Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

TitleAutomated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.
Publication TypeJournal Article
Year of Publication2005
AuthorsHanania EG, Fieck A, Stevens J, Bodzin LJ, Palsson BØ, Koller MR
JournalBiotechnology and bioengineering
Volume91
Issue7
Pagination872-6
PubMed Date2005 Sep 30
ISSN0006-3592
KeywordsAnimals, Antibodies, Antibody-Producing Cells, Cell Adhesion, Cell Line, Cell Separation, CHO Cells, Cloning, Molecular, Cricetinae, Humans, Hybridomas, Laser Scanning Cytometry, Lasers, Mice, Microscopy, Fluorescence, Recombinant Proteins, Staining and Labeling
Abstract

Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

Alternate JournalBiotechnol. Bioeng.
PubMed ID15937942

Location

Location

417 Powell-Focht Bioengineering Hall

9500 Gilman Drive La Jolla, CA 92093-0412

Contact Us

Contact Us

In Silico Lab:  858-822-1144

Wet Lab:  858-246-1625

FAX:   858-822-3120

Website Concerns: sbrgit@ucsd.edu

User Login