5th Annual International Conference on Lyophilization and Freeze Drying

Bologna, Italy, March 2012

New attempt to freeze-drying red blood cells using stevioside as an alternative of lyoprotectant

Virgilio Tattini Jr., Mariana M. Vilhena, Ludmila Moroz, Denise T. Fantoni, Ronaldo N.M. Pitombo

Dr. Virgilio Tattini JuniorPhD Lyophilization Technology

Professor Ronaldo Pitombo

Thomas Jennings

Introduction

• Several attempts were made to improve RBC storage conditions:

Cryo/lyoptortectants;

Process engineering: freeze conditions, temperature profiles;

• Something is missing ?

• More important than lyoprotectants itself !

Aim

Material and Methods

Loading RBC

ElectroporationImmersion

Thermal Analisys

(RBC, Stevioside, RBC + Stevioside )

DSC  Lyostat

Tg´ Tcryst.Tmelt.

Tcol. Tcryst.Tmelt.

FreezingSF (shelves): +25 to -55ºC (± 1,5ºC/min.);FF (N2): +25 to – 150ºC (± 35ºC/min.);

Primary DryingTsh: -35ºCPch: 130 mTorrTco: -80 ºC

Secondary DryingTsh: 25ºCPch: 130 mTorrTco: -80 ºC

Freeze-drying

Hematology analysis and hemolysis measurement

Percent hemolysis (Drabkin’s reagent);

% Hemolysis = 100 x (OD540 of the free Hb)(OD540 of the total Hb)

Radiometer blood gas analyzer

Morphology

Results

Fig.1.Comparison between immersion and electroporation methodologies on the pH values of RBC suspensionincorporated with stevioside at different concentrations.

Optimal pH

Fig.2. Comparison between immersion and Electroporation methodologies on the gasometry of RBCsuspension incorporated with stevioside at different concentrations.

Optimal CO2Optimal O2

Fig.3. Comparison between immersion and electroporation methodologies on the degree of hemolysis ofRBC suspension incorporated with stevioside at different concentrations.

Fig.4. Morphological characteristics of RBCs, incorporated with stevioside by immersion, obtained byoptical microscopy of 500 x magnitude. (A) stevioside 1%; (B) stevioside 3%; (C) stevioside 5% and (D)stevioside 10%.

Fig.5. Morphological characteristics of RBCs, incorporated with stevioside by electroporation, obtained byoptical microscopy of 500 x magnitude. (A) stevioside 1%; (B) stevioside 3%; (C) stevioside 5% and (D)stevioside 10%.

Fig.6. DSC curve showing the glass transition temperature of the maximally freeze-concentratedregion (Tg´) of RBC´s incorporated with stevioside 3% by electroporation.

Tg´ midpoint

Fig.7. Freeze-drying microscopy images of RBC´s with stevioside 3% incorporated by electroporation. (A)Crystallization of the sample at – 14.3ºC; (B) Freeze-drying front formed at – 44ºC; (C) Progress ofsublimation front at – 37ºC; (D) Collapse of the sample at – 34.3ºC.

RBC suspensionRBC suspension CrystallizationCrystallization

Determining the optimum freezing rate during the freeze-drying process of RBC

Fig.8. Comparison between slow freezing and fastfreezing on the pH values of freeze-dried RBC´sincorporated with stevioside 3% by electroporation.

Fig.9. Comparison between slow freezing and fastfreezing on the gasometry results of freeze-dried RBC´sincorporated with stevioside 3% by electroporation.

Optimal pH

Optimal CO2

Optimal O2

Fig.10. Comparison between slow freezing (A) and fastfreezing (B) on the SEM of freeze-dried RBC´s incorporatedwith stevioside 3% by electroporation. Magnitude of 2000and 5000 X respectively.

Conclusion

Stevioside (1 and 3%) – OK

Freeze-drying – not OK

Future

THANK YOU

Dr. Virgilio Tattini JuniorPhD Lyophilization Technology