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Sorbitol Crystallization Can Lead to Protein Aggregation in Frozen Protein Formulations

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Abstract

Purpose

This work examines the cause of aggregation of an Fc-fusion protein formulated in sorbitol upon frozen storage for extended periods of time at −30°C.

Materials and Methods

We designed sub-ambient differential scanning calorimetry (DSC) experiments to capture the effects of long-term frozen storage. The physical stability of formulation samples was monitored by size exclusion high performance liquid chromatography (SE-HPLC).

Results

DSC analysis of non-frozen samples shows the expected glass transitions (Tg′) at −45°C for samples in sorbitol and at −32°C in sucrose. In time course studies where sorbitol formulations were stored at −30°C and analyzed by DSC without thawing, two endothermic transitions were observed: a melting endotherm at −20°C dissipated over time, and a second endotherm at −8°C was seen after approximately 2 weeks and persisted in all later time points. Protein aggregation was only seen in the samples formulated in sorbitol and stored at −30°C, correlating aggregation with the aforementioned melts.

Conclusions

The observed melts are characteristic of crystalline substances and suggest that the sorbitol crystallizes over time. During freezing, the excipient must remain in the same phase as the protein to ensure protein stability. By crystallizing, the sorbitol is phase-separated from the protein, which leads to protein aggregation.

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Abbreviations

10 mM sodium acetate, pH 4.0 with 300 mM sorbitol:

(A4S)

10 mM sodium acetate, pH 4.0 with 320 mM sucrose:

(A4Su)

10 mM sodium acetate, pH 5.0 with 300 mM sorbitol:

(A5S)

differential scanning calorimetry:

(DSC)

fragment crystallizes easily:

(Fc)

glass transition temperature:

(Tg′)

size exclusion high performance liquid chromatography:

(SE-HPLC)

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Acknowledgments

We thank Dr. Michael Treuheit, Dr. Vasumathi Dharmavaram, Alison Butler, Priti Parmar, Dr. Mary Elizabeth Wimer, Dr. David Brems and Dr. Susan Hershenson for a critical reading of the manuscript and support.

Deirdre Murphy Piedmonte and Christie Summers contributed equally to this work.

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Authors and Affiliations

  1. Department of Pharmaceutics, Amgen Inc, MS 2-1-A, One Amgen Center Drive, Thousand Oaks, California, 91320, USA

    Deirdre Murphy Piedmonte, Christie Summers, Arnold McAuley,  Lejla Karamujic & Gayathri Ratnaswamy

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  1. Deirdre Murphy Piedmonte
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  2. Christie Summers
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Correspondence to Gayathri Ratnaswamy.

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Piedmonte, D.M., Summers, C., McAuley, A. et al. Sorbitol Crystallization Can Lead to Protein Aggregation in Frozen Protein Formulations. Pharm Res 24, 136–146 (2007). https://doi.org/10.1007/s11095-006-9131-1

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  • DOI: https://doi.org/10.1007/s11095-006-9131-1

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