NepaChem would like to congratulate Dr. Durga Pokharel for completing his PhD from Michigan Technological University, USA.
Dr. Pokharel worked with Dr. Shiyue Fang on ‘non-chromatographic purification of synthetic bio-oligomers’ producing about half dozen peer reviewed publications and one patent.
He is going to work as a postdoctoral research fellow in the same university from next semester.
Some of his publications are:
Here is the summery of Dr. Pokharel’s dissertation.
- Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization
- Purification of Synthetic Peptides Using a Catching Full-Length Sequence by Polymerization Approach
- Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer
- A Highly Convenient Procedure for Oligodeoxynucleotide Purification
- Synthetic 5′-phosphorylated oligodeoxynucleotide purification through catching full-length sequences by polymerization
Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications.
To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The savings with the new techniques compared with HPLC are estimated to be 70% to 90% depending on purification scale and throughput. We expect these new oligonucleotide and peptide purification technologies to be widely used in academic research labs, biotechnology companies, and pharmaceutical companies in the near future.