A framework to quantify karyotype variation associated with CHO cell line instability at a single-cell level.

Area of Science:

• Biotechnology
• Cell Biology
• Genetics

Background:

• Chinese hamster ovary (CHO) cells are crucial for therapeutic protein biomanufacturing.
• Cell line instability, causing unpredictable changes in productivity and quality, remains a significant challenge.
• Genomic instability and chromosomal rearrangements are suspected contributors to production instability.

Purpose of the Study:

• To investigate the association between chromosomal rearrangements and production instability in CHO cells.
• To develop a model and framework for characterizing these genomic events.
• To elucidate the mechanism driving production instability in biomanufacturing.

Main Methods:

• Developed a production instability model using SEAP-expressing CHO cells (CHO-SEAP).
• Quantified chromosomal rearrangements using karyotyping and fluorescence in situ hybridization (FISH).
• Assessed cell growth rate and SEAP productivity under varying conditions (with/without methotrexate).

Main Results:

• Absence of methotrexate led to increased growth rate, decreased productivity, and altered chromosomal rearrangement ratios in CHO-SEAP cells.
• Re-introduction of methotrexate reversed these changes, indicating reversibility of instability.
• FISH analysis confirmed SEAP gene integration within a chromosomal rearrangement (der(Z9)), correlating with instability.

Conclusions:

• Chromosomal rearrangements are mechanistically linked to production instability in CHO cells.
• Genomic instability can generate cell populations with a growth advantage but reduced productivity.
• This phenomenon, where non-producing cells outgrow producers, explains instability in biomanufacturing.