Transient Protein Self-Association Determined by SEC–MALS

Domain antibodies (dAbs) are the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy (V_H) or light (V_L) chains of human antibodies. Different dAbs can show different degrees of self-association, which drives the need for a cheap, quick — yet robust — technique to study self-association behaviour in drug discovery. This study outlines the use of SEC–MALS to determine self-association, and validates the work by comparison with analytical ultracentrifugation (AUC).

Methods

Shimazdu LC-20AD Prominence was used in series with miniDAWN TREOS and Optilab REX for this study. The columns used were TSK-GEL3000SW_XL and Superdex S200.

dAb_1 was expressed, purified, and dialyzed into PBS, pH 7.4 and submitted to SEC–MALS and AUC. For MALS the sample was concentrated to 2.4 mg/mL, 7.0 mg/mL, 75 mg/mL, and 130 mg/mL and followed by appropriate dilution to obtain nine different concentrations that were run on SEC–MALS (Table 1).

Table 1: List of the samples injected, Mw and concentration as determined by SEC–MALS.

K_2,1 (Propensity of dAb_1 to dimerize) was determined by plotting a graph of M_w versus log₁₀ concentration obtained from SEC–MALS mid peak.

Figure 1: Overlay of UV, dRI, and MALS signal from SEC–MALS for the sample with stock concentration 15 mg/mL.

The K_2,1 values for dAb_1 from SEC–MALS was calculated as 300 μM compared to 391 μM from the AUC experiment.

Figure 2: Overlay of the five different sample results from SEC–MALS highlighting the ’Determined Mw’. Samples with stock concentration 5 mg/mL, 15 mg/mL, 30 mg/mL, 60 mg/mL, and 120 mg/mL represent the smaller to bigger peak, respectively.

Conclusion

While AUC is the gold standard for the determination of self-association in solution, scientists are looking for alternatives as a result of AUC's high costs, low throughput, and expertise requirements. Here, SEC–MALS has shown excellent agreement with AUC results and can be used as a cheaper, easier, and quicker alternative.

This note was graciously submited by M. Burman and O. Schon, GlaxoSmithKline, 315 Cambridge Science Park, Cambridge CB4 0WG, United Kingdom.

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