Biomedical Engineering Reference
In-Depth Information
4.4.5 Pipette Tip Example
Wenger et al. [ 36 ] used PhyNexus tips to mimic a chromatographic process used to
purify recombinant VLP. In the downstream processing following fermentation,
the cells were disrupted by high-pressure homogenisation, clarified by centrifu-
gation and then purified by cation exchange followed by ceramic hydroxyapatite
chromatography. Direct product titre analysis from the ruptured culture medium
was found to be inadequate, as it did not correlate well with the amount of protein
recovered after chromatographic purification. This necessitated an entire purifi-
cation sequence to obtain accurate data about how changing the fermentation
conditions affected downstream performance, using a two-step chromatographic
purification at 30- and 80-mL laboratory column scales for the CEX and CHT,
respectively. This scale meant that assessing the impact of changing fermentation
conditions involved considerable feed volumes, time and labour. Purification
throughput was increased by miniaturising the chromatography by three orders of
magnitude using robotically controlled chromatography tips packed with either 40
or 80 lL of the relevant resin. The tips and the laboratory columns were compared
by examining product recovery for a large number of fermentation conditions and
for multiple product types for both the cation exchange and the hydroxyapatite
steps. Despite the differences in scale and format, the tips were found to be
predictive of laboratory VLP recovery and purity while increasing throughput
tenfold. The comparison in recovery between the two scales showed close linear
agreement.
4.5 Miniaturised Packed Columns
4.5.1 Summary of the Technique
Whereas the dimensions and operating protocols for tips and plates are signifi-
cantly different from normal columns, the appearance, dimensions, matrix
arrangement and principles of operation of packed miniature columns are closer to
conventional columns. Very small columns of up to a few hundred microlitres are
available (up to 600 lL) and can be controlled manually or robotically. Miniature
columns have quite small heights (as low as a few millimetres) and are operated in
a single direction. Especially with the shortest bed heights, this can result in very
low residence times, and hence the potential effect upon breakthrough may be
important. A supplier of miniature columns is Atoll GmbH (Weingarten, Ger-
many), which provides pre-packed columns in a 96-array format that can be
organised as a series of blocks, each containing eight columns arranged side-
by-side. Different blocks can then be packed with different resins. Liquid that has
passed through the columns can be collected in 96-well plates for analysis, and if
required, individual liquid droplets can be pooled to create fractions for generation
of pseudo-chromatograms.
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