Opus v6

FTIR analysis was performed using a Tensor-27 FTIR spectrometer (Bruker, Billerica, MA). For protein concentrations and buffers for each mAb, see Table 1. Protein samples were measured from 800 – 4000 cm⁻¹ with a resolution of 4 cm⁻¹. A total of 256 scans were performed for both samples and buffers at 25 °C. For the thermal melting experiments, 64 scans were obtained, and samples were scanned from 25 to 90 °C using an increment of 2.5 °C/step and an equilibration time sufficient to reach equilibrium (2 min) at each step. Buffer subtraction, water vapor and CO₂ compensation, baseline correction, and normalization of the amide I band (1700 – 1600 cm⁻¹) were sequentially performed on the raw FTIR spectra using the OPUS V6.5 software (Bruker, Billerica, MA). Second derivative FTIR spectra were generated using a Savitzky–Golay filter with a window size of 9.
The bend + liberation FTIR band of water in these protein samples and their buffers were measured by using air as the blank. Samples were scanned from 800 to 4000 cm⁻¹ using a resolution of 2 cm⁻¹. A total of 256 scans were performed. λ_μ was calculated between 1900 and 2300 cm⁻¹ for the water band.

A Tensor-27 FT-IR spectrometer (Bruker, Billerica, MA) equipped with a Bio-ATR cell was used for FT-IR spectroscopic analysis. Liquid nitrogen was used to cool the detector and a continuous N₂ gas flow was used to purge the interferometer. A total of 256 scans were recorded from 4000 to 900 cm⁻¹ at a 4 cm⁻¹ resolution at 10 °C. The background spectra from each experimental buffer condition were subtracted from the sample spectra. Acquired spectra were processed for atmospheric compensation, baseline adjustment, and normalization by OPUS V6.5 (Bruker, Billerica, MA) software. Deconvolution of the amide I region of the acquired spectra from various experimental conditions were performed by Python script where the data set was processed by mixed Gaussian/Lorentzian bands. Similarly, the second derivative of each spectrum was calculated, and peaks were identified for secondary structure content.

FT-IR spectroscopic analysis was performed using a Tensor-27 FT-IR spectrometer (Bruker, Billerica, MA) equipped with a Bio-ATR cell. The detector was cooled with liquid N₂ for 20 min prior to use and the interferometer was purged continuously with N₂ gas. A total of 256 scans were recorded from 4000 to 900 cm⁻¹ with a 4 cm⁻¹ resolution. Buffer/emulsion background spectra were collected and subtracted from the sample spectra. Atmospheric compensation, baseline adjustment and second derivative calculations were applied using OPUS V6.5 (Bruker, Billerica, MA) software. To compare the initial state of the samples, spectra collected at 20 °C were deconvoluted into a set of mixed Gaussian/Lorentzian bands, using the build-in Levenberg-Maquardt algorithm from the OPUS V6.5 software. Thermal unfolding experiments were performed with the temperature ramped from 20 to 90 °C or 99 °C (for AT in SE) at increments of 2.5 °C per step and an equilibration time of 2 min at each temperature. The second derivative of each spectrum was calculated with nine point smoothing. The thermal unfolding curves of AT were constructed by plotting the second derivative signal at 1621 cm⁻¹ (an indication of intermolecular β-structure and protein aggregation) as a function of temperature. The melting temperature (T_m) was calculated by a first derivative method using Origin 2017 (OriginLab; Northampton, MA).

Spectra of rGH solutions (1 mg/mL) with varying concentrations of IONPs (0.42 to 1.47 mM) were acquired with a Bruker Tensor 27 FTIR spectrometer fitted with a Bio-ATR cell (Bruker, Billerika, MA). Scans were recorded from 600 to 4000 cm⁻¹ at a resolution of 1 cm⁻¹. The FTIR spectra were acquired after 10 min, 30 min, 1 h and 2 h incubation periods at ambient temperature. In addition, rGH and rGH-IONP mixtures were incubated at increasing temperatures from 15 to 85 °C at increments of 2.5 °C/step with a 120 s equilibration time, and 128 scans/step. The phosphate buffer-IONP-mixture was used for background subtraction of the FTIR spectra. The Salvitzky-Golay algorithm was used to calculate the second derivative spectra, baseline and atmospheric corrections, and 9-point data smoothing (Opus v6.5, Bruker Corporation, Billerica, MA).