Disposable Systems as a Platform Technology for R&D and Clinical Supply

In this month's column, we continue talking to users about their experience implementing single-use technology. This month, Detlef Eisenkrätzer of Roche talks about Roche's experience deploying single-use bioreactors for early development and clinical supply of mammalian cell-based products.

Miriam Monge

Since 2006, Eisenkrätzer has been the director of the company's non-GMP pilot plant at the Penzberg Biotech Research Center, responsible for process optimization, scale-up, and technology transfer. For this project, carried out over a two-year period, he coordinated technical requirements and user resources among all the departments involved to ensure that all departments, from research to clinical supply, established the same technologies.

Q: What were the main drivers for this in-depth analysis and comparison of single-use bioreactors?

Our objective was to increase the number of new products developed per year. When we calculated how much it was going to cost us to increase capacity based on stainless steel, we realized that this was going to be far too expensive. Cost was the main driver, along with uncertainty regarding the profile of processes to be implemented in terms of scale and the capacity required.

Andrew Sinclair

Q: What were the key problems you faced?

Lack of experience with stirred single-use bioreactors (SUBs) was the key issue. We have a long tradition of building stainless steel pilot plants, so for disposables, there was a lot of uncertainty as to how to go about things.

Q: Did you look to other companies to provide support?

Yes, we used external groups to carry out a number of studies. NNE Pharmaplan supported us with concept studies in terms of layout and infrastructure. Through the use of disposables we were able to reduce our plant footprint by 23%.

Biopharm Services provided us with process modelling and cost of goods analysis with a software package to analyze a particular installation. Here, the analysis demonstrated that significant savings could be achieved through a targeted implementation of disposables.

The lifecycle assessment of the SUBs was supported by ESU Services and demonstrated that CO₂ emissions were reduced by 65%, electricity by 42%, air by 98%, and steam by 80%, whereas chilled water increased by 12% and oxygen 100%.

We also wanted to gain experience from other end users. We made visits to Genentech and Lonza. We developed our own waste management concepts.

Q: How did you develop the methodology for this analysis?

We used our global guidelines for the methodology development of when to look at new technologies. The team defines the approach to implementation. The guidelines require that key stakeholders have input; in this case, this included process R&D, manufacturing, procurement, engineering, and quality assurance (QA).

Q: What areas needed the most analysis?

Single-use sensors. We are collaborating directly with the sensor companies to work on the integration and improvement of these sensors.

Q: How successful was your methodology?

The benefit of a standardized procedure is that you get all the users around the table, and by basing discussions on data, you build a rational consensus. The evaluations gained increased credibility and acceptance as each department evaluated more than one disposable type.

Q: Did the list of bioreactors you compared comprise all the key suppliers at that time?

We evaluated the following configurations:

• two SUB types with rocking motion for seed train cultivation

• four stirred SUB types with sizes from 200 to 500 L

• five automation systems for SUBs.

Whenever possible, automation and bag systems have been combined in different variants.

Our main focus was on vendors with a global presence and global support because these systems would be supplied to our sites in Europe, Asia, or the US.

Q: What criteria did the suppliers have to meet?

The films have to comply with USP Class VI requirements, so we pay attention to whether the suppliers make the films themselves or buy them, and what the manufacturing and quality systems are that they have in place.

An important criteria was that the vendor could supply SUBs from 50 to 1,000-L. There were not many companies who had a 1,000 L bioreactor readily available when we carried out this evaluation, so that limited our evaluation. There may be more now.

SINGLE-USE BIOREACTOR EVALUATION AND COMPARATIVE RESULTS

Q: Is the engineering characterization to compare technical performance of SUBs with stainless steel bioreactors (SSBs) based on standard specifications for bioreactors?

The engineering characterization is based on performance specification for SSBs. All engineering numbers are available and these were the minimum numbers we wanted to achieve with disposable bioreactors.

Q: If we look at the engineering characterization results (Figure 1), it is clear that the performance of the SUBs in terms of power input is poor compared to SSBs, the best being 10% of the SSB. This results in poorer mixing times and mass transfer coefficients. Yet you have ranked the mixing times as acceptable. Why?

Figure 1. Engineering characterization results of a comparison of stainless steel and single-use bioreactors (SUBs). Green shading indicates acceptable results. Yellow indicates acceptable with workaround/additional measures. Red indicates unacceptable.

Although none of the bioreactors achieved an equivalent mixing time as the same size SSB, we used our knowledge of mixing times seen in the large-scale SSBs to define what was acceptable at this scale.

Q: The kLa also has a rating of acceptable with workaround/additional measures. Is this subsequently improved by sparging with O₂?

To achieve an acceptable O₂ transfer rate, we have had to sparge the SUBs with O₂. So we have had to install an O₂ gas system for the SUBs.

PROCESS PERFORMANCE: PILOT-SCALE BIOREACTORS

Q: In your assessment, you found that rockers are suitable for the seed train. Is this because their performance is comparable to SSB and demands on the seed train are not significant?

Rocker systems typically have a lower kLa which limits oxygen transfer and cell densities required, but for the seed train, the required cell density is much lower and achievable in a rocker system. We carried out 17 parallel fermentations in SUB rockers for seed production. These showed identical results for growth and productivity, cell size and distribution, apoptosis, and formation of byproducts (Figure 2). These SUB rocker systems with plug-and-play functionality now replace all our stainless steel seed bioreactors.

Figure 2. Parallel fermentations in single-use bioreactor (SUB) rockers for seed production showed identical results for growth, productivity, cell size, distribution, apoptosis, and formation of byproducts. SSBs: stainless steel bioreactors

Q: What other factors are important for using rockers for the seed train?

Rockers are very beneficial for changeover. We run many more campaigns in rocker systems compared to a SSB. They are more flexible. You can change the inoculation date and then react very fast. We had rockers in place before the project started.

PROCESS PERFORMANCE: BIOREACTORS AT SCALE

Q: In the methodology used to assess process performance, you followed the same methods for both the SSBs and SUBs whenever possible, using the same media lot, inoculum train, and harvest methods. How do the SUBs at scale compare to SSBs from the product quality perspective?

We carried out an analysis of supply campaigns of six different therapeutic proteins from SUBs. The product was always comparable to SSBs regarding ion exchange chromatography and glycosylation pattern. Until now, no differences in product quality between products from classic fermentations and fermentations in SUBs have been observed.

SUPPLIER EVALUATION

Q: What differences did you see in the performance of SUBs from the different suppliers.

Capability of meeting basic Roche specifications: We worked with vendors on bag design and observed differences in reliability and the control systems. Notably, the user friendliness of the control systems differs greatly.

Robustness and reliability: This varied considerably from supplier to supplier. We would not expect this to be the case today, but in 2008, there were quite a few prototypes on the market. Since then, the technology has evolved and in the majority of cases the reliability is comparable.

Quality systems: There are remarkable differences in the quality systems of different vendors. Some companies have very comprehensive quality systems mirroring biopharmaceutical operations, in compliance with cGMPs. There are differences in staff resources at the different companies. These differences impact directly on project timelines and the relative risk of working with a given supplier.

Q: Even with the leading supplier, three different bag types were evaluated. Does this reflect the feedback you were giving them?

Yes, for optimization of some parts of the bioreactor, a different bag design would improve some engineering aspects. Being able to handle the bag more easily, improving the bag systems to better fit our needs in terms of mixing times, and so on.

Q: You say that to maximize reliability, it is important to minimize connections and use tube welders instead. Can you elaborate?

This is related to the fact that in total we have far more than 100 bioreactors running just in our R&D pilot plants in Penzberg. As we install the SUBs in stainless steel facilities, the staff, working in shifts, will be working with both SUBs and SSBs. Operation mode of the tube welders is less operator dependent, and has less risk of errors. Connectors are not an issue for a company who has only SUBs and is used to these connectors.

Q: Your evaluation showed problems using conventional sensors in an SUB and great potential for single-use sensors. Are you happy to rely on the single-use sensors?

Yes, there is poor reliability of class in sensors caused by the absence of grounding and harsh autoclaving procedures. We are working with single-use sensors and the parameters that need improvement with single-use sensors such as DO₂, pH, and CO₂. A lot of progress has been made in the last year. A lot of new products will be coming on the market.

Q: There are many types of single-use sensors available. What would you recommend in terms of essential key features?

Pre-calibrated, ready-to-use sensors should be drift free, have full USP Class VI certification, and have the possibility of being recalibrated. Another important aspect is being able to measure pressure. This is very important as a security measure if, for example, the exhaust filter is blocked because of condensate, clogging exhaust systems, or if you have foaming. A pressure sensor on an SUB is very helpful in routine use.

Q: What are the big areas for improvement in terms of the overall design of SUBs?

We currently buy the bags with the filter preassembled on the bag, and in most cases the filter could be reused. We should therefore look at designs that enable us to connect and disconnect the reusable filter to a second single-use bag. We currently throw the filter away with the bag so there could be substantial cost reductions made here. The other point is that when we want to connect a single-use system to stainless steel, there are not many connectors that are reliable and available in the right dimensions, which means working with hybrid arrangements is actually quite difficult.

PROCUREMENT, SUPPLY CHAIN, AND SINGLE-USE SUPPLIER AUDITS

Q: How was the procurement process carried out at Roche?

Roche started with a technical evaluation and then with the vendor audits. We would not recommend this. When it comes to disposables, you need to do the quality audits first to make sure the supplier meets the basic criteria and then you can carry out engineering characterization with selected vendors. This will save time on the user side. You need to get the input of other departments early such as the procurement people addressing supply chain security issues before the technical evaluation. You need to focus the technical evaluation on parts that are limiting because they are outsourced from the vendors.

Q: How did you compile the manufacturing, quality, and supply chain security standards required for the different pieces of equipment?

Our internal groups developed the URS for the hardware and we worked with the bag system suppliers to define the URS for the consumable part. On audits, we included colleagues from the automation department, and people from our internal disposables group for the consumables part (engineering, QA, and manufacturing). We had two years experience working with prototypes and had identified and removed the weak points.

Q: What problems did you encounter?

One of the issues is the immaturity of the supply chain. At the start of the project, we assumed we would get a reliable supply chain through standardization of disposable components and strategic stockpiling. This varies depending on the type of disposable. It is easier to achieve with bags for liquid handling, for example, where there are multiple vendors and the offering is not that different, and far more of a challenge for disposable bioreactor bags.

Q: In the future, would you validate two different suppliers to do the same duty?

Yes. We would combine the best bag systems with the best automation systems. This way, if there were a disturbance in the supply chain from vendor 1, we would have a second bioreactor that you could plug in.

CONCLUSIONS

Q: Having completed the project and implemented disposables, how satisfied are you with the overall results?

We have started routine production and finished the start-up phase. The experience with the start-up phase was very positive and there is a low failure rate. It is very important to involve operations as well as engineering in factory and site acceptance testing (FAT and SAT). A group of Roche users went to the suppliers for two weeks to get trained on the systems. Having been through this project, we could do it faster next time. [We had] very close cooperation with engineers from the vendors. SAT was done here. FAT was done over there. It worked very effectively.

Q: If you compare your experience with SSBs and SUBs from seed to larger scale, in terms of reliability and performance, are there any differences?

The ability to have multiple processes in SSBs is easier, because you can reengineer by yourselves in a day with stainless steel. You can't do that with disposables; it could take weeks or months to get a new design. Having said this, flexibility and overall capacity is improved with SUBs.

SSBs are more flexible because they typically can be reconfigured for yeast and microbial operations. Our SUBs currently are limited to mammalian use. But in terms of overall reliability, we have not really seen any differences up until now.

Q: Overall, do SUBs deliver on the published benefits? How do they compare to SSBs?

We benefit in terms of faster changeover times, reductions in the footprint of the plant, and the amount of clean steam and pure water required, [which] surpassed our expectations. The smaller footprint was achieved by rearranging the layout with disposables to fit in the existing building even though there were a lot of feedbags around the bioreactor itself. This enabled us to install the pilot plants in existing buildings. If we had stayed with stainless steel, we would have had to build a new plant, so the capital savings here are very significant.

ACKNOWLEDGMENTS

We would like to thank Barbara Jopp Heins of Roche communications for her active collaboration in preparation of this article.

Andrew Sinclair is the managing director and Miriam Monge is the vice president of marketing and disposables implementation, both at Biopharm Services, Chesham, Bucks, UK, +44 1494 793 243, disposables@biopharmservices.comMonge is also the European chair of ISPE's Community of Practice for Disposable Technologies.

REFERENCES

1. Foulon A, Trach F, Pralong A, Proctor M, Lim J. Using disposables in an antibody production process: a cost-effectiveness study of technology transfer between two production sites. BioProcess Int. 2008 June.