Develop HPLC Assay & Impurities Methods For a Prostacyclin Derivative
Challenge: The prostacyclin derivative is a starting material in the manufacturing of a tricyclic benzidene prostacyclin analog with pharmacologic actions similar to those of Epoprostenol. Epoprostenol improves exercise capacity and survival in patients with pulmonary arterial hypertension. The prostacyclin analog is also efficacious by subcutaneous infusion, it is easier to administer and has a longer half-life. Determination of this prostacyclin derivative and its impurities is central to developing a deep understanding of a commercial manufacturing process.
A primary goal in early-stage pharmaceutical development is to prudently establish a scaled-up synthesis process for making acceptable active pharmaceutical ingredient (API). In this regard, analytical impurities methods used for both in process and API release testing must rapidly evolve to account for the detection and quantification of all key impurities.
Tracking impurities in this drug product manufacturing process is very challenging due to the API's complex structure, including several stereogenic centers. The synthesis process also introduces a number of process-related impurities including this derivative and others resulting from side-product formation inherent in the highly reactive intermediate required for the transformation of the intermediate material to drug product. The process-related impurity profiles and API impurity profiles can be complex, with impurities that are difficult to distinguish from each other.
Furthermore, each drug product-related compound can have a complimentary equilibrium isomer created from low-level thermodynamic ratio under reverse phase chromatographic conditions. The prostacyclin derivative and drug product moiety absorbs at UV lambda max 275-280nm, but it was determined that several important degradation impurities were not detected at this wavelength.
With the range and complex nature of the analyte and its impurities (crude reaction mixture from the downstream purification process), development of a single suitable method to comprehensively profile all impurities was not realistic.
Solution: In this project, ways to create a comprehensive process tracking approach that could be achieved by developing a combination of complimentary HPLC-UV and HPLC-MS/MS methods were researched. Strategies included the use of combined analytical tools to achieve the best possible separation of all process impurities, including reactive intermediates and isomers. The resultant studies led to better analytical controls coupled with optimized reaction and purification procedures, providing for more efficient production of the drug product.
Efforts to address these challenges, included the development of a stability indicating HPLC-UV method for the prostacyclin derivativel and related impurity profile studies; an HPLC-MS/MS method for stereoisomer control and targeted reaction monitoring of aqueous-sensitive process impurities; and an HPLC-PDA impurity method for studies of the complicated reaction mixes. Method conditions and example studies involving tracking of key process impurities and their identification were confirmed by NMR.
A reverse-phase HPLC-PDA-MS/MS method was developed and used as the primary procedure for total impurity analyses in the API and raw materials. It was also used as a stability-indicating method that separates most general impurities. However, historically, the separation of closely related isomeric mixtures often requires normal phase HPLC rather than reverse-phase HPLC due to the differences in aligning their polar function groups with adsorption sites. With the range and complex nature of the analyte and its impurities from a crude reaction mixture through the downstream purification process, development of a single suitable method to comprehensively profile all impurities was not realistic.
Since the tracking and mapping of impurities was essential for comprehensive understanding of each impurity's fate including reactive species, a normal phase HPLC-PDA method was selected as an additional research methodology. The second method was used as a screening method and a targeted method that separated isomers of repacking and epimers of the prostacyclin derivative.
Finally, a third method developed that was a reverse-phase HPLC-UV method was used for analysis of highly polar impurities from the crude reaction mixture. All three methods were verified, were capable of MS detection of the process to distinguish and track known and unknown impurities and used for mapping all key process impurities.
Result: Through these efforts, the developed methods assisted the sponsor to significantly understand the synthesis process dynamics with correlation to the API impurity profile and assisted them to further develop this product.