How robust are Optimer-based affinity columns?
As part of the recent Biologics Europe 2021 digital conference our CTO, Dr David Bunka, was invited to present as part of Sartorius’ workshop in the ‘Protein Engineering, Protein & Antibody Production Development, Purification’ stream.
During his talk, ‘Using Optimer™ binders to improve the affinity purification of novel biologics’, David explained how our Optimer technology is being utilised by key partners across the industry to provide novel solutions for the purification of biologics.
After his talk David took some time to discuss how our Optimer technology can be customised to ensure the specific purification of each biologic target.
Below are his answers to some of the pressing questions that arose from the audience.
How robust are Optimer-based affinity columns? How many runs could be completed before the columns need to be regenerated?
Optimer-based affinity columns can be very robust and we select for the best performance according to your needs.
Overlay of 50 repeat cycles of Optimer-target binding traces shows reproducibility & reliability of Optimer interactions
In this example the column was able to be used to at least 50 cycles. We extended the assay up to 200 cycles and we did begin to see reduction in performance after approximately 100 cycles of use. By 200 cycles we had lost about 50% of the activity.
In terms of regeneration of the columns with standard reagents, such as NaOH, Optimer ligands can tolerate these conditions. Though longer incubations will hydrolyze the Optimer ligands, the short pulses used for cleaning-in-place are well tolerated. The Optimer ligands are very robust when it comes to this sort of application.
In this example we regenerated the column following 100 cycles. This would depend upon the functional group used to attach the Optimer ligand to the resin. In some cases if you use for example an irreversible linker you may not be able to do that. However, in terms of regeneration at least 50-100 cycles.
We do offer this kind of validation work as part of work packages with our development partners, to determine the number of cycles your Optimer and column would tolerate. Initially we use proof-of-concept studies using BLI to show binding and elution and then can further validate this using small scale column studies.
Could you elaborate on the potential of identifying Optimer ligands for use with specific binding and reducing conditions? Would it be possible for example to develop Optimer ligands for binding and release at the same pH but with varying salt concentration?
BLI assay demonstrates proof-of-concept purification
Small-scale column test demonstrated a high level of purification in a single pass
In this case, working with Shire, we were able to adjust both the elution pH and salt concentrations to optimise binding and elution of the Optimer ligands to the biologic. However, if your target is particularly pH sensitive, there are many factors that we can modify to tailor development and identification of Optimer binders that offer the desired performance profiles.
Optimer binders are very salt tolerant. So the salt concentration used in affinity purification is dependent upon the tolerance of your target biologic. We could use up to 5M NaCl if we really wanted to. We can also build in requirements for divalent metal ions. We can generate Optimers that require magnesium for target interaction, and elute using EDTA to chelate the magnesium preventing the Optimer-target interaction and allowing elution from the purification column.
The conditions can be changed according to your target requirements. You tell us what your target will tolerate and we will work to find the best conditions for elution.
As part of your custom workflow you offer a feasibility study. Why might a target not pass this stage and what could be the challenges here?
The challenges for Optimer development are largely the same as for any affinity ligand, such as antibodies. The major challenge is that you might not be able to stimulate an interaction with the target.
To limit the chance of this happening in Optimer development, we perform an initial buffer screen. This process analyzes the target interaction with the aptamer library in the presence of a range of different buffers. The aim is to identify the most appropriate buffer for the discovery campaign, which promotes interaction between the aptamer and the target. We use buffers with different salt concentrations to promote interaction with variously charged targets.
The feasibility screen offers two key benefits:
- It helps to screen out targets that we think will have little chance of success in the development campaign and offers a fail fast, fail cheap option for partners to quickly learn whether Optimer technology can offer the required binders for their project.
- It helps to identify the best buffer for use across the discovery campaign to allow optimal selection of the appropriate binder and speed the discovery process.
How do Optimer binders fit into GMP validated processes?
For the custom Optimer binders we have developed, we work with industrial partners to identify and engineer the best Optimer binder and validate this at Aptamer Group. Further scale-up and development takes place at the partners site. Many of the Optimers we have developed for affinity purification with industrial partners are being progressed through scaled-up processes and manufacture, but those Optimer binders belong to the specific partners and we are not currently aware of their specific use in GMP processes.
In terms of GMP production of Optimer binders themselves, transition from a standard production process to a GMP-compliant production process is very simple. Many suppliers can accommodate manufacture of GMP standard nucleic acids, and so can produce GMP Optimer binders easily to fit in with these workflows.
What is known about the immunogenicity of aptamers?
As a general rule, Optimer binders are non-immunogenic. They are nucleic acid molecules, and nucleic acids exist within the body at all times as a basic foundational molecule of life.
Some interactions with the immune system have been noted, for example, some modified aptamers and GC-rich sequences have been shown to stimulate Toll-like receptors. But as a general rule the immunogenicity of Optimer binders is very low.
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