 I consider myself a lucky man and I think, say, thank yous before my talk. I had the opportunity to work together, learn from brilliant people in my career, all over Germany to the US and then coming back to Switzerland. First of all, I think, Professor Jean-Claude Badou, who brought me from US to Lausanne, gave a total ignorant in teaching the chance to talk to students, which was very hard. And then, of course, I met later a wonderful wife, Maria, she's sitting there, you will see her with whom I learned a lot and finally also started this company, Excel Jane. Now this talk covers a lot of years, you will see that. And I have to go back actually here, yeah. This question I'm almost embarrassed to ask you. It has been mentioned here several times. So I say it right away, of course, it's Hela cells, which taught us how to grow cells in a reproducible way and the gentleman behind it was Dr. Gay. Now when you look up show, Chinese-Hambster ovary cells, you'll find here a definition in Wikipedia, we talk about epithelial cells, which is of course not true when you look at them today. But what is quite remarkable when you do Google the two words, Hela and show, it's amazing why so many people talk about show. And I give you the reasons for that. In 1976, two gentlemen, a young man, a venture capitalist, Bob Swanson, and a very famous professor already at the time, her boy was behind the patents cloning genes, by the way, and gave a lot of money to the University of San Francisco due to license fees, said okay, let's start a company. Two years later, that company had cloned human insulin, and another year later human growth hormone and was making money. Audrey and I visited them, there we are, and of course, I'm surprised now how old I look there in comparison to this young Bob Swanson, show cells. They created with show cells a biotech revolution. There's no doubt about it, and I make you believe me when you look at the next slide. Ten of the 15 best selling drugs of the world in 2018, it's an older slide, were made in show cells. 2022, that's out of a recent publication, $250 billion worth of products are made in show cells. Why show? Well, we look now at the proteins which were involved. Insulin is easy. We know it pretty well, but when we look at this protein on the right side, which was actually the very first one, making it to the clinic out of show, much more complicated molecule, which was clear from the beginning cannot be made in bacteria. Now, when thinking about a manufacturing principle, people had to ask the question, what do we know? And one key question which I was involved with was safety. Can you use an mammalian cell safely to make a product which goes into millions of people as a therapy? So these topics up there, tumorigenicity, pathogenic human viruses and so on, were very much in the forefront of letting us do it because in hamsters or hamster cells, very few of those grow. There was a body of literature available already at the time which said, yes, you can use show cells for putting genes into them. The issues of scale up and growth were a little bit murky at the time, but we get to that. I'll give you a short history of the early parts of this knowledge because it is necessary to make you fully understand. Let me go back here. A key in this was Theodora Puk, which created an entire discipline in biological science which we call somatic cell genetics. And he knew, of course, halo cells. They were much more popular at the time already in culture. But he said, no, I don't want to work with halo cells. And he had a good reason for that. He had questions like these on the right side. And of course, most famous for today is because he created show cells. It took essentially a hamster. But why a hamster? Well, again, there was already a body of literature from the 1950s and earlier about hamsters being a useful model as a lab animal. You see here, rabies, virus influenza, virus 1936, 1940. So there was a body of literature on hamsters. And you knew, of course, people in the world who were growing hamsters and said, I want those to look at chromosomes. This is a hamster-carrier type. We had that word today already. And you look at it and say, hmm, that's not too complicated. Only 11 pairs of chromosomes, on top of it, they're big. We can identify each one of them. So Theodor was thinking, wouldn't it be nice to have a reproducible source of a mammalian host system? Halo? It's a genetic mess. You look at this, triploid, 60 chromosomes, small, difficult to differentiate, to even analyze. So he took 1957.1 gram of an ovary, threw it in a bottle of glass, probably, didn't have plastic bottles at the time, threw probably a lot of serum on it, 20% or so, and observed. And they grew. Nine months later, 10 months later, they had changed the morphology a little bit. And he had a cell line which grew. Now, the genetics looks a little bit different. We come to that. He gave, he was a generous man, obviously. It gave these cells to half of the world. The cells went from Denver to Los Alamos to Toronto to New York. And all these people were involved in these early phases let us understand how chromosomes work, how genes are correlating with morphological changes, or metabolic activities, and so on. And Dr. Chasing, Larry Chasing, whom I met personally, he's still active, made out of a chose cell line he got from Pook, one which is DHF minus, which has a deficiency in DHF reductase. That was a key, because in the moment he had done that, other people said, oh, I want to repair that. I want to repair this deficiency. You can grow DHF minus cells when you add a few components into the medium. So the technology to do this was available as well and public knowledge. So take a DHFR plasmid, take another one with a gene of interest, throw it on, selective medium, wait for a little bit, and you have colonies coming up in two weeks. Now I came into this game, if you want, in 1985 and looked at cells after transfection. I had already transfected cells a little bit before, and so I could show here that yes, they go into the chromosome, and you have two chromatids, you have two genes, facefully inherited from one cell to the other, no problem. Now there was another technique which is called gene amplification, which we used also to push expression levels higher, and since I said that before, I worked with a brilliant team around. A genetic Dan Capon is most famous for the first chimeric molecule ever made by mankind, a CD4FC fusion, and since I was part of the team, I'm honored to say that picture was on the title page of Nature. Now, when we compare the hamster chromosomes with Chinese hamster ovary cell chromosomes, this was done already here in Lausanne. You might recognize some similarities here and there, but only here and there. The majority of this is a mess to be clear, a clear mess. So how can we make products out of cells which look so strange? And you look at another metaphase spread and that looks different as well. And it's a clonal cell line. You take 10 clonal cell lines, 10 cells out of it, and you see 10 different pictures. Wow. How can you make products in a cell host like that? Believe me, I and a lot of my colleagues and friends, including Maria, have thought about it a lot and so we published a few papers, which we actually have here. If you're interested, I can share them with you. Cloning of Chosel's productivity and genetic stability discussion, blah, blah, blah, and so on. So it tells you in principle, yes, we can handle it, but it's not easy. Now developing a producer cell line is a long step process. It can take nine months, 10 months, and more. The early part of this is easy. Transfect cells pick many clones, hundreds of clones, maybe 200, maybe 500, maybe 1,000, and find the one which matches your expectation in growth, in productivity, in overall robustness to go eventually into the bioreactor because that's what you need. You need to get a master bank established, which is hopefully stable and maintains productivity over many months, which we test for, of course. And once you have that, you can take a vial, put it in a small bottle, in a bigger bottle, and so on and so on, scale up. At the time when we did this the first time, we had to go from a 50-liter to a 200-liter, from a 200-liter to a, and so on, because sub-cultivation was not possible for one to 20 or one to 50 sub-cultivation ratios. Today we can do this, what I show here. But still, scale up is not easy. I think you believe me that. We were successful in the end to grow cell cells in a 10,000-liter reactor, and we were record holders in yield, 50 milligrams per liter. We needed six 10,000-liter reactors, run around the year to make 50 kilograms of TPA for thrombolytic therapy. Alte Plaza was the name. Just show you a quick picture. This is the top of a bioreactor, goes three levels down, has impellers and spargers and all this other chemical engineering stuff which I learned eventually also some words about. Today we have what we call disposable single-use bioreactors from very small scale to larger scales, but still the dominant products on the market are still produced today in stainless steel bioreactors, large scale. One more word about manufacturing. There is what we call upstream and downstream. Upstream takes about two weeks, three weeks, and then you go into purification which can take another two weeks, but look at the litter indication. 200-liter upstream, 2,000-liter downstream because we produce products at a purity level of 99% and higher. There is nothing in there anymore which is a tough job, I tell you. So anybody who thinks about manufacturing of products in animal cells has to think about where do I get the water from? It's expensive, it's water for injection. I will give you a few more words about technology, how to get to good cell lines producing a lot of product. This is one clonal cell line, clonally derived cell line. It's not a clone, clonally derived cell line exposed to 150 different media formulations. Again, we have to remember the genetic of these clones is different. The next clone would have a different pattern of behavior so it keeps us by the way busy. So some cells grow to 20 million cells per minute and the other only to five million. We want more cells because when we compare productivity and biomass in a reactor from 1986 to 2023, not only did we improve the productivity from then 50 milligrams per liter to now five to 14 grams per liter, we also increase the biomass in the reactor over longer periods of time. That's the key to progress in the industry. We don't publish nature on papers on that, unfortunately, which was my problem with the professor who followed Professor Badu because he wanted me to publish in nature and science all the time. But this is not sexy enough for that journal. Now, I look back at the five decades of what I've observed now and I give you here a summary, yields 1980s, cell density three million cells per milliter. 1990s we got to five and 500 milligrams per liter and so on, one gram per liter, one to three. Now we're in the three to 20 gram per liter and we go to 50. We need to get more cells, which means we also need to understand the biology in the reactor better. We are still not there. It's still a black box, still today. With our little company, we call ourselves a science-oriented company. We do manufacturing sciences. We look at all these parameters, from plasmid to cells, to how we get DNA into cells, the bioreactors, what type of bioreactors do we use? Bioreactors are very threatening to cells. They kill a lot of cells. How do we control pH? How do we provide oxygen, by the way, pure oxygen in steel tanks? We cannot do it with air. There is not enough oxygen in air. The driving force with a slow impeller in the reactor will not get enough into it. Big problem, by the way. Media compositions and ingredients and, of course, the biology of cells in a bioreactor. The title of my talk contains the word tons. And so, I was pretty bored when I came up with this title because I was not so sure, but some of my colleagues and friends who helped me with the calculation. We took blockbuster drugs. These are so-called drugs which are selling more than $1 billion a year. That's public information. We also know how much, what the dose is of such a drug. So we can essentially do a calculation. Sales price divided by price of the dose, we come up to the number of doses. And since the dose itself is public information, you do a simple multiplication, you get a number for accepting. Okay? Now, I consider all these companies mentioned here and came up with a number, a very cautious number. Another approach was, I know a few large-scale bioreactors and I know what kind of yield they have. So I estimated the number of stainless steel and other bioreactors in the industry assumed a titer of only one gram per liter or two grams per liter. We're now at five to 10, as I said before. So with a total volume assumed over the years, I can come up the number. And surprisingly, the numbers of the two matches pretty close. Actually, it's 0.1 gram, not one gram, of the hamster ovary is converted into 400,000 elephants over the history of this industry. 3,600 tons of show sales produce greater than 250 tons of proteins. And again, I emphasize it could be five times more. It's a very conservative estimation. And with this, I hope you enjoyed my short introduction to the history of show sales and I'm open for questions. Questions? Thank you for the nice overview of the show sale. I come closer, my hearing is not anymore as it used to be. I have a question revolving around the media that you use for all of these show sales for production of this. Media question, very good question. Actually, it was mentioned before, how important it is that the media we use in our manufacturing processes are all chemically defined. None of them since more than 30 years, I can say, we use fiddle calf serum or any animal component in our media, which improves reproducibility enormously, obviously, also reduces contaminant in the flow streams of product. We just don't need it. And with this media, we can get to densities of 30, 40, 50 million cells per milliliter which is key to our high productivity. Okay? Thinking probably, this is a very silly question, but thinking out loud when you have these giant bioreactors, how do you deal with the waste? And is it possible to kind of create the fake liver, fake kidney? Yeah, waste removal out of manufacturing plants for pharmaceuticals is a big issue. Obviously, we have still to consider the risk that something can jump into cultures and it has happened. There was a minus, what's it called? Minute virus of the mouse, MVM, once infecting our cell cells at the 10,000 liter reactor. You have probably the largest quantity of virus ever in mankind's existence in one location. So yes, all fluids go through serious killing, no matter what. Now, it costs money. You know, this is the part where I could argue why some of our drugs are so expensive. But the majority of the money actually goes into the development. Before we have a drug, we have spent somewhere between 50 and 100 million dollars. And of course, some drugs never make it. So there's another commercial issue. But yes, downswing processing after harvest of the product includes fluid handling.