A most interesting week in the biological environment of Lonza AG
Lucas Wittwer was one of the winners of the 43th National Competition of "Schweizer Jugend forscht". (Picture: Jan Mühlethaler)
With sleepy eyes I just saw the disappearing ground before it is covered by clouds. It was 7 o'clock and I was on the way to London Heathrow. I landed after a comfortable flight and was fetched by a taxi driver which drove me to the Babraham Research Center outside of Cambridge. It was 9 o'clock and my first full packed day just started.
The first two days I spent at Cambridge with the team around Jesus Zurdo. After a small crash course about the Lonza AG they explained what they are doing here exactly. They test medicinal proteins of customer on their aggregation behavior and optimize them. This is a really important step of a medical protein until it can be used, because the less they aggregate the better the drug works.
The rest of the day I spent in all the parts of the lab so I saw all the steps in the antibody construction process. I was really impressed by all the modern equipment in the lab. I started in the cell culture lab. First we needed to count some cell cultures so we could prepare the transfection of a new antibody-DNA. Different from my final work about gendoping at the gymnasium, they use CHO-cells and not the 293T-cells I've used. Because they work with clients’ products I wasn't allowed to do the transfection. So I didn’t get bored, they prepared some cell cultures and I was allowed to transfect with water. To transfect cells with water is useless but it was great for refreshing my memory.
The second part of the day I spent in the purification lab. Most of the purification processes are automatic so there was a lot of time for theoretical information about the methods they use. With the A protein chromatography you can selectively abstract a protein from all proteins and cellular components. All you need to do is to fill the cell mixture in a column and spin it down. Your target protein bonds to the column membrane, which is laced with the A protein. All other proteins and cellular components just flush out of the column. Then you can change the buffer and the target proteins disconnect from the membrane. The result is a high concentration medium with your protein.
One of the team members took me around after work. He showed me all well known places in Cambridge. After a traditional dinner (well, it was Italian food but England is international) I went to bed, really tired.
The second day was reserved for all the bioinformatics stuff in the lab. This day was the affirmation for my study goals. Most of the proteins optimization work is based on bioinformatics. I started with a small scientific game called “foldit”. You can learn how to optimize a protein and what problems you get. Actually this “game” was created to find out how complex proteins are folded.
After this introduction I had a look at the right software they use. Calculation of the structure of a protein and rendering an image of the structure are hardware requirements, so they need the support of a big server. First we wrote a plug-in script for an automatic translation between two different notations of the strong variable parts of an antibody. I didn’t know the programming language so I wasn't a big help. It was interesting to see how easy it is to customize this amazing software for your wishes and how fast it can handle the big amount of data. After some positive tests of the script we switched to manipulate a protein. First I had to search some regions in the protein by finding the right DNA sequence. Then the assistant explained how I can view the region on the 3D-view of the protein. Further he explained how he can now change some regions so they can not connect with a second protein but he needed to keep the structure of the protein.
In the evening a taxi picked me up and drove me to the Christopher Hotel in Eton near Windsor Castle; a sweet small hotel with nice small rooms, just perfect for me. I spent the evening in Eton discovering the College of Eton and Windsor Castle.
The next three days I spent in Lonza Biologics plc. This site of Lonza AG is one of the biggest protein manufacturing labs in the world. They produce big amounts of proteins for customers. The customer just needs to give them his plasmid or DNA of the protein he wants. All further steps to the product, the proteins, are done by Lonza AG. Like in Cambridge they arranged my program around the production steps of the protein so I started in the microbiology department. Lonza has its own expression system, called GS-expression system. A normal mammalian cell needs Glutamine otherwise it isn't able to grow. In a cell culture lab you have this Glutamine in the growth medium. CHO-cells cannot produce enough glutamine from glutamate so they wouldn't grow. Cells which have assimilated the plasmid (with the DNA of the protein and the GS sequence) can produce enough glutamine from glutamate to survive. That's a selective marker, because every growing cell has assimilated the plasmid and produces enough glutamine. I could assist by a digest of a plasmid but something with the result by the gel electrophoresis was looking strange so the assistant had to repeat it.
The second step is to transfect the cells and after a while you have to select the cells which have the biggest production rate. This is done in small labs (there are lots of them). There is one fully equipped lab for every project so no samples from different customers can be mixed. I gave a hand to prepare some cell counts and measured the sugar content of the different samples. It has a full automatic robot which does the same but only in small amounts. The most productive cell will be selected and increased, first in small bottles and then in bigger bioreactors. These bioreactors are fully controlled, the concentration of all medium components, the temperature and how fast the mixer rotates. They take samples during this step and analyze the protein. Because that's the only way to be sure that it is the right protein it's really important. Sometimes they have two samples from the same protein in two bioreactors so you need to compare them. The analytical machines are really sensitive so you need to calibrate them. This process takes a lot of time, we needed more then half a day, and at the end the results were strange so it was clear that our work was not exact enough during the calibrating. In the meantime we started some deglycosylations and some theoretical approach of the imaginary results. It isn't complicated but with my English background a bit hard to understand but after some illustrations I've got it.
When the proteins are correct and everything was going on the right way the cells come into the big reactors. That means they grow in a reactor with a volume of 2'000 l. As with the small bioreactors everything is changeable and highly monitored even here.
I was pleased have a tour through this part of Lonza Biologics plc. Everything is clean and the rules are really strict. You are not allowed to wear normal dress and in some parts you need to wear something over the work dress. It is important because if something from outside finds its way in these reactors it could destroy or modify the results and this is - in this huge volume - not very pleasant. It was interesting to see how big everything is. You don't need just these big reactors but also the place for producing the medium solution and the buffers.
The last step is to isolate the proteins. This is in done in the purification department of Lonza Biologics plc. Depending on the protein they use different purification method to wash out. From 200 l medium they get something about 300g proteins! I produced some buffer for an A protein chromatography they needed the following week. Simultaneously I learned one more difference between a research institute and biomanufacturing plant. In the biomanufacturing plant you have to document everything in, every nut, bolt and screw. For every buffer you produce you need to write down the date, every bottle number you used, from which flask you have taken the substance X, what the temperature of the water was and so on. At the end you have to sign the form. In a research area you just have to note what you are doing.
Beside this official part of the practical work they had arranged everything for me. The assistants who have shown me what they are doing took me out in the evenings, showed me Eton, Windsor and once Slough and organized the dinner. All in all it was really an interesting week, I have learned a lot and it was my first experience in a biomanufacturing environment. Also the part about bioinformatics was perfect, it affirmed my future plans.
I'm really grateful to Lonza AG and staff, SimplyScience and SJF who made this week possible for me.
Source: Lucas Wittwer and SJf