"Hello and welcome to the Sart 10L's webinar titled Maximizing Efficiency in Drug Formulations with Cenal Use Nitzer presented by Arium Mini and Clarabir.

Please allow me to introduce myself. My name is Miriam Labi and I will be your moderator today.

So now let me introduce our speakers.

Our first speaker is David Menheim.

David Menheim has been working for Sartorius since twenty twenty one as a global product manager in MINI.

He earned his graduate degree in Biotechnology Engineering at Politec Marseille in France.

He also has a Master of Science degree in Management at Cage Business School, also in France.

David started his career in infectious disease diagnostics and has held multiple positions in sales, application support and marketing.

Our second speaker is Karl Bioprinter. Karl Bioprinter has joined Sartoris in twenty twenty one as the Global Product Manager for Sensors and Automation.

He has a degree in industrial engineering with a focus on production from the Royal Institute of Technology in Sweden and the Gallery Mayo Institute of Technology in Ireland.

Karl's entire career has focused on sensor technology. First working in CELLES, he became a technical specialist and then a product manager in petrochemical industry for radar level technology.

With additional experience in fire and gas detection for the marine industry.

So let's get started. We hope you will enjoy today's webinar about single use mixing.

David, Karl, please begin when you are ready.

Many thanks, Miriam, for this nice introduction and many thanks to all of you for attending this webinar.

Karl and I are very happy to present how single use technology and in particular single use mixers can address the challenges of drug formulation.

To introduce the presentation, we start with monoclonal antibody manufacturing process.

Mixing is an important step in the manufacturing process of a drug because we can find mixing applications at all major stages of the process. We can find mixing in upstream for the preparation of culture media for example but we can also found a mixing application in the downstream phases for the preparation of buffers or for viral inactivation.

And finally in the final phases of the formulation of drug substances or drug products.

In the final stages, mixing can be seen as a critical process due to the closeness to the final product and to the patient safety. So today we will focus on the final formulation step.

Single use technologies are more and more popular in the biopharmaceutical industry due to their numerous advantages such as reducing the risk of cross contamination, improving operational flexibility and reducing capital expenditures.

That's also the case of bottom mounted single use mixers.

There are also some challenges associated with their use in drug formulation to ensure product quality and process performance.

The first challenge is related to the product, to the drug itself.

How can we ensure that the drug, the product will not be degraded during the mixing?

The second challenge is related to the process and the key parameters to monitor and control. How can the process ensure consistency from batch to batch?

The last challenges are related to the risk of leakage, the risk of back leakage during handling or processing, the risk of contamination and the process validation.

Let's start with the first challenge of product degradation and how we can preserve the drug quality when using single use ring surfs.

During bioprocessing, biomolecules such as proteins, mRNA may be exposed to different stress that can damage the product and or disturb the process such as the shear stress.

The shear stress is defined as a force per unit area that acts parallel to a surface.

So in other words, the shear acts to change the angle in an object.

In bioprocessing, the shear stress is present in almost all bioprocesses and is known to play a role in protein aggregation and denaturation.

What are the impacts of this degradation of the product?

The degradation of the product may lead to lower yield of aging products but it can also lead to poor performance of the filtration due to the formation of protein particles or aggregates.

So then the entire production batch may be jeopardized leading to a significant financial loss and delays in getting the drug product to patients.

Then what are the criteria to be considered when designing or choosing a single use solution for final formulation?

In terms of application, we talk about homogenizing the purified product and the excipients. So here the objective will be to obtain a homogeneous solution while preventing the product degradation. So the key criteria will be the shear rate that has to be as low as possible and we should also have a good product velocity to ensure homogeneity of the solution.

Here we are describing the mixing technology of the FlexSafe Pro Mixer, a bottom mounted single use mixer.

The mixing technology principle is based on electromagnet in the drive unit interacting with a fully encapsulated permanent magnet inside the mixing bag.

The impeller is held in a part that is welded to the bottom of the bag and this part sits on the drive unit.

When in use, the impeller is stabilized in a position away from the sides and bottom of the bag via magnetic coupling with the rotor magnet.

So there are no moving parts within the motor and no contact with the impeller.

So in the next slides we will see how we evaluated the shear rate while ensuring mixing homogeneity with this mixing technology.

For evaluating the shear rate, we used computational fluid dynamics.

Here we focused on the top of the impeller as it is highlighted in yellow on the screen and we performed CFD analysis in a two hundred liter bag at an agitation speed of four fifty rpm considering viscosity of one centipoise.

You can see on the left the shear. So in red it represents the maximum shear of the order of three thousand three hundred seconds minus one and in blue the minimum shear values. So it's an order of zero point three seconds minus one. So we can see on the left that the bottom of the bag remains blue. So that indicates that the shear in this area is minimal.

But you can see that the high shear rate can be observed adjacent to the blade in the regions where the impeller speed is the highest.

The picture in the middle and on the right correspond to ISO surfaces. So they are surfaces where the shear has a constant value.

In the middle, we can see in yellow color that a very small fraction of the surface of the impeller produce a shear rate about seventy percent of the maximum value.

And on the contrary, on the right, the figure shows how most of the surface of the impeller exerts a shear rate about twenty five percent of the maximum value.

The shear rate was also evaluated by CFD in the lower area of the cup of the impeller.

When in use, the impeller is stabilized in a position away from the sides and bottom of the bag via magnetic coupling.

And we can see that the shear stress is limited to that region.

Now we can see very low value as indicated on the screen. For the free circulation there is no significant shear stress that was found in that part of the of the impeller.

As I said at the very beginning, therapeutic protein formulations are exposed to shear stress during their commercial production.

The magnitude and duration of shear exposure in the absence of the air water interface is unlikely to cause protein aggregation or denaturation in processing operations of bioprocessing. So here we are showing that the maximum shear rate we simulated at four fifty rpm is below common technologies used in the drug manufacturing journey.

We are illustrating at the top of the graph, the shear race needed to degrade a small protein around one hundred amino acids.

However, it's important to note that the specific share rate values that cause protein degradation can vary widely depending on the protein and on the formulation conditions. So for example the pH, the temperature, ionic strains can also play a role in protein stability and can interact with shear stress to accelerate protein degradation.

So the sensitivity to shear rate has to be part of the risk assessment during the process development.

As the shear rate is function of the speed in Newtonian fluid, we evaluated what could be maximum shear rate between one hundred and seven fifty rpm.

We could use this kind of correlation to adjust the mixing parameter to not exceed a certain value of shear rate And that could also help to define the mixing parameters in function of the sensitivity of the drug to shear stress.

So we have seen how the shear rate could be evaluated using computational fluid dynamic, but it's also important to ensure that the mixing is homogeneous.

So for this we also used computational fluid dynamics studies to visualize the movement of the fluid inside the mixing bag.

On the right, on the side view, we can see the evolution of the velocity profile over the time.

So the area in blue are area where the movement is not moving and in red it's area where the fluid moved at a speed around one meter per second. So we can see that there is an absence of blue area on the right, meaning that the fluid is moving in the bag.

The picture of the top view on the left represents the pumping activity of the impeller.

So we can see that the the trades done with the impeller blades.

So this impeller pumps fluid in all directions generating a turbulent zone and a radial flow in the bottom of the bag and that will prevent dead zones. So again it shows that we can ensure a good homogeneity inside the bag.

In addition to CFD, we performed mixing tests at different volumes and different speeds using water but also using viscous solutions to reproduce the viscosities that can be encountered in the final formulation of monoclonal antibodies.

We can see that in all cases the mixing time was less than one minute demonstrating the capability of the system to homogenize the product.

So by combining the technology used to the levitating impeller, the low shear rate and the product homogenization, we preserve the quality of shear sensitive products during final formulation.

Now Karl will talk about how to ensure monitoring and controlling of critical parameters during final formulation with single use mixers.

Thank you David and hello everyone. Yes, so how do we tackle the challenge of variability and keep the process repeatable and efficient?

The best way of doing so is to monitor and control all the vital process parameters for your CCPP.

For buffer preparation, for example, optimal buffer conditions it's important to measure pH.

Conductivity might also be an important parameter to monitor for final formulation. Temperature is very important, but also of course the mixing speed and pH is normally used as well.

So to monitor all these parameters we use PAT or process analytical technology or maybe short just sensors. So we use sensors to monitor what's going on in your process and ensure that you have a GMP production.

It's important also that when handling these sensors that we don't increase any risk of cross contamination, so we want to be able to deliver the data without any increased risk.

And also we want to use the sensors to increase the efficiency, not only the sensors but also automation process to make sure that it's efficient and it goes as fast as possible.

I highlighted some of the challenges for sensors that are especially important perhaps to final formulation.

The first challenge that I wanted to highlight was the sensor accuracy. If we want to monitor these critical parameters, we need to ensure that the sensors can monitor these parameters with a good accuracy or the accuracy that you need.

Another challenge here with accuracy is that we have a lot of different applications in mixing, also in final formulation. So how do we ensure that this accuracy stays the same over a large range of applications?

The second challenge that I want to highlight is that of course handling sensors you might have insertion steps, you have calibration, you have to integrate.

So how do we minimize the risk of any contamination when working with sensors?

The third challenge is that we need to integrate these sensors into the process directly.

How do we make sure that we do not just measure the product coming into the mixing process and then verifying the measurement on the tubes going out of the process? How do we make sure that we get measurement inside the process and can deliver continuous insights to you?

And the fourth challenge, sometimes when working with sensors you have a lot of preparation. You might need to fill the electrolytes in the VH sensor for example that needs to be documented. There is some paperwork related to that. So how do we make the preparation as efficient as possible?

These are the four challenges that I wanted to highlight today.

The tools that we have then for monitoring and control to make sure that we keep the process consistent and that it's repeatable between budgets but assuring quality and efficiency. Well, we have the single use sensors for mixing, which is there to measure all the critical parameters, and it will allow you then to control your process.

And you can say that sensors is the first step of automation because compared to, for example, sampling your product, taking it to the lab or a benchtop to analyze the result, And the single use sensors have sort of automated that process for you.

The second step of automation would then be an IO unit. I'm using a generic name here. It can be called a remote IO or a control box or a product name.

But the idea is that the IO unit is there for several reasons, both to visualize the mixing process. So this is where you read out your parameters and you can follow the process and what is going on inside of your mixer.

It can also enable full process control, so you could have control units inside of the IO units to control the different processes like pumps, etc.

And it's also there because it creates and simplifies the setup because you have one point of connection for all your sensors and parameters and it enables you to have gather all the data in one place and then transfer that if you like.

The challenges that I mentioned on the previous slide.

So in this case we have the accuracy of the pH which is a challenge here.

With sensor that you see on the picture here, you have a built in calibration chamber. It's already integrated to the sensor that is already integrated into the bag.

This calibration chamber then allows you to calibrate this pH sensor with standard pH buffer solutions, which ensures then that you will have an accuracy of plus minus zero point zero eight pH and over a quite large range of pH two to eleven.

Another fine feature with this pH sensor is that it can be recalibrated without breaking sterility.

That means that if you have a long mixing process, spans maybe forty eight hours, you could recalibrate it the second day to compensate for drift, for example, or you could use this feature measurement after the process is done to verify that we kept the promise on the accuracy.

Another point here to simplify the setup as you can see on the picture and all these sensors are integrated into the bag. They're ready to use except you need to calibrate and there's no service or preparation and meaning there's less need for documentation as well.

Another good thing with the calibration chamber especially is that there is no sampling of your product. You don't need to sample your product and insert this into the transmitter in order to get a good accuracy because you have your calibration chamber.

There is also no autoclaving or integration steps, so we minimize all these steps that could be potentially contaminating your process.

And as you can see the final challenge here, all these sensors you can see the conductivity sensor, the pH sensor, and the thermal well they are inserted into the mixing process, which then can deliver immediate insights from the process as it is happening, a continuous measurement of your process. So this is how we tackle these challenges with the sensors.

On the next slide, I wanted to give you an example of the automation layers, how it might look for mixing.

If we start with the third layer there with sensors and actuators, sensors are there as we talked about to monitor your critical parameters. So pH, temperature, weight, speed, mixing speed, etc. You might have actuators there to help your control loops adjust pumps or control temperature control units TCUs.

And then that information is sent then to the IO unit, which then would be the second layer of automation.

It's there to provide then one connection point for all your parameters and this is also where you would calibrate your sensors.

It will help you visualize the process parameters and could if you like allow local control so that you can have your control loops from the IO unit itself.

Another important task of the IO unit is to handle the communication both with the lower layers of the sensors and also to the upper layers, so your control system, which leads us then to the final top layer of automation for mixing which would then be SCADA, DCS, or even a PLC, your control system, how you have built it up, which is then where you create your control and safety loops, and you can execute the sequence or a recipe that you have predefined.

And this was also then where you log all the data and the process parameters to create a report for example.

Just a comment here that a lot of these things could also be done locally in the IO unit, so it's a little bit depending on how you want to set up your process.

So a look at how it might look for mixing a little bit more in detail. So as you can see on the middle picture you have a pellet tank and attached to that is an IO unit.

Here you connect then your pH measurement, you might connect conductivity measurement to measure the concentration of your fluid, You can connect your drug unit to have mixing speed control, weight measurement to tell how much product you have inside your bag. It could be DO measurement, might have pressure measurement on the tubes to monitor the process onto the next step, which might be filtration, pump control for filling and emptying or titration process, and then perhaps a TCU, a temperature control unit, if you have a jacketed pallet tank.

And the good thing about this sort of automation setup is that you can select at what level you want to automate your mixing process. You could have an I. Unit and just use it as the one connection point for all your parameters and then a local readout and telling you exactly what is going on in the process.

Or you can go full out automation and connect the IO unit to your control system using the standard protocols of today Modbus TCP, Ethernet IP, Profinet, Profibus, etc. And with the help of your control system then create a fully automated mixing process, which then basically allows you to push a button and sit back.

The final point that I would like to make is about the data acquisition. So the data acquisition could be done in the I unit or in your control system. And with this data that you gather for your parameters, for example, you would then create your report. So a batch report, for example, it would be the receipt that you create to make sure that you don't vary between batches. That would be your final document on that.

And with that, I'd like to go back to David. Thank you very much.

Many thanks Carl.

In this last part we will focus on the last three challenges that we have grouped under the same theme of integrity of single use solutions and process validation.

As we said at the beginning of the presentation, mixing steps are present throughout the bioprocessing process. The objectives and risks of each of these steps are different.

Especially since the European Commission published in August twenty twenty two, new guidelines on the manufacturing of steroid medicinal products in the Annex one to reflect changes in regulatory and manufacturing environments.

And in particular, there is a dedicated part in the Annex one to single use solutions.

So in the next slides we will focus on two single use system critical attributes. The integrity of the mixing bags and the validation of processes for extractables and leachables.

If we start with the integrity of single use solutions, we can say that integrity starts with having robust single use bags.

The Annex one mentions the fragility of these systems compared to reusable systems as a risk that should be part of the contamination control strategy.

The robustness of a mixing solution is based on three pillars: the film, the validation and qualification tests and the manufacturing process control.

For the film, we use Flex Safe film which is composed of several layers of different materials that provide a strong robust and flexible structure.

To characterize the film and the bag assembly's behavior, we conducted thousands of mechanical tests such as resistance to flexion and fatigue, resistance to uniaxial traction, resistance to puncture and for the mixing bag itself.

When qualifying, we ensure their robustness by conducting worst case tests or customer applications.

So for example we did mixing at low and high temperature for several hours and then we performed a visual inspection of the bag and we also did ink test to ensure there are no defects that could lead to an integrity breach of the bag.

And then quality controls are performed at various stages during the manufacturing process to ensure lot to lot consistent robustness.

The integrity of a bag with respect to the risk of leakage and potentially microbial contamination does not stop at the product itself. Robustness must be ensured throughout the life cycle of the product.

Again Annex one identifies the risk of holes and leakage as a potential risk and must be part of the contamination control strategy.

For critical applications such as final formulation, post assembly mixing bags can be tested upon request with helium test to identify two micrometers holes.

There are studies showing the correlation between liquid liquid and microbial ingress.

And in addition to this, the mixing bags can be also tested just prior to the process to mitigate the risk of holes that could occur during transportation, handling or installation especially with complex bags in order to ensure integrity of the single use bags.

Patient safety and product quality are most important and become even more relevant the closer the single new system is to the patient as it is for final formulation.

So depending on the process parameters and requirements, the evaluation of the extractables and leachables can be done on different levels.

First, we can use the extractables data that we generated on all components of the mixing bags. And then depending to your risk assessment, extractable and leachables evaluation can be taken a step further by performing additional studies and analysis in your process conditions.

If you would like to learn more about AnnexOne and single use technologies, I invite you to visit the Sartorius website where you can access two webinars on demand covering other applications such as storage and shipping and freeze and sew and final feeding applications.

To conclude this presentation, we have seen the challenges faced by drug manufacturers when it comes to using single use mixers for final formulation.

To maximize process efficiency and patient safety, first a low shear mixing solution will prevent product degradation and increase yields.

Secondly, the use of single use sensors and automation will control and monitor critical process parameters and ensure a consistent process.

And finally, the assurance of single use mixing bags integrity and extractables and issuable support will ensure process reliability and process validation.

The presentation is now over.

Thank you all for your attention and Karl and me will be happy to answer your questions.

Thank you.

Thank you Karl, thank you David for this very interesting presentation. So now if you have any questions, please type it in the question box in the control panel.

So I see that we have already received some questions, so I'm going to start with them. The first question is about the slide five.

We are talking about shear stress in the slide five.

The question is, do we only call it shear stress when they are force affecting our sales or product? Or do we also talk about shear stress when it's affecting the bag material and integrity?

I can take this one.

Thank you for your question.

So yes, in the slide number five, when we talk about shear stress, we are talking about the forces affecting the cells or the biomolecules.

As we also said later in the presentation, when it's a question of integrity of the bag, the integrity of the mixer, Here we are more talking about mechanical resistance to the bag and it's also what we described in the presentation with all the tests we did to characterize the film but also to qualify the mixing system to ensure the bag assembly will not present a risk of leakage.

Thank you David. Another question is about the pH sensor design. So is the pH sensor one time use?

Yes, the pH sensors and other sensors that you've seen or parts of other sensors that you've seen are single use sensors, which means that they come fully integrated in into the mixing bag, fully radiated with the mixing bag.

So the advantage then is that there are less handling and preparation of the sensors when they reach you and less risk related to contamination.

With the calibration chamber on the pH sensor, sort of combine the advantages of single use sensors, but also the possibility to calibrate like a reusable sensor.

Thank you, Carl.

Another question is about the product itself, so the mixing bag.

The question is, is Arctorius working on a single use bag for low volume to address batch sizes from one liter to five liter?

I can take this one.

So when using the FlexSafe commixer, the smallest bag size in the portfolio is fifty liters or the nominal volume and the minimum mixing volume for such a size of bag is fifteen liters in aseptic conditions.

So yes, if we wanted to address lower batch volumes, we are looking for new solutions to address these specific needs and requirements.

Thank you, David.

Another question about extractable and leachable. So the question is, does Sartorius provide a service for extra table and reachable evaluation for clients applications? So we'll be addressing the specific process from the clients.

Yes, absolutely. We have expertise at Sartorius when it's a question of extractables and leachables. So we have a dedicated team, a service called Confidence that is able to support first your risk assessment and then your extractables and leasable assessments by doing extrapolation on the current existing data, but they can also do specific studies according to your to your own process and parameters. If you are interested, you can go to the Software's website and you can go to the confidence pages to learn more on that.

Thank you, One question about sensors. So you mentioned online calibration of pH, CAL. How do you control and calibrate the conductivity sensor?

Yes, so the conductivity sensor actually comes pre calibrated with the bag. So the only preparation that you need to do with the conductivity sensor is to insert the CELLCON stand into your transmitter.

So that's the only sort of step that you need to take with conductivity. And upon request, calibration certificate can be shipped with the product as well.

Thank you, Now a question on the mixing system and the mixing technology. Is it a magnetic coupled system or is it a levitation system?

Actually it's both.

We are using managed key coupled because the impeller will move things to magnetic field that is generating by the drive unit. But the impeller is levitating. So there is no contact between the impeller and the other part of the mixing bags or the drive units.

Actually the answer is both.

Okay, then another question related to that is that if the dry unit is using cryogenic based superconductor to levitate the antenna or not?

No, we are not using this kind of technology the ProMixer the FlexSafe ProMixer solution.

Okay, I see that we don't have any further questions, so maybe I can give you a few seconds if you have any last questions.

I see another one coming. So any are there any provision to operate the group of pilot on parallels with the central server?

So maybe I can answer that.

At the moment, no, we don't have any plans to wirelessly operate several pellet tanks at the same time. That's the sort of short answer. The long answer would be that we did have a discussion regarding this and I think this is something that we will continue to discuss for further projects in the future, but not at the moment.

Okay, so another question.

I'd say thank you for participants.

This one maybe it's a good transition to the end of this presentation.

Thank you again, Caroline and David, for this very exciting presentation.

And on behalf of all of us from SARS CoV-two, we would like to thank you for joining us today. And we wish you, to all of you, a great rest of your day.

Thank you Arium. Thank you everyone.

Thank you. Bye bye."