
Increasing Process Development Workflow Efficiency by Integrating High Throughput Technologies
Drivers for increasing upstream process development workflow efficiency
Dr. Frensing explained that his pRED recognized that their upstream process development workflow was inefficient. It was highly manual and the use of 2L bioreactors didn’t permit a high throughput process, so they were not able to run as many fermentations per run as they wanted. They recognized that their workflow could become more efficient by running multiple fermentations at the same time with the use of automated bioreactors. For instance, in their current system samples were collected manually from the 2L bioreactors and then prepared and sent for pH, and cell counting. Samples had to be centrifuged to generate cell free samples that were then sent to their Cedex Bio HT for metabolic and titer measurement. Samples also had to be prepared and sent for osmolarity measurement. Lastly, backup samples were collected and stored. These were all manual processes that consumed a large amount of operator time and resulted in a process was low throughput, time consuming and permitted only a small number of samples and thus a smaller data set.Increasing process development workflow throughput by incorporating automated bioreactors
To increase throughput and improve efficiency, the team decided to incorporate automated bioreactors into their process development workflow. In order to determine their suitability against current operations (2L bioreactors), Dr. Frensing and his team tested Sartorius’ ambr250 and the ambr15 to see how they compared with the 2L glass bioreactor results. In the comparison study, the three systems were run in parallel for one year on every project. The team was primarily interested in evaluating the systems for final clone selection. During the year, 42 CHO lines were evaluated for 5 different antibody formats. The formats chosen were very complex protein candidates, including a brain shuttle antibody fusion and a T cell bi-specific antibody. Inoculation was done with the same pre-culture to ensure a direct comparison.Results of the comparison study
During his talk, Dr. Frensing shared the system comparison data for a final clone screen of 8 clones. The team found that the three systems were comparable in evaluating growth and titer with very minor deviations. However, when they looked at specific parameters, they did find differences between the systems. When looking at ammonium, only one clone showed comparable results. The ambr15 culture had particularly high levels of ammonium and there was also variation among the systems for lactate and viability results. In order to conduct a thorough analysis of the data, the team asked their bioimformatics group to conduct a detailed statistical analysis. They found that in order to fully compare the results, they needed to employ both the two one-sided t-test (TOST) and the model comparison method (Islam et al., 2007). Using these two methods in combination, a statistical analysis was generated that compared each of the ambr methods to the 2L glass bioreactors. Results showed that the ambr250 was slightly better across all parameters, but with the most critical parameters, the ambr250 was much better. The team then evaluated comparability for product quality and all systems were comparable. With glycosylation the three systems were mostly comparable, however there were some deviations with high mannose structures in the ambr15, which Dr. Frensing said, could be do to the higher ammonium levels in those cultures. In clone ranking, with titer as the most weighted value, the ambr250 had comparable results, but there were some differences with the ambr15 system. After all the data was in, they conducted a system comparison where they ranked each of the systems high, medium and low for a set of criteria in order to determine appropriate applications for each system. The criteria and associated rankings are listed below:- Data match and clone ranking compared to the 2L glass bioreactors – ambr250 was ranked good and the ambr15 was ranked satisfactory.
- Instrument reliability – the 2L bioreactors were ranked medium, ambr250 was ranked high and the ambr15 was ranked low. The ambr15 ranking was due to a failure of some fermentation runs, this meant that 24-48 fermentations were lost each time a run failed.
- Lab space and cleaning time – the 2L bioreactor being a poor choice, ambr15 being good and ambr250 medium.
- Cost per fermentation – 2L bioreactors had a medium cost, ambr15 had a low cost and ambr250 had high cost, however Dr. Frensing clarified that when you consider labor, set up and cleaning costs, the ambr250 was less expensive than the 2L bioreactors.
- Throughput – low for 2L bioreactors, high for the ambr15 and medium for the ambr250.
- Flexibility – the 2L bioreactors offered the most flexibility in terms of being able to test different stirrers, spargers, sensors, etc. They also provided the highest sample volume, which allowed more characterization work to be done. The ambr15 offered the least flexibility and the ambr250 offered medium flexibility in that you had enough volume to conduct some additional sample characterization based on the higher sample volume.
Implementing the improved process development workflow
As the team incorporated ambr15 and ambr250 bioreactors into the process development workflow, the number of samples increased. The large number of samples required high throughput sample processing and analytics. To accomplish this, they purchased the Tecan Fluent pipetting robot and used it to link the ambr systems and the Roche Custom Biotech’s Cedex Bio HT Analyzer. Key benefits identified by Dr. Frensing of using the Tecan Fluent to link ambr systems with the Cedex Bio HT Analyzer:- Fast and automated workflow
- 12 – 192 samples can be processed at once
- 48 samples can be processed within 20 minutes (manual processing: 60 minutes)
- Operator only has to spend ~5 minutes hands-on
- Higher and operator-independent accuracy for sample pre-dilution
Summary
In summary, Dr. Frensing stated that the analysis of the three systems led the team to select the ambr15 system for clone screening and process development and the ambr250 system for final clone selection and process optimization. They were able to establish an efficient link between the ambr systems and the Cedex Bio HT Analyzer using a Tecan Fluent system, which permitted them to improve their process development workflow, make it semi-automated, and significantly more efficient. They redesigned their data management platform to handle the large number of samples and data being generated by automating online and offline data transfer to the data warehouse. They are also developing an automated data feedback loop between the ambr systems, the Tecan Fluent and the Cedex Bio HT Analyzer.Recommended Further Reading:
Automated Collection and Analysis of Bioreactor Samples to Enable Quality by Design Initiatives