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A murine monoclonal IgG2c antibody (muAb) was kindly provided by Abbott GmbH & Co. KG. The antibody solution applied in the experiments was diluted to a concentration of 10 mg/mL in a 20 mM histidine formulation buffer at pH 6.0. Compared to the aggregation studies on the human model antibody in Chapter 3, the concentration was enhanced from 4 to 10 mg/mL aiming to generate higher total amounts of aggregates sufficient for preparative separation from accompanying protein species. At the same time the sample volume was reduced from 3 mL in 6 R vials to 1 mL in 2 R vials. Both conditions should have comparable filling volume to head space volume ratios, that otherwise can strongly impact the aggregation behavior of the antibody at the air/water interface. The IgG2c stock solution had an initial concentration of 24 mg/mL and was stored at -80 °C in the deep freezer. Prior to use, the formulation buffer was filtrated using a 0.2 µm sterile syringe filter made of cellulose acetate (VWR International, Darmstadt, Germany). 1 mL of the antibody solution was filled into cleaned and sterilized 2 R vials made from glass type 1 (Schott AG, Mainz, Germany), sealed with Teflon®-faced rubber stoppers (West Pharmaceuticals Services, Eschweiler, Germany) and crimped. All antibody samples were prepared under aseptic conditions. Highly purified water generated in-house was used for the preparation of samples and buffers (PURELAB Plus instrument (USF Elga, Celle, Germany)). All substances used were of analytical grade.

4.2.2 Methods to provoke protein instability

4.2.2.1 Stirring stress

Stirring stress of the muAb was performed in 2 R glass vials filled with 1 mL of a 10 mg/mL antibody solution. The same stirrer, stir bars and incubation temperature as in Chapter 3 were used to conduct this study. To accelerate the formation of particles speed was set to 400 rpm (instead of 240 rpm in huAb studies) and the final sampling time point was set to 24 hours of stirring. One vial was selected and removed after 4 h and another one after 6h of stirring. After the entire incubation time a triplicate of samples was left and used for more comprehensive analytics. For control experiments, the pure buffer solution was stirred for 24 h under the same conditions as well.

4.2.2.2 Shaking stress

Shaking stress of the antibodies was performed in 2 R glass vials filled with 1 mL of a 10 mg/mL antibody solution. A VWR Microplate Shaker (VWR International, Darmstadt, Germany) was used for shaking the vials up to 48 hours. One sample was additionally investigated after 24 h, whereas a triplicate of vials was analyzed after 48 hours. Like in the

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stirring experiments the agitation was accelerated to 400 rpm, compared to the previous huAb studies. The arrangement of the vials on the shaker was identical to the huAb samples. For control experiments, the pure buffer solution was shaken for 48 h under the same conditions as well.

4.2.2.3 Light exposure

The same settings of the Suntest CPS radiation chamber (Heraeus Holding, Hanau, Germany) as in the previous study were used for irradiation. 1 mL of the samples containing 10 mg/mL of protein were filled in 2 R glass vials and placed in the center of the radiation chamber in an upright position. Sampling of the vials was performed after 24 h and 48 h (n=1) and finally after 72 h (n=3). These time points were selected to focus the investigation on conformational alterations and the formation of soluble aggregates and fragments, since the huAb started to precipitate after 4 d (96 h) of light exposure. For size exclusion chromatography analysis 10 µL of light exposed sample were withdrawn of the remaining three vials after 65 hours. Additionally, the formulation buffer was exposed to light as well and sampled at the same time points. For control experiments, a triplicate of vials completely shielded with aluminum foil was included in the study, serving as control for the slight temperature increase (35 ± 3°C) during radiation and was sampled after 72 h of incubation.

4.2.2.4 Temperature stress

Heating was performed in 2 R glass vials filled with 1 mL of a 10 mg/mL antibody solution. The vials were incubated in cabinet dryers of a fixed temperature of 50°C for up to 27 days. Every other week one vial was removed (after 7, 14, 21 days). Finally, a triplicate of vials was incubated 27 days. The melting temperature (Tm) of the murine IgG2c was 65.9°C determined by micro differential scanning calorimetry (µ-DSC) (data not shown) thus several degrees above the predefined incubation temperature. For control experiments, the pure buffer solution was incubated 27 days at 50°C as well.

4.2.3 Methods to determine protein stability

4.2.3.1 Light obscuration

Light obscuration (LO) measurements were conducted with a PAMAS-SVSS-C Sensor HCB-LD 25/25 (Partikelmess- und Analysensysteme GmbH, Rutesheim, Germany) to quantify subvisible particles between 1 and 200 μm. Due to limited volume, the muAb samples were diluted 1:5 using placebo formulation prior to analysis. Please refer to section 3.2.3.1 for detailed parameters of the LO measurements.

4.2.3.2 Turbidity

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Concentrations of soluble aggregates, fragments, and monomeric protein were measured by size exclusion chromatography on an Agilent system equipped with RI and UV detection (Agilent Technologies, Palo Alto, USA) as well as a multi angle laser light scattering detector (MALLS) (DAWN EOS, Wyatt Technology Europe GmbH, Dernbach, Germany). A Superose 6 10/300 GL column (GE Healthcare, Little Chalfont, UK) was used as solid phase. Further settings to conduct SEC measurements are equal to those described in section 3.2.3.4 for the huAb studies.

4.2.3.4 Intrinsic protein fluorescence spectroscopy

The settings for intrinsic protein fluorescence spectroscopy measurements are described in section 3.2.3.5. The sample concentration was changed to 0.1 mg/ml for muAb samples, unless otherwise mentioned.

4.2.3.5 Extrinsic protein fluorescence spectroscopy

The settings for extrinsic protein fluorescence spectroscopy measurements with ANS are described in section 3.2.3.6.

4.2.3.6 Fourier transform infrared spectroscopy (FTIR)

The settings for Fourier transform infrared spectroscopy measurements are described in section 3.2.3.7.

4.2.3.7 High resolution UV absorbance spectroscopy

The settings for high resolution UV absorbance spectroscopy measurements are described in section 3.2.3.8.

4.3

R