Stabilizing Fluorochrome-Labeled Antibodies in Lyophilization with Disaccharide Excipients

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Stabilizing Fluorochrome-Labeled Antibodies in
Lyophilization with Disaccharide Excipients
Meredith Pearcy 2005
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BIOLYPH LLCHopkins, MN
Photo used with permission of Lois Fruen
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BD BiosciencesSan Diego, CA
Photos used with permission of Dr. Homero
Sepulveda
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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.

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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)

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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.

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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

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Procedure
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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

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Procedure
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Lyophilizer
Photo taken by student
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Procedure
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Flow Cytometer
Photo taken by Meredith Pearcy
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Results
Figures 1-4 Retained Antibody Activity of
Individual Antibodies
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Results
Figure 5 Retained Antibody Activity of Antibody
Cocktail
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Results

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

Figure 6 Most Effective and Least Effective
Excipients
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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.

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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.

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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

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Application

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

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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

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