Unified and Versatile Multiplex Platform for Expedited Method Development and Comprehensive Characterization of Therapeutic Peptides.

Agile analytical approaches are needed for fast and comprehensive characterization of peptide drug candidates. In this study, a unified and versatile multiplex platform was developed to expedite method development and enable the routine determination of multiple quality attributes simultaneously. The platform integrates the automation of size exclusion chromatography (SEC), reversed phase liquid chromatography coupled to reversed phase liquid chromatography (RPLC-RPLC), and hydrophilic interaction liquid chromatography hyphenated to charged aerosol detection (HILIC-CAD). Various therapeutic peptide constructs, including macrocyclic peptides and disulfide constrained peptides, across different lots were studied. The effect of the mobile phase acetonitrile content on the impurity profiles was systematically studied using two SEC columns. A prototype MaxPeak Premier SEC 125 Å column packed with BEH PEO particles achieved the separation of impurities (>2.0% area), whereas no impurities could be observed with an ACQUITY UPLC Protein BEH SEC 125 Å column packed with BEH diol particles. Comprehensive impurity profiling and expedited method development was performed utilizing RPLC-RPLC. Each peptide was analyzed by a combination of 12 conditions in the second dimension, using four columns with octadecyl, phenyl-hexyl, and cyano bonded phases, and three mobile phases with various solvents, modifiers, and pH compositions. Additionally, a HILIC-CAD method was developed for the quantification of TFA, commonly present in peptide products.

Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column.

Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column's physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.

Process Robustness in Lipid Nanoparticle Production: A Comparison of Microfluidic and Turbulent Jet Mixing.

The recent clinical and commercial success of lipid nanoparticles (LNPs) for nucleic acid delivery has incentivized the development of new technologies to manufacture LNPs. As new technologies emerge, researchers must determine which technologies to assess and how to perform comparative evaluations. In this article, we use a quality-by-design approach to systematically investigate how the mixer technology used to form LNPs influences LNPstructure. Specifically, a coaxial turbulent jet mixer and a staggered herringbone microfluidic mixer were systematically compared via matched formulation and process conditions. A full-factorial design-of-experiments study with three factors and three levels was executed for each mixer to compare process robustness in the production of antisense oligonucleotide (ASO) LNPs. ASO-LNPs generated with the coaxial turbulent jet mixer were consistently smaller, had a narrower particle size distribution, and had a higher ASO encapsulation as compared to the microfluidic mixer, but had a greater variation in internal structure with less ordered cores. A subset of the study was replicated for mRNA-LNPs with comparable trends in particle size and encapsulation, but more frequent bleb features for LNPs produced by the coaxial turbulent jet mixer. The study design used here provides a road map for how researchers may compare different mixer technologies (or process changes more broadly) and how such studies can inform process robustness and manufacturing control strategies.