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Melting point determination using ultraviolet-visible (UV-Vis) spectrophotometry can be used as a sequence-specific method for identifying therapeutic oligonucleotides in pharmaceutical quality control. This method offers a simple, highly selective approach to differentiate between isomers and ensure the integrity of oligonucleotide active pharmaceutical ingredients (APIs) and drug products.

In this application note, LC separation and MS1 mass identification of a variety of oligos without the use of ion‑pairing reagents is demonstrated. The LC separation allows subsequent positive mode use with little to no flushing or hardware changes. This HILIC-based method uses an Agilent InfintyLab Poroshell 120 HILIC-Z column and MS-friendly ammonium acetate-based mobile phases. The samples were analyzed on an Agilent 1290 Infinity II LC system and a 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (LC/Q-TOF).

Characterization of oligonucleotides requires robust analytical instrumentation and methods as well as ease-of-use data analysis tools. Biocompatibility mitigates non-specific sample binding to flow path and it ensures the integrity of biomolecules and robustness of the system. In this study, two workflows, the Target Plus Impurities (TPI) and Sequence Confirmation workflows in Agilent MassHunter BioConfirm software, were carried out to characterize two oligonucleotide samples.

UV-Vis spectrophotometers have been used widely for nucleic acid quantification and quality control (QC) utilizing the fact that nucleic acids have a maximum absorbance at 260 nm (1). The concentration of nucleic acids can be easily estimated using the absorbance at 260 nm and the established absorption coefficient. Often a background correction is also performed, for example collecting a baseline using a solution containing everything but the nucleic acid or by measuring the absorbance at a wavelength that nucleic acids do not absorb. Double stranded nucleic acids are bound by hydrogen bonds between the base pairs. The temperature at which double stranded nucleic acids denature to become single stranded depends on the: – sequence and length of the nucleic acid – the pH and buffer conditions – and any mismatches in base pairs between the two strands As such, the melting temperature is very useful analytical tool and can be studied by monitoring the absorbance at 260 nm as temperature is increased or decreased. As the temperature is increased, the hydrogen bonds between the strands are broken and the double stranded nucleic acid separates into two

Recent Advances in Oligo Purity and Sequence Determination by LC–MS: Maximizing Throughput, Confidence, and Coverage

Tuesday, November 15th 2022 Morning Session: 11am EST|8am PST|4pm GMT|5pm CET Afternoon Session: 2pm EST|11am PST|7pm GMT|8pm CET Analyzing oligos? Join us to learn about novel, automated, and integrated software to support the analysis of the CQAs of oligos using LC–HRAM MS. Purity and sequencing data will be presented for a multitude of oligos including antisense, aptamer, and long synthetic samples.

Automated High Throughput SEC Native Mass Spectrometry for mAb Screening

Thursday, September 22nd, 2022 9:00am EDT|6:00am PDT|2:00pm BST|3:00pm CEST 2:00pm EDT|11:00am PDT|7:00pm BST|8:00pm CEST See how RapidFire automated size exclusion chromatography (SEC) combined with the power of ion mobility mass spectrometry (IM-MS) enables efficient biotherapeutic development.

Industrializing High-Throughput Glycoproteomics Using AI for Clinical Use

Aberrant glycosylation of proteins has been implicated in key steps of disease biology, including the hallmarks of cancer as well as inflammation cascades associated with autoimmunity and aging. InterVenn Biosciences harnesses the predictive powers of artificial intelligence combined with cutting edge mass spectrometry to discover clinically relevant biomarkers that can only be revealed by high-resolution analysis of the glycoproteome.

Improved host cell protein analysis using highly reproducible and scalable automated sample digestion and fractionation ahead of LC–MS coupled with iterative MS–MS

Oligonucleotide Purification and Characterization - Tips for Success

Wednesday, March 30th, 2022 at 11am SST | 2pm AEDT | 10am ICT | 12pm KST Applying the appropriate tools for oligonucleotide purification and subsequent analysis is a unique challenge requiring application specific solutions. Join Drs. Anne Blackwell and Matthew L. Turner of Agilent Technologies for tips on oligonucleotide analysis and purification with Agilent’s suite of Bio LC solutions.

Glycomics/Glycoproteomics for Biopharmaceutical and Biotechnology Industries

Wednesday, March 16th, 2022 at 11am SST | 2pm AEDT | 10am ICT | 12pm KST Please join us for a series of three intense investigations into the practicalities required to define, identify, and explore the functions of important biological molecules including glycans, monoclonal antibodies, and oligonucleotides. We outline cutting edge and mainstay analytic tools and protocols, with multiple case studies and practical examples from world leading experts.

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