1. Will the increased cycling of columns with MCC affect column performance?
No, as long as the process buffers and sample loaded are filtered and free of fouling contaminants (depending on the resin used) typical columns are designed to run for >100 cycles. Most non-integrated MCC processes can be completed in < 50 cycles.
2. With which resins can the OctaveTM BIO work and what are limitations? (Consolidate with question 5)
You can use resins from any vendor. However, the limits for resins (and column hardware respectively) are:
- Ability to withstand high flow rates/ high linear velocity
- Reproducibility of column packing (ideally almost identical column beds)
- Quality (~stability) of column bed (short bed height columns are usually more difficult to pack, glass columns are not appropiate unless they can tolerate process pressures required)
- Pressure limit under high flow conditions (less rigid base materials like agarose are more limited versus methacrylate in MCC). The maximum operating fluid pressure for the Octave BIO is 270 psi (~20 bar) and Octave PRO 90 psi (6 bar). Pressure for columns connected in sereies within a zone is additive. Therefore, if a single column generates 50 psi and a 3-column capture zone is programmed into the PROComposerTM method that zone will generate 150 psi.
- Very small silica or polymeric HPLC resins <30 microns in size are generally not suitable for MCC on the Octave Systems
3. Are OctaveTM systems compatible with other columns besides SkillPakTM?
Yes, but MCC process columns typically have a different aspect ratio than columns used in batch chromatography. MCC columns will have wider diameters and shorter bed heights to allow the advantageous short residence times and high linear velocity of most MCC processes. These shorter wider columns generate lower back pressure and the multiple columns in series that can be allocated to the capture/loading zone prevent product loss from breakthrough since the secondary and even tertiary column in the capture zone will adsorb product breakthrough even at very high linear velocity.
4. How does troubleshooting with multi-column chromatography (MCC) differ from classic batch systems?
There are different considerations with troubleshooting MCC systems when compared to batch systems that are mainly based on the multiple columns and simultaneous processes occuring. However, OctaveTM software tools like the process flow diagram and process ribbon, along with design simplifications like the valve block, help to make the troubleshooting more familiar to chromatography users.
5. Is MCC really economically beneficial over standard chromatography?
There is no one size fits all answer to that question. For Affinity chromatography it is proven that there will always be productivity gains, which can translate to resin and buffer savings. However, depending on the context of the process in question, economical benefits may vary a lot.
Watch our webinar to learn more about the process economics of MCC.
6. Can the OctaveTM systems do gradient elutions?
Yes, the Octave BIO can perform gradient elutions, but the Octave PRO cannot.
7. What is the reason you have different pumps on the OctaveTM BIO? Which size pumps should I choose?
We have different sized pumps to increase the flexibility and use cases for the system. To choose the right size for you, we have a downloadable sizing guide to help make this clearer.
8. Are the sensors fixed or can they be used “in-between” the columns?
There are 4 each of pH, UV, and conductivity sensors that can be assigned in the PROComposerTM software to any of the 6 process stream outlet lines. The plumbing net list in the controller software allows the user to verify the correct plumbing of the outlet lines to their assigned sensor(s). However, it is not recommended to connect the sensors between the columns. It would be possible to connect the column outlet to a sensor(s) and then connect the sensor outlet line back into the valve block at the column outlet port. Also, the loopback PROComposer feature could be used by plumbing the outlet from a zone through the sensor(s) and then back to a valve block inlet port. The problems with both of these scenarios is that it changes the dead volume flow path length between columns and could affect the separation and at least the pH sensor flow cell and probe are not rated for the pressures that could be encountered when they are plumbed in-line instead of only at the system outlets where pressure is lower.
9. How do I know how many columns are needed?
The number of columns required is determined by the length of time required to load the first column in the capture zone to its desired capacity, compared to the length of time that the non-load process steps take. Such calculations are described in publications like Godawat, R. et al (2012) Biotechnol. J. 1496. The time required to complete all of the non-loading recovery and regeneration steps (tR) must be less than the time it takes to load (tL). Greatest productivity is achieved when tR < tL; At high titer, loading occurs quickly and column number must increase to relieve the constraint.
10. How can I transfer from batch to MCC?
We have tools like the MethodWizard that help simplify the transfer of a batch process to MCC.
11. Why do I sometimes observe air bubbles when there is a valve switch on my OctaveTM?
The main reason that explains the noticed air bubbles in the column lines immediately after a BIOControllerTM valve switch is dissolved gas in the process fluids and columns outgas from the pressure change. Outgassing is a common occurrence in chromatography and is more problematic in HPLC/UHPLC with organic solvents and higher pressures. According to Henry’s Law-At a constant temperature, the mass of gas dissolved in a liquid at equilibrium is proportional to the partial pressure of the gas. The process fluids and columns are under pressure during processing maintaining dissolved gas/air in solution, but pressures between columns and zones can differ considerably.
When a programmed switch occurs the columns and zones can immediately be exposed to a lower pressure than that experienced in the previous zone or column. This results in outgassing as small amounts of dissolved air comes out of solution. The bubble(s) will appear only briefly and will not harm the columns of affect the separation. The bubbles look significant as they travel through the narrow bore column lines, but actually are not a large amount of air. As the pressure increases through the next zone or column, the gas will redissolve. The outgassing bubbles will not be as obvious or numerous in high flow rate separations.
There are a few ways to decrease the observed outgassing:
1) Vacuum filter all process fluids and feed samples and degas with vacuum/sonication, especially if the outgassing is considered a serious problem for your MCC separation or detection.
2) Cold process solutions and samples will contain more dissolved gas than room temperature solutions. If possible allow all process fluids solutions and columns should be warmed to room temperature and then fluids degassed if necessary. Cold solutions can be used, but as aqueous solutions warm to room temperature dissolved gasses become less soluble and more outgassing at switches will be observed.
3) Air bubbles that form in detectors, mainly UV detectors, will cause noise and signal spikes in the chromatograms. These can be reduced or eliminated by degassing as above and by the included 20 psi back pressure regulators installed on the sensor panel pH cell. Finally, if large amounts of air are observed that persist long after the switch occurs, there is likely a connection problem/leak allowing air to enter the flow path or a solvent has run dry at the pump inlet.