Medium optimization case study for continuous upstream process

Based on other mature industries, continuous upstream process is a logical replacement for current fed batch operations. However most industrial medium development has focused on the biological requirements for fed batch and therefore focused on the needs of stationary phase production. There is not an a priori expectation that growth and stationary phase requirements are identical. Yet an ideal continuous upstream process requires some combination of both. An optimal continuous upstream process requires a high cell density similar to fed batch operations. There is also some minimum growth rate required in order to match the combined death and cell removal rate at steady state. Hence, medium optimization yielding high productivity and sustaining sufficient growth is critical. In our work, we first established the minimum metabolic requirements to exceed high cell density at high viability based on our existing cell culture medium platform. Furthermore, the cells were able to reach a high cell density within only a few days post inoculation. Optimization was still required in order to shift from such a rapid growth process to a desirable high productivity continuous process. Fortunately, a continuous system is an ideal setup within which to evaluate multiple effects sequentially. An individual component can be spiked into the culture, and the direct impact can be monitored on cell growth and productivity. As the continuous system will continually wash out the component, the individual component impact is temporal and eventually the system returns to steady state in the absence of the spiked component. This process can be repeated iteratively until an optimal result is obtained. In our case, multiple positive effects were combined into one medium composition specifically optimized for a continuous upstream process

Leveraging Mab cell culture platform to predict product quality

Biopharmaceutical therapeutic development timelines can be reduced by quickly generating material to initiate clinical trials and begin the process of drug development in order to improve the lives of patients. One way Biogen has addressed these challenges is the generation of a high productivity host. The high productivity host enables the use of representative cell pools that produce sufficient material for toxicology studies faster than previous workflows which used an individual clone. In general, one caveat of using cell pools is the risk of generating material that may not be fully representative of the commercial process. As product quality may vary from clone to clone, a less productive clone may need to be selected in order to avoid repeating toxicology studies. Biogen has mitigated this risk through changes in the cell culture platform process in order to have more predictable product quality outputs across pools, clones, and products. Cell culture platform modifications to the host cell line, media composition, and process parameters have enabled a predictable quality profile for both clones and pools, thereby enabling a “fast to tox” strategy that is also consistent with generating a commercially desirable proces

Leveraging a deeper understanding of Poloxamer188 to improve cell culture processes

The industry-wide use of Poloxamer188 (P188) underwent severe scrutiny as a result of lot variability discovered within the past few years. While screening methods have been developed to ensure lot consistency and the root cause of the variability has likely been identified, a fundamental understanding of surfactant-cell interactions has not yet been achieved. As industry continues to push culture densities higher to maximize product yield, higher aeration and agitation are required to supply sufficient oxygen transfer rate. The harsher environment in the bioreactor, depletion of shear protectants, and possible cell physiology change leads to the need of improved shear protection strategies to minimize shear damage in the cell culture process. In this project, novel concentric cylinder mixer (CCM) assay was developed to quantify the relative shear sensitivity of mAb producing Chinese Hamster Ovary (CHO) cell lines in production bioreactors. Compared with other methods to characterize shear sensitivity, the CCM assay requires low sample volume and minimal processing time. Various concentrations of P188 were evaluated using CCM assay to improve shear protection strategies in 3L and 300L bioreactors. Results indicate that cell shear sensitivity dramatically increases upon reaching the cell culture stationary phase, coinciding with viability decline and exponential LDH increase in the bioreactor. With a simple shift in shear protectant concentration, we were able to increase harvest viability resulting in decreased cellular debris, decreased foam stability, and reduction in LDH upon harvest. A strong dose dependent correlation between membrane rigidity and surfactant concentration was also discovered through the studies which provided possible mechanism of how surfactant reinforces cell membrane by decreasing membrane fluidity. The knowledge of cell membrane fluidity combined with the CCM assay contributes to our understanding of cell shear sensitivity and surfactant-cell interactions. These tools can be used to optimize process parameter set points, evaluate media formulation effects on cell sensitivity, and select shear resistant cell lines to improve cell culture process robustness

Application of -omics knowledge yields enhanced bioprocess performance

The classic phrase to describe cell culture is “every cell line is different.” The unfortunate part of this idiom is the actual concealment of a crucial lack of fundamental understanding. Furthermore the phrase ignores the substantial success achieved to date in developing robust industrial cell culture platforms that are applied to all cell lines regardless of their intrinsic variation. At Biogen, our cell culture medium platform is agnostic to CHO host cell line, and the platform can accommodate this inherent genomic variation as cell lines come from different host backgrounds. This is also an opportunity for -omics work then as the differences in cell line performance can be linked back to fundamental differences within those host cell lines. However, the power of -omics technologies to influence process optimization is limited by the difficulty and time scale for execution and interpreting such studies. Our approach to -omics implementation has been to utilize multiple targeted investigations and combine the learnings into an implementation strategy focused on enhancing the efficiency of manufacturing. Metabolic flux analysis was used to establish a baseline knowledge of central metabolism in the Biogen platform. The next step was to incorporate transcriptomics and proteomics with our metabolomics knowledge. With Biogen’s toolbox of CHO host cell lines, this approach identified intrinsic host cell line differences as well as unique limitations in cell culture. Specifically, we have determined sources of novel metabolic inhibitors that suppress cell growth as well as differences in lactate and ammonium metabolism that split according to host cell source. These conclusions ultimately lead to the optimized platform process yielding the desired product quality. Determining these differences led to an increased growth rate in scale up for cell lines from a more sensitive host as well as maintaining robust cell growth and productivity in production bioreactors. Ultimately still “every cell line is different.” Yet the more we know, the more opportunities there are to exploit both the similarities and the differences

Advanced process monitoring and feedback control to enhance cell culture process production and robustness

It is common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply Process Analytical Technology (PAT), we demonstrated three different and novel approaches for implementing online monitoring and feedback control to improve the performance and/or robustness of cell culture processes. First, we describe the first reported fed-batch process utilizing online amino acid measurements (glutamate) to trigger automatic feedback control delivering complex nutrient feed. More importantly, the resulting feed strategy was translated into a manufacturing-friendly manual feed strategy without impact on product quality. Second, we increase the complexity of the control strategy by designing multiple feedback control loops for all feed solutions based on varied inputs (bio-capacitance for cell mass, Nova-Flex for glucose), resulting in a truly fully automatic cell culture process. We then demonstrate the utility of the feedback control system to rescue a batch without manual intervention by automatically adjusting the feed in response to an excursion that was intentionally introduced. Finally, we describe the implementation of a new online monitoring instrument in combination with a logic control module to simultaneously monitor and control glucose and lactate with high frequency, resulting in cell culture process improvement. Together, the three cases presented here illustrate an advanced process control toolbox which can be readily applied to various cell lines, media systems, and processes to significantly increase productivity and improve robustness in manufacturing, with the goal of ensuring process performance and product quality consistenc

Understanding and overcoming process insults through application of ‘omics technologies

Modern industrial process development, at both small and large corporations, usually consists of applying a well-characterized and established cell culture platform. Despite the high productivity available from these process platforms, difficult challenges remain, including with respect to the ability of the process to endure insults or disruptions. We previously demonstrated that overfeeding resulted in an undesirable increase in lactate production late in fed batch culture, which decreased productivity[i]. Here we report on metabolic flux analysis performed utilizing this process and isotopically labeling with multiple tracers (glucose and glutamate) delivered at five distinct time points of the cell culture process. Notably, we identified unexpected behavior within the tricarboxylic acid (TCA) cycle. The corresponding labeling data indicated a significant redistribution of the fluxes in and around the TCA cycle. Understanding the intracellular changes occurring when cells are challenged with a process insult, such as overfeeding, should lead to enhanced process development. Consequently metabolic flux analysis is only the first step in improving the process. We have identified two medium supplements which each independently permit the cell culture to endure overfeeding and result in maintaining or increasing titer despite the process insult. The overfed process and the supplemented processes were utilized to evaluate changes in the cellular metabolism with an untargeted metabolomics approach. Novel findings from the untargeted metabolomics approach when combined with metabolic flux analysis give a complete picture of the cellular metabolism as both reaction rates and relative concentrations are known over the full process duration. With this knowledge in hand, the platform process can evolve to routinely overcome process insults such as overfeeding

Process optimization for high volumetric productivity with product quality control

High commercial demands of biotherapeutics require high volumetric productivities to accommodate their production with the existing manufacturing infrastructure. While titers are exceeding 5 grams per liter in fed-batch processes, it is imperative that these processes result in consistent and desirable product quality. Here we describe a fed batch process optimization effort resulting in significant increased titer than the initial process. During the optimization, we identified a medium component capable of impacting productivity and two different critical product quality attributes. Through complex screening, the component concentration was shown to be proportional to these product quality modifications in opposing directions, thereby requiring a careful optimization of the delivery range. One of these modifications was recapitulated in a cell free system with media and protein indicating that this was not a result of shift in cellular metabolism unlike the other modification. The mechanism of action and strategies to mitigate this issue were also evaluated. Through this work, a well-controlled process without impacting productivity during large scale manufacturing was designed

On-line monitoring and controlling of cell apoptosis in mammalian cell culture processes using dielectric spectroscopy

We investigate a method to control critical quality attributes and apply Process Analytical Technology (PAT) via online dielectric spectroscopy (DS) feedback. This system has been intensively explored and successfully implemented in GMP manufacturing processes at Biogen. The present bioreactor application however, is basic and only allows the prediction of biomass. To further enhance the cell culture process robustness, we investigated the feasibility of using the full-spectrum dielectric spectroscopy scanning function to detect dielectric property changes in the cells associated with shifts in cell health and/or metabolism. In this proof of concept study, we used several CHO cell processes to demonstrate that DS probes can be used to not only measure the biomass but also reflect the cell’s physiological state changes (e.g. cell apoptosis). The results showed that one or more of the key parameters of delta capacitance (De), critical frequency (fc), and Cole-Cole Alpha (a) from the multi-frequency scanning data could reflect the cell’s early apoptosis induced by chemical treatment, nutrient depletion, or shear stress, which were seen earlier than that obtained from off-line methods (e.g. trypan blue exclusion). In some cases, by responding to the earlier detection, the cell apoptosis was reversed in time and the batch was saved. This enables a potential application, transferrable across programs, of full-spectrum dielectric spectroscopy for earlier detection of physiological changes, allowing for timelier bioreactor process adjustments. In addition, the feasibility of the application of multifrequency scanning in cGMP process for monitoring and control was also explored in this study

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein

AbstractRecombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8–15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc