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Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

Abstract

Purpose

The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported.

Methods

The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively.

Results

Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D2O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing.

Conclusions

The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.

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Abbreviations

API:

Active pharmaceutical ingredient

AUC:

Area under curve

BSA:

Bovine serum albumin

CD:

Circular dichroism

DSC:

Differential scanning calorimetry

FCL:

Freeze-concentrated liquid

FTIR:

Fourier transform infrared spectroscopy

HPSEC:

High performance size exclusion chromatography

IR:

Infrared

LO:

Light obscuration

XRD:

Powder x-ray diffractometry

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ACKNOWLEDGMENTS AND DISCLOSURES

This research was funded by an NSF grant (CBET-1335936) to A.A. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

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Correspondence to Alptekin Aksan.

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Jena, S., Horn, J., Suryanarayanan, R. et al. Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability. Pharm Res 34, 462–478 (2017). https://doi.org/10.1007/s11095-016-2078-y

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KEY WORDS

  • crystallization
  • freeze-drying
  • glass transition
  • mannitol
  • trehalose