Continuous Flow Processing

Continuous flow bioprocessing in the Advanced Bioprocessing Centre at Brunel generally takes place along a continuous length of tubing.  The tubing is wound on a drum (more commonly referred to as the bobbin) and the drum/bobbin is rotated in planetary motion.  Liquids in the tubing experience a variable force (CCC animation) which can have different effects depending on whether two immiscible liquids are used for liquid-liquid chromatography/extraction (Liquid-liquid flow processes) or a single phase liquid is used for particle separation involving a form of field flow fractionation (Single phase flow processing).  Both processing methods can be used in different ways for particle purification (Particles and Cells) and complementary processes like filtration (Filtration) can also play a role.  The process can also be used as a bioreactor, where product streams can react under controlled mixing conditions and then get separated as they move on down the column (Flow reactor)

Liquid-liquid flow processes

The unique nature of Brunel’s liquid flow technology enables it to have a number of different operating modes depending on the type of processing that is required.  Immiscible liquids can be stratified along the length of long piece of tubing (or column) in such a way that mixing and settling zones travel along the tubing toward the “head” end of the column (the other end is call the tail).  One of the liquids is held quasi-stationary while the other is flowed passed it. Compounds, from a crude extract pumped into one end of the column with the mobile phase, will travel at different speeds depending on their affinity to the stationary phase.  In isocratic mode (Section 1.1.1) either phase can be mobile (normal or reversed phase operation) and any compounds retained in the stationary phase can easily be pumped out afterwards, so there are no losses. Gradient elution (Section 1.1.2) is also possible using either the upper phase or lower phase as the mobile phase depending on the hydrophobicity of the target compound. Continuous batch flow processing is possible using both isocratic and gradient flow modes.

The mixing and settling zones are set up by up a variable force field obtained by winding the tubing on a drum and rotating the drum in planetary motion.  This causes mixing and settling zones in the tubing which are synchronous with the rotation.  Non-synchronous instruments have also been developed (Section 1.1.3) where the forces for settling the phases and those for mixing them are independently varied. This has advantages when working with low interfacial tension phase systems where the degree of mixing needs to be controlled. All the above instruments are hydrodynamic with “wave” mixing.  When flow stops the phases will redistribute to either end of the column.  The upper phase to the head and the lower phase to the tail. When toroidally wound tubing is wrapped around the drum, the process becomes a hydrostatic one (Section 1.1.4).  Here mixing is “cascade” mixing and when flow stops the stationary phase stays where it is. This has advantages when working with two phase aqueous systems (ATPS) for the separation of proteins (Section 1.1.5).

As both mobile a stationary phases are liquid, it is possible to switch the phase being pumped during a run.  This is known as multiple dual mode (Section 1.1.6 - MDM) which has the effect of increasing the length of the column and enable increased resolution to be achieved.  While MDM can be performed using a single column, the Brunel range of centrifuges each have two columns.  This helps to maintain balance but also allows crude sample to be continuously injected between the two columns.  The main inlet flow is then arranged to switch intermittently between the upper and lower phase flowing in at the “tail” and “head” ends of the columns respectively, to set up intermittence counter-current extraction (ICcE) where hydrophobic compounds elute from one end of the column continuously while hydrophilic compounds elute from the other and the target compound is concentrated in the column for intermittent harvesting (Section 1.1.7 – ICcE). Finally with a specially designed rotor, it is possible to set up true countercurrent flow with each phase being continuously pumped into and harvested from each end of the column simultaneously (Section 1.1.8)