For Research Use Only. Not for use in diagnostic procedures.

Native State Charge Variant Analysis of Commercialized
Monoclonal Antibodies
1 Stephen Lock (stephen.lock@sciex.com) and 2Peter Holper
1SCIEX, Warrington, UK; 2SCIEX, Redwood City, USA
This poster described how a High resolution charge variant analysis can be done in minutes using commercial kits and is :-High resolution charge variant analysis done in minutes using commercial kits
Reproducible: Separations are shown to be reproducible through both replicate injections of commercial mAbs and an inter-company collaboration1
Platform capable: The simplicity of separation allows for application to molecules across a broad pI range between 7.0 and 9.51
High-throughput: High resolution separation can be achieved in less than 5 minutes with only two minutes of buffer replenishment in between injections,making the total sample analysis time under 10 minutes

Versatile: Additional resolution can easily be obtained through use of a longer bare-fused silica capillary, lower capillary temperature or separation voltage.

Further development is possible by modifying buffer pH and levels of additives2

Results of Application Tests
To test the application and the new CZE chemical kit, 4 different commercial mAbs were obtained. Figures 1 through 4 show the charge variant profile of Remicade, Herceptin, Rituxanand Drozitumab biosimilar obtained with the SCIEX CZE Rapid Charge Variant Analysis Kit. The insert in each figure represent the zoomed-in view of the baseline. Ten injections ofeach molecule were also made to show the reproducibility and robustness of the assay. The peak profiles were stable and the %RSDs of relative area percentages of most acidicvariants, basic variants and main peaks were below 2% (shown in Tables 1-4).
Table 1. Statistics for variant analysis
of Herceptin (trastuzumab).
TotalAcidic
Variants(%)

Table 2. Statistics for variant analysis
of Rituxan (rituximab).
Total Acidic Total Total Basic
Variants
Variants
Main
(%)
(%)
Peak (%)

Total Basic
Total Main
Variants
Peak (%)
(%)

Avg

28.7

66.1

5.2

STDEV

0.2

0.2

0.1

%RSD

0.8

0.4

1.4

12.0

78.7

9.4

STDEV

0.2

0.5

0.5

%RSD

1.7

0.6

5.5

Figure 2. Charge variant analysis of Rituxan (rituximab).

Figure 1. Charge variant analysis of Herceptin (trastuzumab).

Table 4. Statistics for variant analysis
of Remicade (infliximab).

Table 3. Statistics for variantanalysis of Drozitumab biosimilar.
TotalAcidic
Variants(%)

Avg

Total Acidic
Total Basic
Total Main
Variants
Variants
Peak (%)(%)(%)

Total Basic
Total Main
Variants
Peak (%)
(%)

Avg

24.2

72.1

3.7

STDEV

0.4

0.4

0.1

%RSD

1.8

0.5

1.5

Figure 3. Charge variant analysis of Drozitumab biosimilar.

Avg

11.1

38.450.5STDEV

0.2

0.2

0.1%RSD

1.5

0.4

1.3

Figure 4. Charge variant analysis of Remicade (infliximab).

Project Background
Biopharmaceutical companies are under increasing pressure to finalize analytical methods earlier in drug development using less material. Finding a robust, stability-indicating methodthat can monitor charge variants is an ongoing analytical challenge. Traditional method development approaches for native proteins involving ion-exchange chromatography (IEX) canspan weeks or months. Capillary isoelectric focusing (cIEF), although quicker than IEX, analyzes proteins in denatured forms that might not perform the same way as native proteins.
Capillary zone electrophoresis (CZE) combines the benefits of a native state analysis with the speed and resolution expected of a capillary electrophoresis method. CZE is the simplest
form of CE and separates based on the differences in a molecule’s electrophoretic mobility, which is directly related to the charge on the protein. Using a widely used buffer, thistechnique requires little to no method development, has a simple sample preparation and a higher analytical throughput than IEX or cIEF. In this project, a commercial available CEsystem is used, fitted with a UV detector, a prebuilt cartridge and a new CZE Rapid Charge Variant Analysis Kit (SCIEX P/N C4479) designed to be used for charge variant analysis ofproteins, to analyze 4 commercial monoclonal antibodies which have been prepared in CE Grade water (SCIEX P/N C48034).

REFERENCES
1. Moritz, B. et.al. J. Chromatogr. B 2015, 983–984, 101–110.
2. Moritz, B. et. Al. J., Electrophoresis 2017, 38, 3136-3146.
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