By Bachem AG
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Bachem harnesses mass spectrometry for highly purified oligomers
As therapeutic usage of oligonucleotide-based drugs grows, so the oligomers are becoming more complex, increasing the already formidable challenges of characterizing and purifying them. At the same time, as a new therapeutic modality, oligonucleotides present pharmaceutical companies with a number of analytical challenges to satisfy regulatory agency demands.
Bachem is meeting these challenges with innovative technologies, such as the use of mass spectrometry (MS) instruments, augmented by leading-edge analytical software.
Experts in MS analysis to support CMC development of oligonucleotides
Bachem’s heritage in peptide development has provided it with formidable MS capabilities, with six high-resolution mass spectrometers in-house and more than 15 years’ experience in using them to investigate ‘tide substances and support Chemistry, Manufacturing, and Control(CMC) development.
Bachem experts have now leveraged these capabilities to develop superior methods to characterize complex oligonucleotides that enables robust and specific sequence confirmation that recognizes chemical modifications.
Because of oligomers’ poor pharmacokinetic and pharmacodynamic properties in the body, it has been essential to add chemical modifications to their different moieties: the nucleobase, the sugar, and the backbone. Over the years, these molecules became more and more complex and highly modified. Furthermore, the modifications add further challenges in the separation of impurities as well as in the development of analytical methods for characterization and quantification.
Together, these factors make it highly challenging to characterize oligonucleotide APIs for impurities using traditional analytical methods, such as polymerase chain reaction (PCR). High-resolution MS techniques present a great opportunity to overcome these challenges in analyzing and identifying modified or non-modified oligonucleotides.
What is Mass Spectrometry (MS)?
Mass spectrometry (MS) is an analytical technique to measure the mass of a molecule or molecular samples based on the mass-to-charge of ions.The results are represented as a mass spectrum that can be used to elucidate the chemical structure of a molecule. Bachem’s powerful equipment and software enables it to characterize complex oligonucleotides and collect the accurate mass of the molecule.
Originally coming from the peptide world, ultra-high-resolution MS has been implemented for oligonucleotide analysis and characterization. However, the methods needed to be adapted because of the complexity of oligonucleotides. The first challenge was to develop a powerful deconvolution tool to make these complex spectra much easier and quicker to interpret. This has been achieved by collaboration with Bruker Corporation, the US-based manufacturer of scientific instruments for molecular and materials research, to shift from a spectrum that is messy and difficult to interpret to a clear and straightforward result.
Supporting CMC development of oligonucleotides
When producing an API, impurities are often a big challenge for manufacturers. Regulatory authorities set strict requirements on the impurities level and their identification. Therefore, having a strong and reliable process to identify the impurity profile is critical in the CMC development.
Ultra-high-resolution MS can also be combined with chromatographic techniques to allow Bachem research scientists to support high performance liquid chromatography (HPLC) with LC-MS/MS peak purity analysis to identify and quantify impurities that coelute with the target molecule.
With the peak purity approach, it has been possible to identify impurities in oligonucleotide APIs. The LC-MS/MS method is also particularly helpful in identifying backbone sulfur (so-called PS) substitution by an oxygen, PO. This PS/PO substitution, is an almost inevitable side-reaction in oligonucleotide synthesis. The new Bachem method enables the identification of this side-reaction as well as identifying the position within the sequence when the PS/PO substitution occurs, while also detecting other side-reactions, such as methylation.
Unique sequencing technique
Ultra-high-resolution MS also provides a powerful and sophisticated method to confirm the sequence of an oligonucleotide. Previously performed by chromatographic techniques (LC) coupled to MS equipment, Bachem’s research team has chosen to dispense with the LC element due to the drawbacks and complexity it presents for GMP validation, and concentrate on MS methods that are faster and easier to validate.
This has meant overcoming a further inherent problem with oligos, whose small size means lower signal intensity in MS. To gain accuracy and precision in the analysis, Bachem researchers have developed an electrospray ionization MS/MS (ESI MS/MS) method using an offline desalting technique and then to directly infuse the oligonucleotide into the ESI source. This innovative method for oligonucleotide characterization is unique in the industry and bring numerous of advantages as a fast and easy technique that enables efficient MS/MS development with robust methods for sequence confirmation. It has been possible to cover the full length of both strand in a siRNA duplex and so to confirm the sequences. The analysis has also been successfully performed as a release analytics of a final API under GMP conditions.
Innovation at Bachem
Ground-breaking techniques for oligonucleotide characterization provide one example of Bachem’s overall commitment to innovation and operational excellence as keys to addressing market needs as well as increasingly stringent regulatory regimes.
From peptides to oligonucleotides, Bachem has become expert in successfully transferring and adapting know-how in developing and validating test methods, especially in mass spectrometry (MS) methods. From this our customers benefit from unique and innovative characterization methods for their oligonucleotide API for a successful CMC development. With its GMP-qualifiedequipment supporting the newest requirements, Bachem is already developing and manufacturing oligonucleotide-based APIs in partnership with customers.
Click on EW Mass Spectrometry (MS) Characterization for an accurate identification of oligonucleotide impurities to learn more.
Click on High Resolution Mass Spectrometry of Oligonucleotides to watch Bachem webinar.
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Drawing of an oligonucleotide showing the different position of chemical modifications: the growing complexity of oligonucleotide APIs make them challenging to characterize for impurities using traditional analytical methods. High-resolution MS techniques present a great opportunity to overcome these challenges in comparison to well established methods such as PCR (Polymerase Chain Reaction).