1. Stabilizing Fluorochrome-Labeled Antibodies in Lyophilization with Disaccharide Excipients Meredith Pearcy 2005

2. BIOLYPH LLC Hopkins, MN Photo used with permission of Lois Fruen

3. BD Biosciences San Diego, CA Photos used with permission of Dr. Homero Sepulveda

4. Introduction Antibodies can be tagged with fluorochromes for fluorescence-activated cell sorting (flow cytometry). Antibodies used for immunology and drug discovery research. Liquid antibodies and fluorochromes aggregate and lose activity. Lyophilization can extend antibody activity.

5. Problem Lyophilization can damage antibodies. Excipients are added to protect antibodies. Excipients used included:  Disaccharides (to protect antibody structure)  Buffers (to maintain pH)  Proteins (to prevent non-specific binding of antibody)  Detergents (to prevent non-specific binding of antibody)

6. Previous Studies Andya et al. (2003) –Sucrose and trehalose cover hydrogen bonding sites, inhibit aggregation. Johnson et al. (2002) –Combination of mannitol and sucrose gives durable cake, protein stability.

7. Goals To develop the most effective excipient solution to retain antibody and fluorochrome activity through lyophilization for: Individual rat anti-mouse antibodies  CD3e-PE-Cy7, CD4-PE, CD8a-FITC, CD8a-APC Rat anti-mouse antibody cocktail  CD3e-PE-Cy7 + CD4-PE + CD8a-APC Higher chance of aggregation

8. Procedure

9. Excipient Formulations Sugar Content  Excipient I 15% sucrose  Excipient II 8% trehalose 7% sucrose  Excipient III 7% mannitol 5% trehalose Base Formulation  0.01% PEG 8000  0.05% CHAPS  20 mM HEPES and150 mM NaCl at pH 7.4  0.5% BSA  0.09% sodium azide

10. Procedure

11. Lyophilizer Photo taken by student

12. Procedure

13. Flow Cytometer Photo taken by Meredith Pearcy

14. Results Figures 1-4: Retained Antibody Activity of Individual Antibodies

15. Results Figure 5: Retained Antibody Activity of Antibody Cocktail

16. Results Figure 6: Most Effective and Least Effective Excipients  Excipient I 15% sucrose  Excipient II 8% trehalose 7% sucrose  Excipient III 7% mannitol 5% trehalose

17. Conclusions Excipient I, 15% sucrose, was most effective in retaining individual antibody and fluorochrome activity. Excipient I was most effective in retaining cocktail antibody and fluorochrome activity.

18. Discussion Sucrose successfully stabilized:  Individual antibodies and fluorochromes  Cocktail antibodies and fluorochromes Possible explanations  Sucrose freezes in form known to protect antibodies (Andya et al.)  Sucrose was the only sugar in Excipient I.

19. Future Work Test samples for long-term stability by simulating 1, 2, 3, and 4 years Test antibodies with different excipient solutions  Experiment with different base formulations Vary detergents, proteins Vary ingredient concentrations  Experiment with sugars Vary sugars Include sucrose in some excipients Test different antibodies and fluorochromes

20. Application Stable fluorochrome-labeled antibodies for use on-demand for immunology research Fluorochrome-labeled antibodies for other uses: Tumor detection and visualization

21. Acknowledgements Dr. Homero Sepulveda Jacob and Fariba Tiffany and Jennifer Dad and Cathy Dr. Paul Haley, Dr. Charles Carpenter, Mr. Jim Sackrison Ms. Fruen Dr. Miller Team Research

22. Stabilizing Fluorochrome-Labeled Antibodies in Lyophilization with Disaccharide Excipients Meredith Pearcy 2005