Antibody analysis using High Performance Capillary Electrophoresis (HPCE)

Label Free Intrinsic Imaging (LFII®) analysis was carried out on the monoclonal antibodies Infliximab and Rituximab for the assessment of stability, fragmentation and charge heterogeneity. This work was performed as part of a study for the UK’s National HealthService (NHS) using a deltaDOT HPCE-512TC Capillary Electrophoresis instrument. Quantifiable differences were observed in the samples before and after heat-stress. Excellent repeatability was also obtained with a peak area Relative Standard Deviation (RSD) of 2.9% and a migration time RSD of 2.3%.

Capillary Gel Electrophoresis (CGE)

mAb samples were diluted to a concentration of 0.5mg/ml in CGE sample buffer and run under reducing and non-reducing conditions. The reduced samples were treated with β-mercaptoethanol and heated at 70°C for 10 minutes. The non-reduced samples were heated at 70°C for 10 minutes with no reductant.

Reducing Conditions

The electropherograms below show the excellent separation of the liberated heavy and light chains achieved in 20 min. Several minor components were also resolved.

Figure 1. Electropherograms of Rituximab reference (stock) and test (saline drip) samples under reducing conditions. The expansion shows minor components present in the samples and demonstrates the power of the signal processing to resolve all peaks present including small ones close to much larger peaks. Without this ability, product quality could not be properly assessed.

Table 1. Molecular weights (kDa), corrected peak areas and % peak areas for the reference and test samples

Table 1. Molecular weights (kDa), corrected peak areas and % peak areas
for the reference and test samples

Non-reducing conditions

Without prior reduction of the disulphide bridges, the intact mAb is resolved from a number of other components.

Figure 2. Electropherograms of Rituximab reference (stock) and test (saline drip) samples under non-reducing conditions. Peak 7 is the intact peak.

Table 2. % of intact mAb compared to other species present in the (a) reference and (b) test samples.

Table 2. % of intact mAb compared to other species present in the (a) reference and (b) test samples.

Capillary Zone Electrophoresis (CZE)

CZE (or free solution CE) was used to assess the charge heterogeneity of Infliximab which can reveal differences in post-translational modification.

Significant differences were observed between the control samples and the stressed samples at the temperatures tested. These changes could potentially lead to major differences in the efficacy of the drug product.

Figure 3. GST electropherogram showing the separation of the Infliximab sample stored at 2-8°C and the sample stored at 40°C for a prolonged period. The main peak is marked with an asterisk.

Further Information

In addition to antibodies, HPCE can be used for a wide range of analytes including:

Proteins
Peptides
Amino Acids
Nucleic Acids
Nucleotides
Carbohydrates
Viruses
Pharmaceuticals
Pesticides
Chiral Molecules
Inorganics

To see what HPCE can do to solve your analytical challenges, please contact me on mark@discoveryanalyticalconsulting.com for a sample submission form plus a full list of what's available at deltaDOT.

Mark PortsmouthComment