Optimization of High-Performance Liquid Chromatography Parameters for Purification of Oligonucleotide-A
- 1 Department of Chemistry, Texas Southern University, Houston, USA
- 2 Department of Chemistry, Texas Southern University, Houston, USA
- 3 Department of Chemistry, Texas Southern University, Houston, USA
Abstract
The modification of high-performance liquid chromatography parameters leads to a more effective oligonucleotide-A purification process. Using various experimental parameters such as buffer, concentration, and pH, a method for optimizing the purification of an oligonucleotide-A on a reverse-phase C18 column was created. To purify oligonucleotide-A, High-Performance Liquid Chromatography (HPLC), Ultraviolet-Visible Spectrophotometry (UV-Vis), Liquid Chromatography-Mass Spectrometry (LC-MS), and lyophilization were used. Chromatographic data were collected with a semi-prep HPLC system, quantified with the UV-Vis technique, and validated with the LC-MS method. The most optimized parameters found to obtain the purity of 93.0% are 40 mM triethylammonium bicarbonate (TEAB) buffer with pH 7, which is approximately 6.0% higher than the reported method of which the purity is 87.0%. However, the yield under these conditions was reduced by about 5%. The worst possible optimized settings that resulted in the lowest purity (84.0%) and yield (69.0%) are 10 mM ammonium acetate (NH 4 CH 3 CO 2 ) with pH 7.
- Biba, M., Jiang, E., Mao, B., Zewge, D., Foley, J.P. and Welch, C.J. (2013) Factors Influencing the Separation of Oligonucleotides Using Reversed-Phase/Ion-Exchange Mixed-Mode High Performance Liquid Chromatography Columns. Journal of Chromatography A, 1304, 69-77. https://doi.org/10.1016/j.chroma.2013.06.050
- Tosi, L., Sridhara, V., Yang, Y., Guan, D., Shpilker, P., Segata, N., Larman, H.B. and Parekkadan, B. (2017) Long-Adapter Single-Strand Oligonucleotide Probes for the Massively Multiplexed Cloning of Kilobase Genome Regions. Nature Biomedical Engineering, 1, Article No. 0092. https://doi.org/10.1038/s41551-017-0092
- Ahuja, S. and Alsante, K. (2003) Handbook of Isolation and Characterization of Impurities in Pharmaceuticals. 1st Edition, Academic Press, Cambridge.
- York, K.T., Smith, R.C., Yang, R., Melnyk, P.C., Wiley, M.M., Turk, C.M., Ronaghi, M., Gunderson, K.L. and Steemers, F.J. (2012) Highly Parallel Oligonucleotide Purification and Functionalization Using Reversible Chemistry. Nucleic Acids Research, 40, e4. https://doi.org/10.1093/nar/gkr910
- Zhang, Q.L., Lv, H.H., Wang, L.L., Chen, M., Li, F.F., Liang, C., Yu, Y.Y., Jiang, F., Lu, A.P. and Zhang, G. (2016) Recent Methods for Purification and Structure Determination of Oligonucleotides. International Journal of Molecular Sciences, 17, Article No. 2134. https://doi.org/10.3390/ijms17122134
- Gong, H., Holcomb, I., Ooi, A., Wang. X., Majonis. D., Unger. M.A. and Ramakrishnan, R. (2016) Simple Method to Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells. Bioconjugate Chemistry, 27, 217-225. https://doi.org/10.1021/acs.bioconjchem.5b00613
- Pinto, A., Chen, S.X. and Zhang, D.Y. (2018) Simultaneous and Stoichiometric Purification of Hundreds of Oligonucleotides. Nature Communications, 9, Article No. 2467. https://doi.org/10.1038/s41467-018-04870-w
- Cohen, A.S., Najarian, D.R., Paulus, A., Guttman, A., Smith, J.A. and Karger, B.L. (1988) Rapid Separation and Purification of Oligonucleotides by High-Performance Capillary Gel Electrophoresis. Proceedings of the National Academy of Sciences of the United States of America, 85, 9660-9663. https://doi.org/10.1073/pnas.85.24.9660
- Guttman, A., Cohen, A.S., Heiger, D.N. and Karger, B.L. (1990) Analytical and Micropreparative Ultrahigh Resolution of Oligonucleotides by Polyacrylamide Gel High-Performance Capillary Electrophoresis. Analytical Chemistry, 62, 137-141. https://doi.org/10.1021/ac00201a010