Effect of Arginine on the Aggregation of Protein in Freeze-Dried Formulations Containing Sugars and Polyol: II. BSA Reconstitution and Aggregation
The current paper continues our study on the ability of l-arginine to prevent/reduce the aggregation of proteins that results from the various stresses during the lyophilisation and/or storage of lyophilized protein-based products. The first part of our study, i.e. formulation development, was devoted to the rational design and optimization of an l-arginine containing lyophilized formulation which can resist the natural tendency of l-arginine to absorb atmosphere moisture. Mannitol and trehalose were chosen among other excipients to be included in the protein-based formulation, as mannitol in a combination with l-arginine has been shown to reduce moisture sorption while trehalose provides a degree of lyoprotection. In the present study, a number of formulations, which comprised bovine serum albumin (BSA) with and without l-arginine, and with five different ratios of trehalose-to-mannitol (from 30:70 to 80:20) were lyophilised and assessed. The internal structures and the moisture sorption/retention of the lyophilized formulations were characterised. To study the effect of l-arginine on BSA solid-phase stability, the lyophilized powder was exposed to accelerated storage conditions (high moisture (75% RH) and temperature (22 or 45 °C)) for up to 24 h. The lyophilized BSA formulations were then reconstituted and solution-state protein aggregation assessed by turbidimetry at 360 nm and fluorescence spectroscopy using the thioflavin T assay. It was demonstrated that l-arginine can be used in protein-based freeze-dried formulations to significantly reduce the aggregation of protein during the manufacturing, storage and subsequent reconstitution. The results also revealed the importance of a sufficient amount of mannitol in the arginine-containing formulations.
KEY WORDSfreeze-drying protein aggregation l-arginine mannitol trehalose
Bovine serum albumin
Differential scanning calorimetry
Scanning electron microscopy
The authors would like to thank Rachel Armitage (De Montfort University) for help with recording SEM images. J. Darkwah acknowledges De Montfort University for PhD studentship.
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