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

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.

But Is It Good Enough for QC?

The high resolution of LC/MS makes it a powerful tool for analyzing oligonucleotide impurities during process development. The knock on this technique is its lack of robustness for quantitative QC determinations. Edward Huber, Ph.D., director, quality control and analytical development, Girindus America, hopes to demonstrate the suitability of LC/MS for both characterization and QC.

Girindus (recently acquired by Nitto Denko Avecia) has a lot at stake in oligo characterization. Last year the company entered into a strategic alliance with Marina Biotech. The deal provides Girindus/Avecia with exclusive rights to Marina’s conformationally restricted nucleotide (CRN) synthetic process. In return Marina gets royalties from the sale of CRN-based oligonucleotide reagents, plus preclinical and clinical cGMP material for Marina and its drug development programs.

CRNs are patented analogs in which a chemical bridge spans from the C2´ and C4´ carbons of ribose, the central component of a nucleotide. CRN locks the ribose within a fixed conformation, which restricts the molecule’s flexibility, thus allowing more specific reactivity and production of putative RNA medicines that were previously believed to be not “druggable.”

Orthogonal Methods

Michele DeRider, Ph.D., lead scientist at Catalent Pharma Solutions, points out the strengths and weaknesses of nuclear magnetic resonance, circular dichroism, and fourier-transfer infrared spectroscopy (FTIR) in oligonucleotide analysis and characterization.

Among NMR’s strengths: it is quantitative, has broad dynamic range, is nondestructive, and versatile. “There are so many levels of information available with NMR, depending on how you use it,” Dr. DeRider says. A big plus for oligos is that the typical formulation buffer is also the NMR solvent of choice. The most-studied nuclei are protons and 32P, the latter being diagnostic for specific base linkages.

“NMR’s weaknesses include expensive equipment and operation,” Dr. DeRider says. NMR uses liquid helium to cool the probe to near absolute zero. The current helium shortage has made the material prohibitively expensive for many research groups.

Circular dichroism, while extremely sensitive to subtle conformational variations, does not analyze molecular structure as deeply as NMR or IR. “You’re limited to looking for minima and maxima, but other than that it is not very feature-rich. It’s most useful for stability or thermodynamic features,” Dr. DeRider notes. “And it can be too sensitive at times to trace contaminants.”

By contrast FTIR instrumentation, commonly used to identify compounds from the “fingerprint region” spectrum, is ubiquitous and easy to use. While some IR bands will be diagnostic for specific linkages, the spectra are extremely complex. This does not help with oligonucleotides, which are all composed of the same four building blocks plus sugars and phosphate.

While Dr. DeRider is partial to NMR, she confirms that spectroscopic characterization of oligonucleotides is a puzzle-like exercise. “You have to piece information from different sources together. Each method yields more and more information.”

Product Specifications: Why We Analyze

The point of oligonucleotide analysis is to assure that products meet specifications. According to Daniel Capaldi, Ph.D., vp for analytic and process development at Isis Pharmaceuticals, “a specification is a critical quality standard, proposed by the manufacturer and approved by regulatory agencies. It is a contract among the manufacturer, the regulatory agencies, and the patient, which describes the quality criteria that the drug substance or drug product will meet consistently.”

Interestingly, therapeutic oligonucleotides, as well as some peptides and radiopharmaceuticals, are excluded from the scope of International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q6A. Evidently the ICH expert working group lacked direct experience with oligos, and time-restrictions prevented their consideration within the guidance.

“We believe that the spirit of Q6A, and that of the related impurities guidelines Q3A and Q3B, which also categorically exclude oligonucleotides, do, in fact, apply to oligonucleotide therapeutics, but that certain details of the guidelines require modification,” Dr. Capaldi explains.

As with small molecule drugs, oligonucleotide specifications are not designed to establish full characterization, but rather focus on critical quality attributes to ensure safety and efficacy. This is achieved by confirming identity and strength and through impurity characterization.

As one would expect from their structure, oligonucleotides present unique challenges. Impurity testing is relatively straightforward with small molecule drugs, but with oligos impurities can only be practically controlled as groups of related structures. “The size and complexity of oligonucleotide drugs often requires the application of sophisticated analytical techniques,” Dr. Capaldi adds.

“The relatively limited data obtainable by analyzing an oligonucleotide drug also enhances the importance of other aspects of the control strategy. For example, at Isis we have invested heavily in process and product knowledge, in-process controls, and process validation, and we view these and other quality-by-design principles, such as risk assessment and adherence to current good manufacturing practices, as equally important components of any strategy designed to assure quality.”

Isis Pharmaceuticals has invested heavily in process and product knowledge, in-process controls, and process validation.

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