Development of Kilogram-Scale Convergent Liquid-Phase Synthesis of Oligonucleotides:
Xuan Zhou, PhD, Senior Scientist, Oligonucleotide Development, Biogen
Dr. Xuan Zhou began his presentation by highlighting the importance of oligonucleotides as a new class of drugs. About 15 drugs have been approved for treatment of rare diseases and one for high cholesterol. There are numerous compounds in development or under investigation for diseases such as cardiovascular, neurological, and cancer. As a result, there is a growing need for large-scale oligonucleotide production to deliver hundreds of kilograms (kg) of oligonucleotides in a robust and economical manner. He explained that current oligonucleotide production relies on solid-phase synthesis, which uses an insoluble support to add nucleotides one at a time. Each cycle requires three to four reactions to add a nucleotide. The process is fully automated and optimized and takes less than 24 hours. However, there are some limitations of solid-phase synthesis regarding the need for specialized equipment, facilities, large volumes, and high number of batches.
Dr. Zhou continued his presentation by focusing on liquid-phase synthesis. The suitability of liquid-phase synthesis for larger batch sizes and numerous facilities available around the world are some advantages. However, there are also some challenges, such as purification, removal of by-products, low overall yield, and difficulty of large-scale synthesis. For example, solid-phase synthesis eliminates the need to isolate oligonucleotide intermediates because they grow on a solid support; however, liquid-phase synthesis is a homogeneous reaction where solid-phase purification strategies do not work. In addition, the accumulation of impurities and the insufficient overall yield of each step dramatically affect the efficiency of the entire synthesis.
To overcome the problems with synthesizing oligonucleotides using a linear approach, a convergent synthesis approach was developed by coupling smaller oligonucleotide fragments. For instance, to manufacture a 20-mer oligo, it is possible to produce 5-mers from nucleotide building blocks and couple them successively to obtain 10-mers and 20-mers. By doing so, the synthesis route is shortened while the overall yield increases. Parallel processing of the fragments leads to a shorter cycle duration. Also, single batch failures are not reflected in the other independent fragment synthesis processes. On the other hand, there are some challenges of convergent synthesis mentioned by Dr. Zhou to be addressed such as the 3’-OH protection group and deprotection, ASO intermediates and fragment purification, long fragment synthesis route, critical impurities control, water control strategy, stability, and so on.
In the next part of his talk, Dr. Zhou shared the solutions that were developed to address the drawbacks of liquid-phase synthesis. For the challenge of the 3’-OH protection group, they showed an increase in overall yield and a decrease in reaction time when they tested tert-Butyldiphenylsilyl (TBDPS), which is a different type of pyridine group in the presence of imidazole. For the isolation of intermediates, an extraction and precipitation method was used to remove hydrophilic and hydrophobic groups respectively by taking advantage of the solubility difference between the product and by-product. The purification of fragments after a coupling reaction is more challenging because the solubility of product and by-product (i.e. 10-mer product and 5-mer by-products) is close to each other. They introduced the 3’-large hydrophobic protecting group (3’-LHPG) to change the solubility of the product to remove the by-products. The other challenge comes from the long synthesis route and the number of reactions required in each cycle, resulting in loss of time, products, and reagents. To combat this, a one-pot method was developed to perform coupling, sulfuration, and detritylation reactions in a common solvent. This allowed them to complete the reaction with 67% fewer isolation steps. As another challenge of liquid-phase synthesis, Dr. Zhou addressed the control of critical impurities such as deamination, n-1 and n+1, and the importance of consulting on how they are formed.
Dr. Zhou focused in on their synthesis route and successful large-scale production. Regarding the 18-mer example, they produced the full-length oligonucleotide from small parts, including an LHPG monomer and four fragments without any column purification. Using a synthesis strategy, they increased the synthesis scale by 10 times from 300 g to 3 kg. He defined the synthesis process as scalable and practical, noting that they are currently trying to improve the UV purity and are also working on 25-kg scale synthesis of 18-mers.
