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

BIOLYPH

LLC Hopkins, MN QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture.

Photo used with permission of Lois Fruen

BD Biosciences

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

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.

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)

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.

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

Procedure

Excipient Formulations

•

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 •

Sugar Content

 Excipient I • 15% sucrose  Excipient II • 8% trehalose • 7% sucrose  Excipient III • 7% mannitol • 5% trehalose

Procedure

Lyophilizer

Photo taken by student

Procedure

Flow Cytometer

Photo taken by Meredith Pearcy

Results

Figures 1-4: Retained Antibody Activity of Individual Antibodies CD3e -PE Cy7 Excipient I II III Percent Retained 100 90 100 CD8a -APC Excipient I II III Percent Retained 96 97 94 CD4-PE Excipient I II III Percent Retained 99 100 92 CD8a -FITC Excipient I II III Percent Retained 100 97 100

Results

Figure 5: Retained Antibody Activity of Antibody Cocktail Cocktail Excipient I II III Percent Retained 99 90 86

Results

Figure 6: Most Effective and Least Effective Excipients Antibody CD3e-PE-Cy7 CD4-PE CD8a-APC CD8a-FITC Cocktail Most Effective Excipient I, III I, II I, II I, III I  Excipient I •15% sucrose  Excipient II •8% trehalose •7% sucrose  Excipient III •7% mannitol •5% trehalose

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.

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 .

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

Application

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

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

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