CONCLUSIONES Y RECOMENDACIONES

A suitable separation method for spider silk proteins such as AF4 was developed to investigate the state of dissolved eADF4(C16) prior to particle preparation. The separation principles of AF4 are reviewed elsewhere [14, 15]. The AF4 system consisted of the main device AF2000 Focus from Postnova Analytics GmbH (Landsberg am Lech, Germany) including focus and tip pump PN 1122, slot pump PN 1610, vacuum degasser PN 7505 and refractive index detector PN 3150. Further components were a SPD-10A UV-detector from Shimadzu (Duisburg, Germany) and a miniDAWN light scattering detector from Wyatt Technology (Dernbach, Germany). The AF4 channel was also produced by Postnova Analytics and had dimensions of 32.0 x 6.0 x 3.7 cm.

For the separation of eADF4(C16) samples this AF4 channel was equipped with a 350 µm spacer and a fixed detection flow rate of 1.0 mL/min was used. The mobile phase was equal

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to the formulation buffer using a 10 mM Tris-buffer at pH 8.0 in order to avoid any influence of the mobile phase during separation. During method development different membrane types and cut-offs were evaluated and the amount of injected spider silk protein was varied from 4 to 60 µg. New membranes were always saturated with BSA before sample analysis was performed. An important difference to HP-SEC analysis was that samples were not centrifuged prior to injection. For data analysis the ASTRA software (Wyatt Technology, Dernbach, Germany), an extinction coefficient of 972 mL*g-1_*cm-1 _{at 280 nm, and a}

differential index of refraction (dn/dc) of 0.185 for eADF4(C16) were used.

2.2.2.2 INCUBATION AT ELEVATED TEMPERATURE

Based on the implementation of higher process temperatures during particle preparation (see section 2.2.3.1), the physical stability of eADF4(C16) at higher temperatures was investigated. For this reason, the most commonly used eADF4(C16) formulation consisting of 1.0 mg/mL eADF4(C16) in 10 mM Tris / pH 8.0 was analyzed. 500 µL of the formulation were filled into 2.0 mL polypropylene tubes and incubated at 80°C for 4 hours. Afterwards, 200 µL of the samples were drawn and diluted with formulation buffer to a volume of 2.0 mL to perform turbidity and light obscuration measurements. The residual formulation was centrifuged and used for size exclusion chromatography.

2.2.2.3 SIZE EXCLUSION CHROMATOGRAPHY

After removing potential insoluble aggregates by centrifugation, the supernatants of the corresponding formulations were analyzed by size exclusion chromatography. The separation was carried out on a Shodex OHpak SB-803 column which was made of a polyhydroxymethacrylate matrix and can be used for alkaline running buffers up to pH 10 in contrast to silica-based columns. As mobile phase 100 mM Tris at pH 8.0 was used at a flow rate of 0.38 mL/min. The column was installed in the aforementioned AF4-system replacing the AF4 channel (see section 2.2.2.1). For elution mode during HP-SEC only the tip pump of the system was necessary. All three detectors were available for the calculation of protein content and molar mass of the eluted protein species.

2.2.2.4 SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS

In order to obtain further information about potential spider silk protein aggregates SDS-PAGE was performed under non-reducing conditions. Samples were loaded on NuPAGE® _{10% Bis-Tris precast gels (Life technologies GmbH, Darmstadt, Germany), which}

had been mounted in the XCell II Mini cell system (Novex, San Diego, CA, USA). The sample preparation included the dilution of the samples to an approximate protein

concentration of 0.025 mg/mL in a Tris-buffer at pH 6.8 and the denaturation of the samples at 95°C for 20 minutes. Finally, 10 µL of each sample were loaded into the different wells of the gel. Separation was carried out at a current of 40 mA per gel and the runtime was about 90 minutes. Silver staining was carried out by using the SilverXPress® _{Staining Kit}

(Invitrogen, Carlsbad, CA, USA). A protein standard (Mark 12® _{Unstained Standard,}

Invitrogen, Carlsbad, CA, USA) was loaded onto the gel for the assessment of the molecular weight of protein species.

2.2.2.5 NEPHELOMETRY

The turbidity of spider silk protein formulations after incubation at 80°C was determined using a NEPHLA nephelometer (Dr. Lange, Düsseldorf, Germany). Turbidity was measured in formazine nephelometric units (FNU) by 90° light scattering at a wavelength of 860 nm. Each value represents the average of two measurements to exclude unspecific scattering effects by rotating the cuvette. As the required measurement volume is almost 2.0 mL, formulations were diluted 1:10 with formulation buffer before measurement.

2.2.2.6 LIGHT OBSCURATION

The size and amount of particles between 1 and 200 µm was measured by light obscuration on a SVSS C-40 apparatus equipped with a HCB-LD-25/25 sensor (PAMAS GmbH, Rutesheim, Germany). The light obscuration system was cleaned with highly purified water which had been filtered through a 0.22 µm filter membrane and was essentially free from particles. Cleaning was performed until the total particle count per mL was below 100 and less than 5 particles larger than 10 µm were detected. This cleaning step was performed and recorded before and in between the individual measurements after flushing the system with 5 mL highly purified water.

The light obscuration measurements were performed subsequent to the turbidity measurements. 0.5 mL of the sample was used for flushing the system. Afterwards, three aliquots of 0.3 mL sample volume were analyzed for subvisible particles. The average value of the three aliquots was calculated and related to the total particle number in 1.0 mL. Typically, the range of subvisible particles is divided into three classes of particle sizes: ≥ 1 µm, ≥ 10 µm and ≥ 25 µm.

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