Extreme Scale-Down Approaches for Rapid Chromatography Column Design and Scale-Up During Bioprocess Development - Measurement, Monitoring, Modelling, and Control of Bioprocesses - page 119

such that little remains occluded inside the tip or on the lumen/outer tip surface.
One key point is to minimise the distance that the tips descend into the slurry
reservoir and to dip the tips into buffer after pipetting to eliminate anything
adhering to the outside of the tips as well as to rehydrate dried resin at the base of
the tip [ 20 ]. To avoid matrix retention inside the tip after dispensing, buffer should
be aspirated prior to the slurry to wash the tip clean during dispensing.
4.3.3 Resin Aliquoting Devices
Another way of generating 96-well batch plates is to use devices that create resin
plaques from loose slurries. Herrmann et al. [ 10 ] described a system in which resin
cylinders were separated from liquid through a mesh by vacuum to form.
A commercial version of this device is available (MediaScout ResiQuot, Atoll
GmbH), creating cylinders of 8, 21 and 51 lL. The device comes with a 96-spike
tool to push plaques into a microtitre plate. Such devices have been used to
measure isotherms [ 8 ] and static capacities [ 17 ].
4.3.4 Mixing Conditions
Plates can be mixed magnetically, by an end-over-end stirrer or in an orbital plate
shaker. Protein uptake depends upon agitation speed, and values of 1,000 rpm or
above are useful as a starting point, using an orbital shaker with a throw radius of a
few millimetres to ensure that resin remains suspended. For intra-particle diffusive
cases where the mixing speed exceeds a threshold value, no significant change in
uptake rate will be observed as speed increases, but if the speed is significantly
below the threshold, uptake will be slow and bulk fluid mass transport up to the
bead surface may be limiting. The threshold depends upon well dimensions, throw
distance as well as matrix and feed properties.
4.3.5 Feed Incubation Times and Theoretical Plate Number Aspects
Choosing the incubation time is critical to acquire column-relevant kinetic and
thermodynamic data. This time may be defined by uptake curves when an
acceptably high percentage of equilibrium has been achieved. Also of relevance (if
known) is the column loading time (contact time) i.e. the total duration for which
matrix is exposed to feed. This is more relevant than column residence time (the
average duration taken by a feed molecule to pass through the bed), because
uptake depends more upon cumulative resin exposure to feed over the course of a
loading step, which in a process column could be several hours. Such a time is
incompatible with HTS, and during early studies, matrix exposure to feed should
be greater than residence times but shorter than column loading times. As protein
challenges increase (e.g. in later development), extended incubations may be
needed to attain equilibrium.