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May 1, 2013 (Vol. 33, No. 9)

Critical Tools for Oligo Characterization

  • 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.

  • LC/MS Reduces Sample Prep

    In-process analysis of purification fractions creates a bottleneck in oligonucleotide manufacturing. Sample preparation and analysis can stall production for two days or more, according to Todd B. Kreutzian, director, analytical development, Agilent Technologies.

    Agilent recently introduced an automated 2D UHPLC UV/MS method designed to relieve the bottleneck. The method combines automated sample preparation of high pH/high salt samples with rapid quantification of N+ and N- RNA impurities. “It’s a key component of process control strategy,” Kreutzian says.

    N+ and N- strands—single base additions or deletions of the full length material—result from over- or under-addition of bases during manufacturing or synthesis.

    The method involves an Agilent 1290 Infinity UHPLC equipped with a binary pump module with solvent degasser, autosampler, heated column compartment, ultraviolet detector, and several other modifications.

    “Our goal was to establish a set of analytical parameters that indicate purity for in-process samples at a pH and at a salt content (pH ~12 and ~1.5M NaCl) not suitable for ion-pair reversed-phase chromatography,” Kreutzian explains. Another objective was to duplicate the current QC method for in-process samples but a higher speed and throughput.

    Further, the method employed a 2D chromatography system to achieve automated, in-line sample desalting and neutralization with minimal operator intervention. The first chromatographic dimension consists of neutralizing and desalting. After neutralization and desalting, the sample is back-flushed off of the desalting column onto the head of the analytical column.

    “The 2D system did not use any custom equipment,” says Kreutzian. “All equipment, the pumps, valves, autosampler, and detector, are off-the-shelf items. Instrument control and data analysis is achieved through Agilent’s OpenLab ChemStation software without custom macros or additional programing.

    The method works as designed for critical validation endpoints:

    • Specificity: Chromatographic specificity for the N- and N+ sequence variants in the presence of full-length product; no significant sample matrix (high pH, high salt) interference in the region of full-length product or synthesis-related impurities elution.
    • Limit of Detection and Limit of Quantitation: As the method is for in-process testing only, specificity and repeatability were given more weight than LOD/LOQ. LOD and LOQ were determined to be 0.06% and 0.3% of the nominal sample concentration, which is adequate for the purposes of the method.
    • Precision: Repeatability and intermediate precision were within acceptable parameters.
    • Linearity: Linearity was evaluated from LOQ to 150% of the method’s nominal sample concentration, and met specification for the coefficient of determination not less than 0.99.

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