Purification of antisense oligonucleotides using hydrophobic interaction chromatography

Area of Science:

• Biochemistry
• Chemical Engineering
• Chromatography

Background:

• Traditional methods like anion exchange (AEX) and reverse phase chromatography face limitations in impurity removal for antisense oligonucleotides.
• Process-related impurities (solvents, small molecules) and product-related impurities (e.g., N-1, P=O1, branchmers) pose challenges in oligonucleotide purification.

Purpose of the Study:

• To evaluate hydrophobic interaction chromatography (HIC) as an alternative impurity control method for antisense oligonucleotide purification.
• To optimize HIC parameters for maximizing yield and impurity resolution in oligonucleotide synthesis.

Main Methods:

• Systematic evaluation of HIC variables including resin ligand, salt types, processing conditions, gradient types, and loading ratios.
• Optimization of column loading (32%-78% DBC) and use of stepwise wash and elution gradients.
• Desorption of purified product using low lyotropic salt concentrations (≤50 mM) and stepwise gradients.

Main Results:

• HIC effectively cleared process-related solvents and small molecules by ≥3 log10 and failure sequences (EEIs) by ≥90%.
• HIC demonstrated significant reduction of challenging product-related impurities like branchmers (LEIs), N-1 impurities, and P=O1 impurities.
• Optimized HIC achieved 90% yield and maximal impurity resolution, retaining non-polar impurities (LEIs) on the column.

Conclusions:

• HIC is a powerful, all-aqueous, scalable, and cost-effective purification method for antisense oligonucleotides.
• HIC can be used as a stand-alone technique or integrated with orthogonal methods like AEX for enhanced purity.
• Properly designed HIC processes provide predictable and high-purity oligonucleotide products.