User guide

Clone scale-up
Introduction
After isolating your clones of interest (previous section), transfer single-cell
colonies from 96-well plates to 24-well plates, and then scale up the volume of cells
every 3–7 days by transferring each clone into the next larger plate or vessel (i.e.,
24-well plates to 6-well plates to T-25 flask and then to 125-mL shaker flasks).
Note
The total clone scale-up process from a 96-well plate to a 125-mL flask takes about
2–4 weeks, depending on the growth rate of each clone. Monitor the protein
production of each clone using your method of choice (see page
53) and carry over
the top-producing clones to the next stage in the scale-up process.
Materials needed
•
Single cell-derived clones in 96-well plates
• CD OptiCHO
™
Medium supplemented with 6 mM L-glutamine
• Sterile tissue culture dishes (24-well and 6-well), sterile T-25 flasks, and sterile
125-mL polycarbonate shaker flasks
• Non-shaking incubator set at 37°C, humidified atmosphere at 5% CO
2
• Shaking incubator set at 37°C, humidified atmosphere at 8% CO
2
, shaking at
130–135 rpm
• Assay for determining protein production
Protocol
1. Perform clone expansion with an appropriate number of top-producing clones
at any stage during screening (i.e., 96-well plate, 24-well plate, or 6-well plate
stage).
2. When individual clones are 20–100% confluent (10–14 days) in 96-well plates,
aseptically harvest the desired clones by pipetting up and down gently and
transferring the entire content of each well into a separate well of 24-well tissue
culture plates containing 0.5–1 mL of fresh growth medium (CD OptiCHO
™
Medium supplemented with 6 mM L-glutamine).
3. Continue expansion; after 3–5 days, transfer the desired clones into the next
larger culture plate or vessel using the same procedure. The final culture
volumes in 6-well plates and T-25 flasks are 2–3 mL and 5–7 mL, respectively.
4. Once the clones are expanded to 125-mL shaker flasks, incubate the cells at
37°C and 8% CO
2
, with shaking at 130–135 rpm.
IMPORTANT!
Prepare frozen cell stocks prior to optimizing protein production. You may then
optimize protein production using different culture conditions, or continue the
scale-up procedure to meet your bioproduction needs. To ensure the stability of the
screened clones, do not use selection reagents such as Geneticin
®
Selective
Antibiotic or MTX during the scale-up process.
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