Biomedical Engineering Reference
In-Depth Information
4.3.9 Pre-Dispensed Resin Plate Examples
Bergander et al. [
2
] used pre-dispensed PreDictor filter plates (GE Healthcare) to
predict column capacity measurements. This involved two different applications
for binding human polyclonal IgG to Protein A and amyloglucosidase adsorption
to a weak anion exchanger. To verify plate predictions, dynamic column binding
capacities were generated by frontal analysis using loading flow rates calculated to
achieve a desired residence time. The impact of this parameter upon dynamic
binding capacity normally involves many tedious and feed-intensive column runs
at different flow rates. Transient batch uptake data at varying feed-resin contact
times offer a useful alternative to understand column dynamics. An uptake curve
was developed in the filter plate, with the incubation data from every well being
used to generate a single data point on the curve. Feed was added to one well at a
time at suitable intervals, such that the first well exposed to protein represented the
longest incubation time and the last one represented the shortest time (Fig.
3
).
After all wells were exposed to feed, liquid was recovered in UV-transparent
plates for 280-nm quantification. Mass transport equations were applied to the data
to calculate column binding capacities.
Batch uptake can be used either qualitatively with limited data to screen search
space trends or with more comprehensive data for quantitative capacity predictions.
Bergander et al. [
2
] used a qualitative approach to acquire trends of how varying the
pH and ionic strength affected dynamic capacities of amyloglucosidase on an anion
exchanger. By contrast, the quantitative method generated a more accurate rela-
tionship between binding capacity and column residence time by combining the
larger data sets with a mathematical model. In this context, Bergander et al. [
2
] used
filter plates to study human IgG adsorption to Protein A. In cases where macropore
diffusion is the dominant rate-limiting step and when the isotherm is sufficiently
rectangular, the shrinking core model can be used to predict binding capacities.
Langmuir adsorption parameters were estimated from 60-min-long uptake curves,
which were then sufficiently shallow to approximate equilibrium. Using these and
fitted pore diffusion coefficients, 10 % dynamic binding capacities were calculated.
Capacities found by the plate method correlated well with column data, and the
authors estimated savings in time and sample of 10- and 50-fold, respectively,
compared with packed columns.
4.4 Chromatography Pipette Tips
4.4.1 Summary of the Technique
There are important differences between packed pipette tips [
26
,
38
] and a normal
column. The height and diameter of the tip column are significantly smaller, and it is
tapered in shape (wider at the top), meaning that the linear flow rate changes along the
bed length. Bidirectional flow is used to bring feed molecules into contact with the
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