Optimize, Adapt and Overcome - 6

Optimize, Adapt, and Overcome: Tips from the Cell Line Development Front * Single-Cell Printing for Establishing Monoclonality

quality. In the process just described, the FDA
specifically requires evidence that a given
mAb is derived from a single cell and that
necessary precautions are taken to minimize
cross-contamination with potentially harmful
agents (e.g., viruses, prions, etc.).
The successful isolation of single cells with
high efficiency and viability is therefore critical
to the cell-line development process, but traditional methods face significant challenges
to meet both needs simultaneously. Limiting
dilution utilizes a series of successive dilution
steps to generate a low concentration of cells,
which are then deposited into a microplate
via manual pipetting or automated liquid handling. At such low concentrations, most wells
will not contain a cell, but those that do will
most likely contain a single cell (as determined
statistically by a Poisson distribution). The
process is simple, cost-effective, and gentle
on the cells, resulting in high viability.
However, the statistical nature of the process
significantly impacts efficiency because at
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best, 30% of wells will contain a single cell.
Furthermore, one cannot assure the presence
of a single cell per well, which often necessitates an additional round of cloning for
regulatory purposes.
Another frequently used method is flow
cytometry, which isolates single cells efficiently
(up to 99% in some cases). Most systems also
possess fluorescence sorting capabilities,
which can be used to screen for productivity,
but is limited in its application, because of the
requirement of a membrane-bound fluorescence signal. The most significant limitation to
flow-based systems, however, is that a portion
of cells are nonviable due to the shear stress
and pressures experienced within the flow cell.
Furthermore, cross-contamination is problematic because it can only be minimized by limiting access to an instrument or by replacing
the flow cell, which is fairly expensive. Finally,
the sophistication of the technique demands
intricate knowledge of the instrument, considerably limiting accessibility to this workflow.

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Optimize, Adapt and Overcome

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