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Characterizing the Freeze–Drying Behavior of Model Protein Formulations

  • Research Article
  • Theme: Sterile Products: Advances and Challenges in Formulation, Manufacturing, Devices and Regulatory Aspects
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Abstract

The freeze–drying behavior of three model proteins, namely, lysozyme, BSA, and IgG, has been studied using a variety of techniques under two different primary drying conditions (shelf temperatures of −25°C and +25°C, respectively) in an amorphous formulation. Manometric temperature measurements were used to characterize product temperature (T pr), sublimation rates, and product resistance (R p) during primary drying. Biophysical techniques such as circular dichroism, fluorescence, and Fourier transform infrared spectroscopy were used to study protein conformation. Size exclusion chromatography was used to monitor the formation of high-molecular-weight species (HMWS) over time on storage, and cake morphology was studied using scanning electron microscopy. The differences in the freeze–drying behavior of the three proteins were more evident at higher protein concentrations, where the protein significantly influences the behavior of the formulation matrix. However, these differences were minimized in the aggressive mode and were insignificant at lower protein concentrations where excipients dominated the freeze–drying behavior. Differences in cake morphology were observed between the two drying conditions employed as well as between the three proteins studied. The stability and the protein structure, however, were equivalent for the protein cakes generated using the two different primary drying conditions.

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References

  1. Wang W. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm. 2000;203:1–60.

    Article  CAS  PubMed  Google Scholar 

  2. Tang X, Pikal MJ. Design of freeze–drying processes for pharmaceuticals: practical advice. Pharm Res. 2004;21:191–200.

    Article  CAS  PubMed  Google Scholar 

  3. Schwegman JJ, Hardwick LM, Ackers MJ. Practical formulation and process development of freeze-dried products. Pharm Dev Technol. 2005;10:151–73.

    CAS  PubMed  Google Scholar 

  4. Manning MC, Patel K, Borchardt RT. Stability of protein pharmaceuticals. Pharm Res. 1989;6:903–18.

    Article  CAS  PubMed  Google Scholar 

  5. Johnson RE, Oldroyd ME, Ahmed SS, Gieseler H, Lewis LM. Use of manometric temperature measurements (MTM) to characterize the freeze–drying behavior of amorphous protein formulations. J Pharm Sci. 2010;99:2863–73. doi:10.1002/jps.22031.

    CAS  PubMed  Google Scholar 

  6. Meister E, Gieseler H. Freeze–dry microscopy of protein/sugar mixtures: drying behavior, interpretation of collapse temperatures and a comparison to corresponding glass transition data. J Pharm Sci. 2009;98:3072–87.

    Article  CAS  PubMed  Google Scholar 

  7. Fonseca F, Passot S, Cunin O, Marin M. Collapse temperature of freeze-dried Lactobacillus bulgaricus suspensions and protective media. Biotechnol Prog. 2004;20:229–38.

    Article  CAS  PubMed  Google Scholar 

  8. Colandene JD, Maldonado LM, Creagh AT, Vrettos JS, Goad KG, Spitznagel TM. Lyophilization cycle development for a high-concentration monoclonal antibody formulation lacking a crystalline bulking agent. J Pharm Sci. 2007;96:1598–608.

    Article  CAS  PubMed  Google Scholar 

  9. Milton N, Pikal MJ, Roy ML, Nail SL. Evaluation of manometric temperature measurement as a method of monitoring product temperature during lyophilization. J Parent Sci Technol. 1997;51:7–15.

    CAS  Google Scholar 

  10. Overcashier DE, Patapoff TW, Hsu CC. Lyophilization of protein formulations in vials: Investigation of the relationship between resistance to vapor flow during primary drying and small-scale product collapse. J Pharm Sci. 1999;88:688–95.

    Article  CAS  PubMed  Google Scholar 

  11. Tang X, Nail SL, Pikal MJ. Freeze–drying process design by manometric temperature measurement: design of a smart freeze-dryer. Pharm Res. 2005;22:685–700.

    Article  CAS  PubMed  Google Scholar 

  12. Lewis LM, Teagarden DL, Johnson RE, Ahmed SS. Rational design of a freeze-dried formulation for a biologic. Amer Pharm Rev. 2008;11(3):91–6.

    CAS  Google Scholar 

  13. Gieseler H, Kramer T, Pikal MJ. Use of manometric temperature measurement (MTM) and SMART™ freeze dryer technology for development of an optimized freeze–drying cycle. J Pharm Sci. 2007;96:3402–18.

    Article  CAS  PubMed  Google Scholar 

  14. Cleland JL, Lam X, Kendrick B, Yang J, Yang T-H, Overcashier D, et al. A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody. J Pharm Sci. 2001;90:310–21.

    Article  CAS  PubMed  Google Scholar 

  15. Han Y, Jin B-S, Lee S-B, Sohn Y, Joung J-W, Lee J-H. Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage. Arch Pharm Res. 2007;30:1124–31.

    Article  CAS  PubMed  Google Scholar 

  16. Wang D, Hey J, Nail S. Effect of collapse on stability of freeze-dried recombinant factor VIII and α-amylase. J Pharm Sci. 2004;93:1253–63.

    Article  CAS  PubMed  Google Scholar 

  17. Luthra S, Obert J-P, Kalonia D, Pikal M. Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer. J Pharm Sci. 2007;96:61–70.

    Article  CAS  PubMed  Google Scholar 

  18. Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G. Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze–drying. J Pharm Sci. 2010;99:2256–78.

    Article  CAS  PubMed  Google Scholar 

  19. Passot S, Fonseca F, Barbouche N, Marin M, Alarcon-Lorca M, Rolland D, et al. Effect of product temperature during primary drying on the long-term stability of lyophilized proteins. Pharm Dev Technol. 2007;12:543–53.

    Article  CAS  PubMed  Google Scholar 

  20. Venyaminov SY, Yang JT. In: Gasman GD, editor. Circular dichroism and the conformational analysis of biomolecules. New York: Plenum; 1996. p. 69–104.

    Google Scholar 

  21. Takeda K, Miura M, Takagi T. Stepwise formation of complexes between sodium dodecyl sulfate and bovine serum albumin detected by measurements of electric conductivity, binding isotherm, and circular dichroism. J Coll Interface Sci. 1981;82:38–44.

    Article  CAS  Google Scholar 

  22. K-y N, Zhao L, Meyer JD, Rittmann-Grauer L, Manning MC. Use of circular dichroism spectroscopy in determining the conformation of a monoclonal antibody prior to its incorporation in an immunoliposome. J Pharm Biomed Analysis. 1997;16:507–13.

    Article  Google Scholar 

  23. Parker A, Rigby-Singleton S, Perkins M, Bates D, Le Roux D, Roberts CJ, et al. Determination of the influence of primary drying rates on the microscale structural attributes and physicochemical properties of protein containing lyophilized products. J Pharm Sci. 2010;99:4616–29.

    Article  CAS  PubMed  Google Scholar 

  24. Costantino HR, Griebenow K, Mishra P, Langer R, Klibanov AM. Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form. Biochim Biophys Acta. 1995;1253:69–74.

    PubMed  Google Scholar 

  25. Liao X, Krishnamurthy R, Suryanarayanan R. Influence of the active pharmaceutical ingredient concentration on the physical state of mannitol—implication in freeze–drying. Pharm Res. 2005;22:1978–84.

    Article  CAS  PubMed  Google Scholar 

  26. Kramer T, Kremer DM, Pikal MJ, Petre WJ, Shalaev EY, Gatlin LA. A procedure to optimize scale-up for the primary drying phase of lyophilization. J Pharm Sci. 2009;98:307–18.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Global Biologics, Pfizer Inc., 700 Chesterfield Parkway, Mail Stop AA5I, Chesterfield, Missouri, 63017, USA

    Lavinia M. Lewis, Robert E. Johnson, Megan E. Oldroyd, Saleem S. Ahmed, Liji Joseph & Sandipan Sinha

  2. Groton Laboratories, Pfizer Inc., Eastern Point Road, Groton, Connecticut, 06340, USA

    Ilie Saracovan

Authors
  1. Lavinia M. Lewis
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  2. Robert E. Johnson
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  3. Megan E. Oldroyd
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  4. Saleem S. Ahmed
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  5. Liji Joseph
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  6. Ilie Saracovan
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  7. Sandipan Sinha
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Correspondence to Lavinia M. Lewis.

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Guest Editors: Lavinia Lewis, Jim Agalloco, Bill Lambert, Russell Madsen, and Mark Staples

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Lewis, L.M., Johnson, R.E., Oldroyd, M.E. et al. Characterizing the Freeze–Drying Behavior of Model Protein Formulations. AAPS PharmSciTech 11, 1580–1590 (2010). https://doi.org/10.1208/s12249-010-9530-9

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  • DOI: https://doi.org/10.1208/s12249-010-9530-9

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