Genetic Engineering & Biotechnology News

AUG 2017

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20 | AUGUST 2017 | GENengnews.com | Genetic Engineering & Biotechnology News as configurable as benchtop systems. It achieves this level of compatibility and scalability by duplicating the geomet- ric characteristics of 3-L vessels in smaller volumes. "We spend a lot of effort on ensuring and demonstrating scal- ability," Poppleton says. One critical aspect of scalability is duplicating the sensor set at small scale. Advanced sensors are common in multi- liter benchtop bioreactors, in part because bioreactors in that size domain serve as testing platforms. "It's a good way to get early adoption without facing the regulatory restrictions of manufacturing scale," says Poppleton. Those sensor technol- ogies that pass muster are then introduced at larger scale, but not always for very small scales because of space constraints. Applikon is changing this upward adoption pattern by in- troducing probes that work at both bench- and mini-reactor scale. Their pH sensor and optical dissolved oxygen sensor has a diameter of 8 mm, as opposed to 12 mm for standard probes. Applikon is also working with Buglab on a biomass sensor that fits into their mini bioreactors. "Mini-scale sen- sors are not as well developed as [they are] for bench scale, but they are catching up," Poppleton adds. Sweet Spot for Process Development Making the case for performing batch or fed-batch cell culture at bench scale is easy. What about perfusion cell cul- ture, which produces product continuously, sometimes for months? According to John Bonham-Carter, director for up- stream sales and business development at Repligen, the very same arguments for batch cultures apply to perfusion-based processes: "Bioreactors of around 3 L in volume are very well understood, and process developers have a great deal of confidence in results at that scale. Three-liter reactors pro- vide almost all of the answers you're looking for during de- velopment or scale down. You can also reach those answers at 50 L, but at vastly higher cost." So, given the performance and productivity of perfusion reactors, and if 3 L cultures work so well and save costs rela- tive to larger sizes, why not go even lower than bench scale? Bonham-Carter explains that smaller sizes, say 250 mL, are not yet as well-characterized as the 3-L scale. "There is much less confidence in predictability [of 250-mL models] with respect to larger scale, particularly in perfusion." Three liters also allows for more space for various ports to measure, analyze, and make alterations (addition of nutri- ents, sampling of fluids, etc.). "These smaller devices provide a good estimate of large-scale work, but you often have some compromises to make," Bonham-Carter adds. For example, smaller versions have less room for probes and sensors, par- ticularly when experimental spectroscopic probes compete for bioreactor real estate with essential dissolved oxygen and cell-density sensors. Another reason why perfusion process developers avoid volumes smaller than about 3 L is that cell-retention devices are hard to come by for reactor volumes lower than 2 L. "Be- low that, you're seeing mostly homebrew or unscalable sys- tems. Standardization of cell-retention devices at very small scale hasn't yet occurred," Bonham-Carter says. One small-volume cell-retention device, the spin filter, uses a rotating sieve to harvest cell-free protein. Product gen- erated within the reactor is drawn through a high pore size membrane to an inner harvest compartment. Spin filters have been around for years and many vendors sell them. But spin filters are not good scale-down models and do not support high cell density, since no large processes use them. Similarly, devices that rely on cell settling and harvest from superna- tants have no counterpart above about 500 L. "The reason you don't see perfusion reactors below 2 L or so in volume is not because people don't want them, but because a scalable robust device hasn't been proven yet," Bonham-Carter explains. Interestingly, bench scale is the earliest development stage where perfusion cell culture reliably mimics performance at pilot or manufacturing scale. Therefore, it is the point at which decisions to go batch or continuous are made. This is, in part, due to the lack of scalability for most perfusion cell culture systems to volumes below 2–3 L. Therefore, even processes that eventually employ perfu- sion cell culture begin with—and learn from—batch or fed- batch culture. "If the intention is to go continuous, then the process development plan will be shaped thereafter toward that goal," says Annelies Onraedt, marketing director for cell-culture technologies at Pall Life Sciences. Materials of Construction While maintaining vessel geometry, agitation, gassing, and form factor among scales lessens the uncertainties in scaling from microscale to benchtop to production, another factor—materials—comes into play with the wide adoption of single-use bioreactors. The need for benchtop or smaller versions of larger single- use bioreactors encompasses a desire for equivalent product contact surfaces, according to Ken Clapp, senior manager for applications, technology and integration at GE Health- care (Marlborough, MA). "Material compatibility should be a foremost consideration." Quality parameters can only be assumed when the vessel's leachables and extractables pro- files are themselves comparable. Suppliers should be ready to assist in this regard. GE's Xcellerex XDR line of bench-scale bioreactors achieve this by using the same polymer family for the pro- cess-contacting bag film as GE uses in all its larger single-use bioreactors. "This allows us to establish whether cells have problems with the polymer early enough in development to make appropriate changes," Clapp says. "You don't get that information by jumping from 10-L glass or rigid plastic bioreactors to 50-L plastic single-use systems. Discovering at that stage that you have cell compatibility or product quality issues is costly." By now, the value of bench-scale bioreactors duplicating the form factors of larger reactors is well appreciated on several levels, including the impact of probes on mixing. "Probes com- ing in through the headspace create a baffling effect unlike the larger bioreactors, so mixing is different than when they come in through the side," Clapp adds. "There will be enough vari- ables associated with media and volume differences, so it's im- portant to limit the number of controllable differences between scales whenever possible." While extractables profiles may differ between benchtop and production scales due to the greater pliability of large surfaces, Clapp says these factors can be minimized using bag-based, smaller-scale systems. The higher surface-area-to- volume ratio provides sufficient information to know what to look for at larger scale. True to Scale: Benchtop Bioreactors Bioprocessing Feature Applikon's MiniBio bioreactors, available in a various sizes (250 m L , 5 0 0 m L , a n d 1 0 0 0 m L t o t a l v o l u m e ) , p e r m i t accurate scale-down modeling of laboratory-scale processes that may be performed by bioreactors in the 1–15 L range. Continued from page 1

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