Hello everyone, welcome to Webina topic best practices of PDA Mac population for a successful by GMP by old Manufacturing. My name is Nandanath pratip sitaan. I will be your moderator today. I'm here today with our two keynote speakers Dr. Thakuro Hosumisan and Dr. Yukit Sunogawasan. Before I hand over to our presenters, I would like to cover a few housekeeping items today. The will be available on demand after the live session and can be accessed through the same link during the session. We love to hear from you and I encourage you to participate the polls. If you have any questions for our presenter at any point, feel free to submit your questions to use given images. We will be answering question at the end. Next we will have prepare some recommendation reading materials in the resource List tab which you can click and easily access to the materials at the bottom of your screen are multiple application which is you can use at the reaches are resizable and movable so feel free to move them around. To get most of our of your desktop space you can expand your slight area or maximize it into full screen. By clicking on the arrow in the top right corner. Lastly, attendees who wish to receive a webinar certification will need to fulfill the criteria of minimal billing time. So without further ado, I would like to introduce our tool keynote speaker today. Our first presenter is Doctor Takuro Hosumi. He is a process development scientist for downstream process based in Japan. He provides technical trial. Process development, consultation and troubleshooting in qualification and TFF. Before joining our organization, he worked as Rd. researcher developing hobo fiber membrane of medical equipment. He has work for production management. He holds a PhD in chemical engineering from the University of Tokyo, Japan and our second presenter is Doctor Yuki Sunogawa. Currently he is downstream technical application expert of non Asia based in Japan. He provides downstream technical application consultation, marketing and troubleshooting for customer developing in biopharmaceuticals. He began his career as academic researcher and prior to join our company he has gained experience at a National Research institute in Japan as a postdoc researcher. He hosts a PhD in human biology from the University of Sukkaba, Japan and Bachelor Sai in the anatomy and history from the University of Sydney, Australia. OK, so now let's welcome our first speaker Doctor Takuro Hosmisan. Thank you Paul. This is Takuro hosmi. Before the presentation, to avoid network issue, we will turn off our camera. In this webinar, we present an application of polymer for grant polydidmac for clarification of high cell density culture feed and regulatory compliance for it. This is the agenda for this presentation. At first the basic of procreation technique will be presented. Next we will show the principle of quality of by design. And the design of experiments with some examples. Then we will move on to the risk assessment, the detection method and finally the effect of fluctuation on the purification step. The first topic is fluctuation technique. Before getting into the topic we will have a poor question. OK, passed to you. Thank you. So well, I would like to welcome everyone to having our first poll question for today. Please let us know your thought on the poll question. We would like to encourage everyone to participate our poll now there are multiple choices for you to select. Let me read the question what is the method of qualification you currently or planning to use in the future? Centifield and normal flow filtration only normal flow filtration, tangential flow filtration, acid precipitation, cation polymers, or others. We will give you about 10 seconds to work right now. And again I would love to hear any feedback from every audiences from live session and also from on demand audience and now I will give a little bit more time and will be shortly to go and off our poll. So I see some moving on the poll answer. So I would like to give a little bit more time for everyone who not family in the poll question. OK. So I think it's will be a good time to move to the end of the poll and the result is here and thank you for voting now I would like to hand over to our presenter. Thank you for. OK, thank you for your cooperation. It seems that this webinar will be helpful to most of you. OK, recently cell density is getting higher because of the development of cell culture technology. Higher cell density leads to the increasing amount of monoclonal antibody production. However, it increases the amount of impurity such as cell debris, whole cell proteins. And host cell DNA. It could be a problem that the larger amount of impurities lowers the product lifetime and process yield. Floculation technique is well known as a pretreatment for high cell density culture feed. Floculant added to the feed forms aggregate with impurities to be removed easily. Acids and. And inorganic salts have been used widely because they are biocompatible and cost effective. However, defects such as low clearance of DNA are also recognized. Polymer Flow Grant was developed to overcome such defects and can form easily removable large aggregates by this mechanism as shown on the light side. Here we introduce polydimethyl ammonium chloride expressed as Peter Mac in this webina. Because Peter Mac has positive charge in the feed, it can aggregate with negatively charged impurities. The aggregate with Peter Mac was larger than acid induced aggregate, so impurity removal could be improved by Peter Mac. The figure shows the relationship between pressure and throughput during filtration. You can see the increasing tendency of three lines are different. If you see the throughput between two and four psi, you can find that the throughput of Blue line is higher than those of orange and green lines. That means that required filter area can be reduced by P dot Mac treatment. Here you can see the example of filter area reduction. In the left column the feed was filtered by Milstack, D0HC and X0HC without pretreatment. In the right column 0.05% of P dot Mac was added. Untreated feed was filtered by Carrizo 40 Ms. the total filter area and the amount of flushing water were greatly reduced by Peter Mac addition. The expected merit of Peter Mac is not only lower cost of flushing water, but also less physical strain by the lower height to bring up the filters. Next I will talk about design of experiments DOE and quoti by design qbd in the process development. As most of you may know, Qbd is described like this. Qbd approach helps us to understand the process and products deeply the deep knowledge. Can contribute to the efficient development of design space specification and manufacturing control. Qbd is not mandatory, but it enables efficient examination of process change in the design space after removal. Doe is a part of Qbd. And usually performed in associated with 40 risk management. Various departments including QA, QC, R&D should work together for risk management process. Qbt is a similar word as qbd. The characteristic of qbt is that manufacturer check whether the product accommodates the regulation after process development. If the product doesn't accommodate the regulation, manufacturer should go back to the process design with much cost. Qbd approach assures the quality by the process in qbd statistical. Method clarifies the relationship between process parameters and product profile. The development strategy prevents wasting time in going back the development flow. In the first step of Qbd, the final product should be defined quality target product profile. Qtpp is a characteristic of the final product. Indication, mechanism, administration, etc. Are defined as qtpp in monoclonal antibodies. Then critical quality attribute cqas are identified. To meet Qtpp, Cqa must be properly identified at the start of the qbd process. Examples of cqa in monoclonal antibody process are shown here. One is the product aggregation. This type of aggregation is a product related impurity and it is the risk of immunogenicity. Another one is hostile protein HCP. It is a process related impurity. And a risk of taking exogenic protein. Other examples of cqa about product and impurity can be mentioned by the risk assessment in qvd approach. Doe is usually utilized to study the relationship between process parameters and cqa efficiently in the example shown on the top left. We cannot see the relationship in whole parameter space. Also in the example of bottom left, too many experiments require too much time and money. For example, we could see data points as shown on the left side. On the right side, sorry, you can see the data points are not too much and distributing in the parameter space. You can choose an appropriate DOE method, such as the optimal design critical process parameters. Cpps are the parameters to be studied by DOE. Candidates of Cpps are identified in risk assessment process, for example. CPP candidates in P dot Mac addition process are field solution, dosage and operation related parameters listed on the left side. Objective reliables cqa could be yield impurities, removal filterability and so on. You should identify critical. Noncritical and negligible process parameters in the risk assessment process. Next I will show the examples of studying the effect of Cpps. First, you can see the effect of cell density, reliability and dosage on the aggregation formation. The. Disability is used as an indicator of aggregation. You can see the optimal dosage is different according to the cell density and valiability. Decreasing Valiability made the dosage window wider, as you can see on the right side. Here I show the effect of holding time on aggregation and filtration. Figure on the left indicates that the impurity formed aggregation immediately and the particle size reached plateau after that. Subsequent filtration was not affected significantly by the holding time, as you can see on the right side. On the left side of this slide, you can see the pressure increasing tendency was independent on the flux and flow resistance was not so different at dosage of plus minus 20% of the optimum. Here shows the data of quality of filtrate with various dosage turbidity. Was decreased, but yield was almost 100%. In this case. That means that Peter Mac could form aggregation with impurity, not with monoclonal antibody product. The effect on the HCP removal was medium, but Peter Mac worked well on hcdna removal. These results represent the desired performance of Peter Mac because Hcdna removal was one of the motivations to develop Peter Mac. In this section I talked about the Qbd and DOE on the Peter Mac edition with some examples. These results are summarized here. Peter Mark worked well on aggregation formation. Also Peter Mark contributed to high product quality. We have been pleased if this information would help your production process development. Thank you. Thank you, Takuro sang. So from here, I will be talking about their risk assessments on impurities. My name is Yuki Tsenakawa and I'll be taking on from here. So before we move on, I would like to take our second poll of this session and I would like to pass it on to you. OK. Thank you, Yuki, sign. So let's start the second poll question for today. We would like to encourage everyone to participate in the poll. So let me read the whole question for you. The question is what is your concerning point for using cation, polymers, quality and performance of cationic polymers, laboratory guidelines, residual polymer and detection methods or nothing much at this point? Again, we encourage everyone to participate this poll and our poll also active for on demand session as well and we see the coming voting in and we will give a little bit time for voting this poll. Still moving around, so I will close the poll shortly and thank you very much for participating the poll. Let me move quickly to the result now, so I would like to hand over to yukisan. Thank you for your responses. Yes. Great to hear that most of you are really excited about the second part of the presentation too. So we will now move on for we'll move forward in the presentation into more of the details of impurity removals. So when defining impurities in biopharmaceutical manufacturing, it is necessary to determine what the product is consists of and what can be considered as an impurity. Impurities and manufacturing are defined by ICH and other guidelines, especially ICH Q6B, which are test procedures and acceptance criteria for biotechnological biological products. ICH Q6B states that biopharmaceutical manufacturing consists of active pharmaceutical ingredients, additives, and impurities. Impurities can be categorized into three types, product related, impurity states, molecular variants related or derived from the target substance, in which are substances that are not effective and have safety concerns. The second is process related, where impurities derived from the manufacturing process, which are substances derived from raw materials or manufacturing materials. Used in the manufacturing process, the others are contaminants, which are impurities that are unexpectedly mixed, such as microbial contamination. Among the three process related impurities can include free substances from cells such as DNA, HCP, or even chromatography resins or enzymes such as benzonase. Pdat Mac is classified as process related impurities as it is a filter aid added after culture impurities and API's and pharmaceuticals require detailed characterization quality assurance to be within appropriate ranges with association to the control strategies to ensure the quality of the drug product. Therefore, regulatory guidelines states. That the specification values of impurities in the drug product must be set appropriately for each impurity or the total amount of the drug. These specifications must be established based on the scientific evidence or impurity, impurity, exposure, safety and toxicity, since preparing these claims would require a very large amount of testing to be performed. Flexibility. And scientific based controls are permitted by the authorities. This means establishment of specification values and test methods may not be required. For example, if it can be removed in the downstream purification process. Specification testing are not required at the final drug product. Instead in process testing is conducted at the purification step. In addition, if the purification process is very robust and can always remove impurities to an acceptable level in process, testing may not be required. However, in order to adopt these methods, removal of the target impurities in the purification process must be shown on a scientific basis and experiments such as clearance tests to spiked impurities must be performed. In order to determine what control strategy to take, whether specification testing is required or not in process testing should be considered or if in process test is also not required because it is known that the impurity can be completely removed, a risk assessment based analysis should be done based on actual clearance test values. As I mentioned, biopharmaceutical manufacturing consists of many impurities where the risk assessment begins with determine determining whether the substance is an impurity or not. According to ICH Q3A regulators required to assess the need of a control strategy on a scientific basis and develop an appropriate control strategy which we can, we can think which we think. Can be determined based on Qbd as previously shown. As clinical trials also investigate the effect of impurity safety on patients, impurity determination should be conducted prior to the nonclinical studies. However, from the objective of developing safe drugs, it is better to develop a process where known impurities are properly removed in the manufacturing process rather than planning a nonclinical study. So how exactly do we evaluate impurities? Figure on the left briefly summarizes the concept of risk and control strategies. If the impurity shows high risk and the small amount of it is contained in the drug product, it is necessary to set to set specifications, lot release decisions and acceptance criteria. In contrast, if the impurity is known to be non hazardous. Control strategy may not be required. If there are some risks, but it's sufficiently removed in the manufacturing process, then this must be demonstrated during process development. However, if the substance has potential hazardous properties or it or if clearance concerns remain, then the necessary controls must be implemented. So how do we determine if an impurity is subjected to be controlled? First, we identify the substance that may occur in the manufacturing process and search for the safety information and the acceptable daily intake i.e PDE value. Based on this, we then predict the safety levels estimated at worst case scenario i.e, assuming that the amount of process in the process is fully contained in the final drug. And determine whether to take action as an impurity or not. In order to determine this, we can use the S value. The S value in the maximum is the maximum allowable amount of an impurity divided by the amount actually contained in the final drug product. An S value higher than one would mean it is a safe amount. Lower than one means develop a process that takes clearance into consideration. However, if safety of the pharmaceutical product is evaluated and it is proven that the sufficient removal is achieved after process development which is supported by the S value being high five or greater, then there is no need to establish impurity specifications etcetera. However, if the S value is still less than one after process reevaluation, process changes would be required. Therefore, the S value is commonly used to assess whether a substance needs to be controlled as an impurity or not. This slide shows the S value criteria mentioned at each development and manufacturing stage. The S value has an estimated safety margin SC and an actual safety margin SM before reaching commercial production. The concentration of the impurity and the clearance capability of the process must be evaluated based on safety and toxicity information, which then leads to assessing the risk of contamination in the final drug product. Let us now proceed to actual risk assessment of PDA Mac Ichq 3A states that most antibody drugs. Must be reported if they contain more than 0.05% impurity out of an antibody dose of two grams per day. This 0.05% is a guideline and the threshold should be set at even lower concentrations for toxic substances. However, as structure determination and safety confirmation are performed at 0.05% or higher anyways, we will consider this as a starting point. Here I show how to calculate the S value if clearance is not performed. I have shown you values of another reagent as a comparison which is also used directly in the bio reactor like P Dad Mac. I will call this reagent reagent a if the amount of P dad Mac and reagent a used in the bio reactor is 0.5% and one and 0.1% respectively. P dot Mac concentration per antibody would be 0.25 to 2.5% for 2.5 grams per liter. Dose of antibody and reagent A would be 0.05 to 0.5% for five grams per liter dose. Therefore, without clearance, this concentration exceeds the 0.05% mentioned earlier. So P dot Mac and reagent A are impurities that must be reported. We then review the safety information of P dot Mac in order to calculate the S value. P dot Mac has no reproducive toxicity or mutagenicity. Furthermore, based on the in vivo toxicity studies, PDE per 60 grams was calculated as 50 milligrams per kilograms body weight per day, which was set as the NOAEL. Limit of maximum exposure without adverse effects. Based on this, a safety assessment would be performed. We can then calculate the SC value based on the safety information of P dot Mac, assuming that P dot Mac is fed into the bio reactor and not removed in the process. Assuming an antibody dose of 100 to 1000 milligrams per dose, the amount of P dot Mac would be 2500. To 25,000 and reagent A would be 500 to 5000 micrograms per dose. Safety information also indicates that the estimated exposure limit would be 6000 for Pdat Mac and 100 and 2000 and 20,000 micrograms per dose for reagent a. Based on these information, the estimated safety margin SE is well above one for reagent a. But still 0.24 to 2.4 for P dot Mac, which may require specification and clearance tests. If the antibody dose is high, evaluation of P dot Mac removal during the preification process may be required. According to the estimated exposure limits shown previously, a clearance precision of up to 4.2 times would be required. To achieve an S value greater than five. However, as this is without clearance and since P dot Mac can be lower than detection limits by purification in the general antibody process, the concentration of P dot Mac after purification would be around 10 to 100 micrograms per dose based on this concentration. The actual safety margin SM value marks 60 to 600, which is extremely high. This suggests that with proper clearance, P dot Max shows low risk and impurity standard setting where testing is not needed. However, I would like to stress that this is just a simulation with a standard antibody purification. Please make sure to calculate your own as values based on your own manufacturing process. To summarize, Pdat Mac is treated as a process related impurity, process related impurity requires a management strategy based on a risk assessment and robust clearance is usually conducted in the case of Pdat Mac. There are cases where specification testing is not required if appropriate clearance is implemented. Therefore, although we have presented a general case study, depending on the clearance process, P Mac is a product that can be used with confidence. We will now move on to how we detect P Mac and the clearance test performed. There are three methods to detect P Mac SPR where P Mac is quantified as the angle of refracted light, HPLC mass which is detecting P Mac by mass spectrum, and qPCR methods which quantifies. By the level of DNA amplification impaired by P Mac, all methods can be used, but each has its own advantages and challenges. The SPR method was expected to accurately detect P Mac, but shows low reproducibility and detection sensitivity. The qPCR method in contrast. Has an extremely high detection sensitivity, but the protocol optimization is challenging. The HPLC method does not have the same detection sensitivity as qPCR, but it has been popular recently because of its high reproducibility and easy protocol optimization. Therefore, we offer AP that mat detection service which uses the HPLC method. Our validation service team offers a service of residual polymer detection using the HPLC method as shown in the table. Our internal data indicates that the amount of residual P Mac in the process after the clarification step being less than one ppm and that this concentration is not toxic as previously mentioned. Our service can quantify up to one ppm using the HPLC method. In addition to confirming the removal of Pdat Mac within the clarification process, analysis can also be performed in post protein a samples API or in the final drug product as well moreover. We have the potential to analyze multiple samples at once, and the full report is provided for your reference. From here we will show you an actual example of HPLC measurement. In this example, P dot Mac was added to the cell culture media, filtrated through a depth filter and to protein a chromatography where the P dot Mac levels were measured by HPLC. To determine detection and quantitation limits, P dot Mac was added to each sample and the signal to noise ratio was calculated as well. Here are the results. Although the samples were processed by protein a chromatography, more than 100 * P dot Mac amount was below the limit of detection and quantitation in all of the cycles. This indicates that P Mac is efficiently removed and that HPLC is a sufficient way for evaluation. In addition, this slide shows the results of residual P Mac after protein a treatment by different detection methods, as you can see. The digital amount of P Mac in the downstream process is infinitely small, regardless of whether HPLC or qPCR is used. Finally, I would like to discuss the impact of P Mac on the purification process and the removal rate of P Mac using real case examples. As discussed in the DOE chapter, the evaluation of process characteristics require an evaluation of P dot Mac removal rate, process and product quality. It is also necessary to show the equivalence and reproducibility before and after scale up. In scale up, it is necessary to show whether the relationship. Between process parameters and outputs such as yield is following the larger scale. At the larger scale, this line shows the comparison results before and after scale up. At the chromatography process, the brown lines show a smaller inner diameter and the blue line shows a larger inner diameter, IE a larger scale. Results show that it there is no significance especially at the chroma chromatogram yield and HCP concentration during scale up even if P dot Mac is present. We also analyzed the effect of quality of the solution treated with P dot Mac for 100 chromatography cycle. Results show that the yield was stable. Over 95% when P Mac was used and that HCP was more stably removed with P Mac. Aggregates were also more stably removed at the chromatography step when P Mac was present. This indicates that P Mac does not affect the performance of protein a chromatography. And can be safely incorporated into the process design. So in summary, Pdat Mac can enhance productivity of clarification of a high cell density cultures where qbd with DOE enables risk based process development. P Mac is safe to use as long as there is an efficient downstream clearance step. However, as there may be a need to evaluate the level of P Mac clearance, implementing an effective detection method may be necessary, which we can support from our residual P Mac detection service. Finally, our internal data shows that. P dot Mac has minimal effect on further downstream process in terms of scalability, yield and HCP removal. In conclusion, P dot Mac is a safe and effective tool which can also be used in GMP biomanufacturing. Thank you very much for your attention as it is such a useful tool, we highly encourage you to try out our P dot Mac. If you have any challenges in your clarification process, if you have any questions or concerns using P dot Mac, please feel free to contact us and we are more than grateful to support you anytime. Thank you. And we are open for questions. Well, thank you Doctor Yukisan and also Doctor Tak Rosan for this great presentation during today. We definitely learn a lot. I would like to move to the Q&A section. As a reminder, if you have question and you have not submitted, please send us in the Q&A. We just and we will get through as many as we can, but if we run out of the time, we will respond you individually via e-mail. OK. I think I see a lot of question coming in. So let me start with the first question. The question is which downstream processing steps are able to remove in the P dot Mac? Yeah. So thank you very much for your question. That will be answered from me, Yuki. It's in the color. The question was which process would be used to remove P dot Mac. Am I correct? Yeah. Correct. Thank you so much for your question. What to start off with, P dot Mac when used would be evaluated at the level of dosage, would be evaluated to be to be adequate for the fluculation to happen in the bioreactor. And this would be based on the deal we of the study itself, which I believe Taco Rosan has already mentioned in his pot. So by that point there will be a stable amount of P dot Mac or just right amount of P dot Mac introduced. Now usually after the clarification step comes the protein a chromatography and as a characteristic of protein a chromatography, it is highly specified to the monoclonal antibodies. So I believe there will not be as much. P that Mac at hiring to the column itself, so most of it would be discarded. Now based on that thought, say if there were the case where P that Mac was at hired or attached onto the protein a column and would be carried on to the further downstream process, it would usually be removed by the ion exchange chromatography, especially the cat ion exchanges. So that would be so there are some multiple barriers going on further from the clarification process and as I mentioned by the time we go through the clarification, we assume that they will not be as much residual or carried over P that Mac anyways. But if I don't to answer your question straightforward, it would be the cat ion exchange chromatography. Hope that answers your point. Yeah, I think very clear now. So I think the the process to remove is very clear from your from your answer. Yeah. Thank you, yukisan. Thank you. OK, so let's move to the second question. Which component in the medium should be considered when Pdap Mac is used? Oh, OK. This this question is answered from me. Takuro Hosmi about the component should be considered in Peter Mark Addition, we don't have any instruction on specific component and the important point is the charge of the components. If your product is negatively charged, the yield can be low. And if, if purities, impurities are positively charged, product quality can be low due to low removal of the impurities. So you should consider the charge, charge of the components rather than any specific components. Is that good for you? Yeah. So you mean the charge component we have to consider? Yeah. Yes. Okay, very clear. Thank you. Thank you. Thank you for the answer. Hope this clear for the audience as well. So thank you to cross sign for the answering this question. So let me move to the third question. Does the residual PDF Mac affect to the chromatography racing such as protein A and then exchange racing and does the effect depend on the number of repetitions? Yeah, sorry. Yeah, I'm not sure who will take this question. I will take it. Thank you. Apple. So I believe the question is does it affect protein A and ion exchange residents? Am I correct and does it affect the, does it affect the, is it dependent on the number of repetitions? All right. Thank you so much for your question. As mentioned in the in the first question, protein a column is specifically is specific to the the monoclonal antibody. So in principle on the polymers or the P dat Mac would not bind to the protein a column so but the impurities as I mentioned if for any circumstances may at higher or be attached to. The protein a column due to DNA or say for example hydrophobic interactions. If that happens then yes there could be an effect in the the capture of protein A's. But as we showed in the in the presentation, there were no effects of the of the. Of the actual performance of P Mac after using P, sorry, there were no effects on the performance of the protein a columns before or after protein P Mac usage. So we can already see that P Mac may not. There's a high chance that it does not attach to the protein a columns and in terms of the repetitions. He also showed that the protein a columns were the sorry. The feed stream has been through the protein a columns over 100 cycles and it did not change the performance at the end. So this data indicates that P dot Mac in the illusion fraction of the antibody was always low or below detection limits and they will know it would not affect the performance of the protein a column itself. I hope that answers your question. Yeah. Yeah, I think it's from the actual performance that I showed, there is no effect, but actually it's low. We can say low or less effect, yes, I would say low. There could be a chance, but we haven't seen it so far is probably the most honest answer, but thinking through the mechanism of protein A. There is a very finite chance that it would affect the protein acons. We just haven't quite seen it before. Yep. OK. So perfect. Yeah, I think. Yeah. Thank you. The use of the P that Mac. Yeah is very good. Yeah. So let's move to the should be the 4th question. Would centrification be unnecessary if P that Mac is used in the clarification process? OK, I will answer. Yeah, I will answer the question about the necessity of centrifugation. It's it's very difficult to say because it highly depends on the field stream. There could be some situations where the combination of field American centrifugation is useful or not. Therefore, we recommend setting a defined goal and learning a small scale trial to study the optimal filter Mark Concentration, Federation profile and fit rate characteristics. Yeah, so if you want us to do a small scale test, please ask our colleagues. Thank you. OK. OK. Yeah. Thank you. Thank you. So anyway, so if any specific question, I think better to reach us, our expert can answer. Yeah, very clear on the specific. Yes, please. Thank you. Thank you. OK. So we still have a little bit time. So let me continue another question. What is the effective cleaning method if Peter Mac remains in the chromatography racing? I think this is very interesting question. Yeah. This is indeed an interesting question. Yes, since this is more about the cleaning pot, I would like to take this question. So does it, does the polymer actually stick to the pipe walls and is validation required? I think that was the question. Am I correct? Yes, correct case. So to be honest, I think there is a possibility that the. See that Mac polymers could stick to the piping, but since there is a possibility to that that it would stick to the wall, we suggest that you should make a judgment require regarding the needs of validation based on what we discussed in the webinar. So more on the residual, the possibility of being residual, any clearance that happens afterwards the. The actual materials of the piping etcetera and discuss with your company's quality assurance department for risk assessment, but we generally think that this is Joe. Polymer concentrations are expected to decrease gradually during the normal purification process as we mentioned and we do believe that. There will be a very small amount of at hired polymers or P dat Mac to the piping wall would be present. I hope that some. I hope I answered your question at that part. Yeah yeah you did. Yeah you did. Yeah I think it is some cleaning method. Yeah need to be. Used, yeah. And I think it's will be good. Yeah, to connect us as of our expert. Yeah, to answer more more in the specific question later on, okay. So I think we still have maybe one more question. The question is would the regulatory authority suggest us to evaluate the PDF Mac concentration in the final product if the product is concentrated in DFF? Even if the Pdot Mac concentrate is less than one ppm after qualification, yeah, thank you so much for your additional question which is very interesting about the concentrated in TFF, I will take this question. So as you mentioned there will be concentration of tea of polymers happening. During the TFF process as we include the polymers at the upstream and it goes through manufacturing. So but what I what I what I believe is I'm going through this presentation that I've shown based on the SM values and the SE values after the TFF process and then the and at the during the purification process you can probably judge. If we have to report this out to the authorities of they or they will be expecting that. So regarding the needs of analysis of this after concentration part, it really depends on how much is actually present after your TFF process. And I think you should be discussing further with the regulatory agents or again have a thorough discussion with your quality assurance department in the company itself. So we will I think having an adequate amount or an ample amount of purification level, sorry, clearance level would support your judgment. So again maybe discussing that with authorities or through your QA department would be necessary. What we showed here is again the general idea on how much would be resolved but. Based on how much it's concentrated in TFF, it could be necessary to be having a discussion and we are always happy to support that anytime. Great. Yeah. Thank you, yukisan. Yeah. Regrettory is very huge and big topic and important. Yeah, I do agree. Yeah. OK. So I think due to the time is limited, we will end of today's seminar right now and the pending question is that not yet answered. We will connect you shortly via e-mail. If you would like us to contact you, you can also go to contact options yeah in here to register for the upcoming webinars or access our archive webina library, please visit our webina website. You can also click on the calendar features at the bottom of the screen to register your next webina. Thank you our presenter Dr. Yuki San and Doctor Takrossan for the great presentation. And more importantly, thank you very much to everyone who joined us today, Webina. Have a wonderful day and goodbye. _1732209858672

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