In bioprocess development, orbital-shaken bioreactors—shake flasks, spin tubes, and microtiter plates—are the unsung backbone powering industry decisions about production strains and media. Yet, when scientists attempt to scale up their textbook-perfect experiments, the results often unravel, leaving teams scrambling for answers.

This episode features Tibor Anderlei, a pioneer who transformed shake flask monitoring from a PhD curiosity into an essential industry tool. After co-founding AC Biotech and leading work at Kühner Shaker, Tibor now steers customer success, translating three decades of hands-on expertise into practical solutions for scientists battling the reproducibility and scale-up puzzle.

Key Topics Discussed

• Lab-scale bioprocesses often fail during scale-up because performance does not translate reliably to production conditions.
• Orbital shaken bioreactors such as shake flasks and microtiter plates are essential for early-stage screening but are frequently under-taught in bioprocess education.
• Reproducibility in experiments is often compromised because critical parameters like shaking diameter and filling volume are not properly reported or controlled.
• Oxygen transfer limitations are one of the primary root causes of failure when moving from small-scale to large-scale systems.
• Different small-scale cultivation systems such as flasks, tubes, and microtiter plates create significantly different aeration and shear environments.
• Operational variables such as shaking speed, vessel geometry, and filling volume strongly determine biological performance at small scale.
• Detailed tracking of small-scale process parameters can enable more predictive and reliable scale-up outcomes.
• Many scale-up failures occur because aeration and oxygen transfer conditions are not properly matched between laboratory and production systems.

Episode Highlights

• Why orbital shaken bioreactors are fundamental to successful bioprocess development [03:11]
• The gap between educational practices and real-world bioreactor expertise [04:00]
• Tibor Anderlei’s journey from the Technical University of Aachen to pioneering online monitoring technology in shake flasks [04:27]
• Reasons why published shake flask and microtiter plate experiments often fail to be reproduced in other labs [09:47]
• Key parameters frequently omitted from publications—including shaking diameter—and their impact on experiment reproducibility [13:10]
• Practical considerations for using microtiter plates and tubes, including automation compatibility and critical shaking speeds [14:13]
• Common scale-up failures due to oxygen limitation and mismatched aeration rates between small-scale and bioreactor systems [22:22]
• The effect of bioreactor geometry, such as neck shape, on process ventilation and performance [24:49]

In Their Words

Automation is a trend in the field of bioprocessing or screening. The advantage, I would say, for microtiter plates is that they follow more or less already a standardized format like the ANSI or SBS format. And a standardized format is very important for us as a supplier for automation equipment like our BELUGA machine or MPS-Z. Automated standardization is key.

Why Your Shake Flask Culture Doesn't Scale: OTR, Shaking Diameter, and How to Fix It - Part 1

David Brühlmann [00:00:30]:
Imagine building the perfect cell culture experiment. Clean data, reproducible results, textbook performance. Only to watch it completely fall apart the moment you scale up. If that scenario sounds familiar, you're not alone.

Today's guest has spent over three decades solving exactly that problem, from microtiter plates and spin tubes to shake flasks and beyond. Tibor Anderlei pioneered online monitoring in shake flasks during his PhD, co-founded AC Biotec, and now leads customer success at Kühner Shaker, offering specialized support in shaking cultivation technology. Welcome to Part one. Welcome, Tibor. It's great to have you on today.

Tibor Anderlei [00:02:31]:
Yeah, thank you. And Grüezi, David—Hello, David—and thank you very much for inviting me. I'm very pleased and excited to be here on your podcast.

David Brühlmann [00:02:42]:
It's a pleasure to have you on, Tibor. It's been a while—we've been talking about it and now we finally made it happen. So I'm really excited. It's like a good old Bordeaux wine—it takes some time to reach maturation, and then when you drink it, it's a good wine. That's the same with having a podcast interview. Tibor, share something that you believe about bioprocess development that most people disagree with.

Tibor Anderlei [00:03:11]:
I like this question, and I would say orbital shaken bioreactors like shake flasks, tubes, and microtiter plates are the most important bioreactors—maybe besides stirred bioreactors, as you know. They are very important because very fundamental decisions regarding production strains and media composition are made in these bioreactors during the screening phase. And in my opinion, also the information the user gets by using these orbital shaken bioreactors is so essential. However, these bioreactors are not treated like that. For example, the topic of cultivation conditions in orbital shaken bioreactors is not always part of education at universities and schools.

David Brühlmann [00:04:00]:
You are making an excellent point. I think I have not heard about these bioreactors during my studies—very spot on. Before we dive further into the technology, I'd like to talk about yourself first. Take us back to your PhD days and tell us what sparked your interest in bioprocessing, and what were some pivotal moments along your long career that led you to your current role at Kühner?

Tibor Anderlei [00:04:27]:
My career started at the Technical University of Aachen in Germany (RWTH Aachen University), where I studied biochemical engineering. Just after I finished my final exam, a new professor started there—his name was Jochen Büchs—and he set up a brand-new lab. This opportunity was very interesting: building up a lab together with a team, and of course working on his new research topic, which was small-scale cultivation.

Professor Büchs came from BASF SE to the university, and he also brought some older equipment from the company, as they had renewed their labs. One system was a prototype of an online measuring device for oxygen transfer rate in shake flasks. He had developed this system during his time in industry.

I can tell you—it was a prototype. It still used very expensive Ingold probes for oxygen sensors, originally designed for stainless steel bioreactors, and even an Atari computer for data acquisition. But for me, the opportunity to start my research with online monitoring equipment was very clear, so I took it.

I further developed the system—expanding the prototype by adding carbon dioxide transfer rate measurements, and of course implementing robust software together with an external company. Looking back, was it a revolutionary technology? At that time, there was essentially no online measuring tool available for shake flasks. So having an online signal already at this scale was very important—and in my opinion, a kind of revolution.

David Brühlmann [00:06:24]:
What made you realize that this technology you were working on could potentially revolutionize small-scale bioprocessing?

Tibor Anderlei [00:06:33]:
Was it a revolution? That is a real question. At that time, there was simply no online measuring tool on the market for shake flasks. So having an online signal already at that scale was very important—and I would say a kind of revolution.

During my PhD, while setting up the lab and working with a company on developing a commercial product, I always had the idea in mind to start a company myself.

At the Biotechnology Conference 2000 in Berlin, I met Simon Curvers. He was a PhD student from the Research Center Jülich, near Aachen, and he had the same idea. Over about a year, we had several meetings and discussions, and eventually founded AC Biotec.

We focused on contract research and contract manufacturing, with bioreactors up to 300 liters. We mainly worked with yeast cultivation, especially Pichia pastoris.
After about five years, I left the company because I realized we were too small for this kind of business, and we did not have our own product idea to attract investors. However, I remained connected to AC Biotec.

After that, I moved to Kühner Shaker. Since the AC Biotec team already had strong expertise in shaken cultures, we had organized shaking seminars together with Markus Kühner, the CEO of Kühner Shaker. I also knew him from earlier collaborations with Professor Büchs.

As early as 1999 in Basel and around 2000–2001 in New York, Kühner and Professor Büchs organized conferences on shake flask and microtiter plate applications. Markus Kühner had a clear vision: to support science not only by building reliable equipment, but also by providing strong scientific support to customers.

I joined Kühner Shaker in 2006, and I’ve now been with the company for about 20 years. I am the CSO, responsible for the customer interface—which includes sales, service, and support. Support in our case includes GMP topics, troubleshooting, marketing, and applied technology. And I think we will talk more about these 30 years of experience in small-scale cultivation.

David Brühlmann [00:09:29]:
Yeah, absolutely. Let's unpack the onion, because there is so much to talk about with this technology, and a lot of people are using shaking cultures in one way or another. So let's tackle the elephant in the room that frustrates many scientists: why do shake flask and microtiter plate experiments that work well in publications often fail when someone tries to reproduce them in their own lab?

Tibor Anderlei [00:09:59]:
I think that's really a nice and important question. And I would say that a lot can be explained by the lack of knowledge about these bioreactors. The reason for this is that cultivation conditions in orbital shaken bioreactors are not a major topic during the education phase, as I said before. This has luckily changed a little bit over the last years, but it's still far from optimal.

I can give you one example. There is a very nice publication from Gesa Brauneck. She is from the Technical University of Aachen, from the lab of Professor Magnus, who is the successor of Professor Büchs. She analyzed publications dealing with aerobic cultivations of E. coli for optimal production strains. She looked at the 15 most recent articles and gathered information regarding their cultivation conditions in shake flasks. And now David, I can ask you some questions—are you prepared for that?

David Brühlmann [00:11:11]:
Yes, you're putting me on the spot now. Let's do it.

Tibor Anderlei [00:11:14]:
Okay. So how many of the 15 most recent publications mentioned the media composition? You can give a number between 0 and 100%.

David Brühlmann [00:11:27]:
I would say media is important—I’d say at least 60% cited media composition.

Tibor Anderlei [00:11:35]:
It's even more—they are really good. It was 100%.

David Brühlmann [00:11:40]:
Oh, excellent.

Tibor Anderlei [00:11:41]:
And how many mentioned the cultivation temperature?

David Brühlmann [00:11:45]:
Well, if media is 100%, I would say it's close to 100%.

Tibor Anderlei [00:11:51]:
You are perfectly right. So now we come to more specific parameters regarding shake flasks. How many mentioned shaking speed?

David Brühlmann [00:12:02]:
I would say it's less than 50%.

Tibor Anderlei [00:12:04]:
Actually, it's higher—80%. And what about the filling volume—how much liquid they put into the shake flask?

David Brühlmann [00:12:13]:
Let’s say 70%.

Tibor Anderlei [00:12:14]:
You are close—65%. And the shake flask size—whether they used 125 mL, 250 mL, or 1 L flasks?

David Brühlmann [00:12:24]:
There I would say close to 100%.

Tibor Anderlei [00:12:27]:
No, it's only 55%. And last but not least—how many mentioned the shaking diameter?

David Brühlmann [00:12:35]:
Oh, I think this is a parameter not many people mention. I would say below 50%.

Tibor Anderlei [00:12:41]:
Now you are far above—it is actually 0%. And that is nothing new to me. I always say in seminars that more than 90% of publications do not mention the shaking diameter. This shows me that the shaking diameter seems to be not an important factor for many users. But I can tell you—it is as essential as, for example, the type of stirrer in a stirred bioreactor.

If you use a Rushton turbine or a pitched-blade impeller, everybody will mention that. But shaking diameter—you don’t see it. And one important point here: you can easily achieve 30% to 50% higher oxygen transfer by using a shaking diameter of 50 mm instead of 25 mm at the same shaking speed. So, coming back to your question—if you do not know the shaking diameter, you simply cannot reproduce the experiments from a publication.

David Brühlmann [00:13:42]:
Yes, this is an excellent point. So details matter—there is no doubt about that. Microtiter plates, tubes, and shake flasks—well, shake flasks have been around for a long time, but the others have become increasingly popular for high-throughput screening. But they come with their own set of challenges. So in addition to what you've just mentioned, Tibor, what other challenges do scientists need to watch out for in order to succeed with these experiments?

Tibor Anderlei [00:14:13]:
That question leads to a long answer. I would say of course you mentioned microtiter plates and tubes, so I would say both systems are quite popular in the moment. And why? Because they can be automated. Automation is a trend in the field of bioprocessing or screening. The advantage I would say for microtiter plates is that they follow more or less already a standardized format like the ANSI, SLAS or SBS format. And a standardized format is very important for us as a supplier for automation equipment like our Beluga machine or MPS-Z. Automated standardization is key.

The tubes are also very nice for automation because they can easily be used for cultivation step, for the shaking and cultivating, and for the first downstream process like separation because of their conical bottom shape. Also suppliers of centrifuges are used to this format and have long time experience with it and provide of course a lot of accessories.

The history of the tubes is also interesting because it started at the EPFL in Lausanne where Florian Maria Wurm used the 50 milliliter TPP tube with a membrane filter to cultivate cells and the tube in my opinion is a very good bioreactor for cell cultivation.

The filling volume is about 10–30 mL and the shaking speed depends on the shaking diameter. Again, shaking diameter is between 180 and 300 rpm in order to increase the oxygen transfer. The filling volume is about 10–30 mL and the shaking speed depending on the shaking diameter is about 180 and 300 rpm. In order to increase the oxygen transfer, some users tilt the tubes. But to be honest, I'm not a fan of it. Tilting is more or less like adding a baffle into the tube and that will create higher shear stress on the one hand and foam creation, and especially foam can on the one hand increase the oxygen transfer when you have a fluffy foam, or on the other hand it can decrease the oxygen transfer if you have a very sticky foam. And this leads, in my opinion, to unreproducible results which should be avoided during the screening phase. So I would suggest to shake the tubes always in vertical position.

Also, just a small side note, a few degrees more in tilting or less can have a huge impact on the fluid dynamics. I have read all the publications and posters on cell culture conferences showing very good results using the tube or this bioreactor to simulate even a perfusion process. The publication compared the results with runs in stirred bioreactors and got similar results.

Nowadays the tubes also get competition with for example a 6-well ultra-deep well plate, which also has a filling volume of about 30 milliliter. And now we come to the microtiter plate topic. So in general, microtiter plates have a very huge range, starting from 96-well plates up to 6-well ultra-deep well plates. In my opinion, mostly used are 96 and 24 deep-well plates.

And we can now discuss of course about different influencing factors like the lid, square or round well shape, or which bottom type is better: round, flat, conical. But to be honest, I also want to focus here more on the shaking speed and the shaking diameter. And here I think it is very important. First of all, there is a critical shaking speed you have to overcome to get good mixing in a microtiter plate. This critical shaking speed depends on the filling volume, on the media composition and on the shaking diameter.

The nice thing is that you can calculate this critical shaking speed which is presented by a friend of mine, Wouter A. Duetz, and you can also find the step-by-step calculation in our science room on our website. I think you will also have that in your show notes. The reason for this critical shaking speed is mainly that you need certain centrifugal force to break the surface tension of the liquid.

For example, shaking a microtiter plate with 300 rpm and a relatively small shaking diameter of 12.5 millimeter. So the reason for the critical shaking speed is mainly that you need a certain centrifugal force to break the surface tension of your liquid. And for example, if you have a microtiter plate, a 96-well plate, and you shake it with 300 rpm and 12.5 millimeter shaking diameter, or you let it sit on the clean bench without shaking, the effect will be the same. The effect will be that you have no mixing at all. So at least here you have to shake, for example, 450 rpm to get a mixing effect in the 96-well plate.

So I think this is very important to know that there is a critical shaking speed. And very often I hear the statement small vessels need small shaking diameter. This is, in my opinion, not correct. In the microbial world where high oxygen transfer rates are essential, we suggest to have filling volume lower than 30% of the nominal well volume of a microtiter plate and you shake it with 25 or 50 millimeter, 300 to 400 rpm, more or less standard.

I would say in the world of cell culture this is different. Here the respiration activity of the cells is much lower, I would say 50 to 60 times lower than a yeast cell. And you can fill up the well of a 96 deep-well plate up to 800 microliter or even 1 milliliter. And in this case, and only in my opinion, in this case, you need a shaking diameter of 3 millimeter and a shaking speed of 1000 rpm to get good mixing. And when you reduce the liquid volume again below 800 microliter, then 25 millimeter and 400 rpm is already sufficient, I think.

Now I give you a lot of numbers, I know, but I hope these examples could show you how important the knowledge on the level of small scale system is.

David Brühlmann [00:21:20]:
Let me pause here for a moment because you’ve delivered a ton of valuable information. Just to get our heads around that—because it’s very important to get this right. I’ve done a lot of experiments using this technology, and what you’ve just said, Tibor, is absolutely essential.

Small-scale shaken systems are great. And if you do them well—if you get all these detailed parameters right—they can be a very powerful system because you can test many different conditions in a very short time.

Now this leads me to the next question. If you do this homework and look at all these parameters in such a detailed way, in most cases you can use the data you’re generating and hopefully even predict behavior at larger scale. But I’d be curious—because you speak to a lot of companies and have seen a lot in the industry—what were some of the most spectacular scale-up failures, and why did they fail?

Tibor Anderlei [00:22:22]:
I would say the most common scale-up issue is that users perform their shake flask screening under oxygen-limited conditions. This is typically due to using too high filling volumes and too low shaking speeds.

Screening under these conditions will lead to issues during scale-up. When the system is transferred into a lab-scale stirred bioreactor, oxygen limitation is often no longer present. As a result, the strain behaves differently—ranging from producing no product to producing more product.

Let me give you another example. I had a client developing a bioprocess in shake flasks. Let’s say he observed 100% of his product yield at shake flask scale. However, when scaling up to a stirred bioreactor, he observed only about 70% of the product yield, which was of course disappointing. He contacted me because he knew I could calculate the aeration rate in shake flasks—that is, how much air is actually transferred through the system. I calculated the aeration rate for his setup, and it was approximately 0.5 vvm (volumes of gas per volume of liquid per minute). Typically, users do not know the aeration rate in shake flasks. In microbial processes, they often set bioreactors to 1 vvm as a standard. That is exactly what he had done as well.

My suggestion was to reduce the aeration rate in the stirred bioreactor to 0.5 vvm to match the shake flask conditions. As a result, he recovered more than 90% of his product in the stirred bioreactor. What I want to emphasize is that, in addition to oxygen transfer rate (OTR), mixing time, and power input, ventilation is also a critical scale-up factor. Ventilation relates to the removal of volatile compounds such as CO₂ or ethanol from the system.

For example, the aeration rate in a shake flask can be significantly affected by vessel geometry. A 250 mL flask with a wide neck can have roughly twice the aeration rate compared to a 250 mL narrow-neck flask. This shows that even small design changes can have a major impact on process performance.

David Brühlmann [00:25:07]:
This wraps up part one of our conversation so far. Tibor Anderlei has unpacked why small-scale experiments fail to translate, how mass transfer dynamics differ across platforms, and how oxygen transfer rate and CO₂ transfer rate monitoring reveal limitations that dissolved oxygen alone cannot capture. These are not just academic distinctions—they are the difference between a process that scales and one that doesn’t. In part two, we go deeper…

For additional bioprocessing tips, visit us at www.smartbiotechscientist.com. Stay tuned for more inspiring biotech insights in the next episode. Until then, let’s continue to smarten up biotech.

Disclaimer: This transcript was generated with the assistance of artificial intelligence. While efforts have been made to ensure accuracy, it may contain errors, omissions, or misinterpretations. The text has been lightly edited and optimized for readability and flow. Please do not rely on it as a verbatim record.

Next Step

Book a free consultation to help you get started on any questions you may have about bioprocess development: https://bruehlmann-consulting.com/call

About Tibor Anderlei

Tibor Anderlei is a bioprocess technology expert with over 30 years of experience in small-scale cultivation systems. He specializes in orbital shaken bioreactors and scale-up strategies, supporting biopharma development from microtiter plates and shake flasks to stirred tank systems. His work bridges early-stage screening with industrial bioprocess implementation, with a focus on improving reproducibility and scale-up reliability.

At Kühner Shaker, he is focused on ensuring customer success and satisfaction through scientific and equipment solutions.

Connect with Tibor Anderlei on LinkedIn.

Further Listening

If you’re interested in this topic, check out these episodes on building a robust scale-up strategy. To get it right, you need to view the process from multiple angles—regulatory, digital, and operational.

Episode 03 - 04: How to Master Biotech Scale-up Without Guesswork with Leonardo Sibilio

Episode 25 - 26: 9 Critical Steps for a Seamless Transition to Large-Scale Production

Episode 231-232: From IND to BLA: The Biologics CMC Decisions That Determine Regulatory Success with Henri Kornmann

Episode 233-234: Why Most Bioprocess Automation Projects Fail with Anthony Catacchio

Episode 237-238: High-Throughput Microbial Screening with Sebastian Blum

Smart Biotech Scientist Toolkit

Below, you’ll find a curated collection of resources, technical guides, and regulatory links shared by our guest.

• Shaking Technology Forum, supported by Kühner Shaker.
• Shaking Technology LinkedIn Group: for users, researcher & biotechnology students
• Kühner Seminars and Trainings
• OTR calculator: Determine the maximum oxygen supply

He is also a biotech technology innovation coach, technology transfer leader, and host of the Smart Biotech Scientist podcast—the go-to podcast for biotech scientists who want to master biopharma CMC development and biomanufacturing.  

Hear It From The Horse’s Mouth

Want to listen to the full interview? Go to Smart Biotech Scientist Podcast. 

Want to hear more? Do visit the podcast page and check out other episodes. 
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