The stability of monoclonal antibodies (mAbs) is a key parameter in their development as therapeutics. The rapid and accurate assessment of antibody stability is of major interest to researchers and manufacturers to ensure the consistency of the mAb therapeutics in terms of their specificity and affinity.
In collaboration with Protein Stable and Malvern Panalytical, Concept Life Sciences has assessed the stability of native and oxidized monoclonal antibodies (mAbs) using Differential Scanning Fluorimetry (DSF) and Differential Scanning Calorimetry (DSC). Both methods enabled rapid identification of stability profiles with high sensitivity, facilitating efficient selection of stable therapeutics. Utilizing DSF and DSC together offers a comprehensive understanding of an antibody’s thermal stability, from initial screening to detailed thermodynamic analysis. A strong correlation between DSC and DSF ensures a reliable assessment of mAb stability.
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The stability of monoclonal antibodies (mAbs) is a key parameter in their development as therapeutics. The rapid and accurate assessment of antibody stability is of major interest to researchers and manufacturers to ensure the consistency of the mAb therapeutics in terms of their specificity and affinity.
In collaboration with Protein Stable and Malvern Panalytical, Concept Life Sciences has assessed the stability of native and oxidized monoclonal antibodies (mAbs) using Differential Scanning Fluorimetry (DSF) and Differential Scanning Calorimetry (DSC). Both methods enabled rapid identification of stability profiles with high sensitivity, facilitating efficient selection of stable therapeutics. Utilizing DSF and DSC together offers a comprehensive understanding of an antibody’s thermal stability, from initial screening to detailed thermodynamic analysis. A strong correlation between DSC and DSF ensures a reliable assessment of mAb stability.
Utilization of Concept Life Sciences Biophysical assay services for assessing monoclonal antibody stability.
To profile the stability of the therapeutic monoclonal antibody candidate, which is crucial for ensuring its efficacy and shelf-life.
Concept Life Sciences offers a tailored approach to assess the stability of monoclonal antibodies using advanced biophysical assays:
Monoclonal antibodies (mAbs) are an important class of therapeutics that address a variety of biological targets of interest. Each product requires detailed physical and chemical characterization during the manufacturing, processing, storage, handling, and distribution process to overcome the challenges associated with mAb degradation. Degradation pathways under conditions of stress may include mAb oxidation, deamidation, isomerization, deglycosylation, glycation, and fragmentation [1].
Here, we report an extensive analysis of the impact of oxidation on mAb stability. Particularly sensitive to oxidation are methionine (Met) groups, which are present in the Fc region of most antibodies. 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.
Chemical modification of mAbs can be studied by generating samples under various stress conditions that are specific to the type of modification. In this study, we treated samples with various concentrations of hydrogen peroxide (H2O2) to produce different levels of mAb modifications through oxidation of H2O2-exposed Met residues. The level of oxidation was assessed for a model commercially available antibody, Trastuzumab (Herceptin®). Chemical degradation was studied using Differential Scanning Calorimetry (DSC) and Differential Scanning Fluorometry (DSF) techniques.
Figure 1: Schematic illustration of methionine oxidation of Immunoglobulin G (IgG) [2].
Formulated Trastuzumab (20 mg/mL in PBS) was obtained from Biosynth. H2O2 was added to the samples to result in final concentrations of 0 %, 0.01 %, and 0.1 % (v/v). The solutions were mixed gently and incubated for 24 hours at room temperature (RT) or 37 °C with protection from light. Next, 20 mM or 200 mM L-Methionine solutions were added to mAb samples oxidized at 0.01 % and 0.1 % (v/v) H2O2, respectively to quench the oxidation reaction. Subsequently, mAb solutions at 0.5 mg/mL (oxidized and non-oxidized) were then buffer-exchanged into HBS-N buffer, pH 7.4 (Cytiva) using Zeba Spin Desalting Columns (Thermo Fisher Scientific). We generated 5 samples (Table 1) which were used in thermal denaturation experiments.
Sample name | H2O2 concentration (%) | Temperature (oC) |
---|---|---|
native_mAb (unmodified) | 0 | RT |
ox_RT_0.01% | 0.01 | RT |
ox_RT_0.1% | 0.1 | RT |
ox_37°C_0.01% | 0.01 | 37 |
ox_37°C_0.1% | 0.1 | 37 |
Table 1: Summary of experimental parameters used for forced oxidation of Trastuzumab.
DSC runs were performed using the MicroCal PEAQ-DSC automated system (Malvern Panalytical). Thermograms for each sample (325 μL at 0.2 mg/mL in HBS-N buffer) were obtained from 20 °C to 100 °C using a scan rate of 1 °C per minute. Thermograms of the buffer alone were subtracted from each mAb sample prior to analysis using the MicroCal PEAQ-DSC software (Malvern Panalytical).
The SUPR-DSF (Protein Stable) was set up to measure the intrinsic fluorescence of mAb samples from 20 °C to 105 °C, with a 1 °C per minute ramp rate. Next, 20 μL of each sample at 0.4 mg/mL in HBS-N buffer was added in triplicate into the wells of a 384-well, black PCR microplate (Bio-Rad: HSP3866). Microplates were sealed with optically clear qPCR adhesive seals (Azenta: 4ti-0560). Intrinsic fluorescence was excited at 280 nm, with fluorescence spectra being captured between 310 nm and 420 nm. Thermal denaturation curves were determined by calculating the barycentric mean of the whole spectra. Denaturation curves were fitted to a three-state thermodynamic unfolding model [3] using the SUPR-Suite v4.0 software (Protein Stable).
Conventional DSF was performed using a QuantStudio5 qPCR machine (Thermo Fisher Scientific) and extrinsic dye (Protein Thermal Shift™ Dye Kit; Thermo Fisher Scientific). Next, mAb solutions were prepared in Protein Thermal 79.8Shift™ Buffer and 1 × Thermal Shift™ Dye. Reactions (20 μL) were prepared in triplicate in a 384-well thin-wall PCR plate (Thermo Fisher Scientific) and immediately sealed with an adhesive PCR seal (Bio-Rad). Fluorescence was monitored from 10 °C to 95 °C at a rate of 1 °C per minute. Changes in fluorescence were monitored at excitation and emission wavelengths of 580 ± 10 nm and 623 ± 14 nm, respectively. The melting temperatures were determined in the Protein Thermal Shift™ Software v1.4 (Thermo Fisher Scientific).
DSC and DSF methods were used to investigate mAb stability after forced oxidation of Met residues. Studies were performed for four oxidized samples at two different concentrations of H2O2 and two different temperatures and compared to the control sample (native mAb).
Investigation of multi-domain mAb folding thermodynamics was performed by DSC. Trastuzumab showed two distinct unfolding transitions (Figure 2) which, based on the signal amplitude and prior knowledge on mAb thermal transitions could be unambiguously assigned to the unfolding of CH2 and Fab (antigen-binding fragment) domains [4]. The latter unfolding is likely to have overlapped with the unfolding of CH3 domain indicating inter-domain cooperativity. Forced oxidation of mAb has resulted in a reduction in the thermostability of mAb, as transition temperature (Tm1) decreased in comparison with the unmodified sample. DSC of the native mAb showed a Tm1 of 71.4 °C and a Tm2 of 81.3 °C. A Tm1 for the oxidized samples decreased by as much as 4.1 – 7.9 °C. In contrast, changes in the Tm2 values for the Fab and CH3 domains were very minor (decrease by 0.1 – 1.5 °C). The change of only one of the transition regions after treatment with hydrogen peroxide indicates a destabilization of CH2 domain by the Fc methionine oxidation and no effect on the Fab fragment and the CH3 domain.
Figure 2: DSC unfolding curves of the native and four oxidized mAbs.
Sample name | Tonset (°C) | Tm1 (°C) | Tm2 (°C) |
---|---|---|---|
native_mAb (unmodified) | 66.1 | 71.4 | 81.3 |
ox_RT_0.01% | 60.4 | 67.3 | 81.2 |
ox_RT_0.1% | 58.7 | 64.0 | 80.4 |
ox_37°C_0.01% | 58.9 | 63.9 | 80.5 |
ox_37°C_0.1% | 56.2 | 63.5 | 79.8 |
Table 2: Tonset and melting temperature (Tm) values for the thermal transitions measured by DSC.
The thermal stability of mAbs samples was measured by intrinsic fluorescence using SUPR-DSF. DSF thermogram (Figure 3) showing the native and oxidized samples revealed two separate transition regions. The first transition (Tm1) represents CH2 domain unfolding and the second transition (Tm2) represents the unfolding of the Fab fragment and the CH3 domain.[4] The SUPR-DSF data of the native mAb showed a Tm1 of 72.5 °C and a Tm2 of 82.0 °C. A Tm1 for the oxidized samples decreased by as much as 4.4 – 8.2 °C. In contrast, changes in the Tm2 values were very minor (decrease by 0.3 – 2.3 °C). The conventional DSF measurements (Figure 4) confirmed two separate transition regions of the native and oxidized mAb samples.
Figure 3: SUPR-DSF measurements of the native and four oxidized mAb samples: fraction unfolded denaturation curves.
Sample name | Tonset (°C) | Tm1 (°C) | Tm2 (°C) |
---|---|---|---|
native_mAb (unmodified) | 67.8 | 72.5 | 82.0 |
ox_RT_0.01% | 61.4 | 68.1 | 81.7 |
ox_RT_0.1% | 59.1 | 65.2 | 81.0 |
ox_37°C_0.01% | 58.9 | 65.3 | 80.8 |
ox_37°C_0.1% | 58.0 | 64.3 | 79.7 |
Table 3: SUPR-DSF measurements of the native and four oxidized mAb samples: Tonset and melting temperature (Tm) values for the thermal transitions.
Figure 4: Conventional DSF measurements of the native and four oxidized mAb samples: First derivative melt curve.
Sample name | Tm1 (°C) | Tm2 (°C) |
---|---|---|
native_mAb (unmodified) | 69.9 | 82.9 |
ox_RT_0.01% | 64.4 | 83.1 |
ox_RT_0.1% | 62.4 | 82.7 |
ox_37°C_0.01% | 62.2 | 81.3 |
ox_37°C_0.1% | 62.0 | 80.4 |
Table 4: Conventional DSF measurements of the native and four oxidized mAb samples: Melting temperature (Tm) values for the thermal transitions.
The results demonstrated very good agreement between the DSC and SUPR-DSF data (Figure 5). The DSF and DSC data showed destabilization of CH2 domain by the Fc methionine oxidation. Thermally-induced transitions for the oxidized mAbs showed a correlation between sample destabilization of CH2 domain and H2O2 concentration, and the incubation temperature.
Figure 5: Comparison between the SUPR-DSF (green), conventional DSF (orange), and DSC (blue) techniques: (Top) Normalized first derivative data of native mAb sample; (Bottom) Comparison between the SUPR-DSF (green), conventional DSF (orange), and DSC (blue) techniques: Normalized (to max value) first derivative data of oxidized mAb sample.
Presented results demonstrate the importance and effectiveness of mAb thermal characterization using DSC and DSF for rapid assessment of mAb stability during therapeutic candidate selection. Stability assays can help in determination of formulations, which are less likely to exhibit long-term stability and aggregation issues. Initial physicochemical characterization can result in more cost‑effective drug production and an increased probability that the mAb will remain active, stable, and correctly folded.
High-throughput fluorescence-based methods are recommended as the primary screen of mAb formulations allowing to rank-order samples. Depending on the material availability, DSC can be used as an orthogonal (medium throughput) or a validation assay of selected mAb formulations. The unfolding profiles of mAbs obtained with DSC also inform the assignment of unfolding transitions detected with DSF. Notably, the order of unfolding transition for an mAb may depend on isoform, presence of mutations, and buffer conditions. DSC experiments are especially important in stability determination as fluorescence-based runs can have artifacts interfering with the assay output and shifting the melting temperature points to a higher or lower value. The results shown here demonstrated a very good correlation between DSC and the intrinsic fluorescence-based DSF technique, which can be used to assess mAb stability in the therapeutics process development.
[1] S. Gupta, W. Jiskoot, C. Schöneich, and A. S. Rathore, “Oxidation and Deamidation of Monoclonal Antibody Products: Potential Impact on Stability, Biological Activity, and Efficacy”, Journal of Pharmaceutical Sciences, vol. 111, p. 903-918, 2022.
[2] Created with BioRender.com.
[3] J. Walters, S. L. Milam, and A. C. Clark, “Practical approaches to protein folding and assembly: spectroscopic strategies in thermodynamics and kinetics,” Methods in enzymology, vol. 455, p. 1—39, 2009.
[4] D. B. Temel, P. Landsman, and M. L. Brader. Orthogonal Methods for Characterizing the Unfolding of Therapeutic Monoclonal Antibodies: Differential Scanning Calorimetry, Isothermal Chemical Denaturation, and Intrinsic Fluorescence with Concomitant Static Light Scattering.
This Application Note was developed in collaboration with Concept Life Sciences, Protein Stable and Malvern Panalytical.