0:00
Thank you for your kind introduction and I would like to welcome you all to my presentation today about orbitally shaken bioreactors.
0:10
When it comes to scaling up a bioprocess on small scale, you're typically using microtiter plates with small volumes and many experiments at the same time.
0:20
You can use tubes, you can use shake glass all the way up to 5000 millilitres and I think it's good advice to take these three scale up parameters into consideration.
0:33
So first, volumetric mass transfer coefficient or KLA.
0:38
If you have fast growing organisms and they are oxygen demanding, it's good to keep that value constant during your scale up.
0:47
Also, what's sometimes forgotten is that the KLA also determines the weight with which volatile components are stripped from the medium.
0:57
That can play a role in some processes.
1:00
And if you're also having fast growing organisms, mixing time plays a role.
1:05
If mechanical stress is a priority for you, you should at least think about keeping the volumetric power input constant when you're scaling up.
1:18
If you want to achieve a process that is viable on production scale, at some point you have to leave the shaken, let's say realm and you have to transfer your process in the stirred tank bio rector, because so far only these types of bioreactors are providing these huge amount of volume.
1:44
In many cases there's a wave bioreactor in between when you're doing the scale up and every time, you're changing fundamentally the way of aeration and the way of how you're doing the mixing.
1:56
You have to think about appropriate scale up parameter for your, for your different type of bioreactor.
2:03
And at least to my knowledge, there's unfortunately not a general scale up strategy out there that can be applied to all cells and all processes.
2:13
So you really have to take your case into consideration.
2:18
And for us, what, what we are advising is when you're doing a scale up that you always start with the question: What are the key process parameters for your process that are likely affect product yield, quality and consistency?
2:34
For example, it could be aeration, it could be power input or mechanical stress, but it's very specific to your process and you have to answer that question yourself.
2:43
And the goal would be of course, to keep those parameters as constant as possible because you cannot keep them all possible.
2:50
That's not possible.
2:52
And then worry about the rest later or fine tune later.
2:57
That's what I mean by iterative process.
3:00
And when it comes to scaling up microbial processes, in most cases it's more straightforward because microbial in general are a little bit more robust towards a broader range of physical environmental properties including mechanical stress.
3:30
In the case of a steel tank bio rector on that scale, there are two sources for mechanical stress.
3:37
First would be the stirring, quite intuitive.
3:39
And secondly, when the bubble is bursting at the surface, there's a very high local energy dissipation rate and that leads to very high elevated stress for the cells.
3:50
And but microbials on average, I would say it's fair to say that they're more robust compared to mammalian cells.
3:59
So, you're not applying the same parameters for an E coli for mammalian cells and because of mechanical stress.
4:09
And, but I have to say it's also not necessary because in for mammalian cells, they do not need as much oxygen.
4:20
So you don't need the elevated stirrer speed and you do not need the high elevation rate.
4:26
Sometimes people are using a bioreactor with no steering at all and just the bubble aeration.
4:36
And of course there are very many viable processes out there that applying stirring bioreactors with mammalian cells.
4:43
So people are reducing the aeration rate and adding some surfactants like Pluronic to prevent formation and also lessen the surface tension of water.
4:58
And that way if the surface tension is reduced, the local energy dissipation rate when the bubble is bursting at the surface is also strongly reduced.
5:08
But there's still cell lines or cells out there that are still making problems when they are scaled up. In this type of bioreactor even if you're doing all these preventive measures, the amount of mechanical stress can be still too high.
5:32
So a couple of years ago the idea came up, why not shake?
5:36
Why not use orbital shakers all the way to production?
5:39
That way you keep the hydrodynamics constant.
5:43
So you have on every scale a surface aerated system and the scale up should be much more straightforward in theory.
5:53
What's necessary for this is that we have no bubbles or the goal should be to not introduce active aeration.
6:02
And this means we have to achieve a comparable KLA compared to stirrer tank biorectors that are currently applied or have been applied in the past.
6:13
So this is the slide.
6:15
You already know the typical scale up in small for small scale cultivations.
6:23
And I would like to take a look at these three parameters.
6:26
And with our orbital shaken bio, just we try to extend this scale up further all the way to what seemed possible.
6:36
And that's how I would like to introduce our orbital shaken bio reactors, starting where the shake flasks left off at a working volume from 1.5 to 12 litres and going in in three further steps all the way to 2500 litres working volume.
6:57
I would like to take a look at the first, the SB10, the smallest one.
7:03
This is the data sheet.
7:04
I don't want to go through everything, just want to highlight some aspects.
7:09
For example, the temperature can be regulated to up to 40°.
7:16
These types of bioreactors work with single use bags and they are equipped with dissolved oxygen and pH sensor spots.
7:25
And what's also special about the SP10, it's still weight wise, it's still manageable.
7:30
So you could carry the module as shown in the picture to the right.
7:34
You can carry that through your lab.
7:36
You could prepare everything under the hood, under the clean bench and then walk through your lab, put it on a shaker, attach the sensor, the optical fibres and then you could start your cultivation.
7:48
As I said, these bioreactors are working with single use bags, so there's no stirrer or additional mixing device inside that reduces the risk of damaging the bag.
8:00
They are gamma irradiated, so you don't need to sterilise your bioreactor.
8:06
Leachable and extractable studies were done by our bag manufacturer Hegewald.
8:12
And just to give you an impression, for the SB10, we offer three different bags, the basic bag which is capable of doing ATF perfusion then the standard bag, that's the basic bag with the optical pH and DO sensors and then the perfusion bag with an additional dip tube and TFF perfusion is possible.
8:36
Now I would like to take a look at the scalar parameters I mentioned before and go through them.
8:42
This is the KLA value over the shaking frequency for different filling volumes for that SB10.
8:48
And you can see first you see the lower the filling volume, the higher the KLA values that you are getting over that system.
8:58
And with just four litres, you get levels above 40 per hour.
9:02
That's even sufficient for higher breathing organisms like yeast or fungi.
9:08
And with the higher filling volumes, you end up way above the 10 per hour threshold for many mammalian cell applications.
9:22
Let's get into the mixing time.
9:23
This was a study done by UCL and they used dual indicator system to determine the mixing time.
9:30
So a fast acid base reaction and presence of a pH indicator.
9:36
And what they did, they recorded an image every time the orbital shaker was at the at the same location.
9:43
And they added these images, put them together in the in the video and that way the time it took until the colour is homogeneous again, that was the mixing time value.
9:58
This is an actual video of that experiment.
10:02
You can see when the base or acid was added and the colour shift took place.
10:08
And you can see how the vortex is forming inside the bioreactor and how the mixing is done in these types of bioreactors.
10:15
What personally struck me at first was I couldn't believe that this is actually done when the orbital shaker is moving.
10:23
But these are really added pictures at the same location.
10:27
So you really see how reproducible the rotation of the liquid inside the orbital shakers.
10:38
In terms of numbers, these are the results mixing time over the shaking frequency for the SP10, the lower the filling volume, the lower the mixing time.
10:47
But you can see if you go beyond 100 rpm with that shaker, you end up below 10 seconds.
10:53
So it's for it's enough for yeast, for example, and definitely sufficient for mammalian cell cultivations.
11:02
Last one, this is the power input over the shaking frequency for different filling volumes again and again, the lower the filling volume, the higher the power input per volume.
11:18
So this this is the three-litre curve.
11:22
And you can see for example, if you're shaking at 140 and let's say we are in the range between 1.5 and 3.5 kilowatts per cubic metre.
11:32
That's admittingly a little bit higher compared to shake flask, but still not by a factor of two.
11:39
But it's quite comparable to shake flask showcasing hopefully that you can expect comparable power inputs with these types of bioreactors compared to the shake flask.
11:51
I would like to go a little bit into one application example.
11:57
This was done by the company, by ProBiogen.
12:00
They wanted to scale up their animal herpes virus production from a batch shake flask to a controlled fed-batch orbital shaken bioreactor.
12:10
And these were the specifics.
12:13
So they started with 4 litres and ended up with 8.5 and they started with 800,000 cells per millilitre.
12:24
You can see the quick results because I'm not saying that the orbital shaker always delivers better results because that is a little bit unfair comparison because the Erlenmeyer flask here was done in batch mode and the orbital shaker was done in fed batch.
12:42
But you can see it's a proper system to scale up from shake flask to a more controlled environment and then even apply the fed-batch and have both higher viability at harvest time and also the amount of viable cells did strongly increase in that example.”
13:04
In terms of numbers, they achieved implication factor by 19,200 and showcased that the SB10 was able to cultivate these large cell aggregates.
13:17
Now take let's take a look at the big boy, the SB2500Z. Here again, the data sheet working volume is between 500 and two 2500 litres.
13:32
I don't want to go through everything, but these types of machines are also equipped with single use bags and they have the dissolved oxygen and pH sensors as well.
13:43
And I would like to point out the weight.
13:45
So the machine is quite heavy.
13:46
Just the machine without the water is 3.2 tonnes.
13:52
And if you add the 2.5 tonnes of the liquid, you end up at 5.7 tonnes.
13:57
So quite big and heavy machinery.
14:00
The respective bags also must be respectively big.
14:06
This time these are manufactured by the company Entegris in the US.
14:12
They contain the sensors and it's a bag in bag concept.
14:16
So basically 2 bags to add an additional layer of security for your process.
14:21
They also of course gamma irradiated just to give you an impression.
14:28
Just a quick look at the mixing time, of course the volume strongly increased. In this case, we ended up with 55 RPMs shortly under a minute.
14:41
So it's admittedly it's much higher, but it's still sufficient for most cell culture applications to have that mixing time.
14:52
Also the KLA over the shaking frequency, we can see that we end up with all filling volumes above the threshold of 10 per hour and at 55 RPMs.
15:07
And I would like to show you what it actually looks like.
15:10
This is the big bioreactor in motion.
15:14
You can see the liquid, I'm not sure how much liquid is in there.
15:18
I would say between 1.5 and maybe 2000 litres.
15:22
And this is a person for scale, just to give you an idea what it looks like, how they work, how they operate in a production environment.
15:32
I would like to give a short overview about the cells or the applications our customers are using so far and applied these bioreactors too.
15:43
Of course, we have a strong focus on mammalian cells, but also plant cells and insect cells are applied.
15:50
In the case of SB10, as I said, fungi and yeast are also possible.
15:55
And besides the life science applications, in recent years we gained some traction in the food market or the cultured meat market as well because they are moving more and more towards production and that that's where we can come to play.
16:11
And also worth mentioning the mixing of mRNA.
16:14
It's quite a nice application for these shakers because these bioreactors deliver very gentle mixing for these delicate molecules.
16:27
If you want to get a closer look at these shaken bioreactors.
16:33
Feel free to go to our scientific poster selection about these bioreactors.
16:38
On our website, you can find a couple of posters and I would like to give a short summary.
16:46
I hope I’ve shown you that the orbital shaken biorectors are efficient and sufficient enough in terms of mixing and KLA values for cell culture applications, that the power input is comparable to the shake flasks due to the similar surface aerated hydrodynamics.
17:06
That counts both for the average power input and also for the local maximum value.
17:11
That's quite important when it comes to cell damages.
17:14
And then so we it's, it's quite comparable and low maximum mechanical stress.
17:22
You need to use no or very little Pluronic because due to the surface aeration of that technology and we assess that the tool is very suitable for sheer stress sensitive and slow breathing cells.
17:36
And of course, because you do not change the hydrodynamic, it's quite an easy and straightforward scale up to 2500 litres.
17:48
That brings me to the end of my presentation.
