Development and biophysical characterization of a humanized FSH–blocking monoclonal antibody therapeutic formulated at an ultra-high concentration
- Center for Translational Medicine and Pharmacology, Icahn School of Medicine at Mount Sinai, United States
- Center for Biomedical Research, Population Council, United States
- Maine Medical Center Research Institute, United States
Figures
Figure 1
Thermal, monomeric and colloidal stability of formulated MS-Hu6 at increasing concentrations.
(A) Protein thermal shift assay confirmed thermostability of formulated MS-Hu6 between 0.2 mg/mL and 100 mg/mL. Compared with MS-Hu6 in PBS, the difference in melting temperature (ΔTm) of the constant (Fc) and antigen-binding fragments (Fab) of formulated MS-Hu6 was <-1°C at all concentrations. (B) Size exclusion chromatography confirmed the monomeric nature of formulated MS-Hu6 between 1 and 100 mg/mL. Monomer loss was <1% in all concentrations. (C) Particle size distribution using dynamic light scattering yielded hydrodynamic radius (rh) and polydispersity index (PDI) of formulated MS-Hu6. Excellent colloidal stability (rh <10 nm, PDI <1) was found at concentrations from 1 to 100 mg/mL.
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig1-data1-v2.xlsx
Figure 2
Thermal and colloidal stability of formulated MS-Hu6 is maintained in the presence of L-methionine and EDTA.
To improve long-term storage stability, an antioxidant L-methionine and a chelating agent EDTA were added to formulated MS-Hu6. Addition of L-methionine and EDTA to formulated MS-Hu6 did not alter its melting temperatures (Tm) in the protein thermal shift assay (A) or the hydrodynamic radius (rh or Z–average rh) or polydispersity index (PDI) in dynamic light scattering (B).
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig2-data1-v2.xlsx
Figure 3
Evaluation of viscosity, turbidity, and clarity of the formulated MS-Hu6 at different concentrations.
Formulated MS-Hu6 showed a concentration-dependent linear increase in viscosity within the acceptable industry standard (<50 centipoise, cP) (A). Formulations were also evaluated for turbidity (opalescence) at 350 nm. Low absorbance (AU, absorbance unit) was noted at lower concentrations; however, there was a concentration-dependent increase in absorbance at 100 mg/mL formulated MS-Hu6 (B). Formulated MS-Hu6 evaluated for clarity at wavelengths of 350, 540, and 595 nm demonstrated that the formulation is clear and transparent even at higher concentrations (100 mg/mL) that correlated well with the turbidity data (C).
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig3-data1-v2.xlsx
Figure 4
Biophysical characterization of formulated MS-Hu6.
Circular dichroism (CD) spectroscopy evaluated the secondary structure of formulated MS-Hu6. Analysis in the far UV region (190–240 nm) revealed that the predominant secondary structure in MS-Hu6 was regular β-sheets and unordered/random coils (A). Secondary structure was also confirmed at higher formulation concentrations (50 and 100 mg/mL) using Fourier–transform infrared (FTIR) spectroscopy. The amide I band peak at 1637 cm–1 (intra–molecular β-sheets) and the random coil (1642–1657 cm–1) did not shift, confirming maintenance of the native conformation in formulation (B). Thermostability was further confirmed using nano differential scanning calorimetry (Nano DSC). MS-Hu6 concentrations of 50 and 100 mg/ml had comparable Tms, indicating that the overall structure is conformationally and thermally stable at high concentrations (C).
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig4-data1-v2.xlsx
Figure 5
Evaluation of freeze-thaw stability of the formulated MS-Hu6 (100 mg/mL) at −80/25 °C and –80 °C/37 °C for three cycles.
(A) Samples were analyzed using protein thermal shift for thermostability. After storage at −80/25 °C, ΔTm values for both Fc and Fab domains revealed no significant difference between each cycle and baseline. However, for samples stored at –80 °C/37 °C (three cycles), while the Fc domain remained stable, there was a small change in Fab ΔTm (within acceptable limit of 1 °C). (B) Samples were also analyzed using dynamic light scattering (DLS) for colloidal stability. The major peak (Peak 1) hydrodynamic radius (rh) was found to be between 3 and 7 nm at both storage conditions (−80/25 °C and –80 °C/37 °C). PDI—polydispersity index. The data suggest no or minimal reversible aggregation (<0.5%; acceptable limit: 5–10%).
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Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig5-data1-v2.xlsx
Figure 6
Stability evaluations of formulated MS-Hu6 at 100 mg/mL at 4°C and 25 °C for 90 days.
(A) Samples were tested for thermostability using the protein thermal shift assay. At 4 °C, there was no noticeable Tm shift for the Fab domain compared to day 0. At 25 °C, there is a slight left–shift in the Tms of both the Fab and Fc domains at day 15, followed by relatively good stability up to 90 days for Fab. However, after 90 days of incubation, there is a loss of thermostability for the Fc domain with a ΔTm of –1.51 °C. (B) Samples were tested for monomer loss using size exclusion chromatography (SEC). After 90 days, all three batches at 4 °C retained an average monomeric area of 99.4%, while the average monomer area retained at 25 °C was 96.8%, which is still within the acceptable limit of >95%.
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig6-data1-v2.xlsx
Figure 7
FSH binding of formulated MS-Hu6.
FSH binding to MS-Hu6 in PBS and formulated MS-Hu6 is shown by a right shift in Fab Tm, which documents higher thermostability of the FSH:Fab complex.
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Figure 7—source data 1
Source data for Figure 7.
- https://cdn.elifesciences.org/articles/88898/elife-88898-fig7-data1-v2.xlsx
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Development and biophysical characterization of a humanized FSH–blocking monoclonal antibody therapeutic formulated at an ultra-high concentration
eLife 12:e88898.
https://doi.org/10.7554/eLife.88898
