Chromatography Challenges in the Purification of Oligonucleotides

Designing an optimal chromatography process for oligonucleotide purification is not easy; many influencing factors must be considered. In this blog post, we detail the features to look out for when selecting a chromatography system.

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Oligonucleotide Production

Oligonucleotides or oligomers are short DNA or RNA molecules. In bioprocesses, they are mainly produced by solid phase synthesis (SPS). Examples include antisense RNA, small interfering (siRNA), microRNA, aptamers, and decoys.

Oligonucleotides have great clinical potential. For example, nusinersen (Spinraza) is an antisense oligonucleotide used to treat spinal muscular atrophy (SMA); inclisiran (Leqvio) is an siRNA used to treat high cholesterol (Figure 1).^[1]

^{Figure 1. Structures of clinical oligonucleotides nusinersen and inclisiran (adapted from Shen & Corey, 2018)[2]}

Oligonucleotide Purification

Vaccine components require a high degree of purity, for which high-performance liquid chromatography (HPLC) is typically necessary.

Several chromatography resins could be applied to purify oligonucleotides, including ion exchange, hydrophobic interaction, and reversed-phase chromatography. These three techniques can be used individually or in combination, depending on the application.

Usually, the solvent employed in the mobile phase is acetonitrile with water, and a high salt buffer is used for hydrophobic interaction chromatography, where a salt gradient increases hydrophobicity in order to elute the product.  

Challenges in Production

The industry must overcome the operational challenges associated with the purification of oligonucleotides to realize their full clinical value. Below, we outline some important considerations when selecting chromatography equipment for oligonucleotide applications.

High Impurity Levels
Oligonucleotide synthesis creates a significant amount of impurities (including shortmers and longmers^[3]) that must be removed during downstream processing. This task is especially challenging because the impurities are often highly similar to the target molecule, making them difficult to separate.  

To maintain a high flow rate and maximize productivity, the chromatography equipment involved in the purification step must resist high pressure levels while maintaining gradient performance.

High Temperatures
Many oligonucleotides require denaturation by increasing temperature to ensure their retention on chromatography matrices. Therefore, temperature is one of the main parameters that must be controlled to ensure high performance and reproducibility. The temperatures required can be very high – some oligonucleotides must be incubated at 60°C to properly denature the molecule and ensure retention on the matrix.  

Such high temperatures require chromatography equipment capable of:

1. Reliably reaching the necessary temperature
2. Accurately maintaining the temperature in the chromatography column for the time required
3. Decreasing the temperature to perform the elution and to protect the operator from high temperatures

Accurate temperature maintenance is critical to ensuring robust process performance.

Aggressive Buffers
Flammable solvents and buffers with high salt concentrations are required during the purification of oligonucleotides. These solutions can be harmful to stainless steel containers, putting them at risk of corrosion over time.  

A chromatography system must be made of materials capable of handling corrosive materials.  Additionally, equipment must be easily cleanable to avoid solutions staying in contact with the stainless steel for longer than necessary.  

Making a cleanable system means:

• reducing the dead leg
• choosing sanitary components like diaphragm valves (rather than ball valves)
• controlling the surface finishing of all stainless-steel components that come into contact with the sample.

Solving Oligonucleotide Purification Challenges

Developing and designing a robust purification process and installing the most appropriate chromatography equipment must consider all these influencing factors and challenges.  

For oligonucleotides, the level of impurities, their interactions, solubility, and clinical goals will directly influence the choice of the stationary phase, mobile phase, and operating conditions, ultimately dictating the most appropriate equipment.

The chromatography system must be rated for hazardous solutions and easily cleanable to minimize corrosion and ensure operator safety. The buffer viscosity and resolution needed will directly influence the pressure rating, so the system must be able to deal with a range of flow rates to maximize flexibility.  

These features should be present without compromising the gradient accuracy and reproducibility, which are fundamental characteristics of a reliable purification process.

^{Figure 2. Hipersep® Flowdrive Process}

Hipersep^® Flowdrive Process M - Uniquely Positioned to Solve Oligonucleotide Purification Challenges  

Now part of the Sartorius family - Hipersep^® Flowdrive Platform is a collection of preparative HPLC systems designed for the purification of peptides, oligonucleotides, insulin, and other small molecules.

For example, the Hipersep^® Flowdrive Process M System can handle a range of conditions, including flow rates from 60 – 500 L/hr, 100 bar pressure ratings, and temperatures from 20–85°C, making it suitable for a range of applications and scales.  

More generally, it is compact, meaning it fits well in low-footprint facilities, and the modular design allows manufacturers to remain agile in their operations. Related to its flexibility benefits, the system is designed to be easy to clean, reducing facility downtime.  

The Hipersep^® Flowdrive Process M offers these features without compromising on gradient accuracy.

Conclusion

The biotherapeutics landscape is evolving, with new modalities emerging and trends moving us towards multiproduct facilities where flexibility is essential. Modular purification systems that can perform at a range of pressures and are easily cleaned are required to ensure their successful operation in diverse processes without cross-contamination.

New modalities bring new manufacturing challenges. Oligonucleotides require unique operating conditions, including chromatography systems that can reach high temperatures and handle potentially corrosive materials.  

Learn more about how the Hipersep^® Flowdrive Platform can solve your purification challenges.

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^References

^{1.     Moumné, L., Marie, A. C., & Crouvezier, N. (2022, February 1). Oligonucleotide Therapeutics: From Discovery and Development to Patentability. Pharmaceutics. MDPI. https://doi.org/10.3390/pharmaceutics14020260}

^{2.     Shen, X., & Corey, D. R. (2018). Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Research, 46(4), 1584–1600. https://doi.org/10.1093/nar/gkx1239}

^{3.     Vanhinsbergh, C. J. (2020). Analytical Separation Methods for Therapeutic Oligonucleotides. LCGC Supplements (Vol. 33). Retrieved from https://www.chromatographyonline.com/view/analytical-separation-methods-for-therapeutic-oligonucleotides}