Bioprocessing References

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Quality control to develop high-quality cell cultures

• Rapid cell counting
• Accurate assessment of proliferation and viability
• Ensuring monoclonality
• Functional and screening assays

Important criteria for clone selection [Gupta and Shukla, 2017]

• Cell growth pattern
• High protein expression
• Appropriate PTMs
• Genetic stability

Figure 1. Wild-type AAV exposure and patient characteristics determine an individual’s response at vector delivery and overtime.

It is critical to assess immune status in patients to monitor gene therapy efficacy [33]. Subjects previously treated with gene therapy may require a second dose as an initial treatment may have activated a patient’s adaptive immune system to eliminate the vector upon reintroduction. This proves that assessing a patient’s immune status is critical to monitoring gene therapy efficacy [33].

To improve the effectiveness of vector delivery, image cytometers can be employed for:

• Rapid screening for nAbs prior to treatment
  • Cell-based in vitro transduction inhibition assays
• Long-term patient monitoring
  • Humoral immune responses for antibodies against the vector and gene therapy protein product
  • T-cell immune responses

Use plate-based imaging to screen patient sera for neutralizing antibodies and monitoring immune responses over time

The Celigo Image Cytometer is a plate-based high-throughput system that simultaneously images and analyzes cells in standard multi-well plates using brightfield and fluorescence technology. The Celigo rapidly screens for sera nAbs that can neutralize AAV vectors for potential pre-existing immunities.

1. High-throughput automated imaging and direct analysis in 96-well plates
2. No trypsinization necessary to count cells
3. Measure antibody neutralization effects in less than ten minutes

This section provides two technical examples of how the Celigo Image Cytometer can be used to develop a robust screening assay for potential pre-existing immunity to AAV vectors.

1. High-throughput neutralization assay of AAV-GFP vectors using sera
2. High-throughput neutralization assay of AAV-GFP vectors using neutralizing antibodies