This evening I was waiting at the airport to catch a flight home and a dangerous thing happened; I started to think. I was thinking whether I could set myself up as a therapeutic antibody supplier, as I discussed in a previous blog. The revenues generated by such biotherapeutics are quite considerable and at the bottom of my garden I do have a sturdy brick-built shed that I had considered turning in to a microbrewery – probably the last time I did some thinking. A microbrewery would have involved some large steel fermenters, much like an antibody bioreactor, and as I had learnt on my Immunology Masters course some years previously how to make monoclonal antibodies, this plan was definitely ON! So how do you produce therapeutic antibodies on a reasonable scale?
The Therapeutic Antibody Production Workflow
What’s my target – first thing first, what disease am I going to treat and what disease specific antigen or pathway is my antibody going to target? Options are to look for disease biomarkers through a proteomics and/or genomics approach, or search the literature for potential targets. I go for the latter to save time.
Engineer my antibody – now I have to use genetic engineering to design an antibody specific for my chosen target. I’ll probably end up making a large number of antibodies and then selecting the one which has the best binding kinetics.
Making the cell line – a cellular factory is now required to make the antibody in the quantities I desire. My antibody’s genetic blueprint is transfected in to a suitable cell line such as CHO (Chinese Hamster Ovary) cells which will then transcribe (or produce) my antibody. The cells are placed in culture media and start to replicate and produce antibodies which are excreted in to the culture media.
Scaling-up – this is where my steel fermenter, or bioreactor, comes in. The cells in their culture media are placed in larger and larger vessels to increase their volume and subsequently the amount of my precious antibody.
Harvesting – my CHO cells have done their work and antibody production has slowed so now it’s time to harvest my crop. The antibodies in the culture media have to be extracted from the fermenter or bioreactor, the cells removed and the antibodies concentrated.
Purification – even with the best extraction and concentration, there will always be more than just your antibody remaining, so now it needs purification by chromatography. This is a multi-step process, typically starting with an initial capture step by affinity chromatography with Protein A and followed by a couple of polishing steps to increase purity, typically using ion-exchange and mixed-mode chromatography.
Formulation and packaging – the final step is to formulate your antibody into the final composition it will be given in, most likely a sub-cutaneous injection, so you have to put it in a syringe as well.
I forgot to say that the process from step 2 has to be done in completely sterile conditions to prevent anything unwelcome contaminating your final product (culture media is very nutritious!). Note to self, better remember to give the shed a good clean before I start.
Analytics, Analytics, Analytics
Now these CHO cells are a little bit fussy and rebellious. If the culture media is not quite the right temperature, pH, wrong oxygen concentration, food not equally distributed, etc., then they are not happy and are likely to protest by making the odd change or mistake in their production of the antibody. With billions of them in the bioreactor the chances of a complex antibody molecule being produced exactly correctly and the same by all over time is remote, so you are going to need analytics.
The analytics are there to ensure that the antibody is produced exactly the same each time, by looking at such parameters as ‘Is the amino acid sequence of my antibody the same?’, ‘Is my glycosylation pattern maintained?’, ‘Has the antibody formed dimers?’ and much more. These analytics are performed using UHPLC and mass spectrometry, so I must make space for these in the shed.
The other side of analytics is making sure that nothing is in your final product that shouldn’t be. There is the potential for components that have come into contact with the cell culture or final antibody to leach chemical entities into your product, a rapidly growing area of concern known as Extractables and Leachables. Your CHO cells may leave contaminants known as Host Cell Proteins (HCPs) or no matter how sterile you have kept your culture, bacteria or viruses may have entered or be present already in your cell line.
Analytics have to be conducted at every stage and are there to ensure the safety and efficacy of the final product and that your production is proceeding as planned.
What’s Stopping Me?
I know how to produce my therapeutic antibody and know what analytics I need to perform so what’s stopping me producing antibodies in my garden shed? Well nothing technically to stop me producing them on a small scale with a few carefully considered equipment purchases, but the chances of a regulator letting me get one of my garden shed antibodies (I call them ‘shedibodies’) anywhere near a patient is zero – I don’t have the facilities to produce a clean and safe antibody at a sufficient scale or the analytical instrumentation for complete characterisation.
It was a nice thought while it lasted; perhaps that microbrewery wasn’t such a bad idea after all. Ah, coming in to land at London Heathrow...