MATERIALS AND METHODS
Bags used for cell-growth testing
The data shown here are derived from three bag vendors. For all graphs, Vendors A, B, and C are the same vendors among each
company's figures. Each company used the bag(s) in use at their facility at the time of testing. The bags are used for either
media storage (and warming) or cell-culture steps (seed train, scale up, and production) in the overall bioprocess.
Preparation of media used in cell-growth testing
The water used to prepare media was from the same source as used to prepare cell-culture media for normal process development
and/or pilot-plant operations.
Companies 1 and 2 employed the following method. Sterilized water was introduced into bags at the lowest volume-to-surface
area ratio normally employed for cell bag (growth in bags) applications. If any company didn't normally use bags for growth
steps (i.e., they only use bags for media storage), they added water at a low volume-to-surface area ratio, similar to that
of a company who would use bags for such an application. Water was incubated in bags for four days at 37°C. As a control,
water was incubated in glass bottles for four days at 37°C. After incubation, both water types were used to prepare cell-culture
media at Companies 1 and 2 according to their normal operating procedures. Following preparation, the media were sterile-filtered
(0.1 μm pore size rating filters of either hydrophilic polyvinylidene fluoride [PVDF] or polyethersulfone [PES] filter media)
into and stored in glass or Nalgene-type bottles. Media were then held at 2–8°C until used for cell culture-growth testing,
up to 30 days. Company 2 also held prepared media derived from water incubation in bags at 2–8°C in glass bottles for 16 weeks.
This case is labeled "aged medium" for the data of Company 2. Company 3 used a similar method to Company 1 and 2, except that
the water incubation temperature was 36.5°C, and the control medium was prepared using the same cell culture-grade water according
to the standard preparation method at the company without four days' incubation at 36.5°C.
Company 4 modified the approach of Companies 1–3 slightly. Medium was sterile-filtered at a low media-to-bag area ratio into
either a glass flask (control) or into a 10-L bag from three vendors. The containers were incubated at 37°C for three days
prior to the media being used in the growth assessments.
The data shown here represent at least four independent media. Because of the proprietary nature of these media, it is unknown
among the authors how similar or unique they are to one another.
The second step consisted of passaging cells using the incubated media. This was done by transferring incubated media into
separate shake flasks, inoculating with cells, and passaging three times, each passage lasting 3–4 days. This procedure was
used to test films from three different vendors, each run in at least duplicate cultures.
All companies used at least one in-house Chinese hamster ovary (CHO) cell line that had been transfected to produce a protein
of interest. CHO lines used dihydrofolate reductase (DHFR [-]) or glutamine synthetase (GS) selection systems. Company 2 also
performed the work with an NS0 cell line. The cell lines in some cases were already known to be affected by this bag application
as discovered by those companies previously. In some cases, the cell lines were tested for growth inhibition for the first
time. All cell lines used for these studies are believed to represent typical production lines within each company.
Cell growth and assessment
Prior to cell-growth testing, each cell line was grown in media identical to or similar to that of the actual test media composition.
Cells were seeded into shake flasks for the first passage in test media following a low-g spin and resuspension step except
for Company 3, where cultures were passaged without this step. The seeding densities used were in the low end of the range
used at each company. Cultures were then maintained for at least 10 generations (doublings) over three passages for the control
condition (water incubated in glass bottles), or for test conditions where there was cell growth. Each passage lasted 3 or
4 days. At each passage, the cells were diluted to the same seeding density as for the initial passage in test media (i.e.,
there were no fixed split ratios). Mixing and CO2 gas levels followed each company's standard practice for the cell line being tested. Cell density and viability measurements
were made using automated image analyzers that employ Trypan blue staining.
Cell growth levels and viabilities were compared between bag-incubated water or media, and glass bottle-incubated water or
media preparations. Cell growth is shown as a multiple of the starting passage density and is labeled as normalized viable
cell density. In all cases, actual viabilities are shown. Data points for cell density and viability in each figure are the
average of at least two shake flask cultures.