During the webinar Automated two-step purification of antibody drug candidates, we collected a number of interesting questions on multistep processing. In this blog post, I have summarized questions related to methods for multistep processing.
Questions and answers
The first five questions are related to principles of multistep processing.
No, there is no need to upgrade the instrument configuration, but you have to open the Admin module to edit system properties and select the added components.
Yes you can, but you need to update methods with regard to added valves and check if delay volumes need to be changed.
How can I concentrate the sample/eluted peak from the protein A column (that I currently do to avoid peak broadening) prior to loading onto the size exclusion chromatography (SEC) column?
The bed dimensions of the affinity column and the elution method used will largely define the elution volume (which defines the concentration of target protein). A small volume of a high-binding capacity chromatography medium in a narrow inner diameter column (as was used in the work presented in the webinar) will provide the smallest elution volume. Furthermore, a step gradient, rather than a continuous gradient, provides a small elution volume.
If needed, two 10 ml loops can be joined. Note, however, that the system backpressure will increase and you might need to run at a lower flow rate. Even larger volumes can be collected if the loops are replaced by flasks and re-injected by using a sample inlet valve and a sample pump.
If you use a loop of large volume (e.g., 10 ml), does laminar flow/dilution occur so that your stored sample is diluted? If so, what can you do?
Yes, dilution will occur. Optimize the loop size. Smaller capillary diameter will give less laminar flow/dilution, but increases the back pressure. The loop should be as small as possible, but remember not to fill the loop completely, as this will result in sample loss.
To neutralize the pH, you have to run a second step using a desalting/buffer exchange column. No pH change will take place inside the loop.
If the proteins need to be kept cool, we recommend placing the ÄKTA system in a cold room.
On slide 35, why is the peak not diverted directly to the injection valve instead of via a mixer valve? In the first part of the webinar, the peak was directed directly to the injection valve via outlet 1. I do not see the need for the mixer valve. Please can you explain?
The mixer valve is used when you are using the system pump for sample application. The mixer valve will work as a bypass to avoid sample going through the mixer when loading the first column. If your system includes a sample pump, you can connect the capillary directly from the outlet valve to the injection valve.
Information for how to download example methods for two-step purifications is available from the Cue Cards. All registrants will receive an e-mail including a link to the Cue Cards.
Although the work presented in the webinar was performed using ÄKTA pure, we have seen multistep methods in the past also for discontinued ÄKTA systems, such as ÄKTAexplorer. However, the hardware components presented during the webinar are intended for ÄKTA pure and ÄKTA avant. ÄKTA avant is the replacement system for ÄKTAexplorer.
ÄKTAxpress has a special functionality that enables you to select which of the collected peaks to use in the next chromatography step. ÄKTAxpress also keeps track of loop collection usage: used loops will be cleaned and ready for reuse in the next chromatography step. ÄKTA pure is a more flexible system, spanning a larger range of applications and flow rates.
HiTrap Protein G HP is available in 1 ml and 5 ml versions. The 1 ml version has a binding capacity of approximately 25 mg IgG, whereas the 5 ml version can bind up to 125 mg IgG. The maximum flow rate for the 1 ml column is 4 ml/min, and for the 5 ml column it is 20 ml/min.
HiTrap Protein L columns (1 and 5 ml prepacked with Capto L) are used to capture Fabs that include the kappa light chain. Useful additional purification steps are, for example, cation exchange (Capto SP ImpRes) and/or size exclusion chromatography (Superdex 75 prep grade). More information can be found from the application note A platform approach for the purification of antibody fragments (Fabs)
If you missed it, you can view the webinar on demand.
In a separate post, I have summarized questions related to principles of multistep processing and the case study presented by Erwin van Puijenbroek and Matthias Hermann, Scientists at F. Hoffman-La Roche.