Extreme Scale-Down Approaches for Rapid Chromatography Column Design and Scale-Up During Bioprocess Development - Measurement, Monitoring, Modelling, and Control of Bioprocesses - page 123

matrix. Automating batch devices may pose certain challenges [ 36 ], such as the need
to move plates from the robot to plate shakers or solid-liquid separation equipment.
Pre-prepared tips and miniature columns avoid some of these issues by passing liquid
through a resin bed constrained between two frits/filters.
In general, tips are pre-packed (e.g. PhyNexus, CA, USA), with resin volumes
varying between a few microlitres and a few hundred microlitres. Feed samples
and buffers can be held in either a multi-well plate or troughs and are pipetted up
and down through the resin at a specified flow rate to achieve a desired contact
time. Selection of the number of up-down cycles and the flow rate needs to be
determined on a case-by-case basis. The time for which matrix is exposed to feed
influences uptake and affects the ability to predict scale-up performance. It is
possible that mimicking a column contact time may be too time-consuming, and it
may be necessary to reduce the contact time and then apply correction factors.
Within this context, there are several considerations for pipette tips:
4.4.2 Contact Time
Since very short beds are used in pipette tips, the per-pass residence time is low
and unlikely to reflect column uptake. Although increasing the number of cycles
will increase adsorption, the chosen number needs to be practical to prevent
excessive sample pipetting. The cut-off point represents a compromise between
uptake and throughput, and it may be useful to create uptake curves to determine
percentage adsorption after successive feed application cycles.
4.4.3 Volumetric Flow Rate
Volumetric flow rates should be lower than normal robotic pipetting speeds i.e. up to
around 20 lL/s (compared with normal speeds of a few hundred lL/s). Depending
upon the resin volume and the volumetric flow rate, it therefore becomes possible to
realise linear velocities between a few hundred and a few thousand cm/h. Generally,
the superficial flow rate is set to around 300-1,500 cm/h, resulting in an equivalent
volumetric flow rate of around 5-20 lL/s.
4.4.4 Impact of Resin Bed Upon Aspirate-Dispense Operations
Compared with an empty tip, the packed bed at the base of the tip hinders liquid
flow and means that, at a specified robotic flow rate, it may not be possible to
pipette the full intended volume over the duration of the pipetting step. This can be
overcome by adding a delay time after aspiration/dispensing to allow the liquid to
'catch up' or by adjusting the robotic flow rate or pipetted volume to compensate.
The delay time is feed and matrix specific, and gravimetric analysis can be useful
in this context [ 36 ].