Exploring the Biophysical Characterization of Monoclonal Antibodies

This article and associated images are based on a poster originally authored by Karolina Tokarczyk, Natalia Markova, and Andrew Scott and presented at ELRIG Drug Discovery 2024 in affiliation with Concept Life Sciences and Malvern Panalytical Ltd.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

Dynamic interactions involving biomolecules drive and regulate all biological processes, making interaction analysis a key area of academic and industrial research and development. The specificity of binding interactions of biomolecules, and thus their function, is tightly linked to their Higher-Order Structure (HOS) and stability.

Among biomolecules, antibodies have naturally evolved as a class of proteins with the unique ability to bind selectively and tightly to diverse targets. This ability has positioned antibodies as one of the key classes of biotherapeutics.

The integrity of an antibody's HOS is critical to its binding specificity to antigens and Fc-receptors on immune cells. These are essential for initiating immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Alterations in HOS can impair these interactions, reducing the antibody's therapeutic efficacy.

Robust product characterization throughout all phases of antibody development is required to secure control over its structure and function. The intrinsic complexity of antibodies' structural organization poses significant analytical challenges and requires domain resolution. By employing biophysical techniques, researchers can probe various aspects of antibody behavior, from assessing conformational integrity and aggregation propensity to determining binding kinetics and elucidating post-translational modifications.

Image Credit: Concept Life Sciences

Antibody Characterization

Chemical modification of monoclonal antibodies (mAbs) can be studied by generating samples under various stress conditions specific to the modification type. Degradation pathways under stress conditions may include mAb oxidation, deamidation, isomerization, deglycosylation, glycation, and fragmentation.¹

Here, an extensive analysis of the impact of oxidation on mAb stability is reported.

Methionine (Met) groups, present in the Fc region of most antibodies (Figure 1), are particularly sensitive to oxidation. Met oxidation may reduce mAb conformational and colloidal stability and decrease mAb interaction with Fc receptors. Therefore, assessing mAb oxidation during the early stages of mAb discovery can improve the engineering process by eliminating oxidation liability and selecting leading therapeutic candidates with maintained binding activity.

In this study, samples were treated with various concentrations of hydrogen peroxide (H₂O₂) to produce different levels of mAb modifications through oxidation of H₂O₂-exposed methionine residues (Table 1). The level of oxidation was assessed for a model commercially available antibody.

Figure 1. Schematic illustration of methionine oxidation of Immunoglobulin G (IgG). Image Credit: Concept Life Sciences

Table 1. Summary of experimental parameters used for forced oxidation of Trastuzumab. Source: Concept Life Sciences

A set of orthogonal biophysical tools were used to probe the effect of stress on the stability and function of Trastuzumab.

Differential scanning calorimetry (DSC) was used to test the impact of stress conditions on the structural stability of individual domains of the antibody molecules. Dynamic Light Scattering (DLS) was used to assess sample heterogeneity. Multi-detector ultra-performance liquid chromatography (UPLC) provided insight into antibody monomeric purity.

Multiplex analysis using grating-coupled interferometry (GCI) biosensor technology was employed to assess the integrity of antibody binding sites specific to both the antigen and Fc receptor.

Sample Homogeneity—DLS

The aggregation of mAb therapeutics is a serious concern that can impact product safety and efficacy. Rapid estimation of size-based heterogeneity in mAb can be assessed by using DLS.

In this study, the DLS assay verified that trastuzumab homogeneity was reduced upon stress (Table 2). Aggregation was also detected for all stressed samples (Figure 2).

Table 2. Summary of DLS data for native and stressed Trastuzumab. Source: Concept Life Sciences

Figure 2. (Top) Size distribution and (bottom) correlogram of native and four stressed mAbs analyzed with DLS. Image Credit: Concept Life Sciences

Monomeric Purity—Multi-Detector UPLC

The fraction of intact monomer, or monomeric purity, is a critical quality attribute in therapeutic mAbs.

The multi-detector UPLC analysis highlighted similar monomeric purity across all samples with a low level of fragments and aggregates detected (Figure 3, Table 3). The DLS data (Figure 2) showed a much higher level of aggregation in stressed samples compared to the multi-detector UPLC results. 

The aggregates detected by DLS may have resulted from weak associations of fragments, which could have dissociated under the conditions used in the multi-detector UPLC analyses.

Figure 3. Chromatograms of native and four stressed mAbs analyzed with multi-detector UPLC. The RI signal is presented in red, the UV/Vis signal at 280 nM is purple, and the right angle light scattering detector is green. Image Credit: Concept Life Sciences

Table 3. Molecular characterization data for native and stressed Trastuzumab. Source: Concept Life Sciences

Thermal Stability—DSC

Stability assays help identify formulations less likely to exhibit long-term stability and aggregation issues. Early physicochemical characterisation can result in more cost‑effective drug production and an increased probability that the mAb will remain active, stable, and correctly folded.

Trastuzumab showed two distinct unfolding transitions (Figure 4), which, based on the signal amplitude and prior knowledge of mAb thermal transitions, could be unambiguously assigned to the unfolding of CH₂ and Fab domains 2. The latter’s unfolding is likely to have overlapped with the unfolding of the CH₃ domain, indicating inter-domain cooperativity. 

Forced oxidation of mAb resulted in a reduction in the thermostability of mAb, as transition temperature (T_m1) decreased in comparison with the unmodified sample. A change in only one of the transition regions following hydrogen peroxide treatment indicates destabilization of the CH₂ domain due to Fc methionine oxidation, with no effect on the Fab fragment or CH₃ domain (Table 4).

Figure 4. DSC unfolding curves of the native and four oxidized mAbs. Image Credit: Concept Life Sciences

Table 4. Summary of DSC data for native and stressed Trastuzumab (total area, total heat, T_onset and melting temperature (T_m) values for the thermal transitions are shown). Source: Concept Life Sciences

Interaction Analysis—GCI

The stability of an antibody greatly influences its performance, including its specificity and binding affinity. The GCI assay can identify the impact of the modifications on the therapeutic antibody's binding to the antigen and Fc-receptor.

Kinetics and Affinity

The GCI assay can detect subtle changes in antibody reactivity, providing domain-specific analysis of mAb binding to both the antigen and Fc receptor (Figure 5).

The assay showed no significant differences in binding levels and affinities between stressed and unmodified mAbs binding to the antigen, consistent with the minor differences in Fab domain stability observed in DSC measurements. However, a decrease in signal was observed with increasing oxidation levels when mAbs were tested against FcRn, aligning with the significant CH₂ domain destabilization indicated by DSC.

Figure 5. Double–referenced sensorgrams of native and stressed Trastuzumab (analyte) samples binding to His-captured FcRn (left) and ERBB2 (right). Image Credit: Concept Life Sciences

Calibration-Free Concentration Analysis (CFCA)

CFCA is a tool used to quantify the active concentration of mAbs, which is associated with the binding properties, without the need for external calibration standards.

CFCA analysis of native and stressed Trastuzumab binding to the antigen confirmed only minor changes in the sample's active concentration (Figure 6, Table 5), which agrees with the binding data (Figure 5).

Figure 6. CFCA of unmodified Trastuzumab binding to ERBB2 at two various flow rates. Image Credit: Concept Life Sciences

Table 5. CFCA of native and stressed Trastuzumab binding to immobilized ERBB2. Source: Concept Life Sciences

Effect of pH on mAb Binding to FcRn

The GCI assay can be used to study FcRn interactions with Trastuzumab at low (~6.0) and high (~7.4) pH to mimic conditions where Fc receptors protect the mAb from lysosomal degradation (low pH) and where the antibody can be released on a cell surface (high pH) (Figure 7).

Figure 7. Double–referenced sensorgrams of native and stressed Trastuzumab (analyte) samples are binding to captured FcRn at pH 7.4 (left) and pH 6.0 (right). Image Credit: Concept Life Sciences

Summary

• A combination of biophysical characterization techniques provides insight into antibody stability and activity. They can be used to characterize the effects of stress during antibody development.
• Changes in HOS observed in stressed mAb samples correlated well with changes in Fc receptor and antigen binding to mAb domains.
• Resolution at the domain level facilitates understanding of the structure-function relationship.
• Early physicochemical characterization of therapeutic antibodies can result in more cost-effective production and increase the probability that the chosen mAb will remain active, stable, and correctly folded during process development and storage.

References

1. Gupta, S., Jiskoot, W., Schöneich, C. and Rathore, A.S. (2022). Oxidation and Deamidation of Monoclonal Antibody Products: Potential Impact on Stability, Biological Activity, and Efficacy. Journal of Pharmaceutical Sciences, 111(4), pp.903–918. https://doi.org/10.1016/j.xphs.2021.11.024.
2. Temel, D.B., Landsman, P. and Brader, M.L. (2016). Orthogonal Methods for Characterizing the Unfolding of Therapeutic Monoclonal Antibodies. Methods in Enzymology, pp.359–389. https://doi.org/10.1016/bs.mie.2015.08.029.

About Concept Life Sciences

Concept Life Sciences is a knowledge-based, science-led and customer-focused contract research and manufacturing organization with world-leading expertise in the pharmaceutical, biotech and agrochemical industries.

We are based across 5 sites in the UK delivering discovery research as well as GMP, GLP and GCP-regulated work. Whether it is delivering whole programs of research or bespoke studies we deliver a collaborative client-centered approach. Our services are built on scientist-to-scientist engagement, deep knowledge, flexibility and extensive in-house resources.

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