Combinations of arginine and pullulan reveal the selective effect of stabilization mechanisms on different lyophilized proteins

Khanh T. T. Nguyen, Daan Zillen, Alessia Lasorsa, Patrick C. A. van der Wel, Henderik W. Frijlink, Wouter L. J. Hinrichs*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

The stability of lactate dehydrogenase (LDH) and β-galactosidase (β-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while β-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, β-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and β-gal may be dependent on the interaction with arginine.

Original languageEnglish
Article number123938
Number of pages9
JournalInternational Journal of Pharmaceutics
Volume654
Early online date24-Feb-2024
DOIs
Publication statusPublished - 10-Apr-2024

Keywords

  • SSNMR
  • NMR

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