As one might expect, single-use bioprocessing equipment was in the spotlight at Bioprocess International's annual meeting in Long Beach, California, last week. Vendors introduced new or improved products. However, there was also some candid discussion of the current drawbacks of existing technologies from those who use, and advocate use of, the systems. Surprise! Biodisposable equipment is subject to the same manufacturing-based variability that most products are prone to. Only now, people are studying more closely the potential impacts that this variability can have on cell culture and other aspects of biopharmaceutical manufacturing.
For a skeptic who prides herself as being unbiased, this was good news. All too often if you follow news from BPSA and other vendor groups, you get the idea that all problems have been solved. In fact, a users group is also following issues closely, while companies such as Boehringer-Ingelheim, are devoting more resources to testing potential sources of variability in disposable equipment.
While issues remain, vendors appear to be working proactively to improve quality control and prevent processing problems. In many cases, they are forging stronger relationships with users, who are providing feedback on issues to focus on.
What follows is a brief summary of only one of the sessions dedicated to single-use technologies.
On November 2, a session chaired by Chun Zhung, senior director of cell culture process development at Shire Pharmaceuticals, focused the lens on biodisposables.
As Mr. Chun summarized, the real growth of single use process equipment began about 10 years ago, with the Wave bioreactor’s introduction a major milestone. He likened the development of new designs to what happened in the 1970’s and 1980’s when new types of stainless steel bioreactors were introduced.
However, accessories and features such as tubing had to be optimized to make this new single-use equipment work, he said. He asked: Where will vendors take this new market? Will the future bring greater simplicity of form, where everyone’s equipment becomes interchangeable, or more diversity. “Industry and vendors must work together to address challenges,” he said.
Charles Sardonini Associate Director of Genzyme’s Cell Culture Development Group, then discussed his company’s experiences with disposables, and challenges along the way to “a 100% disposable cell culture manufacturing process.”
His group has been integrating more disposables in the upgrading of an existing clinical trial manufacturing facility, a plant that has been around for a number of years with legacy equipment and processes. “We’re overhauling the facility to improve process flow and upgrade GMP’s,” he said.
Sardonini noted that disposables can simplify processing and add robustness to a mixed-use facility. There is a reduction in autoclaving, stainless steel parts, utilities and contamination risk. In addition there is less need to track and clean glassware, and less ooperator training is required, he said.
One area where his team is using disposables is in buffer preparation. The plant is using a combination of steel and plastic for solution preparation, but using custom designed disposable manifolds, to allow for different containers and multiple sizing for a single batch of media or buffer.
Mixing containers used for media have already been validated, he said, and won’t be replaced, but disposables are being deployed based on need, unit operation by unit operation “where we find the biggest bang for the buck,” he said. Legacy products will continue to use traditional processing equipment, where newer programs using to disposables….
Closed Systems a Benefit
One of disposables’ greatest benefit, Sardonini said, is the fact that they allow manufacturing processes to be closed. This has allowed air-handling requirements to be changed from the stringent Grade A and Grade C to Grade D (Class 100,000), in some portions of the plant. In turn, he said, this has reduced HVAC, environmental testing and gowning requirements. Testing requirements were significant with Class 100 or better systems, requiring testing every shift.
However, he noted challenges with disposables. “Enter this area too soon and your vendors are educated at your expense,” he said. When one fails to do due diligence, he said, one can expect to see projects delayed and there will also be a need for costly workarounds just to keep work on track.
However, he added, enter too late, and you’ll be locked into old technology.
The key is finding processes where disposables offer ROI. For instance, he said, Genzyme didn’t implement them with solution mixing, because stainless steel tanks are still more cost effective.
However, he noted, manifolds that contain PETG bottles will be used once costs come down. “Concentrating on disposables that eliminate autoclave runs enables the greatest savings,” he said.
Extractables and leachables and ELSIE
He then discussed the biggest industry issue, and the fact that some cell lines are more sensitive to contamination than others. In addition, he said, leachables can vary within films, from lot to lot.
He mentioned the work of the Extractable and Leachable Safety Information Exchange (ELSIE), a users group established in 2007 for biopharm, pharma and medical device companies. Its focus is to develop standardized study protocols and offer another credible source of safety information on leachables and extractables. So far, the group has developed a functional database for 200 identified chemicals. A pilot program is now under way to generate controlled extractables data, he said.
Lack of Standards
Another challenge, he said, is the lack of standards for disposable equipment. “Triclamp connectors have standard specs, and we use standards for stainless steel and elastomers as a model,” he said, “but how about standards for aseptic tube welding and connectors----is tube welding considered a closed process? Can there be a standard aseptic connector?
It is critical to choose vendors and technologies with staying power, and to start with the low hanging fruit, for instance, replacing double bag samplers, working with buffer prep, and disposable filter cartridges.
“For complex equipment, you’ll need a comprehensive technical applications document, then a detailed FAT and SAT must be performed,” he said. “Make sure that any new disposable technology has been sufficiently evaluated. Don’t throw it on the plant floor unless it has been tested.” Shake down and engineering runs are critical before installation, he said.
Also be aware that engineering single use systems may change utility requirements, so your utility plan will need to be updated, as will metrology since temperature sensors may need to be calibrated differently.
Finally, he said, be very careful with user requirement specifications. “Some companies write a single document, but we separate user requirements from technical specifications,” he said. “Overspecifying user requirements can make for a difficult validation plan, so get validation, engineering and metrology teams involved in this process early,” he warned. He noted that his company has agreement with suppliers covering any changes, requiring prenotification of what change is and when to expect it. “We need to get samples of new bags so we can test them in a research environment first,” he said.
Dynamic Vendor Relationships
Masaru Shiratori, Senior Engineer and Group :Leader for media development at Genentech, then discussed his group’s experience with biodisposables. “With qualified vendors, one can have a dynamic relationship,” he said, emphasizing the fact that it is critical to distinguish between vendor and user responsibilities.
His group is using disposables in media prep and cell culture as well as at pilot plant level. At small scale, media were warmed in disposables, and there might have been some variation in handling practices. For larger scales, media are prepared and held in steel tank bioreactors.
In one case, his team noted performance issues for media that batched and warmed overnight in glass scaledown bioreactors. Rresearchers observed lower than expected growth in incoculum trains for CHO cell culture. Data suggested that this was not due to a move to chemically defined media, he said.
The company had to get to the root of this problem to see whether it was due to cell line sensitivity, a one-off problem, or to problems with media. They recreated the problem and tested CHO in batch production at different lines and at different work sites, ranking the best and worst batches at warmed conditions.
They then examined varying media hold times and temperatures, and performed water extraction studies for VOC’s (using purge and trap GC/MS) semivolatiles (GC/MS) and low concentration NMR spectra differences.
Ultimately problems were traced to one type of bag from one vendor, suggesting leachable and extractable problems. For now, they have discontinued use of that bag, and are working with the vendor to better understand the source of the problem. . “We need a transparency of information from vendosr, and information back to the resin, so we don’t have to audit their supply chains,” he noted.
The problem is complex. Are all bags affected, or just bags with similar resins, or just bags with similar contact layers or of the same age?” More testing eliminated the contact layer as the problem. It is likely that the problem was due to volatile or air quenched components generated by or increased due to gamma irradiation of the bag, he said. Another question is whether vendors should be required to change gamma irradiation or aeration practices. “Should vendor’s bioassays be able to detect this phenomenon with a biological test?” he asked.
Thus, another challenge for the user community is establishing reasonable expectations for vendor testing.
Concluding the program was Joachim Bar, Associate Director of Upstream Manufacturing Sciences at Boehringer Ingelheim GmbH’s Biopharmaceutical Operations in Germany.
As he explained, his group experienced some hiccups when applying biodisposables to operations.
One potential issue with biodisposables is variability. Electron microcscopy has revealed variation in surfaces and texture, even within the same contact layer, and the fact that some dirt can be incorporated within the plastics, he said. “The manufacturing process is not fully controlled, at least not to the level of what I would expect for a medical or biotech product,” he said.
He suggested that film extrusion and bag assembly needed to be more closely conorolled to prevent differences in performance of final product.
His group studied these issues in greater depth to determine the potential impact that they could have on cell culture performance. Among factors studied were medium to surface ratio, contact time, film age, process, film, cell and medium.
He presented results of studies of media filtration, involving sterile filtration, both with and without prefiltration. Results with different prefilters were evaluated.
In another case, different filter materials were evaluated, and a huge dropoff in performance was seen with one material.
This posed the question of how to evaluate and cope with the potentially high number of changes that might take place with film material, filter material, tubing and layout. “How much can I translate external biocompatibility study results on my process? Are results representative for my serum free defined media applied in high cell density processes?
New Division Focusing on Biodisposables Testing
Boehringer-Ingelheim is setting up a whole new department, he said, to focus on testing biodisposables. “We’re hiring chemists with background in plastics,” he said.
Processes are expected to be reproducible and to support optimum cell growth. So far, he says the company’s testing program has isolated worst case conditions for processes, and are evaluating short vs. long term storage, differences seen with maximum process and storage temperatures, and worst case volume to surface ratio. Two representative cell lines are being tested, he said, along with process-specific media.
Despite variability in their manufacturing processes, he said, biodisposable equipment vendors are becoming more proactive and interested in troubleshooting and testing, to uncover the root causes of process problems.