Automated Collection and Analysis of Bioreactor Samples by Integration of the Seg-Flow Autosampler with the Cedex Bio HT and HiRes Analyzers
As biomanufacturing processes become increasingly complex, the FDA has stressed the importance of their Quality by Design (QbD) and process analytical technology (PAT) initiatives in an effort to encourage more consistent manufacturing processes and product quality. A QbD approach provides a systematic look at bioprocess development, which attempts to predict unique challenges and address them in advance. A key aspect of QbD is defining critical quality attributes (CQA) and critical process parameters (CPP) and then monitoring these closely through the use of PAT. However a challenge in this approach is to ensure that the culture is being monitored precisely, so that critical process parameters can be maintained accurately.
For QbD to work effectively you must accurately measure and be able to maintain tight control of your bioprocess conditions through input of reliable analytics, characterization of influencing factors, minimizing user interference and early detection of deviation. As a result, innovation in these areas has expanded what is possible in terms of bioprocess monitoring. There are two important tools required for automated culture monitoring, the first is having an automated, scalable and sterile way to collect samples from bioreactors for culture monitoring and the second is being able to analyze these samples to provide routine updates on how the culture is performing. By monitoring cell culture performance in real-time, you can not only gather information about the process, but also utilize this information to make changes to the culture as needed.
Enabling Technologies to Support Quality by Design and Process Analytical Technology Initiatives
At this year’s Boston Biotech Week, Kristin O’Neill and Ankit Mehta of Merck gave a very interesting talk titled, “Experiences automating the Roche Cedex Bio HT and HiRes with the Flownamics SegFlow on-line sampling system.” In the talk, they detailed their process and ultimate success in creating an automated system for cell density, cell viability, and cell culture medium analysis of CHO cell bioreactor cultures. The system consists of 3 components: the Seg-Flow 4800 (Flownamics) automated sampler and the Cedex HiRes and Bio HT analyzers (Roche Custom Biotech). The project was a beta site study developed as a collaborative effort of the three companies
In beginning this project, Merck wanted to improve operational efficiency by looking closely at their in-process analytics. The initial goal described in this presentation was to provide complete, automated analytics for a dual-feed bioreactor culture system. As a ready-to-use solution was not available, Merck partnered with Roche Custom Biotech and Flownamics to design the process and was the beta site for this integration.
In considering the equipment to select, Merck identified some key benefits of each of the components of the process. The Cedex HiRes cell analyzer was chosen for cell density and viability determination because of its state of the art imaging capabilities. A small sample size of 0.3 mL was another key benefit of the Cedex HiRes analyzer. The Cedex Bio HT analyzer was chosen because it had all of the capabilities Merck was looking for in cell culture analytics including: high throughput support, automated analysis, availability of an expanded assay suite, and configuration options to rerun a test with automated dilution if the concentration was outside the measuring range. In addition, the sample size for the Cedex Bio HT is only 2-25 microliters for each test.
The Seg-Flow 4800 met the criteria needed for the autosampler such as: automation, dual sample streams (cell-containing and cell-free), the ability to integrate multiple analyzers, configuration options for sample dilution, and the ability to sample for multiple analytes from a single analyzer.
Once components were selected, the initial phase of the project involved the integration of the cell-containing stream from the Seg-Flow to the Cedex HiRes analyzer. Integration was considered successful if the values obtained were within 10% of those obtained by manual sampling. In order to determine cell density and viability from the start of culture through 20 M+ cells/mL, 1:2 and 1:5 dilutions of cell-containing samples were required. Merck found that 1:2 sample dilutions easily met their criteria for success. Initially, some samples at a 1:5 dilution did not meet their acceptance criteria. Troubleshooting studies showed that this deviance was caused by incorrect mixing flow and not by errors in dilution, thus the issue was quickly resolved by adjustment of the mixing flow rate. The ability to configure mixing flow rate in the Seg-Flow was cited as one of the strengths of the system.
The second phase of the project involved the integration of the Cedex Bio HT analyzer with the cell-free stream. Validation studies used 6 bioreactors sampled at 12-hour intervals over 13-days of CHO cell culture with 6-9 tests ordered for each time point (Glucose, Lactate, Ammonia, Glutamate, Glutamine, LDH, IgG). One assay of particular interest was the lactate dehydrogenase (LDH) assay, a measurement of cellular damage. The LDH assay was successfully integrated into the system, as was configuration to enable automatic dilution for an analyte measured with the Cedex Bio HT, the ability to determine glucose concentrations using both the low and high measuring ranges of the Cedex Bio HT. Merck found that the function of the system was acceptable, as expected, and comparable to manual sampling.
Future goals include validating the system for 1:10 sample dilutions for cell count and viability, use of additional assay that are available for the Cedex Bio HT, and integration of the cell-containing sample with the Cedex Bio HT. Other future goals include, implementation of a more complex, 4-feed bioreactor system, and support for FDA’s Process Analytic Technologies (PAT) guidance for real-time process information.
In summary, this successful effort enabled real-time cell density and viability determination in combination with metabolite and substrte analytics – from start of the bioreactor culture through 20+M cells/ml cell density. This served Merck’s need for complete analysis for a 2-feed bioreactor production system. Additional expanded capabilities planned for the future.