Sponsored content brought to you by
Nucleic acids are emerging as a third generation of pharmaceuticals following the clinical accomplishments of small-molecule drugs and biologics like proteins and monoclonal antibody (mAb) therapies. In particular, the initial widespread success of mRNA therapeutics with COVID-19 vaccines is propelling mRNA therapeutics into new spaces such as autoimmune diseases, rare diseases, and immuno-oncology.
As mRNA based therapeutics move beyond the early vaccine successes to encompass diverse modalities, manufacturers face rising expectations to deliver products with consistent purity and quality. This raised bar is in turn forcing the evolution of traditional purification and quantitation processes to address manufacturing challenges.
Specifically, immunogenic double-stranded RNA (dsRNA) byproducts are a major impurity that complicate the purification of RNA-based vaccines and therapeutics synthesized with in vitro transcription (IVT). Additionally, current UV methods of nucleic acid quantitation require extra sample handling in the form of dilution, which can lead to inaccuracies in measuring mRNA concentration.
In a recent GEN webinar, Repligen senior scientist Nathaniel Clark, PhD, discussed AVIPure® dsRNA Clear, a new affinity resin that removes immunogenic dsRNA byproducts. Clark also elaborated on complementary Slope Spectroscopy technology that accurately measures nucleic acid concentrations, off- and in-line, without dilution.
Characterizing dsRNA
dsRNAs are unwanted byproducts produced during RNA transcription with T7 RNA polymerase. These impurities cause inflammation by engaging virus-sensing innate immunity receptors. Therapeutic applications of mRNA, such as those to treat enzyme deficiencies, cancer, or genetic conditions require very large doses, and therefore control of dsRNA levels is critical.1 Controlling dsRNA levels reduces side effects allowing dosing flexibility.
“Currently there is a lack of regulatory consensus regarding acceptable dsRNA levels in final mRNA products,” noted Clark. “The real question is whether you can remove all immunogenicity from the molecule.”
dsRNA copurifies through oligo DT affinity chromatography. It is not reduced by tangential flow or other diafiltration operations. Solvent-based methods offer some separation, but the reliance on hazardous solvents makes scaling difficult.
Levels of dsRNA are typically 0.1%–0.5% of total transcribed RNA. These low concentration of dsRNAs meant there was very little information on the size of dsRNA byproducts at the beginning of product development. This led to Clark and other Repligen scientists developing a dual-color immuno-northern blot method to measure the size of dsRNA byproducts. Following separation on an agarose gel, the RNAs are transferred to a membrane and probed with the J2 mAb. Residual nucleic acid stain from the agarose gel localizes the primary ssRNA transcripts, whereas the chemiluminescent signal from the J2 mAb identifies the dsRNA byproducts.1
“This technique reveals that most dsRNA byproducts are larger in mass than the primary transcripts,” said Clark.
Removing dsRNA with an Affinity Resin
Repligen developed an AVIPure ligand specific to dsRNA. AVIPure scaffolds are composed of small, highly stable proteins and peptides that are identified entirely through in vitro selection processes. These ligands offer high binding affinities and excellent chemical stability enabling exceptional cleanability, durability, and cost-effectiveness.2
The base matrix is an equally critical component of the affinity resin. Given the high molecular weight and size of dsRNA contaminants identified during characterization, Repligen used a HiPer™ macroporous base bead. This scalable macroporous support is designed to have highly convective mass transfer for rapid flow and binding, independent of target size.2
The AVIPure dsRNA Clear resin captures all dsRNAs, even those larger than 1.8 kilobases, through a protein─nucleic acid interaction. The high-capacity resin is very stable in sodium hydroxide, guanidine HCL, and other common sanitization solutions. According to Clark, the optimal way to start using the AVIPure dsRNA Clear resin is with a high-throughput filter plate screen that allows testing of different salt types, pH ranges, RNA concentrations, and RNA capacity challenges.
“You mix 100 µl of RNA and some resin in each well of a 96-well plate, shake for 30 minutes, spin the plate down, and collect and analyze the flow-through. Control wells without resin test the solubility of the RNA in the varied conditions,” described Clark. “The key is to find the right salt type and concentration. Both sodium and potassium chloride should be tested.” The AVIPure dsRNA Clear resin was demonstrated to bind and remove dsRNA byproducts in mRNA, saRNA, and circRNA products.
To address whether affinity purification is needed with optimized IVT products, Repligen collaborated with etherna, who prepared triplicate GFP mRNA samples and then purified each sample with the AVIPure dsRNA Clear resin.3 “The better the input; the better the output,” emphasized Clark. “With IVT optimization, we saw a 0.73 log reduction of dsRNA. Affinity purification reduced dsRNA 2.3 logs further.” The affinity purification step effectively removed all immunogenicity when paired with optimized IVT. An A549 reporter cell-based assay verified results.
A Better Way to Quantify Nucleic Acids
Quantification of nucleic acids is typically performed with UV-Vis at 260 nanometers. Slope Spectroscopy, an advanced UV-Vis measurement technique, uses a variable pathlength to generate a linear plot of absorbance versus pathlength. The PATsmart™ SoloVPE® spectrophotometer adjusts the pathlength using a fiber optic probe, to provide reliable, accurate measurements without dilution, even with extremely concentrated samples. Each concentration measurement is a result of multiple measurements, typically 10 or more points, at different pathlengths and takes about one minute.
A related in-line process analytical technology (PAT), the PATsmart™ FlowVPX®, uses Slope Spectroscopy to take real-time concentration measurements with virtually no concentration limit during chromatographic or tangential flow operations. “Nucleic acids absorb like crazy and elution peaks can completely saturate detectors. FlowVPX can solve this problem for any nucleic acid purification,” emphasized Clark. Real-time measurements better utilize resin, lead to reduced risks, reduce operational costs, increase process accuracy and productivity, and provide significant time savings.
Together, the AVIPure dsRNA Clear resin and Slope Spectroscopy nucleic acid quantification are critical tools to assist manufacturers in delivering RNA products with consistent purity and quality. The AVIPure dsRNA Clear resin binds and removes dsRNA byproducts in mRNA, saRNA, and circRNA products. This affinity purification step effectively removes all immunogenicity when paired with optimized IVT protocols as verified using an A549 reporter cell-based assay.
References
- Clark et al. An immuno-northern technique to measure the size of dsRNA byproducts in in vitro transcribed RNA. Electrophoresis. 2024 Sep;45(17-18):1546-1554. doi: 10.1002/elps.202400036
- Clark. A scalable purification strategy for removal of dsRNA byproducts following IVT RNA production. Nucleic Acid Insights 2025; 2(5), 117–129, doi: 10.18609/nuc.2025.018
- Clark et al. Removal of dsRNA byproducts using affinity chromatography. Molecular Therapy Nucleic Acids. 2025; 36(2), doi: 10.1016/j.omtn.2025.102549
