Activity–stability considerations of trypsinogen during spray drying: Effects of sucrose

Abstract

The preparation and processing of protein pharmaceuticals into powders may impose significant stresses that could perturb and ultimately denature them. In many cases their stabilization through added excipients is necessary to yield native and active proteins. In this study, the effect of spray drying on the structure and activity of a model protein (trypsinogen) was investigated. In the absence of excipients, spray drying resulted in small losses of its enzymatic activity. Protein conformational rearrangements in the solid state (observed via FTIR) and irreversible aggregation (upon reconstitution) constituted the major degradation pathways. The irreversible unfolding in the solid state was also confirmed by solution calorimetric studies that indicated a decreased thermal stability of the spray-dried protein after reconstitution. The presence of sucrose, a thermal and dehydration stress stabilizer, induced a concentration-dependent protective effect. Protein protection was afforded even at low carbohydrate concentrations, while at specific mass ratios (sucrose-to-protein = 1:1) complete activity preservation was achieved. However, at the high end of sucrose concentrations, a small destabilization was evident, indicating that excluded volume effects may be undesirable during preparation of protein microparticles via spray drying. The profile of both the protein conformational changes and thermal stability in the solid state closely followed that of the incurred activity losses, indicating that protein stabilization during dehydration is crucial during processing of these polypeptides.

Effect of drying methods of microencapsulated Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris on secondary protein structure and glass transition temperature as studied by Fourier transform infrared and differential scanning calorimetry

“…In addition, Schüle et al (2007) found that mannitol at relatively low concentration protected antibodies during spraydrying with inlet and outlet temperatures of 90°C and 50°C, which was similar to our spray-drying procedure. Similar results were demonstrated by Tzannis and Prestrelski (1999) and Liao et al (2002) using different sugars as the protein protectant. The protective mechanism during freeze-or spray-drying of protein models in those studies is taken place through water replacement via H-bond (Maury et al, 2005); hence preservation of protein folding occurs (Garzon-Rodriguez et al, 2004).…”

Mechanisms of protein stabilization in the solid state

“…6,9,10 However, the mechanisms of stabilization in solid state are still not completely understood. Therefore, formulation development in the pharmaceutical industry remains an empirical effort to some extent.…”

“…Above the weight ratio of 1:1, r decreases with increasing sucrose suggesting phase separation in the formulation matrix. 9 That is, the conformation become less native above the weight ratio of 1:1 due to the phase separation.…”

Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies

“…During spray drying, the protein is subjected to nebulization followed by air-drying; thus excipients are chosen to minimize damage due to exposure of air-water interfaces and high temperature [Tzannis and Prestrelski 1999]. Particle morphology and protein stability as a function of spray drying parameters such as input flow rates and temperature have been described [Maa et al, 1997].…”