timeline for purification development is often expected to be minimal, and subsequently, researchers head to the laboratory convinced that a proposed experimental design cannot fail. To the despair and frustration of development scientists, the complex exchange of ions, amphoteric buffer molecules, impurities, and the solubility and stability of the protein of interest may result in experimental data that is often far from predictable.
Many proteins are soluble, stable, and will of course interact predictably with ion-exchange adsorbents. Other proteins, even closely related molecules such as mAbs, however, may demonstrate specific regions of charge, hydrophobicity, or show minor charge heterogeneity. Therefore, an apparently simple IEX-platform purification step becomes complicated.
At least three critical features may need to be considered when designing an ion-exchange step for a therapeutic protein: the stability and solubility of the target protein (and impurities) under the proposed conditions for the purification step; the buffer concentration, composition, and potential interaction of the buffer molecules with the adsorbent; and the physical properties of the base matrix, in particular the effect on nonspecific binding of sample components during the loading, washing, and elution steps. In addition, temperature may also affect adsorption to weak ion exchangers and may need to be evaluated as part of the process characterization.
The surface chemistry of a desired protein may have a dramatic effect on the performance and predictability of an ion-exchange step. Although the pI of a protein defines the pH at which the molecule has no net charge, the protein will undoubtedly have regions of charge on the surface of the protein dependent on the primary sequence, the pKa of the individual amino acid side chains in question, and the complexity of the molecule.
Buffer Composition & Concentration
Although buffer pH and conductivity are usually assessed and monitored when developing an IEX step, the buffer type and concentration is often initially chosen from a combination of historical or published data and large-scale commercial considerations such as cost and availability. Buffer composition should ideally be selected to ensure that the buffer molecules do not have the opposite charge to the bound ligand on the ion-exchange adsorbent.
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