Optimize, Adapt and Overcome - 27

Optimize, Adapt, and Overcome: Tips from the Cell Line Development Front * Enhanced Development of Virus-Specific Hybridomas Using ClonePix and CloneSelect Imager Technologies

Biotinylated peptide was loaded onto
streptavidin surface followed by the addition
of undiluted hybridoma supernatants from 146
samples to measure for IgG binding. Top seven
highly specific sub-clones had exhibited optimal
binding and were chosen for banking.

Pilot Scale Productions of High IgG
Secreting Sub-Clones
Seven high-valued, specific sub-clones were
scaled up in liquid culture for future analysis.
They were expanded and once sufficient volumes
of culture were obtained, five sub-clones were
frozen for future reference studies, while the
remaining two high binding/high IgG secreting
clones were expanded for 2-3 weeks and pilot
scale productions (1 Liter) were completed.

Workflow to Rescue
Poorly Producing Hybridomas
To identify sub-clones for advancing
diagnostic development for ds-DNA virusspecific antibody, 480 FITC positive hybridoma
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clones were screened using ClonePix Technology,
including CloneMatrix Semi-Solid Media and
FITC-labeled CloneDetect Reagent. Of the 146
fastest growing clones as identified by
CloneSelect Imager, seven high producers
were determined to be highly specific for target
peptide binding. Two sub-clones were selected
for pilot production and cell banking. This
workflow reduces hybridoma screening time
by up to 50%, while selecting robust, desired
candidates for additional research or production.

Verification of Monoclonality
and Uniform IgG Secretion
After ClonePix System recloning and
confirmation of specificity, two sub-clones
were expanded and re-plated at 200 cells/mL in
CloneMedia Semi-Solid Media in 6-well plates
with CloneDetect Reagent. Visualization and
analysis by the ClonePix System software
suggested that IgG was being secreted properly
and higher IgG yields were observed due to a
homogeneous population of uniform secretors
(Figure 5).

Production Capacity on
Novel ds-DNA Viral Hybridoma
Sub-Clones
By utilizing ClonePix Technology poor
producing parental clones were re-screened
and higher producing, stable sub-clones were
rescued. mAb productions were purified over
a protein G column and total yields quantified.
Both novel sub-clones had shown a dramatic
improvement in IgG production (17-25 mg/L)
over historic yields of the parent clone (~1mg/L),
shown in Figure 6.

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Optimize, Adapt and Overcome

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