Tomas Kostelec, Rok Sekirnik, Anže Martinčič Celjar, Kristina Šprinzar Nemec, Andreja Gramc Livk, Pete Gagnon, Aleš Štrancar
BioProcess International, June 2021
COVID-19 has focused a spotlight on the ability of mRNA technology to accelerate vaccine development and approval. That same technology can hasten development and approval of other therapeutic classes, including cancer immunotherapy, protein replacement, and gene therapy. Fulfilling those opportunities imposes significant challenges on process developers and manufacturers to improve existing processes. Scale-up to produce millions of doses (tens of kilograms) compounds those challenges. Furthermore, every step of the journey requires high-performance analytical methods, to ensure patient safety and maximize productivity.
Artaches A. Kazarian, Wesley Barnhart, Iain D.G. Campuzano, Jeremy Cabrera, Theodore Fitch, Jason Long, Kelvin Sham, Bin Wu, Justin K. Murray
Journal of Chromatography A,Volume 1634, 2020
The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single stranded oligonucleotide in high purity (92%) under denaturing conditions.
The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.
P. Gagnon, B. Goričar, Š. Peršič, U. Černigoj, A. Štrancar
Cell & Gene Therapy Insights 2020; 6(7), 1035–1046
One of the barriers to development of industrial purification platforms for large mRNA has been an inadequate selection of high-performing capture-purification tools. Hybridization-affinity uses a polythymidine (Oligo dT) ligand to base-pair with the polyadenine tail of mRNA. It can be used for capture but it cannot discriminate dsRNA (double-stranded) from ssRNA (single-stranded) and it supports only brief cleaning with 100 mM sodium hydroxide. Traditional anion exchangers elute only mRNA smaller than about 500 bases unless the columns are heated to 50–70°C. Hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) separate ssRNA from dsRNA and short transcripts, but their sensitivity to fouling by proteins and aggregates makes them better suited for polishing than for capture. Better capture options are needed to meet the needs of large clinical trials, scale-up, and manufacture of vaccines. Beyond that, a new spectrum of gene therapy treatments await. This article introduces two new capture options that both eliminate dsRNA, DNA, and proteins in a wash step, then provide high-resolution polishing of ssRNA in an elution gradient at ambient temperature. One represents a new class of anion exchangers. The other exploits hydrogen bonding. Both support prolonged exposure to 1 M sodium hydroxide. Easy transition to either HIC or RPC provides high-resolution orthogonal polishing.
E. S. Sinitsyna, J. G. Walter, E. G. Vlakh, F. Stahl, C. Kasper, T. B. Tennikova
Talanta 93 (2012) 139-146
Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co- ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure.
The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.
G. A. Platonova, T. B. Tennikova
Journal of Chromatography A, 1065 (2005) 75–81(2005) 75–81
High-performance monolithic disk affinity chromatography was applied to the investigation of formation of complexes between (1) complementary polyriboadenylic and polyribouridylic acids, e.g. poly(A) and poly(U), respectively, (2) poly(A) and synthetic polycation poly(allylamine), pAA. Polyriboadenylic acid and poly(allylamine) were immobilized on macroporous disks (CIM disks). Quantitative parameters of affinity interactions between macromolecules were established using frontal analysis at different flow rates.