Use of Ultrafiltration/Diafiltration for the Processing of Antisense Oligonucleotides
Robert Gronke, PhD, Senior Principal Scientist, Technical Development, Biogen, Inc
Ultrafiltration and Diafiltration Well-Suited to Oligo Product, Says Biogen
Ultrafiltration and diafiltration play a key role in the commercial success of its antisense oligonucleotide manufacturing platform, according to Biogen, the firm behind the recently approved amyotrophic lateral sclerosis (ALS) drug, Qalsody.
In separation science, ultrafiltration (UF) is defined as the use of a semipermeable membrane to separate molecules based on their size. This approach is typically used for purification and concentration.
Diafiltration (DF), in contrast, is the process of adding back a different buffer or solvent after UF with the idea being that it enhances stability.
UFDF is widely used in the production of biopharmaceuticals, primarily as a means of capturing product. But it can also be used in oligonucleotide manufacturing, according to Robert Gronke, a senior principal scientist at Biogen, who spoke about the approach at TIDES USA.
“UFDF is a critical piece of Biogen’s ASO platform. It really does bridge the gap between drug substance and drug product, your purification, and your formulation.”
Biogen’s analysis also shows that UFDF is equally effective for antisense oligonucleotide product, according to Gronke.
“ASOs can be effectively retained on a three kilodalton regenerated cellulose membrane. The rule of thumb is that the ASO needs to be about at least twice of the molecular weight pore size, and with this approach you can get very good yields as long as you apply rinse,” Gronke said.
Another advantage is the ability to achieve higher concentrations of oligonucleotide product using UFDF than with filtration methods more commonly used in the oligo industry.
“We’ve been able to easily hit 100 mg per mil — perhaps 200mg per mil is where we think the limit is. It depends on the molecule, the salts you’re using, the system you’re using, and also the chemistry,” Gronke said, adding that “even though it’s a platform unit operation, there’s experimental space to really optimize around.”
The approach also appears to minimize a process called gel polarization, or membrane fouling, which occurs when product builds up to the extent that the membrane filter through which the process stream is being passed becomes clogged.
According to Gronke, “with UFDF we’re not seeing gel polarization but more of an ionic effect with ASOs at high concentrations. We would need a little bit of salt conductivity in order to prevent this ionic polarization from happening or at least happening at a much higher concentration.”
There are also several advantages associated with using DF for formulation preparation, according to Gronke, who cited the removal process reagents as an example.
“Divalent cations don’t pass through the membrane, and you’ll end up getting roughly a couple of logs of clearance of your formulation, whether it’s a buffer salt that you need to get rid of, residual solvents, or small molecules.”
With the benefits of UFDF for oligonucleotide production established, Biogen is now working to optimize the process, Gronke said.
“Obviously, USDF can be costly. We’re working on membrane reuse, we’re working on sustainability, cleaning procedures, and seeing how high we can go. Maybe we need in some of our processes a much more concentrated drug substance so that we’re now shipping water all over the globe.”