In this study we synthesize oligonucleotides (oligos) and find optimum conditions for purification of oligonucleotides using column chromatography. Read further to find out how:
- A 21-mer oligo was successfully synthesized using the ÄKTA oligosynt™ synthesizer.
- A single step oligonucleotide purification process was developed using the ÄKTA pure™ 25 chromatography system.
Optimized purification conditions were successfully scaled up with the ÄKTA pilot™ 600 chromatography system.
Introduction
Oligonucleotides (oligos), composed of deoxyribonucleotides (DNA) or ribonucleotides (RNA) are indispensable tools in basic research where they enable scientists to probe specific DNA or RNA sequences unraveling the mysteries of gene function, regulation and genetic disorders.
Oligos have also emerged as a promising class of therapeutics. They can be designed to modulate gene expression by either silencing disease-causing genes (gene silencing) or by correcting genetic abnormalities (gene editing). Furthermore, oligonucleotide-based drugs hold potential for treating a wide range of diseases, including cancer, viral infections, and genetic disorders, offering a targeted and precise approach.
Most oligonucleotides today are produced via automated solid-phase synthesis using phosphoramidite chemistry. Our ÄKTA oligosynt™ synthesizer uses this method to sequentially couple nucleotide building blocks (phosphoramidites) creating a growing chain on the solid support, in the 3’ to 5’ direction. Since no chemical reaction is fully efficient the phosphoramidite method includes a capping step that prevents further elongation of any un-reacted 5’ ends of the growing chain on solid support during each cycle. Whenever a nucleotide fails to be incorporated correctly, this approach will inevitably generate several incomplete failure sequences. The amount of these failure sequences in the synthesized product depends on the efficiency of each coupling cycle and the number of cycles performed. The coupling efficiency for DNA commonly reaches 99.5% resulting in a theoretical full-length product purity > 80% for a 21-mer.
The difficulty with purification of oligonucleotides is that all impurities are more or less product related with only slight differences between the correct sequence and shorter variants. As slight variations in the sequence or presence of contaminants can impact therapeutic activity, it is critical to carefully select and optimize the use of appropriate chromatographic resins, buffers and gradients to achieve high purity and yield.
Techniques such as ion exchange chromatography (IEX) or reverse-phase chromatography (RP), and even hydrophobic interaction chromatography (HIC) are commonly employed to purify oligonucleotides. As oligos have a negatively charged backbone it makes them suitable for anion exchange purification whereby separation is based on chain length (directly proportional to overall charge). Seeing as ion exchange utilizes the charge difference it is crucial that all charges are sterically available to the resin, and it is therefore important to avoid higher order structures of the oligo. Purification of oligos consisting of DNA can successfully be performed at high pH, however RNA is a much more sensitive molecule and therefore high pH might not be the best option.
For oligos that are difficult to purify, the hydrophobic trityl group in dimethoxytrityl (DMT) present at the 5’ end can be kept after the synthesis to be used in HIC. However, most applications will require the 5’ DMT group to be removed eventually.
Here we present the work surrounding synthesis of a 21-mer oligonucleotide on the ÄKTA oligosynt™ synthesizer, followed by screening of three different anion exchange resins and an optimized single step for oligo purification using ÄKTA pure™ chromatography system.
Methods and Materials
Oligonucleotide synthesis
The oligos (21-mer DNA with phosphodiester bonds on backbone) were synthesized using the ÄKTA oligosynt™ synthesizer. Initial resin screening and verification for oligo purification application experiments were performed with oligo synthesis at 240 µmol scale. This small-scale synthesis was subsequently scaled up at 12 mmol synthesis scale using Primer Support™ 5G UnyLinker solid support at 353 µmol/g loadings and packed in FineLINE™ 70 column (8 cm bed height, column volume of 308 mL). After cleavage and deprotection with aqueous ammonia a yield of 72% and a concentration of 120 OD/mL) was observed.
Screening of anion exchange resin on ÄKTA pure™ 25 chromatography system
Crude D21-mer from the small-scale synthesis was diluted 1:10 with Buffer A (10 mM NaOH pH 12). Screening of performance was undertaken on three different resins, SOURCE 30Q, Capto Q ImpRres and Capto adhere ImpRres using the ÄKTA pure™ 25 M (Fig 1) with an absorbance measurement at 260 nm. 2 ml fractions were collected using the Fraction Collector F9-C. Purity of each fraction was analyzed using HPLC; the results are displayed in Table 1.
Fig 1. IEX resin screening using ÄKTA pure™ 25 M.
Results
Dynamic binding capacity (DBC), for each resin was calculated using a residence time of 1.25 min at 10% breakthrough and 1 ml column volume for each resin.
Based on the purity and DBC results and the peak shape (see Fig 2), Capto Q ImpRes was selected for further optimization. An example of this optimization was to increase the column load by 80% of the resin DBC. However, increasing the load resulted in earlier elution of the target nucleotides. To accommodate for the earlier target elution, % buffer B had to be decreased in the wash step. In addition, to further increase the resolution and improve the mixing, the concentration of NaCl was reduced from 2 to 1 M.
Table 1. DBC and HPLC purity analysis results for the three resins screened
| Resin | Purity (%) | DBC, QB10 (mg/ml) |
| SOURCE 30Q | 94 | 20 |
| Capto Q ImpRes | 99 | 34 |
| Capto adhere ImpRes | 97 | 41 |
Fig 2. Chromatograms from D21-mer oligonucleotide purification screening using three different resins.
Verification of optimized oligonucleotide purification
A final verification run was undertaken using ÄKTA pure™ 25 M chromatography system and a HiScreen Capto Q ImpRes column (Fig 3). Crude oligo was diluted 1:10 with Buffer A to a final concentration of 1.1 mg/ml. Sample load was calculated to 80% of DBC corresponding to a sample volume of 116 mL. The sample was loaded on a pre-equilibrated column using Sample Pump S9 with an integrated air pressure sensor. Four different fraction pools were analyzed with HPLC and UV at 260 nm to evaluate recovery and purity (Fig 4 and 5).
Fig 3. IEX verification of purification using ÄKTA pure™ 25 M.
Fig 4. Chromatograms from verification purification of a 21-mer oligonucleotide (DMT off) using ÄKTA pure 25 M chromatography system, showing absorbance at 260 nm (blue).
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Fig 5. Four different pooling strategies were analyzed with HPLC to evaluate recovery and purity.
Scale-up of oligonucleotide purification process
A subsequent large-scale experiment was performed on 27 g of crude oligo using a 1 L ReadyToProcess™ column and ÄKTA pilot™ 600 chromatography system. Scale-up of this process was successful with oligonucleotide recovery of ≥ 89% and a purity of ≥ 95%. This oligonucleotide purification process and subsequent results are detailed in the poster, Single-step 21-mer oligonucleotide purification using Capto™ Q ImpRes resin.
Conclusion
In this study we were able to successfully synthesize a 21-mer oligo using the ÄKTA oligosynt™ synthesizer. After screening of anion exchange resins and subsequent optimization we chose Capto™ Q ImpRes to purify the synthesized 21-mer oligo (DMT off) in a single step process using ÄKTA pure™ 25 M chromatography system and UV absorbance at 260 nm.
The purification conditions identified in this study were successfully scaled up on ÄKTA pilot 600™ chromatography system yielding an oligonucleotide recovery of ≥ 89% and a purity of ≥ 95%.
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