TOSOH
CUSTOMER MAGAZINE
MAbs combine high target affinity and specificity with various different
effector functions, such as complement activation. They are in-
creasingly used in diagnostics and therapy. While the number of
approved mAbs for pharmaceutical purposes has grown tremen-
dously, mAb production processes have improved, too. Today,
high mAb titers achieved through advanced expression technolo-
gies have become challenge in downstream processing, especially
with regards to the expensive Protein A chromatography step.
In Protein A chromatography the antibody binds to the stationary
phase while the remaining host cell proteins (HCP) of the expression
cell line flow through the column and are usually reduced by two to
three orders of magnitude. Subsequent mAb elution requires a pH
shift of the mobile phase to pH 3-4. These non-physiological condi-
tions are further applied for acidic virus inactivation but may cause
mAb aggregation. The new TOYOPEARL AF-rProtein A HC-650F car-
ries a recombinant ligand that is stable in alkali solutions applied in
industrial cleaning procedures. It benefits from superior mAb capacity
and increases capturing productivity. Besides higher capacities, the
mAb uptake behavior is a major driver in process economics. High
capacities at high feed concentrations will lead to concerting effects
when it comes to fast and efficient capturing solutions.
PROTEIN A CHROMATOGRAPHY WITH HIGH TITER
FEEDSTOCKS
PROTEIN A CHROMATOGRAPHY HAS BECOME A WIDELY USED PLATFORM IN MONOCLONAL ANTIBODY (mAb) PURIFICATION. IT MAKES
USE OF THE SPECIFIC INTERACTIONS THAT TAKE PLACE BETWEEN THE FC REGIONS OF IMMUNOGLOBULIN G AND IMMOBILIZED PRO-
TEIN A, A CELL WALL COMPONENT OF STAPHYLOCOCCUS AUREUS. HEREIN, WE DESCRIBE THE IMPACT OF COMPARABLY HIGHER mAb
LOADINGS ON AGGREGATION DURING PROTEIN A CHROMATOGRAPHY, AS WELL AS THE BENEFITS OF THE NEW ULTRA-HIGH CAPACITY
TOYOPEARL AF-rProtein A HC-650F CHROMATOGRAPHY RESIN FOR HIGH TITER FEEDSTOCKS.
04
APPLICATION
ANTIBODY PURIFICATION
0
14
12
10
8
6
4
2
Time (min)
100
200
300
400
500
600
0
UV absorbance @ 280 nm [mAU]
50 mg/mL
10 mg/mL
dimer
monomer
FIGURE 2: AGGREGATE CONTENT
Size exclusion chromatogram of two Protein A elution pools. Feed: 4.75 g/L IgG to a
total loading of 10 mg/ml and 50 mg/ml, Elution: 100 mM acetate buffer, pH 3.25, 50 L
of the respective pools analyzed using TSKgel SuperSW mAb HR
FIGURE 1A & B: DYNAMIC BINDING CAPACITY
Breakthrough curves of mAb A on TOYOPEARL AF-rProtein A HC-650F packed into
a 6.6 mm ID X 2 cm L column; residence time 1 min, 2 min, 5 min A: 5 g/L mAb A;
UV
max
:1315 mAU. B: 10 g/L mAb A; UV
max
: >2000 mAU.
0
2
4
6
8
10
12
14
16
volume [mL]
0
40
20
60
80
100
120
140
160
UV absorbance @ 280 nm [mAU]
feed concentration: 5 g/L
1 min residence teime
2 min residence teime
5 min residence teime
0
2
4
6
10
8
12
volume [mL]
350
250
300
150
200
50
100
-50
0
UV absorbance @ 280 nm [mAU]
feed concentration: 10 g/L
1 min residence teime
2 min residence teime
5 min residence teime
