(152ar) Characterization of Novel Oligo(dT) Affinity Membranes for the Purification of mRNA Vaccines

mRNA vaccines have attracted increasing attention as a promising alternative to conventional vaccines due to their high potency, safe administration, capacity for rapid development, and flexible manufacturing [1]. A technology platform and cost-effective manufacturing process is required due to the growing demand for mRNA vaccines.

The ability of nucleic acids to hybridize specifically to complementary sequences represents a key property that plays important roles in biology and molecular biology techniques [2]. Affinity-based separation allows the mRNA to be captured by a single-stranded sequence of deoxythymidine (dT) (Oligo dT) which binds to the poly-A tails present in all mRNA vaccines [3]. After washing, poly-A-containing mRNA molecules are selectively eluted in water or mild base so as to preserve the integrity of the labile mRNA molecule. A number of factors affect the efficiency of hybridization on surfaces including the surface probe density and conformation and length of probe strands [2].

The goal of this work is to determine and model the adsorption isotherms of the binding between poly-A-containing molecules and Oligo dT-grafted membranes as a function of grafting density, temperature, and initial mRNA concentration. This is part of a global study on the development of porous microfiltration affinity membranes for mRNA vaccine purification.

Regenerated cellulose (RC) membranes functionalized with poly-deoxythymidine (dT) oligos were used to capture mRNA from solution through high affinity, high selectivity base-pairing interactions. Twelve different Oligo-dT/G(FAM) ligands were grafted to the surface of RC membranes using Single Electron Transfer-Living Radical Polymerization (SET-LRP) reaction, which was confirmed with fluorescent microscopy. The solution-depletion in fluorescence in the reaction mixture was used as a measure of the FAM labeled oligo dT covalently immobilized on the membrane matrix.

Surrogate vaccine molecules (Oligo-dA₆₀ & fluc-mRNA) were bound to the twelve different Oligo-dT/G ligands grafted on flat sheet RC membranes. Static adsorption binding and elution isotherms were used to help select the best two modification from the 12. The effects of different parameters, such as Oligo-dT grafting amount, initial concentration of mRNA solution, temperature of the binding process and different binding and elution buffers were tested to improve binding capacity. Dynamic binding and elution were conducted in a RPI home built membrane holder (module) called a “Tommy” in the HPLC pumping and detection system (not reported here). Static adsorption isotherm models were studied, and the isotherm data was found to be in good agreement with the Langmuir model ( >0.97) Fig. 1(A, B). Thermodynamic studies on the change in hybridization free energy, enthalpy and entropy were performed. The binding free energy for every unique combination of oligonucleotides/ mRNA was estimated and the values were found at all possible binding interactions based on the nearest-neighbor model [4] Fig. 1 (C-E).

The next steps would be to use Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) open cell module to characterize what kind of binding we are getting and what the ligand density is as well as how much binding we have. We’ll test the twelve different ligands (Oligo dT- poly T and T/G, 20 and 60-mers) and their binding behavior with Oligo dA (20 and 60-mers) and mRNA. Furthermore, we’ll try to develop a simple transport and binding model for hybridization inside a cylindrical pore grafted with a brush of oligo-dT.

References:

[1] S. S. Rosa, D. M. F. Prazeres, A. M. Azevedo, and M. P. C. Marques, “mRNA vaccines manufacturing: Challenges and bottlenecks,” Vaccine, vol. 39, no. 16, pp. 2190–2200, Apr. 2021, doi: 10.1016/J.VACCINE.2021.03.038.

[2] H. Ravan, S. Kashanian, N. Sanadgol, A. Badoei-Dalfard, and Z. Karami, “Strategies for optimizing DNA hybridization on surfaces,” Anal. Biochem., vol. 444, pp. 41–46, 2014, doi: 10.1016/j.ab.2013.09.032.

[3] V. Gote et al., “A Comprehensive Review of mRNA Vaccines,” Int. J. Mol. Sci., vol. 24, no. 3, Feb. 2023, doi: 10.3390/IJMS24032700.

[4] J. SantaLucia and D. Hicks, “The thermodynamics of DNA structural motifs,” Annu. Rev. Biophys. Biomol. Struct., vol. 33, pp. 415–440, 2004, doi: 10.1146/ANNUREV.BIOPHYS.32.110601.141800.