Ribbon structure of an Adeno-associated Virus (AAV) |
On the scientist side of CPI there were three main departments; Upstream, Downstream and Analytical. Simply put, upstream cultures cells to produce specific molecules or proteins, downstream get given the "soup" of everything those cells have produced and are tasked with extraction and purification of a single type of protein and Analytical then test rigorously to make sure that the purity and correct protein or molecule has been collected. In my week I spent time with al three departments, but the main area of the work I was doing was downstream.
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington An industrial scale ion exchange chromatography column |
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington Gold Nanoparticle solution.. different to what I expected. |
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington Centrifuge make sure to balance it out! |
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington JANUS Liquid handler |
(hopefully) GNPs bound to insulin were collected at the bottom and then the robotic liquid handler removed the superb start without disturbing the Pellet at the bottom. They then conducted a BCA protein Assay (bicinchoninic acid assay) was used on the supernatant to assess how much of the insulin was left to then work out how much was bound to the GNPs on the pellet at the bottom of the well. The BCA assay turned green- apparently due to Cu²⁺ and ³⁺ ions- if not much protein was present and a deep purple if there was plenty of protein- you can quantitatively use this data if you enter the plate into a colourimeter and use 562nm green light. They explained to me the importance of balancing out the centrifuge, because if you don't it will apparently begin hopping around the room at high speeds! As shown by the image, some results with hardly any protein in the solution were obtained, so were successful.
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington Bicinchoninic Acid assay (BCA) 1 min after assay |
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington End result of the BCA assay |
Reverse osmosis seen through a scanning electron microscope |
I was introduced to another method of Protein assay, 280 nanometer Absorbance. To demonstrate this, one of the downstream scientists and I made up different concentrations of BSA (Bovine serum Albumin- aka Fraction V) from crystalline powdered, pure protein with water. This test is comprised of a small machine that looks like a miniature George Foreman grill, with tiny metal studs with pores of lights on the base of it, onto which a fraction of a microlitre of the Bovine Albumin serum is pipetted. In every protein there will be some amino acids with an aromatic side chain (such as proline, tyrosine or tryptophan) and these aromatic side chains happen to be very good at absorbing Ultraviolet light at a particular peak of 280nm, so the UV light is shone from the piece of equipment and can be observed in different intensities as it shines through the droplet and is detected on the other side when the lid is closed. Some computer programmes can map this out and tell you the exact concentration of proteins left the drop. The only drawback this method has is that the entire solution needs to be homogenised before pipetting onto the 280nm machine because otherwise you may get a particularly concentrated or weak part of the solution. There is also a coefficient we had to divide our results by which was specific to BSA, but we found that our results were spot on the the concentrations we theoretically had made with a percentage difference of around 5%. The limitations of 280nm absorbance are that it is very sensitive to non-protein matter, for example cell organelles will absorb almost all of the UV light, or even just a mixture of proteins since each has their own specific coefficients (which I assume depends on the quantity of aromatic rings present in the protein structure?).
Protein model of one of the serotypes of AAV |
Next the Analytical department took me on a quick tour of their domain. I never knew there were so many different types of mass spectroscopy- gas chromatography (GCMS), Liquid chromatography (LCMS), and high performance- HPLCMS, even CEMS- capillary electrophoresis. I had not heard of electrophoresis before I saw this machine, so I have done a bit of research around it as I'm sure it will be something which I will be doing at some point in the future. Electrophoresis is defined as a way to separate macromolecules by putting an electric field through a gel. It can be used on proteins, RNA
and DNA, and usually involves a small gel filled chamber with wires attached at either end to create a difference in charge between each side of the chamber, pulling the molecules towards one end at different rates. the faster the rate the further it gets towards the positive terminal and so you can determine the approximate mass of the molecule band seen. Another curiosity I found when researching this type of analytical technique was the unit of mass used, the Dalton (Da). 1 Da is equal to 1 Au or 1/12th the mass of C-12, so the mass of Hydrogen is 1.0007 Da and carbon is 12 Da. Most proteins and DNA are measured in kDa (kiloDaltons). Titin, one of the biggest proteins ever found is 3.86 million Daltons, and around 30,000 amino acids long. so this means the rough mass of an average amino acid in this protein is about 128 Da- a useful conversion if you know the mass of any protein: Mass of protein in Da divided by 128 = approximate number of amino acids. (assuming that all amino acids are in similar ratios to how they are found in titin)
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington Me, all kitted up and pipetting into Eppendorf tubes |
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington JANUS robotic liquid handler |
Centre for Process Innovation's National Biologics Manufacturing Centre Darlington Programming the liquid handler to carry out a set of instructions for dilution |
The suggestion after I showed my findings was that an air blow step was to be added into the instruction commands for the robot when using the disposable tips, in which a small amount of air would be blast through the end of the tip to pop any bubble left and add to the concentration.
Through my entire week at CPI I learned so many new skills and ideas beyond those taught at school which will no doubt be incredibly useful as I continue my scientific studies at university. I can only thank them for giving me the opportunity to do this. It was a great week and CPI is definitely somewhere I could see myself being employed in the future...