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AAPS PharmSciTech. 2005 June; 6(2): E150–E157.
Published online 2005 September 30. doi:  10.1208/pt060223
PMCID: PMC2750526

Freeze-drying of proteins from a sucrose-glycine excipient system: Effect of formulation composition on the initial recovery of protein activity

Abstract

The purpose of this study was to investigate the effect of sucrose-glycine excipient systems on the stability of selected model proteins during lyophilization. Recovery of protein activity after freeze-drying was examined for the model proteins lactate dehydrogenase and glucose 6-phosphate dehydrogenase in a sucrose-glycine-based excipient system in which the formulation composition was system-atically varied. In a sucrose-only excipient system, activity recovery of both model proteins is about 80% and is independent of sucrose concentration over a range from 1 to 40 mg/mL. When both sucrose and glycine are used and the ratio of the 2 excipients is varied, however, activity recovery decreases in a pattern that is consistent with the inhibition of activity recovery by glycine crystals, despite the presence of an adequate amount of sucrose to afford protection. Annealing of sucrose-glycine formulations causes a small but significant decrease in activity recovery relative to unannealed controls, whereas no annealing effect is observed with sucrose-only formulations. Addition of 0.01% polysorbate 80 to the formulation resulted in complete recovery of activity, irrespective of the sucroseglycine ratio or annealing. Addition of the same concentration of polysorbate 80 to the reconstitution medium caused an increase in activity recovery for each formulation, but the overall pattern remained unchanged. The data are consistent with an interfacial model for lyophilization-associated loss of protein activity involving denaturation at a solid/freeze-concentrate interface.

Keywords: interfacial denaturation, protein formulation, X-ray powder diffraction, crystallization, annealing, lyophilization

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
1. Carpenter JF, Pikal MJ, Chang B, Randolph TW. Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res. 1997;14:969–975. doi: 10.1023/A:1012180707283. [PubMed] [Cross Ref]
2. Osterberg T., Fatouros A, Mikaelsson M. Development of freeze-dried albumin-free formulation of recombinant factor VIII SQ. Pharm Res. 1997;14:892–898. doi: 10.1023/A:1012199816852. [PubMed] [Cross Ref]
3. Chang BS, Fischer NL. Development of an efficient single-step freeze-drying cycle for protein formulations. Pharm Res. 1995;12:831–837. doi: 10.1023/A:1016200818343. [PubMed] [Cross Ref]
4. Chang BS, Reeder G, Carpenter JF. Development of a stable freeze-dried formulation of recombinant human interleukin-1 receptor antagonist. Pharm Res. 1996;13:243–249. doi: 10.1023/A:1016043114998. [PubMed] [Cross Ref]
5. Chang BS, Beauvais R, Dong A, Carpenter JF. Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation. Arch Biochem Biophys. 1996;331:249–258. doi: 10.1006/abbi.1996.0305. [PubMed] [Cross Ref]
6. Jiang S, Nail SL. Effect of process conditions on recovery of protein activity after freezing and freeze-drying. Eur J Pharm Biopharm. 1998;45:249–257. doi: 10.1016/S0939-6411(98)00007-1. [PubMed] [Cross Ref]
7. Sarciaux JM, Mansour S, Hageman MJ, Nail SL. Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. J Pharm Sci. 1999;88:1354–1361. doi: 10.1021/js980383n. [PubMed] [Cross Ref]
8. Pikal M, Dellerman K, Roy M, Riggin R. The effect of formulation variaibles on the stability of freeze-dried human growth hormone. Pharm Res. 1991;8:427–437. doi: 10.1023/A:1015834724528. [PubMed] [Cross Ref]
9. Shalaev E, Kanev AN. Study of the solid-liquid state diagram of the water-glycine-sucrose system. Cryobiol. 1994;31:374–382. doi: 10.1006/cryo.1994.1045. [Cross Ref]
10. Shalaev EJ, Malakhov DV, Kanev AN, et al. Study of the phase diagram water fraction of the system water-glycine-sucrose by DTA and X-ray diffraction methods. Thermochimica Acta. 1992;196:213–220. doi: 10.1016/0040-6031(92)85021-M. [Cross Ref]
11. Strambini GB, Gabellieri E. Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys. J. 1996;70:971–976. doi: 10.1016/S0006-3495(96)79640-6. [PubMed] [Cross Ref]
12. Eckhardt BM, Oeswein JQ, Bewley TA. Effect of freezing on aggregation of human growth hormone. Pharm Res. 1991;8:1360–1364. doi: 10.1023/A:1015888704365. [PubMed] [Cross Ref]
13. Chang BS, Kendrick BS, Carpenter JF. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci. 1996;85:1325–1330. doi: 10.1021/js960080y. [PubMed] [Cross Ref]
14. Millqvist-Fureby A, Malmsten M, Bergenstahl B. Surface characterisation of freeze-dried protein/carbohydrate mixtures. Int J Pharm. 1999;191:103–114. doi: 10.1016/S0378-5173(99)00285-9. [PubMed] [Cross Ref]

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