Biomedical Engineering Reference
In-Depth Information
If the resin in each well represents a defined number of theoretical plates found
in a section of a column operated under the same mass transport regime, then
choosing the correct incubation time enables the beads to be saturated to a similar
level as a column. As a guide, a 20-60-min incubation may be relevant, although
column beds contain thousands of theoretical plates per metre and reducing this
number in a batch plate may necessitate use of mathematical models.
4.3.6 Other Mass Transport Aspects
Unlike a column, which uses constant fluid flow to achieve feed-resin contact, a
plate involves agitation of the bulk liquid. Nevertheless, unless very high flow
rates are used, adsorption rates of small proteins at moderate to high concentra-
tions in porous media are governed by internal bead diffusion. The same may not
apply for large macromolecules where bead exclusion reduces internal diffusion
and where convective mass transfer or external film diffusion may be rate limiting.
In such instances, using an incubation time that equals column load time may not
imitate column mass transport [
2
].
4.3.7 Liquid Evacuation
Liquid evacuation can be accomplished in three ways. Direct pipetting for dead-end
plates [
31
] is simple, but it is important to minimise resin entrainment, and manual
pipetting is especially prone to variable liquid hold-up. Vacuum filtration is another
option, but if foaming occurs, there may be carry-over between wells. Coffman et al.
[
7
] found centrifugation to be robust in achieving consistent liquid recovery from
each well with minimal carry-over, while vacuums failed to provide reproducible
hold-up volumes. Hold-up after liquid removal can be measured using high and low
salt buffer [
7
] or by a protein to account for dilution effects in the following stage [
20
].
4.3.8 Self-Dispensed Resin Plate Examples
There are several examples illustrating robotic HTS in self-dispensed plates e.g.
for monoclonal antibodies by ion-exchange purification [
13
,
14
]. Charlton et al. [
4
]
used robotic self-dispensed plates to carry out HTS of different IEX ligands and
buffers. In another study, Coffman et al. [
7
] used a 96-well plate to screen con-
ditions using 50-100 lL of resin per well. A 20-min incubation was used as a fast
way to approximate HIC and AEX column binding conditions for a monoclonal
antibody and an Fc-fusion protein. The HTS generated hundreds of chromato-
grams in under an hour and compared well with millilitre- and litre-scale columns.
Differences occurred due to hold-up and plate number issues, but agreement
between batch and column data was sufficient to predict column yields/purities and
guide subsequent activities.
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