Tosoh
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MAb Aggregate Removal by Salt-tolerant Ion
Exchange Chromatography
Purification schemes for monoclonal antibodies typically consist of three chromatographic steps accompa-
nied by filtration steps. The common Protein A capturing is typically followed by ion exchange (IEC), hydrophobic
interaction (HIC), or mixed-mode polishing steps. Residual DNA, viruses, and host cell proteins are usually removed
by flow-through anion exchange chromatography while aggregates can be reduced through a cation exchange,
mixed-mode, or HIC step.
04
Application
Monoclonals I
The salt tolerant anion exchange resin TOYOPEARL NH2-750F provides
a unique selectivity compared to other anion exchange resins and was
also found to be suited for aggregate removal. In general, anion exchange
resins can be used in bind and elute (B/E) mode as well as in flow-through
(FT) mode. Both options were evaluated for TOYOPEARL NH2-750F. To
increase the amount of aggregates of the test sample, a monoclonal an-
tibody was aggregated by acidic incubation and subsequently diluted to 1
g/L in loading buffer.
The dynamic binding capacity of TOYOPEARL NH2-750F for the mAb used
in this study was evaluated and a value of 95 mg/mL could be reached
with 10 mM Tris/HCl, pH 8.0 for B/E mode. SEC analyses of fractions of
the elution profile of the aggregated antibody on TOYOPEARL NH2-750F
show that aggregates do not elute in the salt gradient and remain bound
until the column is CIPed with sodium hydroxide. Fractions 10 to 14 have
an aggregate content below the limit of detection of SEC.
In order to establish a FT polishing step, buffer conditions were evaluated
to optimize non-binding conditions for the monomer by varying pH and salt
content. Best results were obtained with 10 mM Tris/HCl, pH 7.0 at a sodi-
um chloride concentration of 250 mM. To analyze the aggregate removal,
100 mg aggregated antibody were loaded on a 2 mL column and fractions
of the flow through were analyzed by SEC. All FT fractions are essentially
aggregate free.
TOYOPEARL NH2-750F is ideally suited to develop a polishing step for
monoclonal antibody by either using the resin in BE mode or in FT mode.
For both modes ideal conditions for aggregate removal could be establis-
hed. An additional benefit when using this resin in FT mode is the deliver-
ed excellent viral clearance. Typical virus log reduction exceeds five for
enveloped and non-enveloped DNA and RNA viruses.
Figure 1
Elution profile of aggregated antibody on TOYOPEARL NH2-750F in B/E mode and analysis of fractions by SEC on TSKgel G3000SW
XL
Figure 2 :
SEC analysis of the aggregated mAb sample (red line at
0 mAU) and flow-through fractions in increasing fraction order from
bottom to top.
0
20
40
60
80 100 120 140 160 180
0
2
4
6
8
10
12
14
15
20
10
1
5
Volume [mL]
time [min]
100
80
60
40
20
0
100
80
60
40
20
0
50
40
30
20
10
0
50
40
30
20
10
0
UV absorbance @ 280 nm [mAU]
UV absorbance @ 280 nm [mAU]
% B
UV 280
conductivity
fractions
% B
conductivity [mS/cm]
fraction # 10
fraction # 11
fraction # 12
fraction # 13
CIP
0
2
4
6
8
10
12
14
16
time [min]
100
200
300
0
UV absorbance @ 280 nm [mAU]
