The emphasis on process understanding has been part of pharmaceutical development strategy since FDA’s 2003–2004 promulgation of GMPs for the 21st Century. Oligonucleotides were almost nonexistent in major pharmacopoeias at the time. Now that oligo drugs are nearing late development and commercialization, analytical tools for speeding process development will come to the fore as they have with small molecules and therapeutic proteins.
A new software package from BioSpring for characterizing oligonucleotide synthesis products shows promise for characterizing unknown side products and impurities during and after oligo synthesis.
LC/MS is well established for identifying known compounds and confirming identity from mass peaks. Characterizing small molecule impurities, even unknowns, is facilitated by fragmentation patterns and computerized access to spectral libraries. LC/MS is not as routine or straightforward for oligonucleotides, whose synthesis creates a host of impurities and side products.
“You can’t tell what these are simply from the mass information,” notes Susann Rosmus, Ph.D., head of quality management at the company. Since impurities from each synthesis step carry over, the potential list can get quite large. Each step in an oligo synthesis involves protection, deprotection, and addition of a base. Reactions are never quantitative, so the impurities can add up rapidly after every step. In addition to N+ and N- entities, product may include salt adducts and unremoved protecting groups.
“If you have an N+1, you would detect a mass around 300 higher than main mass,” Dr. Rosmus explains. “But it could also be a protecting group plus something else, or an adduct from the buffer system.”
OligoFrag picks all MS-detectable impurities out and verifies their identity using a “special algorithm,” Dr. Rosmus says. How does it work? Before the synthesis, the identities of all protecting groups, potential salt adducts, and of course the bases, are entered into the program. Since the masses are unique to the spectrometer, the program provides impurity identities by matching up the measured masses with calculated values.
“The software gives all different possible combinations matching the detected mass,” she adds.
Last year, BioSpring became the first European company to obtain a GMP certificate for producing synthetic oligonucleotides. The certificate allows the company to manufacture oligonucleotide active pharmaceutical ingredients. BioSpring has entered a commercial manufacturing agreement with Sanofi for therapeutic oligonucleotide manufacturing.