(T1030-01-03) ATR-FTIR Spectroscopy as an Orthogonal Method for Secondary Structure Characterization of Monoclonal Antibodies Under Thermal Stress

Purpose: Attenuated Total Reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is a fast, low-volume, easy to use, non-destructive, and reliable method to study the secondary structures of monoclonal antibodies (mAbs) in both liquid and solid states(1). Our study explores the changing patterns of ATR-FTIR spectra for a mAb exposed to gradual thermal stress. The results obtained from our study show that T_infl/˚C, a characteristic temperature at which the intrinsic transitioning of the secondary structure of the mAb occurs, can be obtained. We suggest that this method could be used alongside other techniques used to examine colloidal and conformational stability of mAbs in different buffers.

Methods: Monoclonal antibody 1 (mAb1) was buffer exchanged to seven different buffers and concentrated to 30mg/mL. Buffers 1 through 4 included a commonly used buffer with four different pHs within the buffering capacity of the buffer. Buffers 5 through 7 used the same buffer and pH as Buffer 2, with the addition of a commonly used excipient at three different concentrations. ATR-FTIR spectra of the buffer exchanged samples were obtained after thermally stressing the samples from 25˚C to 90˚C at 5˚C intervals. Amide I peak of the sample was deconvoluted to yield two major peaks at 1616cm^-1 and 1638cm^-1. The ratio of Abs_1616/1638 for the samples demonstrated an inflection point at a particular temperature for a sample and was called T_infl/˚C for that sample. The samples were also analyzed for conformational and colloidal stability using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and a fluorescence-based protein stability screening platform. The data obtained from different techniques were compared.

Results: At room temperature, all buffer exchanged samples of mAb1 showed characteristic amide I and amide II peaks at around 1638cm^-1 and 1548cm^-1, respectively. Amide I peak corresponds to the C=O stretching vibration and amide II peak corresponds to N-H bending and C-N stretching in the proteins(2). As the samples were exposed to thermal stress, their amide I peaks exhibited a shift from 1638cm^-1 to 1616cm^-1 and a decrease in absorbance intensity. The absorbance ratio of 1616/1638 demonstrated a characteristic temperature (termed T_infl/˚C) for each sample at which this shift occurred. For the samples tested, this ranged from 70˚C to 75˚C. Wave numbers 1638cm^-1 and 1616cm^-1 have been associated with intramolecular β-sheets and intermolecular β-sheets, respectively(3, 4). Specifically, peaks at around 1616cm^-1 have been attributed to strong H-bonds with lower frequency for C=O stretching(5, 6). By systematically increasing the temperature of each sample, we were able to capture the point at which the samples transitioned from mostly having intramolecular β-sheets to intermolecular β-sheets. The melting temperatures, T_m1 obtained from DSC for the samples represent the temperature at which the CH₂ domain of the mAbs undergo thermal unfolding(7). This ranged from 66˚C to 73˚C for the samples tested. Melting temperatures (T_m1) determined by measuring the unfolding of the protein through intrinsic fluorescence was determined to be from 65˚C to 81˚C. Colloidal stability for the samples were tested using DLS. Their T_agg, the temperature at which aggregation begins were determined to be from 68˚C to 74˚C. Diffusion interaction parameter (kD) which is also related to colloidal stability and corresponds to the attractive and repulsive forces of the molecules showed a range of 8 to 40 for the samples tested. Positive kD values indicate repulsive forces and suggest that the sample are stable(8). Evaluation of T_infl/˚C obtained from ATR-FTIR with colloidal and conformational stability parameters obtained from other techniques yielded mostly similar patterns. For instance, T_infl from ATR-FTIR, T_m1 from DSC, and T_m1 from fluorescence-based screening all showed the lowest temperatures for B01, suggesting that mAb1 is the least stable in B01 under thermal stress among the buffers tested. On the other hand, T_m1 from DSC and T_m1 from fluorescence favored B04 as the most stable buffer for mAb1, whereas T_infl favored B02 and T_agg from DLS favored B06 as the most stable buffers.

Conclusion: ATR-FTIR has been used to detect changes in mAbs after thermal stress previously(3). For the first time, we used a systematic approach to increase the thermal stress on a mAb while capturing the point of thermal inflection from a mostly intramolecular β-sheets state to an intermolecular β-sheets state of the molecule. We successfully devised a term, T_infl/˚C that can capture this transition. While different techniques mentioned above probe different aspects of a mAb under thermal stress and cannot be directly equated, our technique can provide an orthogonal method based on the intrinsic changes in secondary structures of a mAb to screen for potential buffers.

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