de junio: Vocabulario noticia periódico (libre escogencia)

As described earlier, preparation of eADF4(C16) nanoparticles requires preheating of the protein solution to 60°C and 80°C, respectively. Therefore, stability of the spider silk protein formulation was investigated at 80°C over a period of 4 hours. The concentration was set to 1.0 mg/mL and the commonly used formulation comprising a 10 mM Tris-buffer at pH 8.0 was employed.

Figure 3.15. Separation of eADF4(C16) on a Shodex OHpak SB-803 SEC-column. Red: Light scattering at 90°C; blue: refractive index; green: UV-detection at 280 nm

The samples were analyzed using a HP-SEC method developed by AMSilk. The resulting chromatogram of an unstressed eADF4(C16) formulation is shown in Figure 3.15. The order of the eluting peaks was vice versa as obtained by AF4 so that first high molecular weight (HMW) species with a huge light scattering signal were detected. This peak was followed by the monomer peak of eADF4(C16) and a slight tailing of the peak as it was also detected by AF4 (fronting). Throughout the incubation at 80°C the height and AUC of the monomer peak decreased continuously, whereas the amount of HMW-species and the tailing were not altered (data not shown). The total protein recovery decreased to 88% after 1 hour and 63% after 4 hours of incubation, respectively (see Figure 3.16, A). The result from HP-SEC was confirmed by SDS-PAGE under non-reducing conditions (see Figure 3.16, B). A decrease of the coloration of the monomer band was clearly detectable (line 4 vs. 7). More importantly

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and in agreement with HP-SEC results, no further protein species occurred during incubation indicating that no soluble protein aggregates were formed. For this reason, the loss in protein content has to be accompanied by the formation of insoluble aggregates, but light obscuration measurements did not reveal a significant increase of subvisible particles (see Figure 3.16, C) as total particle counts around 2000 particles per mL (1:10 dilution) could not explain a protein loss of approx. 400 µg eADF4(C16). However, in all formulations large visible aggregates were formed, but these aggregates were not of particulate nature as it is usually found for aggregated protein drugs. Figure 3.16, D shows that spider silk fiber formation took place during incubation at 80°C in the 10 mM Tris-buffer at pH 8.0.

4h / 80°C

Figure 3.16. Stability of an aqueous eADF4(C16) solution at 80°C. (A) Total protein recovery determined by size exclusion chromatography. (B) SDS-PAGE (non-reducing conditions) and (C) light obscuration (1:10 dilution) results of the corresponding formulations. (D) Photographic image of the formulation after 4 hours at 80°C (1:10 dilution)

Summing up, section 3.3 of this chapter describes the implementation of analytical techniques for characterizing the physical stability of eADF4(C16) formulations. An AF4 method was developed and established as an orthogonal method to size exclusion chromatography. The results from both analytics agreed concerning detected protein species and revealed that eADF4(C16) formulations contained low amounts of high molecular weight

0 1 2 3 4 40 50 60 70 80 90 100 total protei n r ec ov ery [%] time [hours] t0 1h @80°C 2h @80°C 3h @80°C 4h @80°C 4h @RT 0 1000 2000 3000 4000 5000 cu mul ati ve pa rt icles > 1 µm pe r mL 66.3 kDa 1: t0 sample 1 2: t0 sample 2 3: t0 sample 3 4: 1h / 80°C 5: 2h / 80°C 6: 3h / 80°C 7: 4h / 80°C

Visible aggregates – fibers

A B

aggregates, but no soluble protein oligomers. SDS-PAGE showed only the protein band representing the monomeric eADF4(C16), but due to the large size of the HMW-aggregates it is likely that they were not able to enter the gel and therefore not detected by silver staining. The amount of these HMW-aggregates could only be reduced by long centrifugation times which are not commonly used for HP-SEC sample preparation.

The degradation process of eADF4(C16) induced by incubation at 80°C solely led to the formation of large visible protein fibers. The aggregation pathway is therefore different to globular therapeutic proteins like monoclonal antibodies or cytokines where soluble aggregates and subvisible particles play a major role in protein degradation [39]. The assembly of the spider silk protein eADF4(C16) starts with intrinsically random coiled protein molecules in the aqueous environment. Therefore, it is very likely that aggregates are formed directly by self-association of unfolded protein molecules which act as aggregation nucleus on their own. As a result and dependent on the environmental conditions like ionic strength, eADF4(C16) aggregates are easily formed and were determined in literature as fibrils at low and solid spheres at high potassium phosphate concentrations [19, 36, 40, 41]. In the case of heat-induced aggregation at low ionic strength (10 mM Tris buffer), it is assumed that those fibrils only occur as intermediates which are instantly transformed to larger protein fibers due to the increased kinetics of the aggregation process.

As a consequence for the particle preparation procedure, two different preventive actions can be derived. At first, if a preheating of the formulation is intended, the salting-out process should be performed as quickly as possible. The technical requirements allow a filling of the pump with 100 mL in 5 minutes and the mixing takes 2 minutes at a flow rate of 50 mL/min. Therefore, the time period at which the formulation is processed at elevated temperature can be kept below a total of 10 minutes. Compared to the loss in protein recovery this short time period does not raise a problem for the preparation process. Secondly, in order to achieve a spider silk formulation without or at least with very low amounts of aggregates it is not only sufficient to filtrate the solution, but also long centrifugation times or ultracentrifugation should be applied shortly before particle preparation is carried out.

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