Disposable bags used in cell-culture applications have complex manufacturing processes. Polymeric films in many cases are
sourced from secondary vendors where a similarly complex manufacturing environment may exist. Bag assemblers may not have
detailed knowledge of film manufacturing methods or the authority to influence them if they wished to. Once bags are assembled,
they are typically gamma irradiated, which along with the length of shelf-life post-irradiation, is another potential source
of variability between manufacturing lots as well as between vendors' products with respect to this effect on cell growth
The length of media or water warming used for the testing described here is slightly longer than would likely be used in practice.
However, the method is useful in uncovering growth inhibition effects and reveals an underlying issue with these products.
While industry may not be able to supply vendors with a suitable CHO or NS0 production line to apply this method, the general
nature of the effect across multiple CHO lines suggests that a nonproprietary CHO line may be suitable for implementation.
However, end-users would likely need some degree of bridging data prior to accepting results generated by a bag vendor using
such a CHO cell line. The existence of such bridging data could, in a general sense, also reduce the total in-house effort
required at a given end-user to accept bag vendor cell-culture test results in the future.
To date, the mechanisms that result in cell growth impacts by disposable bags are varied and not completely understood. Medium
component adsorption has been identified as one mechanism that can inhibit cell growth in a cholesterol-dependant cell line
(7). Cell growth impacts resulting from a specific leachable/extractable have been characterized (8, 9). The cell-based assay
was able to identify which disposable bags impacted cell growth and eventually led to identification of the specific leachable
inhibiting cell growth. The vendor was then able to optimize the film composition to ensure biocompatibility.
It cannot be assumed that these are the only mechanisms that can result in inhibition of cell growth. The use of a cell-based
assay like those described here can be used to identify where such impacts exist. Once they have been determined, various
factors such as raw materials, leachables and extractables, irradiation, storage, and other factors can be systematically
investigated. This investigational approach underpinned by a cell-based assay can lead to the identification of the root cause
of cell-growth impacts that can be readily addressed by the vendors once understood. The growth test method described is useful
in troubleshooting and can be applied proactively as a harmonized first step in improving quality control of these widely
used disposable products for both suppliers and customers.
The problem end-users face is not vendor-specific or fundamentally dependent on the end-users' cell lines or media. The growth
test methods described can be applied at bag vendors' own sites. In doing so, vendors may improve the overall understanding
and quality control of their disposable products. While some products may not lead to the same observed phenomena, as polymeric
film manufacturing or gamma irradiation practices change around them, each vendor could use a test such as this to uncover
if those changes have an effect on this particular application. As this issue is still prevalent years after it was first
discussed, the authors and the cell culture-engineering community are eager to see the quality-control testing and practices
for disposables manufacturers improve.
When cell-culture medium or the water used to make it are incubated at operating temperature (37°C) in disposable bags prior
to use, growth inhibition effects can be observed. These effects are not cell-line, cell-type or cell culture-media specific.
The problem is relevant for multiple bag manufacturers. The authors suggest that bag vendors implement the cell-growth test
described here as part of their manufacturing quality control and as a means to understand critical film composition attributes
and manufacturing controls. The question of what cell lines are best used for this testing remains, but it is believed to
be solvable as the effect is observed in multiple CHO lines as well as an NS0 cell line. Having data from such a cell-growth
test together with a deeper understanding and control of film composition and manufacture will facilitate adoption and continued
use of disposable-bag technology. This knowledge will help ensure robust and predictable performance even through the inevitable
changes in bag and bag film manufacturing processes.
The authors wish to thank Robert Kiss, Masaru Shiratori, Thomas Ryll, Reb Russell, and Richard Schicho for their discussion,
review, and comments on this manuscript. Erica Graf and Richard Martel performed cell culture work at Bristol-Myers Squibb
*Brian Horvath is a scientist at Process Technical Development, Late Stage Cell Culture, Genentech, a member of the Roche Group, South San
Francisco, CA 94110; Valerie Liu Tsang is senior engineer III and Weimin Lin is a scientist, both at Cell Culture Development, Biogen Idec, RTP, NC 27709; Xiao-Ping Dai is manager at Cell Culture Science, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Bloomsbury, NJ 08804; Kurt Kunas is a principal scientist and Greg Frank is a principal engineer, both at Amgen, Thousand Oaks, CA 91320.
* To whom all correspondence should be addressed, firstname.lastname@example.org