(MINI WEBINAR) Overcoming genotoxic impurities through a robust analytical approach

30th Sep 2021

Welcome to our mini webinar ‘Overcoming genotoxic impurities through a robust analytical approach’. Thank you for joining us today. My name is Lynda Harrison, Marketing Manager here at Sterling, and today I’m joined by Josh Wagner, Senior Quality Control Manager. and Scott Hugill, Analytical Department Manager. We will also be joined by Adam Kujath, Senior Director of Technical Operations for questions towards the end of today’s webinar, so please post your questions in the chat at the side of the screen.

Today, we’ll start off with an overview of genotoxic impurities and a look at the current regulatory landscape. Then we’ll discuss the importance of robust impurity evaluations and some common analytical methodologies. Next, we’ll give some more background on Sterling and our analytical chemistry capabilities. Finally, we’ll end with a case study that more specifically demonstrates our capabilities around impurity testing. I’d like to hand over to Josh who will go over genotoxic impurities and their recent heightened interest.

Thank you. Let’s start by discussing how genotoxic impurities form and why they have become such a major focus for pharmaceutical organisations and regulatory agencies alike in recent years.

Genotoxic impurities are important to combat because, by definition, they react with genetic material. As a result, they cause mutations and can be carcinogenic, making the removal from drug products critical. Part of the challenge around these impurities is that they can have various sources and arise at various stages of development. They may come from starting materials, catalysts, reagents, and even degradation of a finished drug product due to storage and shipment conditions. A robust analytical approach is critical to not only determining when these impurities may surface but finding ways to mitigate them. Comprehensive testing is necessary for preventing impurities from entering final drug products in harmful concentrations.

To classify impurities and engage their level of risk, we utilise five classes. Class 1 holds the greatest risk with known genotoxic data. These impurities should be completely eliminated whenever possible. If they cannot be fully removed, they should be significantly limited under the threshold of toxological concern, or TTC, which is defined by regulatory bodies. Class 5 holds the lowest risk potential with nothing to indicate genotoxicity. These should be treated as typical impurities that can arise in drug products. You’ll notice that some of these classifications are informed by alerting molecular structures that may indicate genotoxicity. We’ll take a closer look at such structures later on.

So why is testing for genotoxic impurities so important? These impurities are so important to identify and remove because of their carcinogenic risk. When present at high enough concentrations, it can be quite hazardous to human health. As a result, they must be identified and eliminated so as not to end up in final drug products. There have been occasions where genotoxic impurities have been identified in drug products after they’ve already reached the market.

One example was in 2018, when the FDA and EMA recalled drug products that contained valsartan, which is commonly used to treat high blood pressure. In this instance, N-nitrosodimethylamine (NDMA), a carcinogen, was identified in drug products that were already being sold. This caused widespread concern and potentially put millions of people at risk, demonstrating the importance of mitigating genotoxic impurities throughout development and manufacture. In addition to putting people at risk, having such challenges arise late in development, or after commercial launch, can create costly project delays and regulatory challenges. Consequently, accurate impurity assessments and robust control measures are essential in order to avoid such challenges in later stages.

We touched a bit on the role of regulatory measures in limiting genotoxic impurities, but what exactly do the guidelines look like today? In general, guidance focuses not on outright eliminating genotoxic impurities but rather limiting them below the TTC, which the FDA and EMA have both set as 1.5 micrograms per person, per day. Regulatory guidance around such impurities is relatively new, with the EMA and FDA releasing guidelines in 2006 and 2008 respectively.

These were preceded by multiple papers that discusses controlling impurities and classifies them into the five categories we saw earlier. Later, the ICH released its M7 guidelines. this is the main regulatory focus today as it has rectified some inconsistencies between previous guidelines. It provides detailed recommendations for testing impurities and controlling them so as to minimise the level of carcinogenic risk in end products. It’s also important to note that potential and known genotoxic impurities are treated differently. Known impurities should always be included in the API specification. Potential genotoxic impurities on the other hand are theoretical and their inclusion depends on at what point in synthesis they occur.

To give some more context, this table provides an overview of major regulatory guidance that has been released around genotoxic impurities to date. As you can see the ICH M7 guidance is quite recent, which has contributed to the heightened focus on genotoxic impurities we see today. When developing an analytical approach for genotoxic impurities, these guidelines take precedent. Adhering to ICH M7 is satisfactory for the other major regulatory bodies.

Now that we have looked at the nature of genotoxic impurities and the existing regulatory landscape, let’s discuss exactly how they are assessed and controlled. As we’ve mentioned, comprehensive analytical testing for impurities is important because of their substantial health risks, as well as their ability to impact regulatory approvals costs and project timelines.

It’s necessary to consider and test for genotoxic impurities throughout every stage of a product’s development because as we saw they present several key challenges. First, the lack of a singular source makes them particularly challenging to pinpoint and overcome. Genotoxic impurities can turn up at any stage, even through degradation in storage and shipment as a product prepares to go to market. It’s important to test for their potential to form early on as well as to continue testing throughout a product’s lifecycle. They also require very low levels of detection, typically based on the product’s maximum daily dose.

Next, regulatory requirements become more stringent as a product moves into later stages and nears commercial approval. This is another reason that it is so important to remain on top of PGI testing through each stage of development and manufacturing. It can also be challenging to develop appropriate analytical methods for genotoxic impurities because of their relative unpredictability. For the best chance of overcoming impurities, it is critical to develop a thorough analytical approach very early on to proactively overcome challenges that can surface later. In addition, it is imperative to continue testing throughout the lifecycle of a product, as it can be nearly impossible to predict if and when impurities may arise.

Impurities can be identified in a variety of ways, including recognition of a known genotoxin, Ames test, certain structure activity software programs that can deliver genotoxicity alerts, and structural similarities with known genotoxins. Below, you’ll see a diagram of an Ames assay which is commonly utilised to assess mutagenic potential. This test uses a salmonella strain to determine whether revertence forms when reacting with the potential mutagen, which would indicate the substance in question results in mutations. In short, a positive Ames test indicates genotoxicity. Structural alerts are a key indicator of genotoxic potential.

There are a range of structures that should prompt further testing but some of the most common ones are aromatic nitros, aromatic amines, Michael acceptors, and alkylating agents. Some examples are shown here. The large range of possible alerting structures and different variations of each creates another challenge in analysis. These tend to be reactive which can make finding methods with good recovery in an appropriate quantitation limit challenging. This again highlights the importance of utilising a highly tailored analytical approach to carefully control genotoxic impurities of all kinds.

So, what are our requirements for analytical methods? First, any analytical testing must be capable of identifying potential impurities at concentrations well below the TTC or known impurities under the permitted daily exposure. It is important to be aware of the presence of any impurities, even if just in trace amounts. This can help to determine whether they may present a higher risk as development progresses.

Next, testing should consider both actual and potential impurities. As we’ve discussed genotoxic impurities can arise at any stage, and having an idea on whether they may form later on can help mitigate added challenges in the long run. Finally, selectivity is important because of the variety of organic compounds that may be present in a product. Testing should be able to weed these out to solely detect genotoxic impurities. As a result, it is important to develop methods which are compatible with HPLC low sensitivity detectors, such as UV Vis or UV, as well as high sensitivity detectors, like mass spectrometers, as a project grows in scope through phases of analysis and development. Gas chromatography with FID detectors is also a common approach. sometimes a combination of analytical methodologies may be needed to gather the necessary data on genotoxic impurities.

To provide a clearer image of what testing approach to leverage at what stage in development, this flowchart developed by ready explains some of the rationale for analytical methodology based on when potential genotoxic impurities surface. If the impurity is introduced in the final step, it is critical to test the API itself for the presence of impurities and impose a limit on impurity concentrations. If it is introduced in a penultimate step and found at a level of concern, the same testing approach should be utilised as if introduced in the final step. If it is not present at a level of concern, then it is important to demonstrate its absence with robust supporting data and analysis. If an impurity surface is earlier in development, further out from finished API, then it is important to show rationale for its removal. If no PGIs are identified, then it is important to generate supporting data that proves the absence of genotoxic impurities. All of this will help to mitigate project challenges in the long run and to deliver the evidence and analyses needed to gain regulatory approval.

Now that we’ve surfaced some key considerations when assessing geotactic impurities, I’d like to hand it over to Scott, analytical department manager here at Sterling, who will look at how our analytical team at Sterling is well equipped to aid our customers in assessing and controlling impurities.

Thank you, Josh. Here we’ll look at some of the key strengths in analytical chemistry here at Sterling. Our analytical work is supported by years of expertise in complex chemistry, a team of highly skilled and innovative analysts, we have a 24/7 dedicated shift team who support your projects through their lifecycle, a range of specialised equipment in LC-GC and mass spectrometry that are able to support the full lifecycle of your product with analytical capabilities and specialised software. All of this enables us to help our customers overcome complex analytical chemistry challenges in their programs and to generate the data needed for regulatory filings.

Now let’s take a closer look at some of our analytical capabilities at Sterling which support our work in genotoxic impurity testing. As you can see, we deliver a wide range of analytical services throughout each stage of the molecules lifecycle, from pre-clinical, phase one, phase two A and B, phase three, to commercial manufacture. Our state-of-the-art analytical equipment and experienced team members enable us to support a wide range of projects requiring investigation and identification of potential genotoxic impurities. With strong capabilities in different types of chromatography as well as mass spectrometry, we help our customers identify and mitigate impurities to ensure the safety of their product and to meet regulatory requirements. Whenever process changes are necessary, we deliver supporting evidence needed to maintain quality compliance with strength of method development and validation on our fully qualified lab equipment which is qualified and supported in GMP manufacture.Our analytical strengths can be best summarised into five critical success factors that can help our customers overcome genotoxic impurities in their project. Let’s take a closer look.

First is our analytical expertise. Our world-class equipment and highly experienced analytical team enable us to support a wide range of projects for our customers. Our team is passionate about what they do and entirely focused on affirming quality in our customers products. We also have the state-of-the-art equipment needed to carry out a wide range of analytical testing for genotoxic impurities and we have a wide range of equipment such as HPLC and GC with a wide range of detectors including mass spectrometers.Next is our tailored approach to analytical chemistry. Analytical requirements can vary significantly from project to project, and we appreciate that the one size fits-all approach doesn’t work for our customers. Our wide range of equipment, team, and multi-disciplinary experts enable us to develop an analytical program that is suited to your project’s requirements, gathering the data you need at each stage.

Data Integrity is another key aspect of our analytical approach. We know that data is critical for both regulatory filings and the overall success of the project, so we make sure we deliver our customers the precise and accurate data they require. When it comes to genotoxic impurities the data that validates their absence is just as critical to gather and present as the data validates their presence.In addition, we’ve served customers around the world for more than 50 years and have extensive knowledge of varied regulatory requirements and nuances in different regions. This enables us to develop an analytical approach that will stand up to scrutiny no matter where the customer intends to file. For genotoxic impurities in particular, we have an in-depth knowledge of M7 requirements and the expertise to overcome such impurities in our customers projects. We know exactly what it takes to identify and mitigate the genotoxic impurities from early stages to enhance our customers chance of success.

Perhaps most importantly we serve as a true scientific partner to our customers throughout the whole lifecycle of the product. We know that continuity is critical, and our customers work with the same dedicated analytical team throughout the duration of their projects. With expertise supporting customers throughout their entire lifecycle, we proactively anticipate challenges that may surface and work to overcome them while containing costs and adhering to our customers desired project timelines.

Throughout these five qualities we empower our customers to navigate the complexities of genotoxic impurities and prevent them from entering the final product. In every project our one little team delivers our customers full confidence in the quality of their product and processes.

Now that we’ve looked at Sterling’s analytical capabilities, we’re going to go over a case study that can provide some more insight into our analytical capability approach to genotoxic impurities here at Sterling.

In this case the study looks at a project that began at our Cary site in the pre-clinical phase and was then transferred to our Dudley facility for manufacturing. The customer had concerns about one identified genotoxic impurity and the addition of free PGIs during the micronisation process. This initiated the detection and the development of them PGIs, initially via a HPLC method using UV as the detection method. It was imperative our team worked quickly to ensure that the customer could get the product into phase one clinical trials as soon as possible.

After investigation via the HPLC UV method, it was determined that it was not suitable for two reasons. The sensitivity required for recovering all the impurities via the UV detector was not suitable and also one of the impurities consistently had low recoveries. We decided to move to an LC-MS single ion monitoring method using an electrospray ionisation source. Each PGI was separately infused into the system allowing for optimisation of the impurity to allow for sensitivity to be increased. Once the method was set, we used a combination of the HPLC to separate out the PGIs which was then ionised via the electrospray into the mass spectrometer and then each ion was monitored separately via their mass. Mixing different methodologies was challenging we knew the potential complexities could arise later on, and the customer at this point requested that the method be validated to phase one requirements. One of the impurities fragmented while in the LC-MS thus meaning that we had to adjust the mass collected to give a true representation of that PGI in the solution.

It was important that our team worked together. Cary, who had the understanding of the chemistry, and the customer was kept regularly updated with teleconferences and presentations which were given on a weekly basis. It was important that our team worked together to maintain close alignment with the customer in order to proactively overcome challenges, ensure the necessary level of sensitivity and effectively recover all the impurities in the validation. Eventually, all impurities were recovered in a limit test. The material was micronised in two parts. All PGIs were below the limit set by the customer. Overall, the project was deemed a success, the micronisation produced the desired particle size for the material while maintaining PGIs below the set specification limit for the daily dose for the customer required. The material has entered phase one clinical trials at this point. and we’ll be supporting the customer throughout the lifecycle of the product.

Thank you, Scott. Feel free to reach out with questions at enquiries@sterlingpsl.com or via the contact us page.

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