Application Notes

Chemical and thermal stability screening of an IgG1-antibody

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2 Material and Methods Preparation of protein formulations By ultrafiltration/diafiltration (UF/DF), nine formulations containing an IgG1-Antibody at a protein concentration of 25 mg/mL were prepared. All formulations were used both for thermal and chemical unfolding experiments. Table 1: overview of the buffers and their pH-values used in the screening Buffer pH-value 20mM Na 2 HPO 4 ·2H 2 O 5.5 6.5 7.5 10mM/10mM Histidine/Glycine 5.5 6.5 7.5 50mM TRIS-buffer 6.5 7.5 8.5 Thermal unfolding experiments The capillaries were filled with the different antibody formulations and placed onto the capillary tray of the Prometheus NT.48. Start and end temperature as well as heating rate were defined (1 °C/min, from 20 °C to 95 °C). Chemical unfolding experiments Chemical unfolding of the IgG1-Antibody was induced by constantly increasing the concentration of the denaturant, while keeping the protein concentration and pH constant. This was done by combining three solutions as presented in Table 2: i. a fixed amount of protein stock solution (concentration 25 mg/mL) ii. varying amounts of 8 M guanidine hydrochloride stock solution (denaturant) iii. buffer stock solution Before data are recorded, it is important that the folding/unfolding reaction reaches an equilibrium state. Therefore, the twenty experimental solutions were given an equilibration time of 24 hours. The solutions were measured using the Prometheus NT.48 employing the same instrument settings as already mentioned (1 °C/min, from 20 °C to 95 °C). The changes in the fluorescence ratio (F350/F330) of every experimental solution were recorded versus increasing temperature. However, for this Application Note the fluorescence ratio at a fixed temperature of 20 °C was used for detecting the chemical stability (C m ) to have only the denaturant concentration as varying parameter. Table 2: experimental solutions were prepared volumetrically from GdmCl, buffer and protein stock solutions following this schema Number GdmCl stock solution [mL] Buffer stock solution [mL] Protein stock solution [mL] GdmCl experiment tal solution [mol/L] 1 0.1 1.9 0.5 0.32 2 0.2 1.8 0.5 0.64 3 0.3 1.7 0.5 0.96 4 0.4 1.6 0.5 1.28 5 0.5 1.5 0.5 1.60 6 0.6 1.4 0.5 1.92 7 0.7 1.3 0.5 2.24 8 0.8 1.2 0.5 2.56 9 0.9 1.1 0.5 2.88 10 1.0 1.0 0.5 3.20 11 1.1 0.9 0.5 3.52 12 1.2 0.8 0.5 3.84 13 1.3 0.7 0.5 4.16 14 1.4 0.6 0.5 4.48 15 1.5 0.5 0.5 4.80 16 1.6 0.4 0.5 5.12 17 1.7 0.3 0.5 5.44 18 1.8 0.2 0.5 5.76 19 1.9 0.1 0.5 6.08 20 2.0 0.0 0.5 6.40 Stability upon 6 weeks storage Furthermore the Prometheus NT.48 was used for a stability experiment to generate more data on the stability of the IgG1-Antibody in the different buffers. For this, all formulations were stored at 2 °C-8 °C as well as in a heating chamber at 40 °C. Thermal unfolding curves were then measured at T 0 and after 7, 14, 21 and 42 days of storage. Results and Discussion Measurements of the chemical and thermal unfolding with the Prometheus NT.48 reveal typical sigmoidal curves with a left plateau representing the fully native state and a right plateau representing the fully denatured state of the protein. The inflection points, which are plotted between these plateaus, correspond to the maximum value of the first derivative. They indicate the temperature or GdmCl-concentration, at which the amount of folded and unfolded molecules are equal. Due to

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