Shaken Disposable Bioreactors
Disposable bioreactors for cell culture have recently taken over the intermediate volume component of the industry, given their convenience and ease of regulatory compliance. For high-throughput screening and other tasks requiring low volumes and many samples, microtiter plates are the receptacles of choice.
“Working with UNIL-EPFL, we have evaluated shaker technology for cell cultivation going all the way from microtiter plates to 2,500 L bioreactors,” states Tibor Anderlei, Ph.D., head of science and business development at Adolf Kühner.
Although Wave Disposable Bioreactor Bags (GE Healthcare) have dominated cell culture disposable technology for a number of years, there are alternatives that tender important advantages. According to Dr. Anderlei, orbitally shaken cylindrical bioreactors offer superior mixing and gas transfer, thus avoiding concentration gradients that can be harmful for mammalian cells. Scaleup calls for disposable 50 mL Erlenmeyer flasks, then to 1 L, 10 L, 100 L, and 250 L.
“However, we can go much higher, and we have pilot disposables that hold 100 L and even 2,000 L,” adds Dr. Anderlei. The EPFL team has published evaluations of the technology, demonstrating that the mixing time decreases as the agitation rate increases until a minimal value is reached. As the shaking diameter was reduced, a higher agitation rate was needed to reach the minimal mixing time. These studies have established that the mixing in orbitally shaken cylindrical bioreactors ensures homogeneity for mammalian cell cultures at scales up to 1,500 L.
“We strongly endorse the shaken bioreactor as an alternative to disposable bag systems,” Dr. Anderlei explains. “The easy scaleup, low shear stress, and straightforward handling are known advantages of shaken bioreactors. In addition, they provide performance, flexibility, and cost-effectiveness when compared with more conventional technologies. Further scaleup is already being considered, and initial successful experiments have been carried out with a 2,000 L shaken bioreactor prototype.”
Larry J. Cummings is a consulting scientist involved in antibody purification at Bio-Rad Laboratories. “Bio-Rad has collaborated with the pharma industry, going back to 1981, working with ascites fluid. As the technology has evolved over the years, the favored strategy for antibody purification is to do a capture step with protein A, followed by an ion-exchange step and the final purification on our turnkey product, CHT™ Ceramic Hydroxyapatite media.”
According to Cummings, protein A has long been favored for antibody capture, given its abilities to bind immunoglobulins from cell culture harvests; reduce host-cell proteins, nucleic acids, endotoxins, and viruses; as well as its flexibility over a wide range of conditions. However, the elution conditions may promote aggregate formation, and a major shortcoming is its tendency to leach into the product.
“Protein A developed a high visibility in the industry because it provided high affinity and selectivity, while capturing a moderate load of antibody,” Cummings continues. “Very few materials had that capability.”
However, the demands of higher antibody-expression levels coming from the pharma industry, combined with the shortcomings of protein A, have driven companies to search for more productive alternatives. A number of companies, including EMD Chemicals, Tosoh Biosciences, Bio-Rad, and Life Technologies, have developed high-capacity cation-exchange resins. Given the long-term standing of protein A in antibody processing, these relatively new products will take some time to be accepted by the industry.
Cummings argues that his company’s product, Nuvia™ S, offers a number of important features, principally its performance as a high-capacity cation-exchange medium, providing antibody binding of 100 to 200 mg/mL.
In Cummings’ view, Nuvia S is a good candidate for antibody capture from cell culture harvests, as well as for intermediate purification steps. Although most of the DNA, endotoxins, and viruses flow through unbound, residual impurities including host-cell proteins must be removed following capture. Given that the cleaning conditions for the Nuvia S product are much less harsh than those required for protein A, concerns over antibody leaching and aggregation are eliminated.
“Moreover, antibody aggregates can be separated from monomeric antibody at loads up to 100 mg/mL, with a high recovery of the monomer,” he advised. “Fragments of light chain have been separated during antibody capture, using an intermediate wash step prior to the elution of the intact antibody.”
Additionally, industry practice is to conduct a viral inactivation step and then remove the inactivated virus, residual host cell protein and DNA with an anion-exchange resin. This step is given over to the Nuvia Q, which has a dynamic capacity for proteins of about 200 mg/mL. The Nuvia Q ligand, whose functionality is a quartenary ammonium group, is recognized for its viral and DNA reduction properties. The base chemistry is a proprietary polymer, consisting of rigid particles with large pores to accommodate strong flow properties and fast mass transit.
The final polishing step removes residual aggregates with CHT Ceramic Hydroxyapatite in the cation-exchange mode. While the complete platform is designed for whole IgG antibody purification, Cummings indicates that it could also be used for both minibody and antibody fragment-purification protocols.
Newly Modified Ligands
Hans J. Johansson, Ph.D., a senior scientist at GE Healthcare, focuses on downstream processing, in particular the capture step. “We are continuously working to further increase the capacity of our MabSelect™ product line.”
MabSelect is an improved resin with an engineered protein A ligand. This design provides an oriented coupling resulting in an affinity medium with enhanced binding capacity for IgG molecules. It has the capability to capture mAbs from large volumes of feed by packed-bed chromatography, with sufficient affinity for a one-step purification process. Furthermore, it is compatible with the high flow rates and high pressure required when scaling up.
MabSelect SuRe™ has been developed from the same highly cross-linked agarose matrix used for MabSelect. However, it contains an alkali-tolerant recombinant protein A ligand, resistant to harsh cleaning agents such as sodium hydroxide, resulting in significant cost savings. MabSelect Xtra™ was developed to deal with high levels of upstream antibody expression. MabSelect Xtra™ uses the same recombinant protein A ligand as MabSelect, but has a smaller particle size and greater porosity, which ensures increased dynamic binding capacity. The medium provides a lower overall production cost due to the possibility of processing concentrated feedstocks in fewer batches.
Dr. Johansson stressed that his group is also pressing investigations on new multimodal resins, capable of ionic, hydrogen bonding, and hydrophobic interaction. “These ligands are gaining an increasing interest throughout the industry, in that they meet demands of a wide range of protein purification needs.”
The latest product launch is MabSelect SuRe LX™, a resin with a capacity designed to handle the very high titers (5–10 g/L) that now are reaching regular production.