Protein Purification Principals
Proteins (and other molecules) can be purified from crude extracts or other complex mixtures by a variety of methods, most of which involve some form of chromatography. In these separations, the target molecules in solution (mobile phase) are separated based on chemical interactions (e.g. charge in Ion-exchange or hydrophobicity in reversed Phase Chromatography) or physical interactions (e.g. size-exclusion chromatography), with a stationary material (solid phase). These purification approaches are not specific to the target, meaning that multiple passes, using several different column types are required to achieve a highly purified sample. As each of these steps has inevitable loss in yield, a multi-stage purification process may not be viable. This is especially problematic where highly purified material is required (such as therapeutic proteins), or where the initial yield is low.
Target specific affinity chromatography utilises a specific binding interactions between molecules. A specific ligand (e.g. antibody) is chemically immobilised to the solid support so that the target molecule is captured when the sample mixture is passed over the column. Wash steps are included to remove other sample components, before eluting the target from the support. As this process is target specific; fewer purification steps are often required to give a highly purified final product.
Key Benefits of Aptamers
- Aptamer is isolated in complex matrix (e.g. cell lysate) and will discriminate between your target and the other proteins (including customer defined counter-selection targets)
- Selection processes can be tailored to include customer defined ‘loading’, ‘wash’ and ‘elution’ buffers.
- Built in protein compatible elution buffer conditions avoids the need for harsh elution conditions (e.g. denaturants)
- Increase the yield of your product by reducing losses.
- Aptamers maintain function after 50+ cycles of binding, wash and elution.
Aptabind™ – Aptamer Mediated Affinity Chromatography (AMAC)
As with other applications, aptamers can be isolated with protein purification goals ‘in mind’. The binding conditions used during the selection process can be adapted to include cell-lysates, culture supernatants etc. ensuring that our aptamers will recognize and bind to the customer target as it will be ‘presented’ in the end application. Customer defined buffer criteria can also be included for the ‘wash’ and ‘elution’ steps.
Customer defined elution buffer conditions are a significant advantage of our Aptabind™ process, as this can be tailored to ensure that the chosen buffer is compatible with the protein of interest i.e. gentler elution conditions can be used to avoid protein losses through denaturation etc.
As with our standard aptamer isolation and characterisation processes; we start with the end application in mind and confirm that our aptamer populations (and individual aptamers) perform as expected in a functional assay
In the case of the AptaBind™ processes for Aptamer Mediated Affinity Chromatography (AMAC); aptamers are immobilized onto a biosensor surface and then monitored for interactions with the application matrix (e.g. cell lysate), with and without target. Washing and elution conditions are also tested, to ensure that the aptamer releases its target when required.
AptaBind Aptamers Show Target Specific Binding, Wash and Release
Aptamer populations and individual aptamers are assessed to demonstrate their performance in a functional binding assay which mirrors the end application. Individual aptamers are immobilised onto a biosensor and interactions monitored with the target (in complex matrix). Washing and Elution conditions are also assessed to ensure that the aptamers both bind and release. Aptamers which do not possess both features are not taken forward. A blank sensor is also included (green trace) to ensure that the interaction is aptamer dependent.
Aptabind Aptamers Achieve ~90% Purified Protein in a Single Pass
Aptamer Mediated Affinity Chromatography (AMAC) was used to purify a customer protein from a culture harvest stock. Aptamers isolated using our AptaBind™ approach are readily immobilised on commercially available purification resins to give a target specific affinity resin. The tailored elution conditions give a significantly purified product, without the risk associated with using antibody-based purification.
AptaBind Aptamers are Reusable
As customers would generally prefer to be able to reuse their AptaBind™ columns, we tested the reusability by exposing the aptamer loaded biosensor, to repeated cycles of binding (to buffered cell culture harvest), wash (in customer defined buffer) and then release (in the target-compatible elution buffer).