The last day of talks at BPI featured more great speakers and topics. I chose to spend most of my time in the manufacturing track and I selected the following three talks to highlight.

Michael Goodwin, Associate Director, Development Engineering, Thermo Fisher Scientific, gave a talk titled “Development, Qualification and Application of Single-Use Technologies for Bioprocessing.”

Mr. Goodwin did a great job providing an example of how important it is for vendors and end users to work together to understand new technology and mitigate risk. The example he provided clearly demonstrated that implementation of single-use systems requires a close partnership between both parties. He stated that each step in the disposable supply chain is complicated and each step can impact quality and control.

The example Mr. Goodwin provided was a case study done in conjunction with Amgen. Amgen had seen poor cell growth for two sensitive cell lines where media was being used that had been held in bags.

After analyzing results, one extractable compound stood out as the issue. The leached compound was antioxidant related and had come from one of the stabilizers used in the bag. They later narrowed it down to bDtBPP that had an impact on both viable cell density and cell growth at very low concentrations. For this particular clone impact was seen as low as 30 ppb.

After the compound was identified and it was traced back to a particular stabilizer, the question then became how to solve the problem and how much stabilizer is actually needed. bDtBPP was linked to an antioxidant connected to the polyethylene resin. The typical prevailing thought across most industries where polyethylene is used is that more antioxidants are better, however this may not be the case in biomanufacturing where cell lines can be particularly sensitive.

The solution was to reduce the polyethylene antioxidant and then test to be sure that the reduction did not have a negative impact on the bags. After the reduction, the bags were tested across a number of different performance variables and it was found that there was no difference between the test bag and the control. There was a difference between the films at a molecular level, but it did not impact performance on a macro level. This solution was the result of both companies working closely together to identify then correct a problem. This demonstrated the importance of transparency between supplier and end user.

Mr. Goodwin presented the following conclusions:

• Science based understanding of the single-use system is a combined effort between supplier and end user.
• Risk of use is reduced with transparency.
• Control along the entire supply chain must be clearly defined
• Understanding variability in raw materials and processes along the entire supply chain is foundational to ensuring form, fit and function of single-use systems.

Rajesh Krishnan, Ph.D., Associate Director, Cell Line and Upstream Process Development, Gilead Sciences, gave a talk titled “Investigation and Reduction of Performance Variability in Single-use Cell Culture Bioreactors.”

In the talk Dr. Krishnan provided a very interesting case study in which Gilead was seeing performance variability in two cell lines in their single-use bioreactors. Both lines were from the same CHO parental host and were producing antibodies. Gilead was working on scale translation of the two monoclonal antibody processes (mAb 1 and mAb2).

Pilot scale for both mAb 1 and mAb2 went well but scale translation to pilot single-use bioreacators resulted in significant decrease in performance. After additional testing they discovered that performance was partially restored in bags conditioned with media and cells prior to production, but a media rinse alone with no cells had no impact.

They initially thought that it must be the single-use bag causing the poor performance. Gilead then began examining potential root causes, but nothing jumped out as a cause. They did notice that there had been a great deal of variability in set up and operation of single-use bags during normal operation. So they decided to set up a modified operational strategy for single-use bioreactors including delay sparging and pH control after media change. In mAb 1 the optimized gassing and agitation strategy restored the single-use bioreactor performance. In mAb 2 they followed the same process but also added an exogenous stabilizer which conferred extra protection to cells.

When they ran the process with the old strategy they got similar failures to the original failure prior to process failures. So even though they initially thought that the bag was the problem and there was bag lot variability found, they were able to restore performance by optimizing their process.

Ying Zhu, Ph.D., Lab Head, Cell Culture Technology, Boehringer Ingelheim, gave a talk titled “Implementing Fully Disposable Upstream Bioprocessing Systems for GMP Clinical Supply.”

Dr. Zhu gave an interesting talk about Boehringer Ingelheim’s experiences using single-use bioreactors. In both their Freemont and Germany facilities they have a cell culture train up to 500 liters, a GMP cell culture train at 100 liters and 500 liters (both single-use bioreactors) and a GMP downstream train. In their China facility they are establishing equivalent systems.

For inoculum and fermentation they follow the process below:

Vial to Shake Flask to WAVE 10L to WAVE 50L to Single-use 100 liters to Single-use 500 liters

Dr. Zhu did present some of the challenges and solutions to their use of single-use bioreactors. In one case they experienced a deformed bag during a GMP run due to back pressure in the exhaust line. This was not an issue in non-GMP because there was no back pressure in the non-GMP exhaust line.

In another similar case the vent filter clogged and caused high pressure in the bag. The bag became increasingly inflated due to increasing gas flow. As a result the bag became deformed around the motor and agitator seal where the bag then became thinned and ruptured causing a small break. The vent filter clogging caused the bag leakage. The resolution was to increase the height of the filter holders and improve the setting with critical process interlocks for shutting down at high pressure.

In another case they found that cell growth and viability in bags was impacted by the age of the bags. They found better growth in bags older than six months olds. Their hypothesis is that volatile substances generated during gamma irradiation diffuse out concentrations after storage.

They also found in one instance that media/solution storage in bags was causing low cell growth. This was identified as the leachable compound bDtBPP. The resolution was to change to an alternative vendor for media solution storage that don’t contain bDtBPP.

Overall in their production and product studies they found that in a disposable upstream process the scale-up growth, cell viability and production are all comparable to using stainless steel systems. In addition, product quality was comparable in all bioreactor systems.

Even though there have been some challenges, they still believe that single-use systems are important to getting a GMP facility commissioned quickly (within 5 months). Also key in multiproduct manufacturing, clinical trial material supply, and having global facilities due to the easy technology transfer across multiple locations.