Once you have decided on the optimal conditions for producing your recombinant protein target in baculovirus-infected insect cells, your thoughts may turn to scaling up its production prior to purification. You may be fortunate enough that the yield of recombinant protein is very high and your requirements fairly modest that only a small volume of virus-infected cells is required. On the other hand, you may have lofty ambitions for your protein target and you need to produce many litres of starting material prior to extraction and purification. In this blog, we discuss some simple options for achieving the scale of protein production you need for your particular project.
Firstly, it is worth reiterating the value in thoroughly optimizing recombinant protein production in baculovirus-infected cells. We addressed this issue in the last blog we posted. Secondly, you need to scale up protein production using essentially the same methodology as you used in the optimization experiments. This means that if you optimized protein yields using monolayer cell culture it is unwise to assume that the same conditions will apply in a suspension cell culture. Optimize in a suspension cell culture to mimic your scale up conditions. It can be argued that even moving from a 20ml suspension cell culture to a 1 litre suspension is not reproducing conditions exactly, but very few of us could afford to optimize protein yields at the volumes we intend to use in our scale up process!
The most likely equipment you will use for scaling up protein production will involve the use of shaking suspension cultures. These can range in volume from 20mls to over 1 litre. You can incubate the cells in a standard orbital shaker set at 28°C. The rotation speed (revolutions per minute [rpm]) is determined by the radius of the circular motion – the lower the radius, the higher the rpm required. Consult the manufacturer for advice. However, if your shaker/incubator is very old and you can’t get any guidelines drop us a line (firstname.lastname@example.org) and we will try to help out where we can. If your incubator has been used previously for bacterial (or worse, yeast) work then you will need to give it a thorough clean before setting up your insect cell cultures. There is no reason why different cell types can’t coexist, but it is better if they are kept out of the same flask!
If you haven’t got a shaker/incubator then you can use a very simple laboratory shaker set up in a warm room at 28°C. This may cause problems as warm rooms are generally set at 37°C, which insect cells don’t like – they tend to go into a heat shock state where virus replication is compromised. Your colleagues working with mammalian cells probably won’t like you messing around the temperature, so you will have to be creative. If you can find a store cupboard, clear out the old junk and add a simple fan heater. With a bit of patience, you can regulate the temperature of the room to within a degree of 28°C, which will be fine for amplifying your cells and virus for protein production. For a last resort, you can grow insect cells at room temperature. Virus infection also works well at this temperature; it just takes a bit longer for the replication cycle to be completed. However, if you use room temperature, don’t forget that you need to have optimized everything using the same conditions. This capacity for cell growth at lower temperatures probably derives from the fact that insects can’t regulate their own body heat and so have to survive quite low temperatures in the environment on occasion.
If you are scaling up your cell cultures to 1 litre volumes and require multiples of these cultures then consumable costs can start to mount. It is possible to reuse plastic culture 1 litre vessels by thorough washing and autoclave sterilization. However, it is wise to purchase new caps rather than attempt to recycle these components. Consumable costs can also be minimized by using 1 litre glass flasks in much the same way as they are used to grow bacteria. Simple cotton wool plugs capped with foil is sufficient to seal the flasks, followed by wet heat sterilization. We would just advise selecting a set of glass flasks that are reserved for growing insect cells. You don’t want your fellow scientist’s baked on Escherichia coli reside contaminating your culture, even if it is sterile. Our CEO advises us that in his early days of working with insect cells, this was the only option for scaling up their growth. This was a time when vinyl music still ruled and you bought an LP or Album rather than a download! He is also getting tiresome reminding us that vinyl is back – and even being sold in Sainsbury’s again.
If you are a regular reader of our blogs you will know that in addition to our useful tips, we also shamelessly plug some of our products or services. This one is no exception. We would like to highlight our scale up service for insect (and mammalian cell culture). Although you may be able to meet your protein needs with multiples of 1 litre cultures, sometimes it just isn’t enough. We can offer our WAVE Bioreactor scale up service that can handle culture volumes up to 10 litres (soon to be 25 litres). This service operates with a range of mix and match options. These include giving us your recombinant virus (already titrated or you can get us to do it) to infect the cell culture volume of your choice. Once the protein production run is complete, you can either receive the cells and/or culture medium from us for further processing. Alternatively, we can perform some or all of the protein purification procedures that you require. These might include concentration of a secreted protein from the cell culture medium – sending 10 + litres of liquid can be inconvenient – and/or affinity purification of a tagged product. We are very flexible in how we structure these services so if you have any queries just drop us an email or give us a call to discuss your requirements further.
Having touched upon protein purification in this blog, we will return to the topic in more detail next time and offer some tips on how best to isolate your favourite protein from either virus-infected cells or cell culture medium.