Trends in Biopharma Analysis: Oligonucleotides, mRNA Characterization, and More at HTC-18

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A morning session on Wednesday 28 May 2024 at the International Symposium on Hyphenated Techniques in Chromatography and Separation Science (HTC-18) in Leuven, Belgium focused on biopharmaceutical analysis. The session was chaired by Huiying Song from Johnson & Johnson, Belgium.

The first presentation was an illuminating talk by Davy Guillarme from the University of Geneva, Switzerland entitled “Innovative Chromatographic Strategies for Oligonucleotide Characterization.” Guillarme stated that DNA and RNA-based therapies are increasingly being used to treat genetic diseases, particularly with the success of therapeutic oligonucleotides, and are effective because of high target specificity and enhanced pharmacokinetic properties. Although oligonucleotides are categorized as small molecules by regulatory agencies, they share traits with therapeutic proteins, making oligonucleotides a unique class requiring specialized analytical approaches, Guillarme highlighted. To ensure the safety and efficacy of therapeutic oligonucleotides robust analytical methods are essential reverse phase liquid chromatography with ion pairing agents (IP-RPLC) is the standard method, while hydrophilic interaction liquid chromatography (HILIC) offers unique analytical characteristics.

Guillarme described four key analytical strategies to enhance sensitivity, throughput, and selectivity in IP-RPLC and HILIC for oligonucleotide analysis: The importance of bioinert columns to reduce oligonucleotide adsorption, the benefits of alternative column chemistries to improve selectivity; the practical value of ultra-short columns for high-throughput separations; and application of pressure as a parameter to adjust selectivity were highlighted.

This presentation was followed by a talk from Koen Sandra from RIC Group, Belgium called “Advancements in mRNA Structural Characterization – The Coming-of-Age of the Analytical Toolbox.” Sandra started the talk by emphasizing that the global success in managing the COVID-19 pandemic was (and is) largely due to the development of mRNA-based vaccines.

MRNA-based vaccines have paved the way for extensive exploration of mRNA technology for both preventive and therapeutic purposes, according to Sandra. The recent advances are based on three decades of solid research, he emphasized, with key discoveries including the use of naturally modified nucleotides in mRNA to suppress immune responses and the packaging of mRNA in lipid nanoparticles (LNPs) to protect it during administration and improve cellular uptake.

Sandra highlighted that the rapid emergence of mRNA therapies has created a need for sophisticated analytical methods to study various structural properties, including 5’ capping efficiency, mRNA sequence, 3’ poly-A tail length, post-transcriptional modifications, mRNA integrity, fragmentation, double-stranded RNA (dsRNA), and aggregation. Sandra described in detail a range of analytical techniques based on liquid chromatography (LC), particularly the importance of ion pair reversed phase liquid chromatography mass spectrometry (IP-RPLC–MS) and hydrophilic interaction liquid chromatography (HILIC), as well as the role of multi-angle light scattering (MALS) detection, and mass photometry to comprehensively analyze the properties of these important therapeutic molecules.

Quang-Dong Bui from the Vrije Universiteit Brussel, Belgium then discussed a project he worked on in collaboration with Janssen Pharmaceutical, Belgium entitled “Oligonucleotide Analysis with Bio-Inert Chromatography and Ion Pair Agents for MS Detection.” This project focused on the analysis of oligonucleotides (ONs) that are polymers of nucleotides used to treat genetic diseases. Bui highlighted that ion-pair reversed-phase liquid chromatography coupled with mass spectrometry (IP-RPLC-MS) has become essential for ON quality control. His research aimed to examine the impact of different organic modifiers, ion-pair agents, such as TEA, DIPEA, HA, and counter ions, including HFIP, HFMIP, NFTBA, on chromatographic resolution and MS sensitivity using bioinert columns with varying particle types and sizes.

Bui highlighted that HFIP increased detection sensitivity, while HFMIP slightly improved chromatographic resolution. Rapid analysis of 15-35 polyT ladders was achieved with a short core-shell column. The study also successfully separated full-length products (21-mer) from impurities (20-mer and 19-mer) and phosphorylated oligomers using a coupled-column system.

Bui then described the development of a membrane-based microfluidic stripper to remove ion pair agents, such as TEA. before MS detection. This stripper used a Nafion anion-exchange membrane for selective ion transport to replace TEA with positively charged ions. The effectiveness of this approach was confirmed through off-line GC-FID analysis. The design of the stripper was optimized, considering regenerant composition and membrane thickness, and its ability to enhance ON intensity in MS direct infusion experiments. Bui concluded that the approach he described optimizes chromatographic and detection methods for oligonucleotides and introduces a novel technique to improve MS sensitivity by removing interfering agents.

Ravindra Suryakant Hegade from Johnson Innovative Medicine, Belgium then presented on “Automated QbD Method Development for Pharmaceutical Impurity Separation.” Hegada highlighted that efficient separation of impurities is vital in pharmaceutical production and Quality by Design (QbD) methods streamline this process by systematically designing and refining techniques to ensure product quality and regulatory adherence. Hegade highlighted that automation plays a key role in speeding up and standardizing method development and that automated QbD strategies enable pharmaceutical firms to enhance the efficiency and reliability of impurity separation. These methods use sophisticated software algorithms and robotic systems to methodically assess the impact of various factors on separation effectiveness. Automated platforms also enable swift screening of multiple variables, pinpointing the best conditions for impurity separation. Combining automation with QbD principles not only accelerates method development but also enhances its resilience and transferability to allow real-time monitoring and data analysis, he concluded.

The session concluded with a talk called “Online Coupling of Size exclusion Chromatography to Raman spectroscopy for the Analysis of Therapeutic Proteins and Disaccharides Jana Thissen from the Institut fur Umwelt and Energie and Analytic (IUTA), Germany. Thissen commented that the efficacy of therapeutic proteins depends on molecular structure and techniques to verify structural integrity are therefore crucial. Raman spectroscopy (RS) is effective for structural analysis, especially when combined with size exclusion chromatography (SEC) in capillary enhanced Raman spectroscopy (CERS). SEC–CERS allows for structural assessment and protein identification in biological samples or formulations, according to Thissen. This approach was used to successfully analyze proteins such as h y37bovine serum albumin and monoclonal antibody rituximab in medicinal products and identified serum albumin and IgG antibody in complex samples such as porcine blood serum. Additionally, CERS quantified the excipient sucrose in biopharmaceuticals to provide an alternative to UV/vis absorption detection. However, online SEC–CERS requires advanced data post-processing, and this has led to the development of a software platform for automation. Thissen concluded that online SEC–CERS holds promise for biopharmaceutical development and quality control by simultaneously elucidating protein structure and quantifying excipients.