Answering the Call with Compliance-Ready LC-MS Solutions for Oligonucleotide Analysis

Oligonucleotides, or short strands of DNA or RNA, continue to increase in popularity. In addition to their essential role as primers and probes in the expanding clinical DNA testing and diagnostics market, they’re also seeing great success as new gene-based therapeutics that hold tremendous promise.

With a string of recent drug approvals, including the first RNAi drug (Patisiran) from Alnylam, pioneers in this field have now proven to regulators and the rest of the industry that these DNA- and RNA-based drug modalities can be stable, safe, and highly effective. In turn, this has spurred even greater investment into their development across the industry, with many large pharma companies now funding their own development programs. Currently, there are now more than 300 oligotherapeutics in the clinical pipeline¹.

Oligonucleotides drive faster development of targeted gene-based therapeutics

Unlike most traditional small and large molecule drugs, which bind to protein targets in the body to achieve a therapeutic effect, oligotherapeutics (excluding aptamers) work upstream at the genome and transcriptome levels to either block, repair, or alter the mRNA instructions that code for a specific protein. And since they interact with their DNA or RNA targets via sequence homology (i.e. Watson-Crick base pairing), they don’t require a protein target and can avoid much of the target validation work required for traditional drug development. In a very real sense, with gene based therapeutic approaches, every gene sequence and the protein that it codes for can now be a potential target. This has opened the door for drug developers to go after many rare and intractable diseases where an accessible protein target has yet to be found. Additionally, because oligotherapeutics interact via homology, they can be designed in silico based on DNA sequencing data. All of this means that they can be developed much more quickly than traditional drugs.

Meanwhile, in the burgeoning field of DNA diagnostics, GMP validated oligonucleotide primers and probes are essential reagents needed to facilitate the amplification and detection of target DNA, whether it be to confirm the presence of infectious disease agents or to determine the genetic profile of a human patient. The growing use of PCR (polymerase chain reaction)-based diagnostics and the eventual move of targeted next-generation sequencing into the clinic is all about providing physicians with greater patient data to inform their diagnosis and treatment strategy.

Taken together, the increased interest in oligonucleotide analysis is easy to understand, as is the desire for robust, compliance-ready analytical tools and solutions that can support their accelerated development, growth, and expansion in the years ahead. From a manufacturing and quality control (QC) release standpoint, the vast majority of oligonucleotides produced today are made via solid-phase synthesis. The main difference between one oligonucleotide and another is its nucleotide sequence and the degree to which they incorporate chemically modified nucleotides, or are conjugated to other molecules. As such, oligonucleotides are well suited to a streamlined, platform-based approach to manufacturing and analysis, which makes them even more attractive to drug developers.

Satisfying regulatory authorities

To satisfy regulatory requirements for method and process development – along with QC release testing, including confirmation of product ID, sequence verification, and impurities analysis  – robust liquid chromatography-mass spectrometry (LC-MS) analysis is needed. This applies to both upstream characterization work and downstream monitoring and QC release. Traditional sequencing technologies don’t have the ability to confirm the site of each modified nucleotide, or conjugation, and no other technology can meet this need today other than LC-MS analysis.

Answering the oligonucleotide challenge

 At Waters, we are proud to have built a strong legacy of innovation in oligonucleotide separations and analysis over the past two decades. From our leading BEH column chemistries and UPLC separations technology, to our fit-for-purpose MS tools and unmatched compliance-ready informatics to ensure data integrity and streamline regulatory submissions, we provide the tools and expertise to advance your oligonucleotide development projects.

The SmartMS features incorporated into the BioAccord LC-MS system ensure that scientists of wide-ranging skill levels can operate the machine with confidence and generate high-quality, compliance-ready data. Additionally, our expert support team is available to qualify the system and can provide on-site training as well. Moreover, the BioAccord LC-MS system is designed to work under our waters_connect software, which supports both characterization workflows using our more advanced, research-grade Xevo G2XS and Vion MS instruments, and also monitoring and QC release workflows using our BioAccord LC-MS system. These “harmonized” LC-MS workflows all benefit from the software’s compliance-ready architecture and automated data processing tools that are actively being enhanced to meet the needs of tomorrow.

By leveraging the unique capabilities of the BioAccord LC-MS system, we’ve worked hard to ensure that scientists can meet regulatory requirements and accelerate their oligonucleotide-based therapeutics and diagnostic reagents to the market.

BioAccord delivers fast and accurate oligonucleotide separation

In a recent study using the Intact Mass Analysis workflow of the BioAccord system, a diverse range of modified and unmodified oligonucleotides were run on the system to assess how well it could separate the full-length product from its truncated n-1 impurities, and to determine what mass accuracies could be achieved for each. In short, the BioAccord system passed with flying colors. It was able to separate each of the oligomers from their truncated impurities in under 8 minutes, and with waters_connect providing compliance-ready data acquisition and processing, all oligonucleotides were positively identified with excellent low ppm mass accuracy (most below 5ppm; none more than 15ppm).

These results and those from previous studies clearly demonstrate the utility of the BioAccord LC-MS system to provide compliance-ready oligonucleotide analysis across a wide range of chemically modified species and oligonucleotide lengths (15-100mer).

Paving the way for next-generation oligonucleotide analysis

 As scientists continue to develop and unlock new applications for oligonucleotide-based therapeutics, and as demand for clinical DNA diagnostics continues to expand, the need for robust, compliance-ready LC-MS analytical workflows to support the development, manufacturing and QC release of oligonucleotides has never been greater. Drawing on our considerable experience in the field, our team at Waters have purposely-built the BioAccord LC-MS system to meet this need and to support a range of routine biopharmaceutical analyses in regulated development, manufacturing and QC labs, including oligonucleotide analysis.

Among key BioAccord LC-MS development goals, we wanted to:

• Ensure the system could easily be set up and run by non-MS experts
• Design a compliance-ready system
• Enable highly reproducible separations and accurate mass performance

We were also mindful of the need for it to be compact, robust and easy to maintain so it can be deployed across an organization.

Having met these goals and more, we’re confident the BioAccord LC-MS System can play a key role in assuring the identity, quality and purity of oligonucleotides for both therapeutic and diagnostic applications, allowing for more streamlined operations that fuel productivity gains for our customers.

Watch our Select Science webinar from December 2, 2020 to learn more about the diverse range of modified and unmodified oligonucleotides we’ve analyzed using the BioAccord LC-MS System.

References:

1. https://www.bachem.com/service-support/newsletter/oligonucleotide-trends-september-2020