Tips for purification method editing – Learnings from the webinar

During the ÄKTA club Webinar on 14 June, Jens Widehammar presented the topic Tips for protein purification method editing in UNICORN software. Jens presented the basics of the UNICORN 7 software and provided useful tips and features on how to develop methods that are easy to maintain, reuse, and share. View the webinar on-demand. 

The live Q&A session after the Webinar provided many interesting questions that Jens and his colleague Lena Nyholm responded to. Below you can find a selection of questions.

Tips around method creation

Q: We have UNICORN 4.12 set up on our computer. Is it similar to UNICORN 7 in the way you tailor the experiments?

A: Yes and no. The text instructions in UNICORN 4 and UNICORN 7 are very much the same.  UNICORN 4 includes the method wizards to support method creation while UNICORN 7 includes preprogrammed phases, which makes method creation more flexible.

Q: Is there a way where I can create a method with gradient wash (i.e., gradient wash from 0.2 M to 1.5 M NaCl) using a pump?

A: Yes. Most ÄKTA instruments support gradient formation and it is easy to program a gradient. In this case, I would use a buffer A with no NaCl and a Buffer B with 2 M NaCl. Set the start value to 10% B (corresponds to 0.2 M NaCl) and run up to 75% (corresponds to 1.5 M NaCl).

Q: Is “Fill in the system with selected buffer” the same as “Pump Wash”?

A: No, it is not. The purpose with "Fill System" is to fill the flow path from inlet tubing all the way to the outlet valve with the current mixture of buffer A and B, for example, 95% A and 5% B. A "Pump Wash" is used to flush the pump using the buffer connected to the selected inlet and will fill the flow path to the injection valve. Note: a “Pump Wash” will consume a larger buffer volume than the “Fill System”.

Q: How can I set up fractionation by UV Watch command independently in two different blocks? First block in "Wash" and second block in "Elution".

A: By text-editing the method, it is possible to set up fractionation by watch commands independently in different blocks. By having a Watch in each phase that triggers fractionation it is possible to set different parameters for each fractionation.

Q: Is there an optimal range in percent (%) that you would recommend using the BufferPro for the stock buffers?

A: When you use a BufferPro recipe, UNICORN will automatically calculate the concentration range and pH range that it can be used for. This takes several factors into consideration such as:

- optimal operational range for the pumps for accurate mixing

- buffer capacity of the prepared buffer

If you create your own recipes, you will see that the concentration of the stock solutions will affect the operational concentration and pH range.

You will not be able to operate outside the concentration range since such solution is not possible to mix with good accuracy. You can however use a buffer recipe outside the recommended pH range but it is not recommended since the buffer capacity will be very low.

Q: Is there any way to change fraction collection size during the run?

A: Should not be a problem. Just send a new fractionation instruction with the new volume and this should override the old command.

Q: When reviewing your results, you can use the presentation button to copy the graph. Is it possible to copy the table underneath too?

A: Yes, right click in the peak table and choose to copy into, for example, Excel®.

Q: Can you give advice on tuning the proportional-integral-derivative (PID) function? When in use, the function does not respond fast enough to prevent an over-pressure alarm. This is controlled by the P value, as I understand it. Should I try doubling the P value?

A: The P value regulates how fast the response of the flow regulation will be and the parameter regulates how fast the re-adaption to the original flow rate will take. In this case, I would recommend increasing the P parameter to see if this helps. I should also mention that the most important parameter to protect the column is the flow, NOT the pressure. Using the recommended flow rates will give better protection than the pressure limits BUT you need to adjust the flow rates depending on the viscosity of the buffer or sample.

Q: I heard about single software for chromatography/tangential flow filtration (TFF)/filtration. Will UNICORN 7 have these options?

A: Yes, UNICORN 7.1 and later can be used to control chromatography, cell cultivation, and filtration systems, for example, ÄKTA avant, ReadyToProcess WAVE 25, ÄKTA readyflux, and many other systems.

 

Troubleshooting

Q: When performing gel filtration (size exclusion chromatography) with Superdex 200 Increase, I see a consistent fluctuation of the baseline (UV280 and conductivity) that almost looks like a sine curve. The amplitude is rather low and it does not seem to affect the purification. However, I was wondering if that is normal?

A: Great to hear that you are using the Increase columns but sad to see that you are experiencing problems, which are not normal. I would have a service technician look into this since a component of the system might not be working properly.

Q: When we work with hydrophobic interaction chromatography (HIC) column, is it normal to see very small bubbles coming through the sample pump line from sample? How can I avoid it?

A: Air bubbles in the sample are not uncommon and can often arise when the sample is taken directly from the fridge. When liquid is warmed to room temperature, the air in the liquid is transformed into bubbles. In HIC, it can be even trickier since you most likely have the sample in a viscous buffer with high conductivity and the sample can easily precipitate. I suggest looking at one of these solutions:

1) let the sample reach room temperature before applying it to the column.

2) try lowering buffer concentration in the sample preparation if this can be performed while still maintaining good results.

But first - do the air bubbles interfere with your separation? If not, I would not bother trying to remove them.

Q: When running a protein A column, our eluted protein is aggregated or degraded. Any advice?

A: This could be due to several reasons. If any NaOH has been used prior to starting the purification run, residues could be the reason. Before starting the purification run, flush the flow path with buffer to ensure that the NaOH is washed away properly. I assume that you elute your protein with low pH. Perhaps the pH is too low and therefore bad for the protein? Have you tried to elute with a somewhat higher pH? Increase the pH as soon as possible after the elution since this could help the stability of the protein.

Q: We are using our ÄKTA pure system at 4°C. Is it necessary to slow down the flow rate?

A: Yes, it is. This will typically increase the viscosity two-fold compared to room temperature and you will need to lower the flow rate to half of what you normally use. If you are also using high viscosity buffers, you would need to lower the flow rate even further.

 

Do you have additional questions related to the protein purification or ÄKTA system? Post them on our discussion forums.

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