User guide

Genomic amplification by MTX addition, continued
One round of MTX
amplification
The productivity of each clone depends upon the integration locus of your
expression construct, the response to amplification using MTX, and the nature of
your protein. Depending on your protein production needs, your available time,
and your resources, you may perform one round of MTX amplification at various
concentrations (such as 50 nM, 100 nM, 250 nM, 500 nM, and 1 µM). Additional
rounds of MTX amplification may be performed using higher concentrations (such
as 2 µM and 4 µM) to potentially increase your protein production.
Protocol for MTX
amplification
1. For each cell pool, centrifuge cells at 300
×
g for 5 minutes, then aspirate old
medium.
2. Seed cells at a density of 3 × 10
5
viable cells/mL in 100–300 mL of media
containing various concentrations of MTX in 0.5–1 liter shaker flasks.
3. Incubate flasks at 37°C/8% CO
2
with shaking at 130–135 rpm.
4. Passage cells into fresh medium containing MTX at 2 × 10
5
–3 × 10
5
viable
cells/mL in shaker flasks every 3 or 4 days. Spin down cells and re-suspend
cells in fresh medium if the dilution factor at passage is <2.
5. Passage cells at 2 × 10
5
–3 × 10
5
viable cells/mL when the viability starts
increasing. The cells amplified with low concentration of MTX recover faster
than that with high concentration of MTX.
6. When cell viability is >90%, freeze cells and start protein productivity
analysis.
Next steps
•
Because MTX amplification produces a polyclonal population of cells, you
must always perform clone selection (page
37) prior to clone scale-up.
• Optional: You may perform further evaluation of the MTX amplified pools to
decide which amplified pool to choose for clone selection and/or protein
production.
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