Liquid-Phase Oligonucleotide Synthesis: Past, Present, and Future Predictions Alejandro Gimenez Molina 1,3 and Yogesh S. Sanghvi 2 1 Nucleic Acid Center, Department of Physics, Chemistry & Pharmacy, University of Southern Denmark, Odense, Denmark 2 Rasayan Inc., Encinitas, California 3 Corresponding author: molina@sdu.dk Therapeutic oligonucleotides have emerged as a powerful paradigm with the ability to treat a wide range of the human diseases. As a result, we have wit- nessed more than one hundred oligonucleotides currently in active clinical trials and eight Food and Drug Administration (FDA)-approved drugs. Until now, the demand for oligonucleotide-based drugs has been fulfilled by conventional solid-phase synthesis in an effective manner. However, there are products in advanced stages of clinical trials projecting a collective demand of metric ton quantities in the near future. Therefore, large-scale manufacturing of these products has become a high priority for process chemists. This article sum- marizes the advances in liquid-phase oligonucleotide synthesis (LPOS) as a possible alternative strategy to meet the scale-up challenge. A review of the lit- erature describing major efforts in developing LPOS technologies is presented. Gratifyingly, serious attempts are under way to develop an efficient environ- mentally benign green chemistry protocol that is scalable and cost effective for the manufacturing of oligonucleotides. A summary of the most innovative LPOS protocols has been included to provide a glimpse of what may be possi- ble in the future for large-scale production of oligonucleotides. C  2019 by John Wiley & Sons, Inc. Keywords: DNA  large-scale synthesis  liquid-phase oligonucleotide syn- thesis  oligonucleotides  phosphoramidites  RNA  scale up  soluble supports  solution phase How to cite this article: Molina, A. G., & Sanghvi, Y. S. (2019). Liquid-phase oligonucleotide synthesis: Past, present, and future predictions. Current Protocols in Nucleic Acid Chemistry, e82. doi: 10.1002/cpnc.82 INTRODUCTION Oligonucleotides (ONs) are chemically synthesized in high yield and purity via an ef- ficient and easy to operate automated DNA/ RNA synthesizer using a four-step cou- pling cycle via phosphoramidite chemistry on a solid support. With the success of the first Food and Drug Administration (FDA)-approved oligonucleotide-based drug formivirsen (brand name Vitravene; see Crooke, Witztum, Bennett, & Baker 2018) in 1998, the number of oligonucleotide ther- apeutic drugs in the market has increased gradually during the last two decades (Stein & Castanotto, 2017), for example, pegap- tanib (brand name Macugen; 2004), mipom- ersen (brand name Kynamro; 2013), nusin- ersen (brand name Spinraza; 2016), eteplirsen (brand name Exondys 51; 2016), defilnotide (brand name Defitelio; 2016), rHBsAg-1018 ISS (brand name Heplisav-B; 2017), in- otersen (brand name Tegsedi; 2018), and patisiran (brand name Onpattro; 2018). With >100 oligonucleotide-based drug can- didates currently in different phases of clin- ical development (Current Protocols arti- cle: Sanghvi, 2011), it is anticipated that this trend will continue and will generate a Current Protocols in Nucleic Acid Chemistry e82 Published in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/cpnc.82 C  2019 John Wiley & Sons, Inc. Molina and Sanghvi 1 of 17