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Effect of Arginine on the Aggregation of Protein in Freeze-Dried Formulations Containing Sugars and Polyol: II. BSA Reconstitution and Aggregation

  • Ellen Hackl
  • Joseph Darkwah
  • Geoff Smith
  • Irina Ermolina
Research Article

Abstract

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 WORDS

freeze-drying protein aggregation l-arginine mannitol trehalose 

Abbreviations

BSA

Bovine serum albumin

Arg

l-arginine HCl

ThT

Thioflavin T

FD

Freeze-drying/freeze-dried

RH

Relative humidity

OD

Optical density

TGA

Thermogravimetric analysis

DSC

Differential scanning calorimetry

SEM

Scanning electron microscopy

Notes

Acknowledgements

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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Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Ellen Hackl
    • 1
    • 2
  • Joseph Darkwah
    • 1
  • Geoff Smith
    • 1
  • Irina Ermolina
    • 1
  1. 1.Leicester School of Pharmacy, Faculty of Health and Life SciencesDe Montfort UniversityLeicesterUK
  2. 2.School of PharmacyUniversity of ReadingReadingUK

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