Michael McGinley, bioseparations product manager at Phenomenex, describes the pharmacokinetic analysis of oligonucleotide drugs as “a brand new field” and “a growing area of concern.”
Oligo therapeutics are essentially small polymers. “They are highly polar and most have been massively modified,” he says. This makes even traditional reverse-phase liquid chromatography a challenge. For pharmacokinetic studies that require analysis of oligo drugs isolated from biological samples, the difficulties include purifying the oligos from serum or plasma and getting rid of the lipids, organelles, and other large molecules present in tissue samples.
Oligos do not adhere well to reverse-phase chromatographic media because of their high polarity. “You have to add ion-pairing reagents, which tend to suppress the sensitivity of mass spectrometry.”
Adding yet another level of complexity are the modifications to the oligo backbone intended to enhance the stability, bioavailability, and activity of oligo therapeutics. Many of the drugs in development “are more RNA than DNA and the modifications are getting even more esoteric these days,” McGinley says. Further complicating the picture is the attachment of peptide or lipid leaders to facilitate entry of the oligos into cells.
With recent improvements enabling the synthesis of larger quantities of therapeutic oligos, many of the past manufacturing challenges are being overcome. However to bring down costs, some manufacturers are looking to overseas amidite vendors, primarily in India and China, and “we are starting to see some impurities in these amidites” making the need for LC/MS analysis even more critical for monitoring the impurity profiles of oligo APIs.
Phenomenex’ Clarity® BioSolutions portfolio of products for synthetic oligonucleotide purification and analysis has grown and evolved to include Clarity Oligo-RP™, reverse-phase HPLC columns launched in 2006 for achieving greater than 90% purity at both small and large synthesis scales, to Clarity QSP™ for high-throughput trityl-on purification in 96-well plates formats. Clarity Oligo-WAX™ ion-exchange columns and bulk media are designed for preparative purification on HPLC or FPLC systems.
At “TIDES” in 2009, the company introduced the Clarity OTX™ solid-phase extraction system for cost-efficient isolation of oligo therapeutics from biological fluids and tissues in 15 minutes. And most recently, it unveiled Clarity Oligo-MS™, which McGinley describes as an ultra-high resolution reverse-phase column for rapid and efficient LC/MS analysis on both HPLC and UHPLC systems.
State-of-the-art oligo synthesizers can produce oligos at millimolar scale, putting pressure on purification technology and strategies to be able to handle gram quantities of oligo drugs. McGinley sees a need for “better ion-exchange preparatory and analytical solutions” and continued development of HPLC solutions. “People started out treating oligos like proteins, using low pressure LC for purification. Now they are taking advantage of the benefits of HPLC.” He also notes a shift toward the use of stainless steel dynamic axial compression columns in place of glass columns.