Abstract
The temperature-dependent secondary structure of two monoclonal IgG antibodies, anti-IGF1R and anti-TSLP, were examined by transmission mode Fourier Transform Infrared (FTIR) spectroscopy. Anti-IGF1R and anti-TSLP are IgG monoclonal antibodies (mAbs) directed against human Insulin-like Growth Factor 1 Receptor for anti-tumor activity and Thymic Stromal Lymphopoietin cytokine for anti-asthma activity, respectively. Differential scanning calorimetry (DSC) clearly indicates both antibodies in their base formulations have a lower temperature protein conformational change near 70 °C (Tm1) and a higher temperature protein conformational change near 85 °C (Tm2). Thermal scanning dynamic light scatting (TS-DLS) indicates a significant particle size increase for both antibodies near Tm2 suggesting a high level of protein aggregation. The nature of these protein conformational changes associated with increasing the formulation temperature and decreasing sucrose concentration were identified by transmission mode FTIR and second derivative FTIR spectroscopy of temperature controlled aqueous solutions of both monoclonal antibodies. The transition from intra-molecular β sheets to inter-molecular β sheets was clearly captured for both monoclonal antibodies using FTIR spectroscopy. Finally, FTIR Spectroscopy was able to show the impact of a common excipient such as sucrose on the stability of each monoclonal antibody, further demonstrating the usefulness of FTIR spectroscopy for studying protein aggregation and formulation effects.
Highlights
Protein aggregation is considered a major formulation problem for therapeutic biologics and vaccines
The protein unfolding temperatures (Tms) for the two monoclonal antibodies studied in this paper were first identified through two methods: differential scanning calorimetry (DSC) and dynamic light scattering (DLS)
The Differential scanning calorimetry (DSC) results demonstrate that each monoclonal antibodies (mAbs) has two Tms, with each mAb having similar Tms: the first occurring at approximately 70 °C and the second occurring at approximately 84 °C
Summary
Protein aggregation is considered a major formulation problem for therapeutic biologics and vaccines. Biologics and vaccine formulation studies often quantify increasing protein particle diameters using techniques such as static light scattering (SLS), dynamic light scattering (DLS), size exclusion chromatograph (HSEC), and microflow imaging (MFI) [3,4,5]. These and other particle sizing technologies can clearly identify protein aggregation and are very useful screening tools for formulation excipients such as surfactants that inhibit protein aggregation [6,7,8]. Most particle sizing technology does not provide insight into the specific molecular interactions involved in protein aggregation. Some bioanalytical techniques such as tryptophan/ tyrosine intrinsic protein fluorescence (IPF) and differential scanning fluorimetry (DSF) using extrinsic fluorescence probes can detect general molecular events associated with protein aggregation such as protein unfolding [6, 7]
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