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European Biopharmaceutical Review

Rapid Reaction


The growing take up of single-use bioreactors over traditional stainless steel reactors has prompted new research on their suitability for process development and clinical facilities. Results show they are a viable alternative to traditional bioreactors

In the biopharmaceutical industry, singleuse bioreactors have become increasingly accepted due to their advantages over traditional stainless steel reactors. The benefits include increased flexibility and reductions in cleaning, crosscontamination, sterilisation, equipment lead time, validation and labour. This article compares single-use bioreactors that incorporate different agitation and vessel design technologies, such as stirred tank, orbital shaking and rocking bioreactors. It evaluates aspects of mixing, mass transfer, cell culture performance, product quality attributes and efficiency, compared to traditional stainless reactors.

Materials and Methods

The disposable bioreactors evaluated ranged from 3L to 200L in working volume and incorporated different design elements as well as varying agitation, sparger, heating and dissolved gas control. Table 1 describes these characteristics.

Mixing Studies

Mixing time in all bioreactors was evaluated using a pH equilibrium standard when the final pH reached ±0.1 pH units with agitation speed recommended by the manufacturers. Concentrated base was added to a solution of phosphate buffered saline (PBS) either at the top or bottom of the bag, and pH was measured via electrochemical pH probes or fluorescent-based single-use probes, as per the manufacturer’s specifications.

Mass Transfer Determination


Oxygen mass transfer (KLa) was assessed for each bioreactor. A solution of purified water at 37°C was used and agitation speed was determined by the manufacturer’s recommended settings. Gas flow was normalised to 0.003 vvm (L/L/min) for each sparged reactor using the ‘open pipe’ non-microsparger, with the exception of the Mobius CellReady reactor (EMD Millipore, Billerica, MA). For the non-sparged orbital reactor and rocking reactor, gas flow was normalised to 0.0125 vvm. KLa was calculated using the standard methods. Maximum KLa was also measured.

Temperature Mapping

Temperature mapping was performed using the agitation recommended by the manufacturers. Purified water was used and filled to the maximum working volume for each reactor and standard non-optimised proportional-integral-derivative (PID) settings were used. Standard temperature control units (TCU) for each manufacturer were also featured. Units with blankets versus jackets are indicated in Table 1.




Cell Culture and Analysis

Chinese hamster ovary (CHO) cells producing a highly glycosylated Fc fusion molecule were selected from an internal clone that is well characterised. A typical fed-batch production cycle was used, with one feed sampled daily for off-line analytical results. Cells were grown in disposable seed trains and inoculated for all bioreactors at the minimum working volume provided by the supplier. After minimum growth performance was observed (approximately two days), the working volume was increased to the maximum, keeping a consistent cell density among all bioreactors. This effectively allowed the vessels to serve as the N-1 and N production reactors. Temperature was regulated at 37°C and agitation speed was fixed by the supplier. Dissolved oxygen was set at 50 per cent and controlled by purified air using a normalised flow rate for each vessel. pH regulation was set between 6.8 and 7.2 and controlled with sodium hydroxide as the titrant and dissolved CO2 in either the overlay or sparger. Off-line DO, CO2 and pH were monitored daily. Molecule quality was measured on the last day.

It should be noted that some vessels have different agitation, sensing, temperature control and sparging mechanisms. In all cases, care was taken to normalise these parameters; however, due to the divergent vessel design, not all parameters could be normalised exactly.

Results and Discussion

Bioreactor Characterisation
It is important to understand the differences between the bioreactors on the basis of their fundamental engineering characterisation. This includes, but is not limited to, mixing studies, mass transfer determinations and temperature mapping of the vessels.

Efficient mixing in the bioreactor vessels is important for a well-suspended cell culture and good mass transfer of dissolved gases. Figure 1 shows the results of the mixing studies. The 20L paddle and 50L orbital style bioreactors had the longest time to reach stability, which was over 80 seconds. This is not surprising given the agitation mechanism employed in these reactors – they have no impeller mechanism.

The rocker and stirred tank reactor (STR) followed with a mixing time of 55 seconds. It should be noted that the rocker style reactor only works when the working volume is 50 per cent or less of the total volume.

Finally, the Mobius CellReady 200L and 3L reactors demonstrated the shortest time to achieve final pH +/0.01 unit. The 200L reactor has a bottom mount and off-centre agitator which allowed for efficient and low shear agitation with a wide turndown down range (5:1). The 3L reactor is smaller and thus at the recommended agitation range the mixing time was less than 20 seconds.

It should be noted that the paddle mixing system has classical electrochemical probes, whereas the orbital shaking and stirred tank mixing bioreactors have optical probes. Bioreactors with disposable optical probes may have a higher mixing time than traditional probes because they have a greater response time.

These results as a whole suggest that there are no great deficiencies with any of the bioreactor designs with regards to mixing. Most suspension cell cultures will be ‘mixed well’ even at high cell densities with these systems, however it is possible that the paddle style and the orbital shaking agitation designs may be deficient at very high densities due to the lower mixing efficiency. It should also be noted that P/V determinations and requirements should be optimised for each vessel to ensure adequate mixing and mass transfer.

In addition to mixing, mass transfer capabilities should be determined in order to ensure that the reactors can efficiently transfer dissolved gas. All reactors were filled to the maximum working volume and gas (air) flow was normalised to appropriate platform conditions for cell growth. In addition, the non-micro sparger was employed for all reactors with the exception of the Mobius CellReady bioreactor. Figure 2A on page 67 shows the results of the normalised gas flow KLa studies.

Surprisingly, the orbital shaker style vessel demonstrated the higher KLa at the normalised conditions, reaching values of approximately 3.5 hr -1. The 3L Mobius CellReady bioreactor followed with values close to 2.5 hr -1, and the rest were between 1.5hr -1 and 0.5 hr -1. These KLa values are all within our accepted range. In addition to the normalised standard gas flow, we also wanted to demonstrate the maximum KLa values for the highest recommended gas flow the manufacturer specified for each reactor (Orbital = 0.4 vvm, Mobius CellReady bioreactor = 0.2 vvm, STR = 0.1 vvm, Paddle = 0.09 vvm and the Rocker = 0.0125 vvm), see Figure 2B. The highest KLa reached was from the Mobius CellReady 200L reactor, which achieved a KLa of 11 hr -1 with the micro sparger.

Again, all the reactors achieved KLa values that are in the acceptable range for our standard processes. It should be noted that KLa values are relative to several factors, including gas flow, bubble size, agitation rate (or P/V) and the solution used. Purified water was used for these KLa studies for convenience and consistency with our characterisation philosophy, but higher values would be possible with mock media or actual cell culture media. In summary, each reactor, given the individual vessel designs, is capable of delivering good mass transfer of dissolved gas.

Tracking Changes


The glycosylated molecule process evaluated for these studies employed a temperature shift. Therefore, temperature mapping of the vessels is another critical function of the bioreactor characterisation. Figure 3 summarises the temperature mapping studies; the results outlined in Figure 3A indicated there was higher heterogeneity during heating compared to cooling action. Figure 3B shows the maximum difference between the value and set point during heating and the temperature gradient during heating. Conversely, Figure 3C shows the maximum difference between the value and set point during cooling and the temperature gradient during cooling. Tuning of the PID loops was not fully investigated, and more controlled heating and cooling could have been obtained by more careful tuning exercises. The ‘stirred tank 50L’ vessel showed a greater degree of heterogeneity in these experiments, presumably because temperature transfer of the partial jacket is less than a fully jacketed system.




Cell Growth and Production

After the engineering characterisation has produced the requisite information from which to start cell culture operations, a mammalian CHO cell process was run to produce a highly glycosylated molecule. This molecule is well characterised within our facility and we chose to evaluate the reactors based on our knowledge of historical process trends. Each bioreactor was inoculated according to the materials and methods, and process kinetics were monitored over time.

Figure 4 demonstrates the population doubling levels (PDL), which is a way to gauge the growth rate of each cell culture over the process duration. As shown in Figure 4A, PDL for the glycosylated molecule cells was similar for all bioreactors evaluated. Additionally, doubling time (in hours) shows that the reactors were similar to each other (data not shown). During the production phase of the run, there was low variability among the bioreactors. Figure 4B shows the final integral viable cells (IVC) between the vessels. There was low variability observed across all disposable bioreactors and growth was similar to that of our historical stainless vessels (data not shown). It should be noted that lower growth was observed in the bioreactors, with non impeller style agitation (orbital shaking and rocking motion bioreactors) compared to stirred bioreactors.

Product titre and quality attributes are also critical; to further evaluate bioreactor performance, final titres and glycosylation patterns were assessed. Figure 5 shows final titre and glycosylated forms for each vessel noted. Final titres from the disposable bioreactors were similar to that of our historical stainless steel bioreactors, and all bioreactors demonstrated the ability to produce a glycosylated molecule with our minimum specifications for glycosylated forms.

Efficiency of Production

The study also explored whether the use of single-use bioreactors can accelerate process efficiency for the glycosylated molecule. Table 2 on page 70 provides a comparison of lead and sterility hold times for process development, seeding and production reactors for both traditional stainless and single-use bioreactors. With a single-use bioreactor, one can expect reduced cycle times and increased overall throughput. These trends hold for process development, seed trains and production reactors. By considering process development (three runs/ bioreactor), analytics, two weeks for tech transfer, cell amplification and manufacturing runs (one preclinical and three GMP runs), we calculate that the use of disposable bioreactors would require 144 total cycle time in days, compared to 177 for stainless steel/traditional systems. This time savings speaks to the potential speed to clinic advantage of singleuse systems.

Conclusion

This work encompassed our efforts to evaluate potential single-use bioreactors for inclusion into our clinical and process development facilities. We looked specifically at the general engineering characterisation, including mixing studies, mass transfer capabilities, temperature mapping and cell culture performance, as well as product quality. The results demonstrate that the capability of each reactor evaluated fell within our minimum specifications. However, aspects that we did not directly discuss that elevate certain reactors over others include: flexibility; security of supply; ease of use; and robustness. These attributes are, of course, necessary to evaluate for proper selection of a bioreactor platform.

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Lee Madrid is Consulting Biomanufacturing Engineer at EMD Millipore Corporation. He received his PhD in genetic and molecular biology at the University of North Carolina and did a post-doctoral fellowship at the Fred Hutchinson Cancer Research Center. His areas of expertise include cell culture and bioreactor technologies, purification systems, TFF and NFF filtration, nano-filtration and aspects of disposable manufacturing.

Aurore Poles-Lahille is USP and New Technology Manager at Millipore SAS. She started as an intern at Merck Serono Biodevelopment, where she worked on scale-down models of manufacturing bioreators and protein purification platforms. She has also presented and published articles about disposable bioreactors. Prior to this role, she worked as a new technology and manufacturing support specialist, and a USP new technologies engineer.
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Lee Madrid
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Aurore Poles-Lahille
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