Caracterización de la palabra según Porto Dapena

4.3.1 Lysozyme

In this study, the lysozyme/trehalose formulations were composed of 80.0-90.9% trehalose by weight. The trehalose serves to protect lysozyme integrity, thus minimizing the formation of larger structures (e.g., dimer, trimer). In this study, the lysozyme activity and

structure was fully retained after reconstitution of the processed materials (Fig 5, Table 4) and only lysozyme monomer was observed by size exclusion chromatography (Fig. 6). Perez-Moral et al. [46] have demonstrated that increasing the percentage of trehalose or sucrose in

a freeze-dried formulation inhibited the thermal aggregation of β-

lactoglobulin. A significant decrease in β-lactoglobulin dimers was

observed when the amount of sugar was greater than 50% in the formulations.

While the reconstituted solutions showed no visible insoluble residues (no optical density at 350 nm), they were also analyzed for sub- visible particles, the results of which are shown in Fig. 7 and 8. The numbers of sub-visible particles in protein formulations are considered important quality attributes [47]. According to the US and European pharmacopeias, there is a limit to the number of sub-visible microparticles that may be present in a protein solution, when observed by light obscuration (LO). Briefly, parenteral therapeutic proteins should contain fewer than 6000 particles/ml larger than 10 µm and 600

particles/ml larger than 25 µm using the LO method, which reports the

concentration and size of particles in liquid samples in the range of 1-600

µm. For this study, we have used flow imaging microscopy, where the

particles in the range of 1-400 µm are captured in individual

photographs. The number of particles above 1 µm, 10 µm and 25 µm for

each sample are reported in Table 5, and in all cases are within the above-mentioned limits.

In order to observe the particle concentration and size in the range of 30-1000 nm, NTA was used. With NTA, the individual particles are tracked by Brownian motion. Although there are currently no

regulations concerning particles that are less than 1 µm in size, the results

from the NTA can provide information regarding the protein aggregation in the sub-micron size range [40].

While the pathway of protein aggregation is presently unclear, induced conditions, such as heat stress or pH shift, enable the smaller aggregates to agglomerate and present larger particles. This has been observed in the case of conventional spray drying, where increasing the temperature induced changes in the secondary structure and aggregation of an immunoglobulin (IgG) [17].

It should be noted that in this study, a significantly large number of particles were detected in the dried pure trehalose after reconstitution. So, it appears that the observed particles are not the result of protein aggregation alone but may also be due to contamination. This is highly likely as the products were not prepared in

a clean room facility. Moreover, the negligible loss of monomer in HP- SEC (Fig. 6) indicates that the level of proteinaceous particles was very low.

In order to evaluate the stability of the protein formulations, the dry samples produced on the 10L scale were stored in a desiccator at

room temperature (22°C) for 1 year. After this time, the formulation 1:10

retained full preservation of the lysozyme activity and structure. The number of particles in range of nanometer and micrometer after redissolving in pure water was not significantly different when compared to the freshly prepared products. For the formulation 1:4, however, a decrease in lysozyme activity was found. Consequently, it appears that in the case of the formulation 1:4 that there is insufficient trehalose to protect the protein from degradation during storage. These results suggest that the stability of the protein is not only dependent on the residual moisture content, but also the formulation composition.

4.3.2 Applicability of the developed method to other proteins

In order to gain insight into the applicability of the scCO2 drying

process for other proteins, the best drying conditions found for lysozyme

were used to prepare dried powders of α-lactalbumin, α-

chymotrypsinogen A, and monoclonal IgG. The same formulation as implemented for lyosozyme was also used for these proteins. Although no attempt was made to optimize the formulation for the specific proteins, in terms of the residual moisture content, protein structure and aggregation, the results for these three proteins were comparable with those of the processed lysozyme.

The sub-visible particle content of the processed α-lactalbumin

formulation was comparable to that of lysozyme. However, a large

increase of sub-visible microparticles was found for the α-

chymotrypsinogen A and monoclonal IgG formulations. This could be due to the non-GMP environment and the suboptimal formulation of each protein. For instance, inclusion of a buffer in the formulation was

shown to increase the recovery of soluble polyclonal IgG after scCO2-

processing [26], likely because it limited CO2-mediated acidification

during the drying process. Therefore, we expect that the loss of soluble protein and the formation of sub-visible particles, as observed for some of the protein formulations, can be inhibited by tailoring the formulation to the needs of each protein. To this end, the mutual influence of processing and formulation effects on protein stability should be studied. This was beyond the scope of the present study, but is currently under investigation.