A re-usable wave bioreactor for protein production in insect cells

Graphical abstract


Method details
Preparation of bioreactor 1. As a cultivation container, a polycarbonate box 500 U Eurostandard Typ IV S, TECNIPLAST, Italy, Size 480 Â 375 Â 210 mm, Volume 38 L, originally made for animal housing was used. The material is transparent and autoclavable repeatedly at 121 C. The 1st Cooper model was autoclaved and used 35 times without loss of performance. 2. The lid was constructed in-house. It consists of polycarbonate at corresponding size and is sealed with a layer of rubber foam. Six metal clamps on four sides guarantee tight attachment to the base container. 3. Within the lid, seven ports with connectors to both inner and outer sides are inserted. These insertions are screw threads manufactured in-house. a. Two openings are standard 13.5 Pg ports for optional pH or pO 2 sensors (not used in our settings approximately those values in order to maintain high quality and reproducibility of experiments. 2. Cells were grown in shaker incubators (Infors, 50 mm rotating diameter) at 27 C in Erlenmeyer EM or Fernbach FB glass flasks covered with Silicon Caps (Hirschmann, VWR) at the following combinations of culture volume À flask volume À shaking speed: 30 mL À 250 mL EM À 120 rpm; 150 mL À 1 L EM À 120 rpm; 200 to 400 mL À 1,8L FB À 90 rpm; 400 mL to 1 L À 5 L EM À 90 rpm 3. Baculovirus expression was performed according to the Bac-to-Bac 1 protocol (Life Technologies).
Bacmid transfections in Sf9 cells were harvested after 3-5 days. Virus titer was determined using the SF9-ET easy titer cell line [1]. Virus was either amplified in two subsequent steps to generate Passage 1 and Passage 2 virus stocks or used to generate Baculo Infected Insect Cells (BIIC) as described previously [2]. BIICs frozen at À80 C served both for protein expression and as virus storage. For protein expression, Hi5 and SF9 cells were adjusted to 1 Â10 6 cells/mL and infected with virus stock or BIICs at different dilutions, typically in the range 1:1000-1:10,000.

Method validation
For protein production at 1 L scale we are using 5 L Erlenmeyer glass flasks (modified in shape, see Fig. 1) and have also tested Thomson Optimum Growth Flasks (Cat No 931116) filled with 2 L culture volume. In order to evaluate the upscale to 5 and 10 L, protein expression was first compared between flasks, Wave disposable Bags (GE Healthcare, Cat No CB0010L10-01), and the newly developed bioreactor, named Cooper. Both containers were shaken on a Wave System 20/50 EH. Parameters previously optimized at small scale were: temperature 26 C; production time 72 h; cell density at infection 1 Â10 6 cells/mL. The optimal rocking rates and angles were optimized individually for both bioreactors based on cell viability and productivity of a target protein Zyx-LIM-eGFP. For Wave Bags, a rocking rate and angle of 28 rpm and 9 respectively, were recommended by the manufacturer and resulted in best productivity (Table 1). For the Cooper bioreactor, the rocking rate and angle were adapted to achieve a fluid wave comparable to the Wave Bag as well as maximal cell viability and Zyx-LIM-eGFP productivity. The resulting parameters were 30 rpm and 7,5 . In summary, productivity of Zyx-LIM-eGFP was comparable in all cultivation containers tested as shown in Table 1.
Encouraged by the results, productivity at 5 L scale in Wave Bags versus Cooper Bioreactor for three more proteins was compared next. Protein yield, concentration and purity in the re-usable bioreactor were comparable in Cooper and Wave Bags (Table 2 and Fig. 2). Comparisons of cell parameters at harvest show higher cell viability in three out of four Wave Bag productions. Despite having omitted any antifoam reagent in the ExCell 420 medium, no foaming was observed in both bioreactors, as illustrated in the Graphical Abstract showing Zyx-LIM-eGFP production in the Cooper bioreactor.

Additional information
Space is rapidly becoming limited when scaling up insect cell suspension cultures for production. One Infors shaker can accommodate 6 Â 5 L Erlenmeyers flasks, giving 6 L production capacity. There are different solutions to that space limitation, the choice of which depends very much on the individual equipment available. Most common are bioreactors especially in the pharmaceutical industry, albeit less represented in academic laboratories. In the past few years, alternative choices have emerged, like high cell density protocols [4] or culture vessels that can be filled up to 5 L [5]. Having Labfors stirred-tank bioreactors and Wave Platforms available, we invested efforts in bioreactor cultivation. Upscale to 5 L was first performed in a stirred-tank bioreactor (Labfors, Infors, 7.5 L) adapted to insect-cell cultivation with integrated pH and pO 2 measurement and regulation. Expression levels were comparable to flask expression (data not shown). Most interestingly was the fact that pH and pO 2 was very stable throughout the entire cultivation and protein production process and did not need any correction. As a consequence, the rather labor-intensive setup of the Labfors stirred-tank bioreactor His-mZyx-LIM-eGFP (Lim domain from mouse Zyxin, Uniprot Q62523, fused to eGFP;) was produced for 72 h in Hi5 cells. Protein was purified by one-step immobilized metal affinity chromatography. Cells were infected at 1 Â10 6 cells/mL. Proteins were produced at 5 L scale for 72 h. All proteins were His tagged and purified by one-step immobilized affinity chromatography. ODC1 (Homo sapiens Ornithindecarboxylase, Uniprot P11926; modified); Spd-5 ( [3],C. elegans, Uniprot P91349). was discontinued and replaced by Wave Bags. The advantage of Wave-mixed agitation is the efficient nutrient distribution, off-bottom cell suspension and efficient oxygen transfer without the damaging shear forces induced by mechanical stirring and gas sparging in stirred-tank bioreactors [6]. There are a variety of systems commercially available, that differ in bag design, sensor types and type of platform movement (reviewed in [7]). The mixing is driven by the oscillating movement of a platform and the mixing rate is controlled by modifying the rocking rate, rocking angle, filling volume and aeration rate of the shaken bag. With the development of a re-usable Wave-shaken bioreactor 1 that can be operated on these platforms, we exploited all these advantages without increasing our running costs for consumables. Apart from that cost reduction, some more advantages of our re-usable Wave Bioreactor were identified during the evaluation process: 1. Despite a filter heating integrated in the Wave system, the air outlet filter of a Wave Bag was occasionally wetted by condensing water in the tubing and as a consequence, air transfer was blocked. To circumvent this problem a wide-mouth port covered by a Silicon cap was instead inserted as air outlet. This guaranteed undisturbed air circulation and also assured sterility. 2. The filling-volume of the box is very flexible in the range of 2-10 L 3. A disposable Wave Bag obtains its shape by filling it with air and the shape is maintained by constant air circulation. In contrast, the re-usable polycarbonate reactor has a rigid and robust shape which is insensitive to errors in air exchange like filter wetting. 4. The geometric plane shape together with the grip-like lid facilitates handling especially in transport.
In summary, this new re-usable bioreactor is most suitable for academic institutions and productions of low-value products, whereas for high value products, single-use bioreactors will still be preferred. At the Biochemistry Core Facility of MPIB, this bioreactor enabled the cost-effective upscale of protein production in insect cells to meet the increasing needs of the users. Todate we have performed fifty Cooper productions at 2-10 L scale, among them Spd-5 [3] and ATG1 [8], with a contamination rate of 0,5% and 35 life cycles of the 1st generation Cooper bioreactor.