The technique of the separation of a mixture of compounds into its individual components is known as chromatography. When talking about the biochemical mixtures or compounds, a modified chromatographic technique is used known as affinity chromatography.
In the most basic definition, affinity chromatography depends on the interaction of one biomolecule with another (hence the name affinity) and hence utilized this interaction to carry out the separation of the molecules. The types of molecular interactions on which this technique has been developed involve interaction between
- Antigens and antibodies
- Receptors and ligands
- Proteins and nucleic acids, and
- Enzymes and their substrates
Apart from the advantages affinity chromatography offers like high sensitivity and selectivity, maintenance of quality and purity, and reproducibility, it also bids some disadvantages which are important to consider if someone is planning to work with this technique. Let us have a look at them one by one with a relevant example where applicable.
Affinity chromatography requires some good analytical as well as hands-on expertise for its successful execution. These include media selection and preparation, buffer preparation, sample preparation and application, elution, maintenance of flow rates and lastly analysis of the results.
Overall, all these steps require one to be good at calculations, organized with respect to time management and must have a neatness in carrying out the process as a whole.
Good technical skills are also required for proper column packing for affinity chromatography. So this becomes a tedious and time-consuming technique especially in the case of larger samples.
Moreover, constant attention must be devoted while performing the experiment such as the continuous addition of the solvent to keep the adsorbent covered and frequent switching of vessels for sample collection.
Sample Volume Limitations
The sample volumes need to be judiciously adjusted while performing affinity chromatography. For example, sometimes an experimenter has a sample containing a lower concentration of the component to be separated. In such cases, it is necessary to cut down the sample volume via concentration techniques so as to make it more efficient.
On the other hand, if one has a sample with highly concentrated amounts of the substance to be separated, it is logically a good practice to dilute the samples at concentrations at which the experimenter gets a good and efficient separation of all that is required.
Purification Challenge for recombinant and Therapeutic Proteins
Therapeutic proteins and vaccines are most commonly produced utilizing recombinant technologies. For this reason, they often lack a common structural element required to capture them via affinity chromatography. This poses a unique challenge for the purification of such proteins via affinity chromatography.
One way to overcome this problem is to specifically structure and make ligands for such purposes. However, for many such targets, it is extremely difficult (in some cases impossible) to prepare viable affinity candidate ligands. Moreover, in some cases, artificial ligands can be utilized. Still, this is a major disadvantage associated with this technique.
Sample / Protein loss
Sample or protein loss is considered to be a major disadvantage sometimes associated with affinity chromatography. It happens in such a way that in some cases that the affinity column or wells of the plate become clogged due to cell debris which might be present in a sample.
In such scenarios, the protein/sample to be purified remains attached to the cellular debris instead of their ligands. Therefore, a considerable amount of sample is lost during clarification/elution steps leading to major reductions in final yield.
Affinity chromatography relies on the specific interaction of a sample with a ligand bound to the resin used to make the chromatographic column. These ligands and their production is a costly procedure.
For example, in most of the case, the ligands are antibodies against a specific protein. These antibodies must be manufactured before starting the experiment of purification of the protein.
Overall, the demands for specific ligands make the process of immunity chromatography an expensive technique.
Ligand leakage is a process in which the ligand attached to resin is detached from it, resulting in decreasing the efficiency of the resin ultimately making it useless.
It is generally observed in the affinity matrices and its extent depends on various factors. For example, one influencing factor for ligand leakage is its coupling efficiency with the resin.
The lower this efficiency, the higher would be the leakage. Other factors affecting ligand leakage can be special conditions of the process used to carry out affinity chromatography.
Excessive leakage of the ligands leads to contamination of the final product being purified resulting in the impairment of its quality. Then it becomes imperative to separate the sample from this ligand impurity via expensive downstream processing.
Ligand Degradation and solid support Degradation
It is vital to take the utmost care while handling the resin so as to minimize ligand degradation. Since most of the ligands used in affinity chromatography are proteins (i.e., antibodies), they require specific temperature conditions to be maintained for their longevity.
For example, most proteins are stable at 4 degrees Celsius and degrade at higher temperatures. Not only this is an adverse consequence for purification purposes but also the life of the resin gets severely affected as a consequence of ligand degradation.
This is because the resin is useful until it contains the active ligand, and when the ligand is inactivated or lost, the solid support also becomes useless.
Metal-ion transfer and Leakage
Metal ion affinity chromatography utilizes metal ions bound to the resin. Often this leads to the transfer of metal ions to the protein being purified either directly or via leakage in the elusion steps. This way, further purification of the final product becomes a requisite demanding some extra efforts afterward.
Affinity chromatography presents the limited lifetime of the resins used when the ligands are antibodies. Other ways through which their life is affected are the reasons already discussed such as ligand leakage and degradation.
The major reason being the shelf life associated with the ligands being used in the process which is the major limiting factor for this technique.
Although affinity chromatography is a powerful technique being extensively utilized in a variety of applications. Care should be taken not only to consider the technical issues associated with its low productivity but also one should be careful to gain comprehensive knowledge beforehand so as to tackle the arising problem in time.