Impurity identification in Small-Molecule APIs

Timely identification of impurities is critical for maintaining clinical and marketing timelines, and it requires a unique combination of process chemistry knowledge and considerable analytical capabilities. At Grace Fine Chemical Manufacturing Services (FCMS), we keep customers on track by combining decades of pharma research and development experience with teams who closely collaborate on all elements of process development, from the kilo lab to commercial production.

These teams are formed early in process development and then maintained through all phases of commercial development. This ensures that when an impurity arises, the pieces are already in place for prompt identification, and the team just needs to execute.

Impurities of principal concern are often first discovered through final product purity analysis, which for most small molecule drug substances utilizes high-performance liquid chromatography (HPLC) with UV detection. However, tracking and identification of impurities observed during in-process control testing, along with purity analysis of raw materials, is the strategy most likely to lead to successful management of a drug’s total impurity profile.

Understanding Customer Needs

Projects cannot be completed successfully unless the CDMO truly understands the short- and long-term goals of the customer. That understanding helps the CDMO establish the deliverables and determine what is needed to achieve those goals. The kickoff meeting allows the CDMO and customer teams to get acquainted.

It is also critical that all CDMO and sponsor team members agree at the end of the meeting on the scope of the project, the deliverables, and the timeline for those deliverables. It is essential for the CDMO to be as flexible as possible in establishing robust, commercial-ready processes and methods, so that important customer milestones can be achieved without issue.

Technology is Just One Piece of the Puzzle

While necessary for structural elucidation of impurities, technology alone cannot solve problems associated with identification and control. However, prompt access to modern analytical technology is essential for quickly identifying impurities. Grace FCMS has invested in top-tier technological equipment, including HPLC coupled with high-resolution mass spectrometry, nuclear magnetic resonance (NMR) spectrometers, gas chromatography – mass spectrometry (GCMS), liquid chromatograph (LC) fraction collectors, and semi-preparative LC. Our lead analytical chemists have proven experience in applying these technologies to solving chemical impurity problems.

Advances in modern mass spectrometry make it possible in many cases to obtain a mass of an impurity quickly. With the increased sensitivity of high-resolution instruments with tandem mass analyzers, such as a quadrupole time-of-flight (tof) mass spectrometer, a skilled analyst can isolate a parent mass along with several fragments, all with accurate mass, in a matter of hours. Our analytical and process chemists then collaborate to identify possible impurity formation mechanisms and propose potential structures for the impurity, based on available knowledge. If necessary, the same mass spectrometry techniques used to determine the mass of the unknown impurity can be used to generate in-process control samples and raw material samples to help identify upstream sources of impurities.

If additional information is still required, both process and analytical chemists work to further isolate the impurity or chemically synthesize lab quantities of proposed structures through unambiguous routes. While fraction collection and preparative chromatography are valuable tools, it is worth considering that a 0.1% impurity is present at 10 mg in a 10-G sample of API, assuming similar response factors. These separation tools can be time consuming, and often do not yield sufficient material to perform further characterization. In these cases, it is often valuable to look at mother liquor streams for the impurity of interest, as these impurities frequently are enriched in such streams. These increased concentrations can help to isolate the impurity of interest or obtain better quality mass spectrometry data.

Most often, however, we propose structures from the mass spectrometry data available, and when straightforward, synthesize these compounds from known, unambiguous routes. This approach has multiple advantages. First, synthesis from a known route improves the confidence in the identification. Second, it produces enough material for the work that comes after identification, including possible toxicology and response factor determinations. Once a potential impurity is synthesized, its structure can be confirmed by mass spectrometry and NMR.

Putting the Pieces Together

After an impurity has been synthesized and its structure confirmed, the impurity should then be analyzed by the chromatographic methods that initially found the impurity, confirming the right entity was identified and synthesized. Occasionally, these impurities are already toxicologically qualified, or a sponsor may wish to qualify the impurity. Most often, though, there is minimal toxicological coverage for new impurities, and therefore the formation of the impurity must be prevented. In these cases, the source of the impurity must be investigated. This can involve assessing raw material manufacturers and/or production campaigns, and developing new impurity control strategies; often, all three are necessary. Ultimately, it is the collaboration and commitment of Grace FCMS’s analytical team that produces results, helping clients maintain timelines for their new medications to reach the market.