Oligonucleotide purifications: optimization and scale up
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ar. oavi oayanade*
Regional Dir. Bus. Dev., India
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Mr. Jan Berglöf
Co-Founder & Sr. Technical Advisor, Bio-Works, Sweden AB, Uppsala, Sweden
Introduction
Solid-phase synthesis of oligonucleotides generally give material of rather high purity. Peptide therapeutics requires purification to remove incomplete or erroneous sequences. Anion exchange chromatography (AIEX) provides efficient purification giving high purity and yield in a single step. Process optimization of the binding and elution conditions is needed to meet required purity and yield prior to scale-up. We have investigated the correlation between purity and yield, transitioning from low to high sample load.
Purification optimization
The resolution of oligonucleotide purifications on WorkBeads™ 40Q was compared for two different NaCl-buffers as outlined in Fig. 1 below. Each individual fraction was analysed for yield and purity on a DNAPac PA200 analytical IEX column (Thermo Fisher). Capto™ Q ImpRes resin (Cytiva) was also used for comparison.
The dynamic binding capacity (DBC) was determined by frontal analysis at 150 cm/h to 48 mg/mL resin by applying the crude oligonucleotide preparation from the solid support without further adjustment of the feed.
Purity and yield
Selected 1-mL fractions were combined to assess the effect of pooling on purity and yield. See Fig. 2 and Table 1.
No significant difference was seen for the two buffer systems used for this oligonucleotide batch. A purity of 95.6% with 74.2% yield was obtained with the broadest pooling using Tris-buffer (Fig. 2B). Higher purities could be obtained by a narrower pooling, as illustrated in Fig. 2B. WorkBeads 40Q gave both higher purity and increased yield compared to Capto Q ImpRes (Table 1).
Scale-up
When the process conditions giving required purity and yield have been obtained, scale-up can be done. To investigate scale-up conditions, sample load of 80 % of the resins DBC, i.e., 132 mg of the oligonucleotide preparation was loaded to the column.
Fig. 3A shows a typical recovery shape of the full-length oligonucleotide for a process purification run with a sample load corresponding to 80% of DBC. In the beginning of the elution gradient the N-x species are eluted, and the full-length oligonucleotide starts to elute later in the gradient. The histogram in the chromatogram in Fig. 3B shows the purity in individual fractions. This demonstrates the separation of the full-length oligonucleotide.
Conclusions
Purification of a 20-mer oligonucleotide using WorkBeads 40Q gave excellent purity with good yield in both NaOH- and Tris-based buffers. Both purity and yield were higher on WorkBeads 40Q compared to Capto Q ImpRes. The rigidity of WorkBeads 40Q allows for efficient purifications of full-length oligonucleotides also at process scales.