40
MX
MASS TRANSFER PARAMETERS
The mass transfer properties of a resin influence the
economics of loading and elution and the degree of reso-
lution. In keeping with the exceptional target binding and
elution properties of the TOYOPEARL GigaCap
®
resins,
the new TOYOPEARL MX-Trp-650M also shows a narrow
elution peak width to complement its higher capacity. The
mass transfer properties also minimize peak broadening
and contribute to the excellent peak shapes observed
when comparing a separation of standard proteins on
TOYOPEARL MX-Trp-650M versus the agarose based multi-
modal cation exchange material (Figure 5).
Purification of Immunoglobulins
The resin benefits from a selectivity which is in many
respects similar to hydrophobic interaction chromatog-
raphy. Possible applications include mAb polishing, as the
resin allows favorable resolution of closely related protein
species, such as aggregates and monomers. This can be
achieved by applying a pH gradient.
Antibodies captured by protein A affinity chromatog-
raphy might be loaded at pH 4.5 and eluted with increased
salt concentration and pH. Elution of various IgG mono-
mers and their well separated aggregates was successful
using an overall salt concentration of 0.5 mol/L and pH 5.5
(Figure 6). Monomer purities of up to 98.5%can be achieved.
ToyoScreen prepacked columns for process
development
ToyoScreen columns packed with TOYOPEARL MX-Trp-
650M are available in 1 mL and 5 mL resin volumes.
ToyoScreen columns provide a convenient way to perform
early resin screening for both target retention and recovery.
Multiple columns can be connected in series for additional
capacity or resolution. ToyoScreen RoboColumns are
miniaturized chromatographic columns for operation with
a robotic liquid handling system, such as the Freedom
EVO
®
from TECAN. This approach allows automated high-
throughput, small-scale biochromatographic separations of
protein samples by running up to eight individual columns
simultaneously. ToyoScreen RoboColumns packed with
TOYOPEARL MX-Trp-650M are available with 200 µL and
600 µL resin volumes. Please see the ordering information
at the end of this section or contact us for more information
on these products.
GOOD PEAK SHAPE AND HIGH RESOLUTION
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0
20
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140
0
10
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mV
Time (min)
Toyopearl MX-Trp-650M
Brand M
Resins:
TOYOPEARL MX-Trp-650M, Brand M
Column size:
7.5 mm ID × 7.5 cm
Mobile phase:
Buffer A: 20 mmol/L phosphate (pH 7.0)
Buffer B: 20 mmol/L phosphate +
1.0 mol/L NaCl (pH 7.0)
Gradient:
30 min. linear gradient from buffer A to buffer B
Flow rate:
1.0 mL/min
Detection:
UV @ 280nm
Sample:
trypsinogen (6.6 mg/mL)
Resins: TOYOPEARL MX-Trp-650M, Brand M
Column size: 7.5 mm ID × 7.5 cm; Mobile phase: Buffer A: 20 mmol/L
phosphate (pH 7.0); Buffer B: 20 mmol/L phosphate + 1.0 mol/L NaCl (pH
7.0); Gradient: 30 min linear gradient from buffer A to buffer B; Flow rate:
1.0 mL/min; Detection: UV @ 280 nm;
Sample: trypsinogen (6.6 g/L) cytochrome C (3.6 g/L)
lys zyme (6.6 g/L); Sample volume: 25 µL
Column: TSKgel G3000SW
XL
(7.8 mm ID X 30 cm);
Mobile phase: 0.1 mol/L phosphate buffer (pH 6.7) + 0.1 mol/L Na
2
SO
4
;
Flow rate: 1.0 mL/min; Detection: UV @ 280 nm
Sample: pooled monoclonal antibody fractions eluted from
TOYOPEARL MX-Trp-650M @ 100 mmol/L acetate buffer pH 5.5 +
0.4 mol/L NaCl; Injection Vol.: 20 µL
purity check for mab fraction
figure 5
figure 6
mixed-mode
chromatography
