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Tosoh
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Tosoh bioscience
ANALYSIS
PROCESS
INSTRUMENTATION
The existing downstream plant footprint becomes a constraining factor
as higher volumes of water are demanded. The number of manufacturing
cycles will therefore need to be expanded. Existing tanks/bags for buffer
preparation and in-process hold steps may be increased in size or
number. Inline buffer dilution could be used, but the increased volume
of WFI needed would remain at an elevated level. At some point, larger
chromatography columns could be incorporated, or resins changed to
higher capacity versions, to accommodate the increased requirements.
Toyopearl GigaCap resins can reduce WFI consumption through high
dynamic binding capacities (DBCs). DBC for the target molecule is the
first performance parameter evaluated when chromatographic resins
are screened for capture or concentration steps. Higher binding capacity
allows more feedstock biomass to be processed per liter of resin. When
considering several resin candidates with equivalent final purity and yield,
the one with the highest capacity is selected. Having a smaller resin
column volume also normally leads to reduced buffer/WFI consumption
for equilibration, wash, CIP, and later downstream steps. However, all of
the benefits can be undone for further downstream steps if the selected
resin’s target elution peak tails.
Toyopearl GigaCap resins minimize peak tailing. When purity and yield
are equivalent among the screened resins, selecting the resin with the
highest capacity for the capture step makes sense. The resin’s design,
however may be such that it has poor mass transfer properties or
non-specific interactions between the target and the matrix. This causes
the target elution peak to tail (Figure 1), requiring the use of more WFI
buffer for elution, and resulting in larger pool volumes and lower titer
fractions. This could ultimately impact subsequent processing steps. If
the next downstream unit operation is a flow through chromatographic
step or ultrafiltration/diafiltration step, a smaller elution volume reduces
their inprocess costs. In some cases, a lower DBC resin with the best
elution properties may be selected because of its overall effect on the
downstream economics of the process.
Figure 2 shows the full binding and elution curves for Toyopearl
GigaCap CM-650M, Toyopearl GigaCap S-650M, and a competitive
medium pressure agarose cation exchange resin. Figure 1 shows the
elution volume of the medium pressure agarose resin as 38 mL and the
elution volume for Toyopearl GigaCap S-650M as 8 mL. Using Toyopearl
Gigacap S the antibody elutes in less than 25% of the buffer volume of
the agarose resin, resulting in a significant product concentration.
The improved elution dynamics of the polymeric Toyopearl GigaCap ion
exchange resins, when compared to commercial agarose based resins,
result in significantly less tailing in the elution process with up to 75%
less buffer volume required for target fractions. The use of these resins
can considerably contribute to the reduction of downstream purification
bottlenecks.
Reducing Downstream WFI Consumption
Advances in upstream expression have significantly increased protein titers. Many of today’s processes have
their expressed biologics in the 2-4 g/L concentration range. By optimizing the expression systems, upstream
engineers can increase the quantity of active biologic produced in their pre-existing bioreactors. For down-
stream purification, the given equipment may not be readily able to handle the increased biomass. A critical issue
is the proportionality of protein amount to the volume of WFI (water for injection) needed for purification.
05
TOYOPEARL
BIOPURIFICATION
⇒Figure 1: Peak tailing comparison
Column size: 6 x 40 mm; Sample: polyclonal human IgG (1 mg/mL); Elution: linear
velocity: 424 cm/hr; Detection: UV@280nm
⇒Figure 2: Breakthrough and elution profile comparison - cation
exchanges resins
Conditions see Figure1
0
20
30
40
10
desorption time (min)
TOYOPEARL GigaCap CM-650M
TOYOPEARL GigaCap S-650M
1000
800
600
400
200
0
mV
Medium pressure agarose S
96
96
Recovery (%)
38
8
Elution Volume (ml)
96.
20
Medium pressure agarose S
TOYOPEARL GigaCap° S-650M
TOYOPEARL GigaCap° CM-650M
Time (min)
TOYOPEARL GigaCap CM-650M
TOYOPEARL GigaCap S-650M
1000
800
600
200
0
-200
mV
1200
400
100
50
0
150
200
250
At 5% breakthough value:
GigaCap CM elutes in 17.7 CVs
GigaCap S elutes in 7.7 CVs
agarose S elutes in 33.6 CVs
Medium pressure agarose S