A Generic Growth Test Method for Improving Quality Control of Disposables in Industrial Cell Culture - Independent data, using several different cell lines and growth media, reported growth inhibition

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A Generic Growth Test Method for Improving Quality Control of Disposables in Industrial Cell Culture
Independent data, using several different cell lines and growth media, reported growth inhibition resulting from the use disposable bags and suggests a method that can be implemented for quality control at disposable-bag vendors.


BioPharm International
Volume 23, Issue 6, pp. 34-41

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 37C. As a control, water was incubated in glass bottles for four days at 37C. 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 2–8C until used for cell culture-growth testing, up to 30 days. Company 2 also held prepared media derived from water incubation in bags at 2–8C 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 36.5C, 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 36.5C.

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 37C 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.

Cell lines

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.


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