Don’t Risk Protein Quality: Your Questions about Automating Buffer Exchange Answered

Buffer exchange is a ubiquitous part of protein formulation and many sample preparation requirements. Whether you work on developability screening, sample prep for analytics, biologic characterization, protein conjugation, activity studies or formulation development, you will be familiar with the challenges of buffer exchange.

In a SelectScience webinar, Donna Chen and Joe Barco, of Unchained Labs, introduce a brand-new solution for buffer exchange automation. Big Tuna provides a more uniform sample handling approach and can additionally provide degrees of process control that are otherwise inaccessible by manual methods, helping you to overcome challenges with manual methods and to simplify your workflow.

Read on for highlights from the webinar Q&A session or register to watch the webinar on demand.

How long does it take for Big Tuna to finish buffer exchange for one plate?

For a 96-well plate, the low is somewhere around 2.5 hours, and for a 24-well plate it’s about 4 to 4.5 hours. It is dependent on the protein concentration and the viscosity of the solution being transferred, but at a lower concentration, say, 10 mg/mL or below, or simple buffers, it's about that time.

Can I use part of a plate in a single run, or do I have to use the whole thing?

You can use part of the plate. The GUI (graphical user interface) for Big Tuna will allow you to choose certain groups of wells or a single well and do transfers on those. You can then put the plate aside and use it a second time for the unused wells.

How can it handle samples with volumes over the maximum volume? I sometimes have samples that are 20 mL - 25 mL to exchange.

You can put those in multiple wells of the plate. You can tell the software that it can either address those as individual wells and just track the percent exchange and the volume removal as individual wells. Or you can identify within the software that those are a pool of samples so that if you're going to a certain percent volume exchange, it will keep track of those and then when you hit the average across those wells, it will then stop the buffer-exchange process.

Does Big Tuna measure protein concentration?

The instrument does not measure protein concentration directly. It requires the operator to put in an approximate protein concentration so that the system can then provide expected parameters as to how long to do the pressurization and how to run the plate. So that's how the information is used on Big Tuna.

For all of the data that we've generated and shown on this presentation and in our application notes we've used the Lunatic, which is another low-volume-protein-concentration analytic instrument that we sell to do those measurements. We use the Lunatic measurement and the Big Tuna volume to confirm recovery and percent exchange.

Is online protein concentration Lunatic-included, or is it possible to add on?

Within the space of the instrument, it's not possible to have the Lunatic physically integrated into the instrument. We do have our software package called LEA which allows you to integrate both Big Tuna and Lunatic into a single experiment file, but you could also run those things offline. I think for the speed of the liquid handling, it may be more efficient to load the Lunatic chip offline regardless, because we have a 6-tip on the Big Tuna.

Can I run samples at different volumes at the same time?

Yes, the instrument allows you to do that. However, if you're looking at efficiency and best results, you'd want to limit the variation. So, for example, if I have an 8 mL plate or if I'm using the 24-well plate, I don't want to mix samples that are 8 mL and 1 mL, because everything is going to be gated by how long it thinks that 1 mL sample is going to run. So, you'd want to separate that out from an efficiency standpoint.

What about samples at different viscosities or concentrations at the same time?

It’s a similar thing with samples of different viscosities – it will handle it, but in general course of use, you probably want to keep things sort of grouped together. So, you don't want to transfer protein that's in 50% glycerol at the same time as you're transferring something in PBS or something at 10 mg/mL and 200 mg/mL. It ends up being inefficient for the slower protein to run. So, you'd want to separate that out as a course of normal operation anyway.

Are there any challenges with buffers containing high levels of sugar or high-viscosity samples?

Yes. We addressed this a little in the presentation. We've tested buffers with high levels of sugar on our buffer-exchange systems and it just runs a little bit slower. High-viscosity samples result in a similar thing, so we highlighted changing the parameters and how we actually do the buffer exchange to make a higher-viscosity sample run a little bit faster and more efficiently.

For the concentration step, how do you control the speed of concentration and how do you make sure it's uniform across each well?

The way that the concentration step operates is, you do your buffer exchange – the buffer exchange process works by measuring volume all along, in between every cycle of buffer exchange. When it's calculated that everything has reached the exchange percentage, it starts the concentration process.

The concentration step works through three steps of pressurization. It does pressurization for a few minutes, stops, measures volume, and then does that two more times. It uses that first check to approximate how long it should be pressurizing for the next couple of cycles. You get to the point when you have some wells running at different viscosities than others, simply by a function of the buffer it's in. Big Tuna will re-add that buffer back in, so that every time you start that next cycle, things that are running a little bit faster are not going to overconcentrate, and it will allow time for the things that are running a little bit slower to concentrate to the target.

Find out more about how Big Tuna and how it can help you to automate buffer exchange by watching the full webinar on demand >>