Aptuit has successfully completed several anti-sense oligonucleotides projects in various phases of clinical studies for three pharmaceutical companies. These projects required method development, phase appropriate validation and longterm stability studies of their drug substances and drug products, and testing of the in-process samples.

Aptuit has extensive experience in analyzing large molecules such as proteins, peptides, and oligonucleotides using innovative techniques. Employing state of the art instrumentation, Aptuit’s highly qualified analysts were able to develop and validate appropriate methods for the analysis and full characterization of anti-sense oligonucleotides and their impurities.

Aptuit’s scientific expertise, integrated method development and validation services enabled the successful launching of the Phase I clinical trial of multiple anti-sense oligonucleotides.

The Challenge

Anti-sense oligonucleotides are single strands of DNA (or analogs) that are complementary to a target sequence. They are chemically modified to improve in vivo stability, which is crucial for therapeutical purposes. During synthesis of these oligonucleotides, various oligo-related impurities are formed due to failure sequences, addition sequences, adducts, depurination and depyrimidation. The impurities also include phosphodiester analogs and partially de-protected sequences (trityl-on and trityl-off). Most of the oligo-related impurities co-elute with the main peak during HPLC/UV analysis and thus create difficulties in obtaining sufficient resolution and complete identification. The formation of an-Bn, bn, cn, wn, xn, and yn series adds complexity to the characterization. (Figure 1)

Adopted from reference: Scott A. McLuckey, Gary J. Van Berkel, and Gary L. Glish; Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides; Journal of American Society of Mass Spectrometry, 1992, 3, 60-70.

The Aptuit Solution

Aptuit was able to develop and validate appropriate methods for the analysis and full characterization of anti-sense oligonucleotides and their impurities. The following methods were employed:

• A full characterization of the anti-sense oligonucleotide included sequencing using ESI/MS/MS, determination of phosphorous backbone using 31P NMR, extinction coefficient, physicochemical characterization, CGE, PAGE, UV absorbance, molecular weight, specific optical rotation, moisture content, ICP for determination of sodium and phosphorus, TGA and residual solvents (Table 1);

• Various method development, method transfer and phase appropriate validation activities to support these programs;

• Stability profile, assessed by setting up short term and long-term stability studies for both drug substance and drug product. Study programs included analytical testing support, such as product and package appearance, pH determination, container closure integrity, osmolality, particulate matter, assay using ion-pairing reversed phase HPLC with mass spectrometer (IP-HPLC-MS), oligonucleotide profile, impurity profile, ID, sterility, endotoxin.

• GC/MS methods were developed and validated to provide quantization of genotoxic impurities (GTIs) in the final drug substance.

• For impurity analysis, an ion-pairing reversed phase HPLC method with MS detection in the negative ion mode was developed. Appropriate ion-pair reagents were identified to achieve mass spectrometric sensitivity and optimum separation of the full-length product from the failure sequences, which can be quantified using UV detection and identified with mass spectrometry detection. Co-eluting species were also identified and quantified using mass spectrometry. Accurate mass measurement was used to analyze the full-length product, failure sequences, synthetic impurities, and degradants.

• Deviations from the expected full-length product molecular weight always suggest modification of structure such as oxidation, deamidation, and backbone fragmentation. Combination of UV detection and mass spectrometry detection allowed for quantification of impurities eluting outside of the main peak and under the main peak. The impurities eluting outside of the main peak were reported in UV impurity profile. The impurities co-eluting within the main UV peak were reported as MS impurity profile and were determined by detection of the appropriate molecular ions (Figure 3).

Conclusion

Aptuit successfully applied validated methods for identity, assay, and impurity profile of multiple anti-sense oligonucleotides. These methods were implemented in multiple development phases, in support of analyses for synthetic intermediates, formulation development, stability study, and release testing. Aptuit’s scientific expertise and integrated method development and validation services provided the successful launching of the Phase I clinical trial of multiple anti-sense oligonucleotides. The breadth of integrated capabilities and high quality services offered by Aptuit enabled the comprehensive oligonucleotide projects to be conducted from a single source. The clients reported substantial satisfaction with the outcome of these projects, and attributed the positive outcomes to the professionalism and collaborative efforts of Aptuit teams. At Aptuit, it’s all about the science.