Mammalian Cell Scale-Up

Translating the small quantities of cell culture used

in the laboratory to a large production setting

Often involves intermediate stepping stone

volumes to work up to the final production

quantities
mammalian cells scale-up is different than for

bacteria
Not all products are successful at scaling-up

Cell Bank - LN2 Cryogenic

Storage

Initiating a new working cell line begins by

rapidly thawing a vial stored in liquid nitrogen

(-160 to -174C )

Inoculum Train
If this little vial of cells is placed into a

15,000L culture vessel filled with media

would they thrive?
Cells need buddies too!
Cells like to be in contact with other cells and

their metabolism conditions the media gets

them out of lag phase!

Inoculum Train
initial cell density may be only 1 x 106 cells/ml

or often less.
Gradual expansion into larger and larger culture

vessels can lead to high cell densities 4 x 106

cells/ml or more!
Thats 4,000,000 cells/ml!
or 4 billion cells/L
or 4 trillion cells/1000 L
or 40 trillion cells/10,000L

Spinner Flask

Bacteria may simply be grown in shake flasks

but mammalian cells (such as CHO cells) cant

withstand the shear force of agitation and
need gentle mixing with an agitator.

Alternatives
Tissue culture flasks (T-flasks) or roller

bottles are common in cells that are

adherent (stick to surfaces)

Scale-Up
Spinners of increasing size will be used over a

period of several days until volume and

density is sufficient to be transferred to a seed
bioreactor (about 40L).

Scale-Up
Once the cells reach a target density of 4 x

106 cells/ml in 2 to 3 days, they will be used

to inoculate a larger bioreactor of
approximately 600-800L.
The final production bioreactors are large

typically 10,000 to 15,000 L in size.

Scale-Up

Production
Once the cells are scaled up to production

volume the cells need to be subcultured in the

growth (continuous culture) vessels every few
days Why?
Need to add fresh media, remove waste,

reduce cell density!

Continuous Culture
Depending on the cell density a calculated

amount of culure (thousands of litres) is

literally sent down the drain or to a second
tank for product production.
Fresh media will be added to compensate for

the volume removed.

Typical target cell density is 1 x 106 cells/ml

after subculture (a.k.a. splitting or passaging)

Perfusion Bioreactors
There are also perfusion bioreactors that trap the

cells inside of hollow fibers.

There is a continuous removal of old media and

equal additions of fresh media.

Advantages are there is no subculture shock thus

no lag phase.
Disadvantages are the complexities of the system.

Wave Bioreactors
Wave bioreactors are becoming more

common as they are disposable

Less worry of maintenance and

contamination, may actually be cheaper in

long run.
Cant scale up to thousand liter volumes

though

Wave Bioreactors

Production
To make a production batch, the growth vessels

are subcultured (some not all of culture) is

transferred to a production vessel by pressure.
The production vessel then has a different type
of media (production) added that doesnt
stimulate cell growth but makes the cell focus
on protein production.
Chemicals are sometimes added to stimulate
protein production (sodium butyrate) and limit
growth.

Harvest
After a few days of production the protein is

ready to be recovered or harvested

Harvest activities often include filtration

(microfiltration, diafiltration, and TFF

tangential flow filtration)
Centrifugation is also an important part of

harvesting product.

Harvest
The advantages of using mammalian cells to

produce protein:
Bacteria do a poor job of proper folding of the

protein produced Mammalian cells (CHO in

particular) do a great job with complex proteins
Unlike bacteria, mammalian cells secrete the

protein outside the cell making protein purification

much easier allowing higher yields of product.

Harvest
Disadvantages:
Mammalian cells not as hardy, slow growing,

prone to contamination, and may harbor

viruses dangerous to humans.

Harvest
Since Mammalian cells produce extracellular

proteins our job here is simple

Separate the cells from the media.
What do we keep the cells or the media?

Harvest
The point of centrifugation skids are separate

the cells from the media.

Cells go down the drain and the media is

recovered as it contains the protein.

Production centrifuges are large, loud, but can

go through CIP, SIP processes.

Filtration
Filtration follows centrifugation and helps

concentrate the product and reduce the

volume of media from thousands of liters to
hundreds or less.
This is done by recirculating the media

through (microfiltration) or across a filter (TFF)

trapping proteins (retentate) but letting
supernatant (fluid) though to waste

TFF Skid

TFF Skid

A centrifuge is an instrument that rotates

samples around a central axis

This centripetal acceleration separates

substances in the sample according to

density

The heavier or more dense the substance

is the greater its sedimentation rate

The more dense the substance is the

closer it will be to the bottom of the

centrifuge tube.

The force the centrifuge exerts on the

sample is measured in gs or the # of

times greater than the force of the earths
gravity
The speed of the centrifuge can also be

monitored in RPMs or revolutions per

minute.

Generally the greater the force exerted by

the centrifuge and the longer the cycle is

run the greater the separation
You need to be careful with delicate

samples that they are not crushed or

sheared apart
So use only as much force as is necessary

for separation

The samples are loaded into numbered

wells in the rotor of the centrifuge

The rotor needs to be balanced so it

operates smoothly
Unbalanced centrifuges are dangerous,

breaking samples, spilling chemicals and

hurling pieces in addition to damaging the
centrifuge