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Size exclusion chromatography (SEC) is commonly used in the QC of
protein therapeutics to separate aggregates and monomers. Accelerating
SEC has turned out to be a hot topic for shortening analysis time
per sample and keeping up with growing production. Especially for
routine analysis it might be favorable to sacrifice maximum performance
for a significant reduction of analysis time. Focusing on the total aggregate
content, one might accept to lose resolution between higher molecular
weight aggregates.
Figure 1 shows an aggregated mAb sample analyzed on TSKgel
SuperSW3000 4.6 x 150 mm. Resolution for aggregates and
mAb monomer remains high, while analysis time is cut down to
4 minutes. This is only a quarter of the conventional analysis time,
including fragment elution. To accomplish a less time-consuming analysis,
column dimensions were changed and the linear flow rate was increased
compared to standard columns. Even when using high flow rates
pressure doesn’t exceed 130 bars. Higher pressure might cause frictional
heating inside the column, although the solvent as well as the column are
temperature controlled. Sensitive samples like proteins might then suffer
from aggregation or denaturation on the column, resulting in incorrect
results.
Besides reaching for faster analytics, a back-up method for the verification
of SEC results would be desirable. The most common methods for the
determination of aggregate contents are based on difference in size for
aggregates and monomers. However, different hydrophobicities offer an
additional separation criteria. Aggregates are more hydrophobic than the
corresponding monomers, leading to a diverging retention behavior in
hydrophobic interaction chromatography (HIC). Aggregates, monomers,
and fragments can easily be identified, as the relative hydrophobicity
for these molecules is well known. Figure 2 shows an aggregated mAb
sample analyzed on 2.5 µm TSKgel Butyl-NPR 4.6 x 35 mm. The total
aggregate content of this sample is about 11%, which was confirmed by
SEC on TSKgel G3000SW
XL
7.8 x 300 mm.
Besides the quantitative aspect, the qualitative separation performance
of HIC is enormous. In contrast to SEC, HIC does not rely on varying
hydrodynamic radii. We know from dynamic light scattering and viscosity
measurements that the hydrodynamic radius does not correlate linearly to
the molecular weight. There are only slight differences for high molecular
weight aggregates, whereas the monomer and dimer differ significantly.
Therefore, SEC has a natural disadvantage regarding the separation
of high molecular weight aggregates whereas HIC enables precise
determination of miscellaneous aggregate species. However, HIC must
not be considered to replace SEC in standard analytics. Time consuming
method development is inevitable. Hydrophobicities of mAbs can
vary tremendously. This means that screening for the appropriate salt
concentration is crucial for a successful analysis. Further, the chosen
salt type or salt mixture has a great impact on resolution and efficiency.
This can be utilized to optimize resolution and modify selectivity for
different aggregate species. But for the same reason, HIC does not meet
the requirements of daily routine in a high throughput lab. Nevertheless,
HIC with nonporous particles is a very useful tool to overcome the natural
limits of aggregate analysis methods based on size. It opens up
opportunities to get a more accurate analysis of certain mAb samples and
to distinguish also glycosylation variants of the antibody.
Monoclonal antibodies are a growing segment within the biopharmaceutical industry. An increased utilization
and extended scope of application have trended production towards growing scales. However, high expression
rates also bear the risk of protein aggregation. This needs to be closely monitored because aggregates often
show increased immunogenicity.
04
PROTEIN
ANALYSIS
Figure 1: Aggregated mAb sample on TSKgel SuperSW3000.
Various flow rates are applied: 0.35 mL/min, 0.5 mL/min and 0.7 mL/min
Figure 2: Aggregated mAb sample on TSKgel Butyl-NPR.
Fragments elute first, followed by monomers and the aggregates.
New Approaches in Antibody Aggregate
Analysis – SEC and HIC
0
2
4
6
8
Time (min)
140
120
100
80
60
20
40
0
-20
A280 (mAU)
0,7 mL/min
0,5 mL/min
0,35 mL/min
monomer
monomer
monomer
10
12
14
8
Time (min)
0
emission @ 348 nm (counts)
conentraion eluent B (%)
-1x10
6
1x10
6
2x10
6
3x10
6
5x10
6
-60
-40
-20
0
20
40
60
80
100
120
fragment
glycosyation variant
monomer
aggregates
4x10
6