How to analyze antibody aggregation and molecular weight using BeSEC

Monoclonal antibodies (mAbs) are highly specific proteins that can recognize and bind to antigens and pathogens, such as viruses and bacteria, enabling their rapid detection through assays such as immunofluorescence and ELISA. 

Beyond diagnostics, mAbs have a crucial role in oncology as they can identify tumor-associated antigens and support early cancer screening.

As therapeutics, monoclonal antibodies target tumor cells directly. For example, Rituximab binds CD20 on B-cell lymphoma cells, which catalyzes immune-mediated clearance. Antibodies can also be conjugated to radionuclides and chemotherapeutic agents to create antibody-drug conjugates (ADCs), which deliver cytotoxic payloads to tumor cells while reducing any off-target effects.

During formulation, monoclonal antibody solutions often contain dimers, trimers, and higher-order aggregates. These species pose safety concerns because they can increase immunogenicity upon administration. As a result, the accurate quantification of oligomers and aggregates is crucial for formulation development, quality control, and aggregate characterization.

Experimental section

This study used a size-exclusion chromatography (SEC) system equipped with light-scattering (LS) and refractive-index (RI) detectors. The chosen LS detector was the BeSEC LS2 from Bettersize Instruments, which has detection angles of 90 ° and 7 °. The BeSEC workstation integrated light scattering with UV or RI signals to calculate molecular weight distributions and averages, including Mn, Mw, and Mz.

System configuration:

• Detectors: Light Scattering (LS) + RI
• Column: Shodex PROTEIN LW-803
• Mobile phase: Phosphate-buffered saline (PBS)
• Flow rate: 0.7 mL/min
• Injection volume: 100 μL
• Column temperature: 40 ℃
• dn/dc: 0.185 mL/g

Sample preparation:

Three monoclonal antibody samples (A, B, and C) were prepared for analysis. The samples were each weighed, dissolved in PBS to a concentration of 2-5 mg/mL, stirred until completely transparent, and filtered through a 0.22 μm PES syringe filter.

The filtered solutions were transferred into autosampler vials for the SEC injection.

Results and discussion

Figure 1. Elution profiles of the multi-detector signals for Sample A. Image Credit: Bettersize Instruments 

Figure 2. Elution profile of the molecular weight for Sample A. Image Credit: Bettersize Instruments 

Figure 1 shows the elution profiles of the multi-detector signals for Sample A, while Figure 2 shows the corresponding molecular weight distribution.

The light-scattering signals at 90° and 7°, along with the RI trace, reveal multiple distinct peaks, suggesting that the sample contains multiple aggregation states.

The molecular weight of each species was calculated using peak-based integration, as summarized in Table 1. The elution volume versus molecular weight plot shows that as elution volume increases, molecular weight decreases.

This is consistent with the SEC separation principle, in which smaller species elute later than larger ones. Each peak shows a molecular weight plateau, which reflects the comparatively narrow molecular weight distribution of the individual oligomeric states within the protein sample.

Table 1. Molecular weight results for peaks in Sample A. Source: Bettersize Instruments 

The molecular weight of Peak 1 was approximately 150 kDa, compatible with the theoretical value of a monoclonal antibody monomer. Peaks 2 and 3 scaled as clean multiples of Peak 1, correlating to the dimeric and trimeric species.

Quantitative analysis showed that the monomer accounted for 75.80 % of the total protein, while aggregated species collectively made up more than 5 %. For therapeutic applications involving injection, these aggregates are particularly concerning, as they could increase immunogenic risk and compromise product safety.

Figure 3. Elution profiles of the multi-detector signals for Sample B. Image Credit: Bettersize Instruments 

Figure 4. Elution profile of the molecular weight for Sample B. Image Credit: Bettersize Instruments 

Figure 5. Elution profiles of the multi-detector signals for Sample C. Image Credit: Bettersize Instruments 

Figure 6. Elution profile of the molecular weight for Sample C. Image Credit: Bettersize Instruments 

Figures 3 and 4 show the chromatogram and molecular weight profile of Sample B. A smaller peak is also seen with an earlier elution time. This suggests that higher-order aggregates were present.

Figures 5 and 6 present the chromatogram and molecular weight profile for Sample C, which exhibits a single dominant peak, suggesting minimal aggregation.

Table 2. Molecular weight results for peaks in Sample B. Source: Bettersize Instruments 

Table 3. Molecular weight results for peaks in Sample C. Source: Bettersize Instruments 

Table 2 shows that, in addition to the primary monomer peak, sample B also contains a dimer. The dimer’s molecular weight is approximately twice that of the monomer, and its relative abundance is low at just 1.34 %, suggestive of a low level of aggregation.

In contrast, Table 3 shows that Sample C only contains the main peak, with no detectable oligomers or higher-order aggregates. This profile reflects exceptional structural integrity and suggests that Sample C is more stable than the other tested formulations.

Conclusion

This article has detailed how the BeSEC LS2 detector was used to measure the molecular weights of monoclonal antibody samples.

The presence of aggregates in biopharmaceuticals may substantially impact patient safety and therapeutic performance, as large aggregates could trigger immune responses. Therefore, understanding the type and level of aggregates that are present in monoclonal antibody formulations is crucial.

The BeSEC system demonstrates that static light scattering can effectively resolve monomer, dimer, and higher-order species and quantify their content. This is a valuable ability for the development, stability assessment, and quality assurance of biological drugs.

Acknowledgments

Produced using materials originally created by Zhibin Guo from Bettersize Instruments.

About Bettersize Instruments

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