Expert Insight: New analytical methods for impurity detection

Watch this on-demand webinar to learn how risk-based approaches can be implemented to improve the control of impurities

09 Jul 2020



Dr. Mark Argentine, senior research advisor at Eli Lilly and Company

The presence of impurities, even at trace levels, may affect the quality, safety and efficacy of drug products. Impurities in a drug substance or drug product can arise from chemical synthesis, degradation, manufacturing, storage conditions, packaging, excipients or contamination. Analytical method development for impurity detection and quantitation is a necessary and often challenging task for pharmaceutical manufacturers. Risk-based approaches such as analytical quality by design (AQbD) and method lifecycle management (MLCM) can be implemented to develop fit-for-purpose and robust methods that improve the control of impurities.

In our on-demand webinar, Dr. Mark Argentine, senior research advisor at Eli Lilly and Company, describes the following within the context of AQbD and MLCM:

  • Method lifecycle management concepts applied to impurity analysis
  • Impurity method development strategy leveraging AQbD principles
  • Concepts and considerations toward monitoring method performance
  • Case studies highlighting analytical knowledge gathering for analytical method development, method transfer, and routine method controls

Read on for highlights from the Q&A discussion at the end of the live webinar or register to watch the full webinar on demand:

Q: The case studies that were shared appear to provide focus on chromatographic aspects of small molecule drug substances. Can you comment on the applicability to drug products? Are there any additional considerations that you might be able to share?

MA: Yes, certainly. Hopefully, I’ve highlighted that these strategies are readily applicable to drug products and methodology, particularly small molecule ones. Perhaps it’s worth mentioning that the methods usually focus or change to focus more on drug products as highlighted in the webinar and/or formulation-related components or impurities that are more important than drug product impurity control strategies. I should also stress the importance of sample preparation and drug product methodologies and the recommendation for thorough robustness studies regarding sample preparation. It is also very important to think about drug product in addition to the chromatographic robustness aspects, as mentioned.

Q: The tools that have been shared, for example, chromatographic modeling and design of experiments software for development aren't necessarily new. Where do you see opportunities for advances in these tools?

MA: I agree. ACD Labs and more recently S-matrix offer some opportunities for peak tracking. Automated peak tracking is tremendously valuable. Currently, most of that software, particularly from an automated perspective, is limited to a single quad mass spec, whether that's a QDa or a single quad itself and the integration and automation. Clearly, there's opportunity to expand that support for more automated capabilities, particularly in the area of peptide and protein development. For example, ACD software for ACD Labs can import some high-resolution mass spec data, it currently can't control or manage those systems in an automated format. There have also been enhancements in the user interface and reporting, these have come a long way.

But I think, as we continue to look toward the future and evolution of varied modalities and studies, obviously, with peptides, the opportunity to greatly reduce time spent with development through verification on peak tracking can't be understated. I think efforts in that area will certainly be encouraged and found. As well as this, leveraging these enhanced chromatographic prediction models to really account for things like peptide-protein sensitivities and folding, which I know some modeling programs have, will only continue to become more important. Alongside empirical models for that type of work.

Q: In your last couple of slides, you indicated the ability to leverage advanced chromatographic technology for increased lab efficiency. One of the greatest barriers to this appears to be managing global regulatory changes. What ways are there to facilitate enhanced regulatory flexibility and the method changes for enhanced lab efficiency?

MA: Indeed, scientific kinds of strategies or method comparisons for moving from one to the next oftentimes make good scientific sense and can be adequately supported. However, regulatory management of those changes might not always be. I think that's been one of the conversations and goals regarding analytical target profiles and this advanced method lifecycle topic. It's certainly an opportunity with the evolution of ICH Q14, which is currently being developed, perhaps in step one. And hopefully, we'll see more of that in the future.

But I think there's opportunity to leverage those concepts to support multiple acceptable methodologies that might be capable of an intended purpose. One good example of where that might be seen is in the recent example in Pharmeuropa 32 on nitrosamine detection in sartans as a limit test. There were three different methods: an LC-MS/MS method, a GC-MS method, and a GC-MS/MS method. All three appropriately validated for one or several of the common small nitrosamines in the very sartan matrices to a 30-part-per-billion limit. Again, I think this provides an opportunity to say that there's not just one right analytical methodology to meet an intended need. And as we continue to work through these concepts to help with regulatory flexibility, that will be beneficial.

Q: For the ICH Q14 Analytical Procedure Development and Revision of Q2(R1) Analytical Validation dated 14 November 2018, is there any news about this document?

MA: I suppose the only update I can share is that the ICH expert reading group has begun to discuss this topic. They met in Singapore back in November and were originally planning to prepare a step 2 document. They were supposed to be meeting again in May of this year, but this meeting has now been postponed due to our current global circumstances. However, I do believe they're working as quickly and efficiently as they can toward getting a document out for step 2 later this year if possible.

Q: How can you decide to use the limit test instead of a connotation procedure?

MA: That will depend upon what the need for your control strategy might be. In some instances, the examples I shared today, maybe limit tests, particularly when you're trying to demonstrate the absence of a particular component at a low level. I've mentioned some examples with mutagenic impurities, and one might find that as an effective strategy.

But there may be other good reasons when a limit test to simply demonstrate that a component is below a certain level is adequate for its intended purpose. In which case, a limit test may be appropriate versus a quantitative test, which might be more helpful, particularly when tracking and trending results from batch to batch over periods of time.

Q: While you spoke of making analytical method control changes, how was that practically implemented?    

MA: Scientific method comparisons are straight forward. However regulatory management of changes might not always be. Goals of analytical target profiles (ATP)s and advanced method lifecycle topics (hopefully with Q14 to reward knowledge space with flexibility) can assist in that regard and accentuate options such a pre-approved comparability protocol. As a recent example in Pharmeuropa 32 on nitrosamine detection in sartans as a limit test, three different methods; LC-MS/MS; GC-MS, GC-MS/MS were validated for analytes in matrix to a 30-ppb limit. This acknowledges several method approaches to achieve one defined ATP meaning the control of nitrosamine impurity in sartan matrix to ensure levels are below 30 ppm limit.

Q: After establishing design space and transferred to a manufacturing location, I observed there is a need to change the method because of new process impurities. Do I need to revalidate if the changes are within the design space and there are no major changes to the original impurities?

MA: If new impurities are identified (or expected) from process changes, one needs to understand the analytical method capability to resolve and quantify these new impurities, together with the expected process impurities and potential degradation products. The answer may depend upon how appreciable the method change is. If the change is a very minor and the change is already well within the known design space with good robustness and the existing method controls remain suitable for accurate quantitation, a rationale could be provided to limit the amount of any additional validation. One would want to have a strongly documented method understanding and rationale for this position.

Q: During stability, my formulation developed one impurity, we have since identified that impurity but we couldn't isolate or synthesis it. How do I perform prove specificity, accuracy, and detection/quantification without the physical impurity?

MA: If you have a matrix with this impurity (e.g. degraded product sample), through linearity experiments you can assess a relative response factor for this impurity. Presumably, you can and have also used this sample to illustrate method selectivity. There are approaches such as fraction collection to trap and collect impurity components if observed on a method other than your intended control method to demonstrate selectivity. If you have been able to identify the impurity, perhaps you can isolate small quantities. If you know the impurity identity, it may be possible for you to use such tools as NMR for assisting with accuracy assessments.

Q: What would be the optimum conditions to test a forced degradation sample?

MA: This answer will depend upon your sample. Assuming that you are assessing for potential impurities from a forced degradation condition for a synthetic small molecule, I'd recommend that you use a broad gradient screen or screens on columns that you feel would give the best opportunities for separation of impurities from the matrix. I'd also recommend variable wavelengths (e.g., DAD use), and possibly alternate detectors (MS) if using UV detection, to best detect for unknown peaks and look at mass balance assessments to have some confidence in your detection. 

Q: Are there any advanced manufacturing technologies to decrease process impurities?

MA: There are a variety of approaches to decrease process impurities. These can include but are not limited to extractions, crystallizations, filtrations, and chromatographic purification operations.

Q: Can you suggest any quality by design (QbD) tools that are available free of cost and can be used most effectively?

MA: Design of experiments simply requires an understanding of the concepts and benefits and limitations of each design. While I highlighted some tools that can have integrated controls in both the design and analysis for efficiency, these can easily be done separately and manually without any additional expense.

Q: Do you ever consider sample preparation as part of your method robustness studies? How do you factor these into your design of experiment (DoE)?

MA: Yes, sample preparation should be evaluated through robustness studies, especially for more complex preparations often used with drug product matrices. In many cases, these may be studied in separate robustness studies from the ‘instrumental analysis’ robustness assessments.

Q: Do you have experience with electrophoretic methods? Do you see any adaptation of what you said about DoE when we move from chromatography to electrophoresis?

MA: Yes, the concepts are not limited to chromatography. One is simply looking at understanding variables in the analytical method and identifying sensitivities (or robustness) to perturbations in operational variables.

Q: Can selection and optimization of column chemistry be a part of QbD?

MA: Absolutely. This is part of well-designed method development.

Q: Are QbD approaches only applied during method development or can it also be applied during method validation?

MA: Quality by design can be applied to analytical methods and can be applied throughout the analytical method lifecycle from development through validation and continuous verification and improvements. One potentially thinks of QbD as simply DoEs, but it is really having thoughtful intentionality around method design, development, and implementation. There is no magic to QbD.

Q: Earlier in the slide, you spoke of using photodiode array (PDA) and mass spectrometry (MS) in tracking impurities. Do you have experience with Waters QDa? If so, I'm curious about your experience. It seems not necessary to have a highly sensitive MS-MS. Did you observe any limitations with using the QDa for the purpose described in your presentation?

MA: The QDa is a single-quad mass selective detector. It has a limited upper mass range and nominal mass resolution but that is generally adequate for synthetic small molecule work. 

Q: For robustness study, do we perform system suitability before, in between, and at the end of the sequence?

MA: During robustness studies, it is a good practice to monitor the system suitability during each condition. This can provide a basis for establishing/justifying meaningful system suitability limits.

Q: Could you please explain how the ATP help in QbD?

MA: In my mind, an ATP provides clarity around the intended analytical measurement requirements. It should be technology independent. In that way, it provides a framework for looking at potential method options and also for potentially making method changes in the future as long as they meet the ATP.

Q: My question is about the application of statistics toward setting acceptance criteria, for example, t-test, ANOVA, tolerance tests, etc. Do you typically apply such statistics to the development of acceptance criteria? For example, in one slide, there was an acceptance criteria of NMT 10% relative standard deviation (RSD), how was the number 10% selected? In your opinion, at what point should such acceptance criteria be statistically determined?

MA: This is a great question and will depend upon the required level of control. For example, does one need a high confidence level in the analytical result? Or is the control established such that the targeted control level is well within demonstrated process capabilities for removal such that lesser rigor in the estimated actual value is needed?  

Q: May I please know if any software was used for modeling in the third example? Can you please suggest software other than Fusion QBD and Drylab/ ACD Lab suite?

MA: The third example used DryLab. There are a variety of chromatographic modeling software tools available. I simply mentioned several that I am most familiar with through my use in LC development. I have additionally used ProEZGC for GC development. There are other method development/validation automation software packages also available from instrument vendors such as Waters and Agilent.

Q: Have you tested several of the analytical QbD software packages available (Fusion, Drylab, ACD, etc.)? Do you have a preference for one in particular? If so, which and why?

MA: Through the presentation, I tried to illustrate the use of a few of these available tools.   There are many excellent development software packages, hopefully from the presentation you can see we use multiple tools. Each has its current strengths for our particular needs.

Q: Can QbD be applied to further fine-tune a method or is it only applicable during initial screening?  Also, when do I carry out the sample preparation DOE?

MA: Thoughtful experimentation (such as DoE) can be performed at any time, in both screening development and in optimization efforts. Like chromatographic separation development, sample prep design studies can be carried out when both developing and optimizing conditions. 

Q: How much % of active pharmaceutical ingredients (API) degradation is required for a related substances method for impurities?

MA: For forced degradation studies, one often hears of an ~10% degradation target to help define primary degradation mechanisms. I refer you to this reference for further guidance on forced degradation testing: "Pharmaceutical Stress Testing: Predicting Drug Degradation, 2nd Ed."  by Steven W. Baertschi, Karen M. Alsante, Robert A. Reed. This should provide you with the relevant information. 

Q: What is DS Control?

MA: In this presentation, DS was an acronym for ‘drugs substance’ and reflected controls placed at the drug substance stage.

Q: What is GTI?

MA: GTI is an acronym for genotoxic impurity. Perhaps the more appropriate term is mutagenic impurity in alignment with ICH M7.

Q: How can I identify the genotoxic impurities (GTI) actually present in my drug substances in new drug application (NDA) molecules and would they be in trace amounts?

MA: Work with your process chemists and toxicologists to identify potential mutagenic impurities. You may then need specific method development to evaluate for detection, including mass spectrometry or other sensitive detection if control at very low levels is required.

Q: How do you verify the design space?

MA: One can certainly select points within and around a predicted design space from a model for verification. If the design space was defined through experiments at extremes of the space, those experiments provide verification of experiences at those points in the design space. 

Q: How many batches are needed to be tested as part of a method transfer?

MA: There are not specific batch number requirements for a method transfer. The work must be suitable to ensure confidence in the proper operation of the method in the receiving laboratory and may involve using representative samples, impurity-enriched samples, system suitability samples and/or other samples that might assist with the overall assessment.

Q: How does the selection of column length and type of column play a role in method development?

MA: This is an initial part of method development if one pursues chromatographic techniques for analysis. Column type and length are important for overall selectivity assessments.

Q: How can this QbD approach help in assessing phytoconstituents with complex structures? 

MA: The approach is simply a scientific, methodical one that leverages thoughtful experimentation and tools (e.g. modeling) to guide the definition of "optimal", robust conditions and a fit-for-purpose method.

Q: How can I identify an impurity in a standard degradation product?

MA: For identifying impurities, you would leverage the same principles as for identifying any unknown impurity, regardless if it is from a degradation or a process-related impurity. Usually for unknown impurities, if one can find conditions that enrich/enhance the impurity, this may aid with isolation and identification. 

Q: How do I identify critical quality attributes (CQAs) with respect to analytical methods?

MA: This is part of an overall risk assessment from method understanding and can include prior knowledge in addition to experimental results to guide that assessment.

Q: How do I finalize the impurity method?

MA: Once you feel that the method has been suitably defined to be fit for purpose and you understand the method operation and desired set points and controls, it can be finalized with meaningful controls (e.g. system suitability) and validated to support routine use.

Q: How do I separate the UV additive hindered amine light stabilizers (HALS) with the help of HPLC?

MA: Do you know the specific compound or compounds being used as your HALS? If so, you may be able to obtain and use authentic samples and/or use tools like MS to assist.

Q: I am working with LC/PDA/MS quantitative descriptive analysis (QDA). Can you please name a module of automatic peak matching tools that is available in a chromatographic software like Empower?

MA: I am aware that ACD Labs has excellent peak tracking capability with PDA and single-quad MS systems, although it does not operate through Empower. Recently, S-Matrix Fusion software has begun to include QDA for mass-assisted peak tracking method development. 

Q: If we use impurity-rich sample during method transfer, would it be possible to overlook the validation of the sensitivity of the impurity on the receiving lab?

MA: It is important to assess your overall method transfer plan for the ability to confirm important elements for a meaningful transfer. For example, you may have sensitivity controls in your system suitability tests to evaluate for sensitivity. An impurity-rich sample might be used as one or more samples that are analyzed in the transfer study to be able to meaningfully compare analytical quantification between labs if impurities were present in a sample or perhaps simply to evaluate an HPLC profile for expected chromatographic selectivity. 

Q: In force degradation study, do all degradants needs to be identified?

MA: Not necessarily. The goal in forced degradation studies is to identify degradation mechanisms and primary degradation products.  While you may also identify secondary degradation products, you would hopefully learn of environmental sensitivities through the forced degradation studies and develop suitable controls to limit any potential degradation so that secondary degradants become ‘non-relevant’.

Q: In protein product purification there are issues in getting a good yield for crystal structure determination. For instance, a membrane protein serine/threonine kinase presents a difficult task as it is tricky to optimize its yield for downstream approaches. So, the fit-for-purpose approach for this task of impurity detection and subsequent automation will be ELISA or chromatography based?

MA: This question is probably one to have with your process and purification chemists. Are there specific, likely impurities that you believe are causing the greatest difficulties?   

Q: In your earlier example with GTI of 2.5 ppm at an S/N (signal/noise) =3, how was it possible to have a valid quantitative method at such a low S/N limits of detection level?

MA: This was a limit test method. You simply need to ensure a method's ability to detect the impurity at a particular level and show its absence. It was not intended to be quantitative.

Q: Is it essential to take a statistical approach while doing the DoE approach to method development?

MA: A statistically-based DoE will provide an efficient way of collecting data and defining contributions from varied factors in a methodical manner.

Q: Is the separation for impurity peak 4 acceptable?

MA: Which chromatogram are you referring to? If associated with the method comparison slides (slides 43-46), the peak 4 remains baseline resolved from the main peak and remains acceptable. If referring to slides 40-41 with the impurity-rich sample, this was also still acceptable to allow capture of overall impurities. Acknowledging limited resolution of this impurity from the main peak but also the magnified view of this chromatogram and degradation-related controls for this material.    

Q: Is this chromatogram acceptable over a period of baseline change?

MA: While I am not sure of the specific chromatogram that you are referring to, while baseline changes in an analytical region are not preferred, they can be acceptable. You may need to ensure adequate sensitivity, which can become more limited in changing baseline regions, and consistently predictable baseline changes for appropriate integration settings to support effective data processing in such regions.

Q: It is necessary to have a QbD software to develop a method according to the method lifecycle management principle?

MA: Not necessarily. However, several of the modeling and analysis tools help to facilitate efficient implementation of these concepts, not only some mentioned but also other statistical programs such as JMP that allow experimental design and analysis tools.

Q: It is necessary/mandatory to calculate the mass balance in a force degradation study during analytical method validation. What will happen if the mass balance value is not equal to the value of the impurities developed during the force degradation study?

MA: It is wise to look at mass balance in forced degradation studies to ensure that data is being interpreted appropriately. Missing mass balance could highlight gaps in knowledge and limit meaningful interpretation of the data. For further guidance on forced degradation testing, the following is a useful reference: "Pharmaceutical Stress Testing: Predicting Drug Degradation, 2nd Ed."  by Steven W. Baertschi, Karen M. Alsante, Robert A. Reed. 

Q: Could you describe the effect of column length and particle size in impurity peaks resolution?

MA: These attributes affect overall resolution based upon impact on efficiency. Please also refer to fundamental chromatographic theory discussions including those available from tools like the book "Practical HPLC Method Development".

Q: Should I refer to/investigate old analytical methods when wanting to apply a lifecycle approach?

MA: All the principles for monitoring method performance equally apply to historic or ‘new’ analytical methods. You may also leverage historic method and process knowledge to provide continuous verification and/or implement additional controls to aid in consistently desirable method execution.

Q: When the method is developed and registered for a product, if I have change in the process, can I change my method, keeping my designing space defined? Is it true I wouldn’t need a new validation because I have knowledge about the design space?

MA: With a process change you will need to ask and answer the question regarding continued relevance of the existing analytical method. What was the nature of the process changes? Are there any new potential impurities? If so, you will need to assess the continued capability of the method against fit-for-use expectations and appreciate how any potential impurity profile changes can impact the analytical method performance and results. It may be possible that the process change had no impact on the analytical attributes of the isolated product such that the existing design space and validation might remain suitable/appropriate. If so, appropriate understanding and justification should be captured.

Q: Please specify for a new development method how to set principle peak concentration to detect all impurities (least sensitive impurities). 

MA: While a broad question, I will assume that this is an HPLC-UV method being installed for impurities and try to address it with some general comments. You will need to look at several factors in the development of the method including detector wavelength, UV spectral profiles, and responses. Response factors must be considered to ensure adequate sensitivity for all desired analytes in addition to important impurity resolution principles. If very different impurity response factors exist, it may mean developing the method for the least sensitive impurity or perhaps developing multiple methods for quantifying different impurities. Chromatographic performance based upon column loading may also play an important role in both method design and quantitative strategy (e.g., peak area versus total, external standard). 

Q: Can QbD be performed using our existing Empower 3 software or does it need other particular software?

MA: The Empower CDS simply enables chromatographic systems control, data collection and reporting. You can manually program varied chromatographic conditions to be executed and analyzed in Empower or you might choose to leverage additional software tools that interface well with existing chromatographic instruments and data systems to enhance efficiency with designed experimental studies.

Q: Referring to monograph methods (e.g: USP or BP), how do I differentiate whether it is a quantitative method or limit test?

MA: Please refer to the individual monograph method. Most impurity methods are quantitative. However, there may be some instances where limit tests are suitable (e.g. for potential mutagenic impurity control tests such as the example mentioned in Pharmeuropa 32 on nitrosamine detection in sartans as a limit test). Limit tests may generally be noted as such and/or have method reporting information to support its use as a limit test.

Q: What is the simplest low-cost technique or method to determine fine particulates present in air?

MA: I would refer you to general references such as this pdf from the WHO for varied approaches for monitoring particulates in air.

Q: Slide 50 in the final method, the actual impurity cannot be separated/quantitated. Is this right?

MA: Slide 50 is a cartoon to illustrate how analytical and process development complement each other and how final control methods are defined based upon process and product knowledge in addition to analytical controls. In this cartoon case, the ‘control space’ methods detect all relevant, expected impurities with the implemented process and product controls. For example, potential degradation impurities from caustic solutions might never be observed in practice for a solid oral dosage form that is made via direct compression with an excipient matrix that is acidic to neutral. 

Q: Could you please comment on the Fusion software? Can it model and predict like DryLab?

MA: The Fusion and Drylab programs are both excellent tools for modeling but do have differences.   The DryLab program offers chromatographic modeling predictions and simulations based upon limited inputs, while Fusion offers more empirical modeling through well-controlled designed experiments.

Q: Is there is any good software to optimize method lifecycle?

MA: If you are looking for a single software tool that can do everything from development to optimization to validation and routine method performance monitoring and control, I am not aware of such.  However, many of the chromatographic modeling and statistical software tools continue to develop instrument automation capabilities and are improving integration with chromatographic data systems. Several CDS platforms can aid with routine performance monitoring and control.  For method development, I recall that these tools give you landscape information, but it is up to the user to define important or relevant impurities. Also, sample preparation is often an important aspect and several tools can guide toward sample preparation development and robustness. In short, there are a lot of tools to assist but an entire method lifecycle covers a lot of territory.

Q: Does the software help you design some of the tests? And can you tell how much it helps, or is it just trial and error to find out the best methods? Which software do you find most useful?

MA: Well-designed experimental plans and modeling can certainly save time over trial and error and give one much more knowledge and confidence around the selected final ‘optimum’ conditions. 

Q: The successful use of in silico techniques for method optimization that you mentioned depends on good-quality input data. Selection of mobile phase and stationary phase chemistries for evaluation is key. Could you share your thoughts on column screening?

MA: Indeed, the use of thoughtful column screening programs that perhaps leverage columns offering different selectivity, the evaluation of varied organic modifiers, and the consideration of varied pH conditions can methodically help one to select the best avenues to further pursue for development/optimization.    

Q: This question is about using appropriately stressed samples such as for transfer. It seems difficult to know when is it enough to stress a sample? How do you know when to stop stressing a sample so as to use it for development or transfer?

MA: Stressed samples are not required in method transfer but may be helpful to ensure that the lab can reliably quantify degradation, if it does occur, and provide education to the receiving lab on what impurities might be possible. To that end, I would not recommend stressing material beyond what might be reasonably expected in that receiving lab. Overstressing can lead to secondary or tertiary degradation products that may not be very relevant.

Q: What about the orthogonal techniques? Could you elaborate on what GTI control is?

MA: GTI is an acronym for genotoxic impurity. Perhaps the more appropriate term is mutagenic impurity in alignment with ICH M7. So GTI control is specific control for mutagenic impurities, which often require high sensitivity to achieve the necessary low detection limits. Orthogonal techniques can always be useful for added confidence in data, for example providing some assurance that you are not missing any impurity peaks.

Q: What are some experiments that can be done to assess column lot variability?

MA: I'd recommend getting columns with distinctly different gel lots. Many companies now offer validation kits that will provide at least three columns from distinctly separate batches. You can also look at new and aged columns as well.

Q: What are your thoughts on including tolerance interval in the ATP?

MA: The ATP must be defined with appropriate requirements for the necessary control. That could involve the inclusion of a tolerance interval.

Q: What do you think is the best method to use in order to evaluate or quantify different response factors in gas chromatography of samples including paraffins, cycloparaffins, and aromatic groups?

MA: The best approach is the use of authentic standards for each compound, although that is often not practical or possible.

Q: What is the best strategy to qualify the method after a development by analytical quality by design (aQbD) and the determination of the method operable design region (MODR). How do I qualify the MODR?

MA: Your actual strategy or approach will likely depend upon several factors. For example, what equipment/systems are available? You do not need to use automated, integrated systems for design study work although that is a convenience, if available. 

Q: What is the general degradation study procedure for validation of highly volatile compounds using gas chromatography with flame ionization detection (GC-FID)?

MA: I expect this to depend upon each matrix and what information is needed. Studies to evaluate oxidation/air exposure versus inverting? Studies to assess light sensitivity? Studies to evaluate some thermal stability relative to intended storage?  Studies around container and closure for perhaps material compatibility concerns? There should be a rationale around a ‘stability-based’ study plan to identify reasonably possible degradation impurities for your matrix and potential exposure conditions.   

Q: What is the mechanism of model predictions?

MA: It depends upon which model you are referring to. Are you speaking to chromatographic models that leverage chromatographic principles?

Q: What is the stand of regulatory authority on method development using quality by design principles? Do they really insist MLCM implementation?

MA: In my opinion, it is good science to always deliver robust analytical control methods that are fit for purpose. I think that regulators would agree. Regulators do not expect MODRs in a submission, as the traditional regulatory submission approach is acceptable. However, if you want more regulatory flexibility associated with slight method changes, then an enhanced submission approach might be a mechanism for such conversations.

Q: What does it mean if the R value changes during different sequences like at initial vs ending of sequences?

MA: Are you speaking about R as in ‘correlation coefficient’ in an HPLC-UV run? If so, this indicates variability and/or departure from linearity in a Beer's Law response. Are analyte peaks fully resolved from any interferences? Are concentrations at a level that does not invoke self-absorption? Is the UV/Vis lamp providing a stable output? Consider the many reasons why one might observe a departure from maintaining Beer's Law.

Q: What software did you use for QbD in analytical methods development?

MA: I hope that this presentation showed that there are many software tools that have been used and are useful for impurities method development.

Q: What type of mass spec detector do you typically use when doing development? Have you used Water’s QDa? If so, is that sufficient for method development, or at least for peak tracking during optimization studies?

MA: Mass spectral detection may depend upon the matrix. For small molecule method development, nominal mass peak tracking, and such, the QDa is generally sufficient. This may not be the case for peptides, proteins, or other potential modalities.

Q: When mass balance does not add up during a forced degradation study, what would be an effective way to describe or analyze the samples?

MA: You can use forced degradation studies to identify degradation mechanisms. If mass balance does not add up, then additional studies may be needed to better understand why. Here are some key questions to consider:

  •  Are key degradation products not observable by the method? 
  • Do degradation products have very different spectral responses? Or even no appreciable chromophore? 
  • Can these be probed with alternate orthogonal evaluations, such as different tools like NMR or different detectors like MS?  

Q: Where and how do you document and record your ATP requirements? Is this simply in an electronic lab notebook (ELN), or is it recorded in a more formal document and stored in a structured document repository?

MA: Many different systems and approaches can all be acceptable and will depend upon your local business preferences to establish and manage or maintain. Such a system should acknowledge that, in some cases, ATPs will evolve as more process or product knowledge is gained during the development of a process or product.   

Q: Which particular software would you recommend in terms of method development using UPLC-UV/MS to get fast and reliable results including peak tracking? What are the pros and cons of the different software?

MA: I am aware of several very good systems that I have mentioned. There may be even more or better systems available now and in the future. 

Q: Will the design differ with the type of impurities present, e.g., organic from inorganic impurities?

MA: The methodology may differ, and this may lead to different study designs around method parameters for evaluation.

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