Welcome to our webinar, Planning Your Testing to Identify Unexpected Impurities in Medicines. The webinar will be presented by Christoph Diefenbach, our Laboratory Manager, Research and Development at SGS. At the bottom of your screen are multiple widgets that you can use. All the widgets are resizable and movable. If you have any questions during the webinar, you can submit them through the Q&A widget. We will answer them at the end of the presentation in our Q&A session. If we do not have time to answer them all, Christoph will contact you separately after the webinar. Christoph, the stage is yours now.
Many thanks, Aurelie, and welcome to all participants. The reason why we talk about the impurity is that there are a lot of guidance are available, which rules our companies how to handle impurities during all the processes we have to handle. I listed on this slide three examples, but please understand it, this list is not full, where you can read something about impurities in guidelines. At the end of the slide, you see how important it is to understand when we have to elucidate an impurity, which is recommended during any process in our production line. You can see which challenge is behind that when we have to clarify a structure of an unknown with less or around 0.1% content in a sample.
On the next slide, I will give you a topic or an overview, which and where it could happen, that an impurity will be present. After that, I show you two examples from our laboratory, how we had handled this point of interest for our clients. One in a general information, and a second, which is, of course, in the overview of our presentation. Thank you. I think the most of you is familiar with the situation that an unknown arrives during any process of your production line. This could happen during a quality control testing, as well as in a laboratory of a supplier of you, or during a long-term and stress testing investigation. Good advice is now urgent.
You have to react fast, but not headless, because all products of you bound a lot of money, resources of personnel, and you have to solve this problem, problem properly and fast, because it decides what you can do with your product afterwards. At the beginning, I will give you an overview where an unexpected impurity can be arise. As I mentioned, the most important thing when you get an impurity is to keep a cool head. Concentrate you on the important steps to elucidate the structure of your unknown, be aware of the regulations by the authority, and don't do anything which don't bring you forward. The available time is the most important regular, which is aware, and sometimes your product can stay in the store as long as you need to clarify the structure of the unknown. Start with the brainstorming. Collect all the information you can get.
Even small details could bring you forward to understand the nature of the impurity. Discuss with the supplier of your products, because sometimes they change their production processes and could give you an information which is helpful. Talk with the chemist in the synthesis laboratory. Very often, these people have information about the reaction processes, which could bring you forwards. How many batches are infected? What is the worth of the affected batch? Calculate this sum, and make you clear, is it possible or necessary to solve the problem? Or sometimes it could be easier to destroy the batch because the money you can spend or must spend, differs. Additionally, it is helpful to be aware of the reason of this event. Think about what has changed: the supplier, the quality of the products which you use. Is there a change in the reaction conditions?
Has anything in the system been replaced? Could you clarify if the unknown is part as a byproduct of the synthesis product? Which analyses were used where the impurity arise? Is there an investigation of materials used? Have you unusual influences, for example, in the weather change or pH value is not constant? Collect all the information. This will help. On this slide, I will give you some helpful information, which could be respected. At first, how much time is available to clarify the unknown? Because if you don't have much time, it's not possible to clarify the structure of the unknown. Repeat, how much is the good worth? Because if the affected worth very less, don't implement an expensive structure elucidation process.
The structure determination, you can see on the right side, what I mentioned with that, how detailed must the structure determine? Is there a method transfer necessary? Have you got your own laboratory, which supports you with the determination of the unknown, or must you look to a supplier laboratory, and then you have to transfer the method first? Is the method where the impurity arise compatible to the technology, which is necessary for structure elucidation? And at the end, what information is required? All GMP laboratories are really familiar with risk analysis. In this case, a risk assessment could help you, too. Do it to clarify the nature of the unknown, when it arise, where it arise, and how it arise. You can collect all information, bring it together, and at the end, the best case, you have a result.
A further step is to collect the information about the impurity. The analytical method, which was used, can give you a lot of information, and this could bring you forward to understand the impurity. Very important is also to make an OOE process or something like that, to reproduce the result. An estimation about the quantity of the unknown substance is necessary. Think about the slide two, that the 0.1% clarification is present. With the used method, you can get a lot of information, for example, the polarity of the substance, or when you have a GC investigation, the boiling point. The color of the sample could help you. The substance class, where it is, will be rare. Also, the solubility of the unknown can give you an information or an estimation of the mass of the unknown substance.
Have you got an evaluation process for your suppliers? Because the quality of the raw material is really important for the quality of the end product. Is there any process being inspected during production process? Are the laboratories using suitable methods for the investigation? Check the method details, process materials. Now, the risk assessment is finished, and the laboratory work can start. At first, you have to transfer the method from the laboratory where the impurity arise, to that laboratory, which support you by the structure elucidation. Important is to readjust the result from, for example, the quality control laboratory, and that laboratory which you support. It, it is absolutely necessary to ensure that the test result is verified. Product analysis methods are often in use, which are not usable for structure determination. This can be for HPLC method, for example, salts, solvents, or ion pair reagents in the eluent.
Changing an unsuitable method into a suitable method could cost you a lot of time, and it's always a challenge. In addition, it could be very difficult to clarify the correct analyte. There is more than one method present on the marketplace to clarify your impurity. The choice of the method is crucial. The implemented equipment has an enormous influence on the cost, and you can see in the table a quick roundup of some, and the list is not full or complete, of possible methods, the price, and the information you can get. When the lab work is done, the analyst has to generate a structure proposal. Be it that the reliability of this depends on the quality of the existing data. More data, and better data will generate a better result.
It's really important that you have an analyst on the other hand, who is a prophy and has the necessary expertise to fulfill all these needs. In principle, the process of the determination of the structure can be broken into five essential steps, and on the next slide, I will give you an overview about this process. We can break down the process to determine the structure into five steps. First step is the method transfer. Takes up to a week for a successful transfer from one laboratory to the next laboratory. Second step is the choice of the analytical analysis method. Normally, it takes only one or two days to decide which type of method will be. Gathering the analysis data is the next step, and this takes from one week to two months. The next step is the assessment of the analysis data.
This can take up to two weeks. At the end, the verification of the analysis data might be necessary. You can understand that a synthesis of a substance takes a lot of time, and this process, or the time of this process, is open. Realistically, a timeframe from a few weeks to several months, months, absolutely normal. When the work is done, and I hope your unknown impurity is now a known substance, there might be a lot of work necessary. For example, is a toxicological assessment necessary? Or do your quality department tell you, "Please write a catalog." Have you got updates on other pieces? Normally, I expect a personal training. Is that the quality of the processed material insufficient, and you have to provide an extractable study to avoid the replacement of the item.
A tip from my side, at the time of developing a method for quality control, it's important to already think of any problems that may arise. Make a better method at the beginning of all processes. A mass spectrometer could not operate with a mobile phase that contains, for example, ion pairing agents. An NMR spectrometer requires greater substance quantities. Respect it in your plan. As conclusion, I can say when an impurity arises, it's not possible to foresee the complexity of the problem. But when you follow the way I described, chance of success is present. But be aware, there's no guarantee. For example, when the amount of impurity is not sufficient, you can get a result. When the unknown is identified, source of the entry must be determined. Any anomalous process parameter found must be counteracted.
An impression of the workload should give you this slide, but there's no claim for completeness, and I don't want to go in detail, because I discussed all these points forward, and it's only a visible overview of the workload. Now, let me show you two examples which were present in our laboratory. Here you can see a typical HPLC chromatogram after a one-dimensional separation on a C18 column. The following MS investigation gave us an unclear result, and then we decided to make a further separation. Our surprise was really big that a further separation on a C18 column with another eluent gives us two impurities with nearly the same peak area. So we can say that the concentration of both is nearly identical. And now think back to slide two, that impurities up to 0.1% must be clarified.
In this case, when the sum of both not higher than 0.2%, you don't have to clarify any of them. So this can help you to decide as we go further on, to clarify an entity identification, or can we finish the steps yet? And now the MS spectra we get, they're really good. Here you can see the MS spectra of impurity A. The determined sum formula was C20H27N3O5S. The ionization was performed with AVD in positive mode. Here you can see the MS spectrum of impurity B. That's a mistake on my slide. The determined sum formula was C20H27N3O4S, and the ionization was also made in AVD positive mode. Between impurity A and impurity B, there was only difference by one oxygen.
With both impurities, further MS-MS investigation were executed, and this MS-MS result gave us a deeper impression about the nature of the impurity. The data information were interpreted, and with the information from the client, both structures could be proposed. Be aware, there was only a difference between both impurities by one oxygen. The elution of both impurities were the same in the first HPLC chromatography, and this might be a challenge very often, that there's not only one unknown behind one peak. There could be several peaks behind one peak. Be aware about that. At the end, let me give you a short information about the used device. We have coupled an HPLC system with an ion trap mass spectrometer. We have used an AVD source, using or operating under positive voltage. This is general information about the system.
I think this system is a good compromise between cost and used amount of the impurities. For the first example, I couldn't give you more detailed information about the structure of the impurity, because we have a secure purity agreement with our client. In the second example, I can go a little bit deeper in detail, with the example of an oxazolidinone drug product. We also start with an HPLC-UV chromatogram. This chromatogram was performed by our customer, and the sample represents a stability test. Three impurities were assigned to identify, and with this information, we start our work by SGS. We stress the drug product with different conditions, and in the chromatogram, every color represents a stress under different conditions. In summary, we can say all impurities which have to be clarified were found, and the method transfer was successful.
As I suggested, very often the methods from our clients are not compatible with our equipment, and that was in this case, too. The first step was to transfer the method from our client to an MS-compatible method in our laboratory. We changed the mobile phase into an MS-compatible mobile phase, and afterwards, the chromatograms are really different. For example, impurity one was a broad, weak peak, and impurity two and three were outstanding. But now we are ready for structure elucidation. We collected the three impurities by an HPLC fraction collection system and measured all three extracts afterwards on a high-res MS device. Here you can see impurity one by an MS/MS into the investigation with three different collision energies. You can see that the first collision energy is too low to fragment the impurity, but with a higher collision energy, you get more information about the molecule.
The same was done with impurity two, and you can imagine that with 30 volts collision energy, the amount of the impurity was not really sufficient. At last, the investigation for impurity three was done. You can see on all three spectra that the impurities are really close together. A stress test of Linezolid generates a manifold structures of the degradation products. Take a deeper look onto impurity two. The position of the hydroxylation of the degradation product could not be clarified final, but the toxicologist of our client, they say, an all clear. So the client have all the information which is necessary, and we have done our lab work properly. As summary, I can say, in this case, the investigation was successful. The client received all necessary information to close this process.
The MS/MS investigation was sufficient enough, in this case, for a structure interpretation, and a final toxicological assessment could be done based on this information. Now, let me ask you, do you plan to outsource structure elucidation in the future? I hope you enjoy the presentation, and it helps you in the future. I hope you feel a little bit more confident with that background. And now, I think we are ready for questions.
Thank you, Christoph, for the presentation. We will now start the Q&A. So we have a first question, which is: Which amount of sample is necessary for a structure test investigation?
Yes. At first, let me thank you to you all, and I have heard that the sound was not really good during all the presentation, so I have to apologize for this bad news. But, yes, thanks to you all. And now to the questions. Yes, the first question is, which amount of sample is necessary for structure test investigation? And this is a really important and manifold question because it depends on several points. At first, you have to decide which equipment do you want to need for structure elucidation. For example, when you want to use an NMR device, you'll need at least 2-3 mg of purified substance when you want to make an LC experiment.
For the test I showed in the presentation, the two examples, there only were a few microgram substance necessary for all the MS and MSMS investigations. You can see there's a big difference between the two different techniques. Let me make a further comment. This is valid for small molecules. In case of your molecule is very heavy, you have to need more substance because the count of molecules per milligram is corresponding lower. In addition, for an MS investigation, and mostly this will happen, you generate multi-loaded molecules, and this results in a complex data interpretation and in a higher necessary substance amount. It could be that you need further cleanup steps in this case, and this even results in a loss of substance, and you need more substance. Can give you a tip.
When you have an estimation about the molecular weight of the impurity, think about it, very small molecules are volatile or semi-volatile, and this can also happen with a molecule with 250 or 270 Dalton. And when you have spent a lot of time in sample preparation, you're really disappointed when your substance is blowing in the wind. So keep careful in this process and work really careful.
Thanks, Christoph. We have the next question. At which level do impurities have to be labeled as specified impurities?
Yeah, thank you for this question. I think in principle, there's no limitation... But there are parameters available which reduce the chance of success. And, I have to repeat that there is no guarantee for positive results. For example, an insufficient sample amount or a bad ionizable molecule can reduce the chance of a success, or the molecule is not stable during the preparation of them, or the molecule is not soluble in a solvent, which is proper to use for the experiments. And so there are a lot of points which can, yes, reduce the chance of success, but in principle, there's no limitation.
Thanks. There is also a question which says: Are there limits with limits in investigation? I guess it's kind of answered at some point.
Yeah. So let me comment that we are in a laboratory, we are working for other clients, so we have we don't have to decide which impurity have to be clarified. This is a point where the client tell us, do this or do that. So normally, our clients are under regulation for ICH Q3, but very often they have further information which are not available in our laboratory, so we are a little bit pending on the information of our clients, and they tell us, do this or do that. So yes, this is a process for every client, is very special.
Okay. Does this approach apply also to generic products where we have identified unknown impurities?
So I think it's a little bit going into the answer of the first question. I'm not sure what is the difference between this, at which level to, to be labeled? Yes, I think that the regulation, for example, under ICH Q3, and there's a 0.1% limitation, but be aware that I'm not a regulation pro here. When you want to discuss about that point, other people can bring you forward. That's not my best topic, I have to say. So keep you on the rules, and yes, when you have to decide one more or one less, use one more impurity to clarify.
Okay, we have a question. Normally, what kind of impurities are you talking about that are not HPLC?
So.
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Yeah.
I'm sorry.
Yes. Okay, in most cases, I think you have smaller molecules and molecules with no double bond in the molecule. So, yes, small and very polar compounds, which you can detect on a GC system, for example. And let me comment that a GC system can also be connected with a QTOF detector, and you can generate the same results as I presented in the examples.
Thanks, Christoph. Do we have more questions from the audience? We will give them one more minute to ask further questions. Okay, so I don't see anything else coming. Is there any additional information that you wish to add, Christoph, or should we be closing the webinar?
Yes, from my side, we can close the webinar, and at the end, I will say thank you for your attendance and the attention, and in this time, I wish all a good time and stay healthy.
Thank you.
Thanks a lot.