Understanding the thermal stability of a monoclonal antibody and its modified version using dynamic light scattering

Executive summary

Dynamic light scattering (DLS) provides a powerful and sensitive approach to investigating the colloidal stability of biotherapeutic proteins under formulation conditions.

Here, we demonstrate that  DLS thermal ramp and isothermal incubation studies reveal significant differences in the thermal stability of a template monoclonal antibody (mAb) and a modified monoclonal antibody (mmAb) – providing significant insights  that would not be fully captured by bulk aggregation assays or turbidity measurements alone.

 In particular, DLS reveals that the lower thermal stability of the mmAb, evidenced by early oligomerization at approximately 50°C, can be tracked in detail through Z-average size, polydispersity index (PDI), and size distribution data – demonstrating the value of multiple complementary DLS parameters in characterizing complex aggregation behavior.

DLS-based colloidal stability profiling of this kind forms the foundation of rigorous early-stage biologics characterization. The integration of DLS with orthogonal structural stability techniques, including static light scattering (SLS) and intrinsic differential scanning fluorimetry (DSF), in unified, high-throughput platforms represents the next step in delivering this depth of insight at the speed and scale that modern biologics workflows demand.

Introduction

In developing biopharmaceutical products, characterizing thermal stability is an important step in the process of learning about and optimizing monoclonal antibodies (mAbs) and their formulations. Differential scanning calorimetry (DSC) is the standard technique used to characterize thermal behavior, providing an endotherm from which one can derive stability and thermodynamic properties. In order to minimize aggregation during heating, DSC samples are run at low concentrations (0.5 mg/mL–1.0 mg/mL). The technique is therefore not well suited to testing the stability of higher concentrations that are typical of formulated biotherapeutic products. Furthermore, although the assumption is that no aggregation occurs as a result of heating these low-concentration samples, there is no way to confirm this from DSC data alone, and samples with visual evidence of aggregation are often the end product of a DSC run.

DLS enables the study of thermal stability at formulation concentrations and with excipients present. Critically, DLS is able to detect the earliest signs of aggregation and oligomerization that bulk techniques such as turbidity and SLS cannot resolve, thanks to its sensitivity to size distribution and polydispersity. When combined with structural stability readouts, DLS data can also help differentiate unfolding events from aggregation events: a distinction of significant mechanistic and formulation relevance.

In contrast to globular proteins (see Inform White Papers on use of DLS/Raman to characterize BSA and Lysozyme) in which secondary structure changes induced by thermal stress are often significant, the secondary structure in mAbs is known to be quite stable. This is due to the fact that the structural framework is rich in β-sheet, with covalent disulfide bonds providing additional stability. Thermal stress alone is not anticipated to significantly perturb secondary structure. In this context, DLS provides particularly valuable early warning of colloidal instability, detecting oligomerization and aggregation events that reflect the tertiary structural vulnerabilities of individual candidates.

In the case study presented in this white paper, we compare the thermal stability of a mAb that has been structurally modified (i.e. a modified monoclonal antibody or mmAb) and the corresponding template mAb upon which the modification has been performed.

Materials and methods

Malvern Panalytical’s Zetasizer Advance instruments integrate proprietary non-invasive backscatter (NIBS) detector technology with dynamic (DLS), static (SLS) and electrophoretic (ELS) light scattering to measure the hydrodynamic radius of proteins from 0.15 nm–5 µm, from 0.1 mg/mL to ≥ 100 mg/mL. The mAb and modified mAb (mmAb) were provided at 50 mg/mL in 10 mM His buffer at pH 5.5. The samples were stored at 4 °C until the studies were conducted. Sample aliquots (~120 µL) were placed into a 3 mm quartz cuvette and positioned in a compartment providing temperature control from 0 °C–90 °C ± 0.1 °C. Thermal ramp studies were conducted by collecting DLS data over a series of pre-defined 0.1 °C – 5 °C stepwise increments. Isothermal incubation studies were conducted by collecting a series of DLS data over a predefined time interval at the desired temperature set-point.

Results and discussion 

Dynamic light scattering

Figure 1: Z-average size (A) and polydispersity index, PDI (B) derived from dynamic light scattering (DLS) plotted for the monoclonal antibody (blue trace) and its modified version (red trace) as a function of temperature

Figure 1A demonstrates that the aggregation onset for the mAb occurs at a higher temperature than for the mmAb, again demonstrating its high thermal stability. The mmAb exhibits a double transition, specifically for the tyrosine H-bonding environment. The DLS Z-average size data shows that the lower temperature transition is an oligomerization event at ~50 °C. The higher temperature transition for the mmAb (~65 °C) is a rapid and major aggregation event, as seen in the mAb sample >70 °C.

The PDI trend, shown in Figure 1B, is consistent with this interpretation. For the mAb sample, the PDI fluctuates around 0.1 until a single significant increase is seen at ~60°C. The PDI evolution for the mmAb sample shows two peaks at ~50 °C and ~62 °C, indicating transitions from a relatively homogeneous size distribution to a polydispersed distribution, and then dropping back to relatively homogeneous before finally becoming quite polydispersed again. This suggests that the mmAb samples form oligomers during the first transition, and that these oligomers then form the large aggregates indicated in the second transition.

Figure 2: Intensity and volume distribution data derived from dynamic light scattering data for the monoclonal antibody (A) and its modified version (B). Data collected at different temperatures plotted in different colors

DLS determines not only size itself, but also size distribution, which is not obtainable using static light scattering or turbidity measurements alone. Figure 2A1 illustrates that for the mAb, a peak of ~100 nm appears at ~65 °C, while for the mmAb (Figure 2B1), a population of particles with this size is present at ~50°C. It should be noted that due to the extreme sensitivity of DLS, the presence of 0.1% (by volume) large aggregates will generate the equivalent scattering intensity as 99.9% of particles 1/10th of their size. Therefore, the transition temperature derived from the volume-weighted distribution is higher than the same value derived from an intensity-weighted distribution. For the mAb, the volume distribution shows a clear increase at~70 °C (Figure 2A2), while for the mmAb, the peak shifts to ~57 °C (Figure 2B2).

The PDI appears to be the metric which is most sensitive to aggregation, compared to Z-averaged size and volume distributions. In Figure 1B, the aggregation onset temperature from Z-averaged size is ~65 °C, but analysis of the PDI puts the onset temperature as low as ~60 °C.

It is important to note that the determination of hydrodynamic size from dynamic light scattering is based on the Stokes-Einstein equation, which requires the solution viscosity to be known. At relatively low concentrations (~5% by weight), using the viscosity of water to approximate that of the solution is reasonable. However, once the protein is aggregated, precipitated, or gelled, this approximation is most likely no longer valid. Under such circumstances, the size values derived are likely not precise, but it can be assumed that the trends they exhibit are qualitatively correct.

Isothermal incubation

The above results strongly indicate that the differences in the thermal stability profiles of the mAb and mmAb are due to oligomerization of mmAb, driven by tertiary structure (side chain) unfolding. However, what is not clear is the kinetics of this process and how stable the oligomers are. Further experiments to investigate the kinetics of oligomerization of the mmAb were conducted. Specifically, isothermal incubations at pre-transition (46 °C), in-transition (53 °C) and post transition (60 °C) temperatures were conducted. The results are shown in Figure 3.

Figure 3: An isothermal incubation study for mmAb, Z-averaged size derived from dynamic light scattering data (A); Tyr peak position (B); and Trp peak position (C). The different incubation temperatures are presented as blue (46 °C), red (53° C), and green traces (60° C), plotted as a function of time

Incubated at the pre-transition temperature, 46 °C, the size slowly increases. At 53 °C, however, the size quickly increases from 10 nm to ~35 nm and then stabilizes.

Concurrently, the Tyr peak position drops, while the Trp peak position shifts slightly. More specifically, the Tyr peak position shifts from 856.8 cm^-1 to 855.5 cm^-1, the value seen for the first transition in Figure 3B. At 60 °C, the size quickly increases toover 40 nm within 3 hours, and both Trp and Tyr peak positions quickly shift lower, and again the Tyr peak position shifts to ~855.5 cm^-1.

These observations further support the suggestion that the newly added or modified tyrosine residues trigger oligomer formation at lower temperature, which ultimately leads to lower thermal stability for the modified monoclonal antibody. Even more interestingly, on returning to the starting temperature after the isothermal incubation, the Tyr peak position remains lowered, but the peak position of the Trp reverts to some extent (data not shown).

As mentioned above, the final peak position of Tyr following the 53 °C incubation is close to that seen after the first transition has occurred during the thermal ramping experiment, shown in Figure 3B. Therefore, we stopped the incubation at 60 °C, considering the first transition completed, and cooled to 20 °C to test the thermal reversibility of the oligomers.

Figure 4: The isothermal incubation study for mmAb with dynamic light scattering data (A), Tyr peak position (B) and Trp peak position (C) along the incubation time at the specified temperature

In Figure 4A, the initial size is ~50 nm and remains as such until the final aggregation onset close to 64 °C. Trp and Tyr do not exhibit the characteristic double transitions, just a single transition at ~64 °C, as was seen for the size transition. This indicates that the oligomers formed at ~50 °C are stable and do not dissociate by cooling. Dilution experiments (data not shown) also indicate that the oligomerization is not completely reversible.

Conclusions

Using DLS thermal ramp and isothermal incubation studies, this work demonstrates clearly that:

1. The tested monoclonal antibodies have different secondary structure transition profiles compared to model globular proteins.
2. The thermal stability of the modified mAb is lower than the mAb, evidenced by the formation of oligomers at ~53 °C.
3. The mechanism of oligomer formation appears to be driven by a change in the tyrosine side chain.
4. The oligomers formed are stable and not reversible by cooling or diluting.

Methodologically, multiple DLS-derived parameters can be used, including Z-averaged size, PDI, and size distribution. However, PDI appears to be the most sensitive early indicator of aggregation onset, and size values require careful interpretation if the solution viscosity changes significantly from that of water.

This work illustrates the diagnostic value of DLS in characterizing complex aggregation behavior in biotherapeutic candidates: sensitivity that is not achievable through bulk turbidity or static scattering measurements alone. The ability to detect early oligomerization events, track size distribution changes across temperature ramps, and assess the reversibility of aggregation through isothermal studies makes DLS an essential tool for early-stage stability screening.

While DLS provides powerful colloidal stability insight, the full picture of biotherapeutic stability requires orthogonal structural data. The integration of DLS with complementary techniques that can deliver a range of stability data represents the logical next step, enabling the depth of characterization demonstrated here at the speed and scale that modern formulation screening workflows demand.