Product Survey: Bioreactors and cell bags

The Art of Mixing
by Harald Zähringer, Labtimes 03/2014

Levent Yilmaz (third, right), world champion in show-barkeeping, shows how to shake cocktails. Photo: Europapark Rust

The decision making of potential bioreactor buyers may be boiled down to two simple questions: shaken or stirred and single-use or re-usable?

Barkeepers and cocktail drinkers may get into fierce debates when it comes to the question whether a certain cocktail should be shaken or stirred. As a rule of thumb, cocktails are usually shaken when they include fruit juice, cream liqueurs or other thick mixers and stirred when they contain distilled spirits or light mixers. The question “shaken or stirred” is not only a hot topic amongst cocktail drinkers. Life scientists looking for a new bioreactor may argue about the very same question. Should they go for a traditional stirred-tank bioreactor (STR) or opt for a shaken plastic cell bag?

STRs are essentially cylindrical reaction vessels made of glass or stainless steel and closed with a stainless steel or plastic lid. A motor-driven impeller system installed through a sealed void in the lid agitates the culture media, while gases such as air, oxygen or carbon dioxide are flushed to the bottom of the vessel via spargers. Additional sensors integrated into the lid may control crucial cell culture parameters, such as temperature, pH or dissolved oxygen. Easy scalability is one of the major advantages of stirred-tank bioreactors. Hence, they are available with working volumes varying from a few dozen millilitres in small research reactors up to 20,000 litres, or even more, in production plant reactors.

Magnetic paddles

Spinner flasks are basically slimmed-down versions of stirred-tank reactors devoid of impeller motor, spargers and control units. They usually come with simple plastic screw caps and a magnetic paddle system, or a hanging stir bar mounted underneath the cap that is rotated by a magnetic stirrer. Two angled sidearms permit access to the interior of the flasks and allow a firm gas exchange.

Exploding gas bubbles

Paddles and impellers of spinner flasks and STRs allow an easy mixture of suspension cultures. They may, however, also cause considerable damage, especially to delicate cells such as mammalian cells due to shear stress. Another issue of STRs are “exploding” gas bubbles, stemming from gas sparging, which may also harm sensitive cells.

In their quest for bioreactors, allowing homogeneous liquid mixing and a high mass-transfer rate without detrimental effects to the cells, researchers came up with the idea for cultivating cells in plastic bags – which are shaken, not stirred – in the 1960s, already. Since the gas exchange through the plastic material was pretty lousy and mixing wasn’t very efficient, the culture bags soon fell into oblivion. It took until 1996, before Vijay Singh, then working for the US Pharma company Shering-Plough, solved the mixing and gas exchange issues of plastic bags with two simple modifications and a clever shaking technique.

The basic parameters of orbitally shaking liquids are rather simple, the physics behind it, however, is devilishly complex.

Simple and efficient

He connected two short flexible tubes to the disposable gamma-irradiated plastic bags: one serving as the inlet for air (oxygen), the other as an exhaust pipe for carbon dioxide. His brilliant idea, however, was to fill the inflated cell bag only partially with culture media and place it on a rocking shaker platform, to create a media wave running back and forth inside the bag. The media wave ensures a homogeneous, shear-stress-free mixing of the cells and facilitates the transfer of oxygen into the media.

Disposable wave bioreactors, which have been on the market since 1999, have turned into serious competitors of traditional STRs. They are available in different sizes, ranging from 100 millilitre working volume for small scale applications to 1,000 litre monster bags, designed for biopharmaceutical production lines. Upscaling the pillow-shaped wave bags beyond the 1,000 litre frontier, however, is limited – which might play well into the hands of disposable orbital shaken bioreactors (OSRs) developed by Florian Wurm’s group at the École ­Polytechnique Fédérale de Lausanne.

The Swiss group got the idea for OSRs during an extensive screen for cell culture parameters in CHO cells under “bioreactor-equivalent” conditions in 2004. They initially took the simplest approach and filled the suspension cultures into 50 ml centrifuge tubes, with slightly loosened screw caps, to allow gas exchange and ran them at 130 to 250 rpm on an orbital shaker.

To their surprise, the cells grew to similar densities as in stirred-tank bioreactors equipped with sophisticated control units. Obviously, the orbital shaking mode enables a thorough and fast mixing of the media as well as an efficient aeration. Prototypes of orbital shaking bioreactors have already been upscaled to 1,000 litres, using cylindrical disposable bags fixed into ­orbital-shaken stainless steel vessels. But that’s not the end of the line, Florian Wurm has plans for even bigger OSRs with 2,500 litres.

Single-use boom

The recent boom in cell bags has sparked a general trend towards disposable bioreactors and single-use systems (SUS). For good reason: they save researchers from cumbersome cleaning and sterilisation processes, and may reduce production costs. However, single-use systems made of plastic not only place an extra burden on the environment. A paper published by a group from Amgen’s product development unit in Thousand Oaks, California, early this year, also suggests that leachable compounds found in some disposable cell bags may also harm your cells (Hammond et al., Biotechnol. Prog., 30, 332-37).

Matthew Hammond and his co-workers grew CHO cells in six different bag types from five vendors and checked their viability. Cell growth was impaired in three bags, with almost no viable cells in one bag type. Further investigations revealed that the cytotoxic compound bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP), leaching out of the bag films, was responsible for the reduced cell growth. Obviously, bDtBPP is generated by the degradation of tris(2,4-di-tert-butylphenyl)phosphite (TBPP), which is added to the plastic film as an antioxidant stabiliser.

Everthing is fine now?

The cytotoxic effects of certain bags could be traced back to mistakenly high TBPP concentrations applied during film production, which has led manufacturers of bag films to take action and control the TBPP concentrations more closely. That’s certainly not a bad idea but probably not enough, as Hammond points out, “I am not ready yet to say that the only important parameter is the concentration of TBPP in the film – there may be complex interactions with other additives, polymers and the manufacturing processes used.”

Asked for some advice to researchers working with disposable cell bags he says he would “call the vendor of any SUS bags and specifically ask if the product I was considering for use is known to have an issue with leaching this compound. The other suggestion I would make is to follow some of the recommendations we made in our Biotech Progress paper: if there is a worry that bDtBPP leaching might be problematic, avoid using SUS bags at very low working volume (relative to the capacity of the bag) or very low initial cell density”.

New bag material

The manufacturers of single-use systems and cell bags are also aware of the problem. Sartorius Stedim, for example, announced on June 1 in a notification paper that it has changed the material of the polymer film used for SUS and introduced a new product line that will succeed the CultiBag RM system (which will be discontinued after December 31, 2016). According to Sartorius, the new bag material has been validated by an independent round-robin study conducted by the DECHEMA temporary working group on “single-use technology in biopharmaceutical manufacturing” (Eibl et al., ISBN 978-3-89746-149-9).

However, changes in plastic production processes are still an immanent problem of SUS. Or as Hammond puts it, “SUS vendors will have to work hard with their suppliers to make sure that there are no unannounced changes to the formulations (or other processing parameters) of the plastics they receive.”

And researchers should keep their eyes open when deciding to work with disposable bioreactors.

First published in Labtimes 04/2014. We give no guarantee and assume no liability for article and PDF-download.

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