Decreasing drug development timeline via upstream process intensification

Decreasing drug development timeline via upstream process intensification A scalable, high-intensity perfusion process was developed at Boehringer Ingelheim, Fremont Inc which is 10x more productive for producing recombinant proteins than comparative fed batch processes in the same 14-day run duration. By eliminating wasteful cell bleed we were able to achieve cell densities up to five times greater than standard “steady state” perfusion culture previously used. In order to sustain such large cell masses at manageable media exchange rates, concentrated media feeds were developed which effectively allow for optimization of nutrient delivery and dilution rate. We believe this system is scalable up to 1kL; the process has already been demonstrated successfully at the pilot scale (100L), where bioreactor productivities averaging over 5 g/L/day have been demonstrated. We begin development with new cell lines for the high intensity perfusion process by adapting spin-tube and shake flask models that others have used for fed batch. These methods are used to test for important control parameters to allow full development in a 2L bioreactor. AMBR250 bioreactors can be used, though not optimal, as will be discussed. Due to the simplicity of the process design, the integrated downstream is developed at small scale using classical batch chromatographic techniques, including high throughput process development and standard chromatographic steps. The virus inactivation step is developed by accounting for viscosity and titration of the product and buffer in the Protein A elution peak, which differ slightly from product to product. With these simple development techniques, we believe the highly productive process could be commercially viable at Phase I, with limited to no Phase III process development

Workshop 3: Business Cases for Integrated and Continuous Biomanufacturing

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Development of highly intensified cell culture perfusion media and process with tremendous productivity potential, while having a low cell bleed requirement for maintaining an overall high yield

Process intensification leveraging perfusion offers immense opportunities for yield improvement over fed-batch processes for the production of monoclonal antibodies. In the context of continuous processing, the goal is to achieve highly intensified perfusion processes that allow substantial footprint reduction and enable flexible adaptation in new facilities. Developing a productive and efficient perfusion process requires not only the application of the “push-to-low” concept for reducing the perfusion rate requirement, but also requires in-depth mechanistic development of medium formulations in order to decrease byproduct waste generation, reduce unproductive cell growth and increase productivity. Specifically reducing the usage of cell bleed is particularly desirable for improving the overall yield, since as much as 30% of the generated product may be lost through the use of cell bleed. In this work, we share case studies of perfusion medium development studying classical components such as vitamins and salts that can be manipulated to have profound effect for controlling the cell growth and reducing the use of cell bleed. In one case, the cell bleed rate was reduced down to as low as zero, while still being able to maintain a highly viable culture. Furthermore, in some cases, significant increase in the cell specific productivity (qp) was achieved when the perfusion culture was switched to a growth suppressed mode. In one example, the qp increased from 30 pg/cell/day to as high as 115 pg/cell/day when the cell growth was arrested. This led to increased daily volumetric productivities of 3 to 5 g/L/day compared to the control of 1 g/L/day. Cell cycle analysis of the arrested culture by flow cytometry also revealed an induced state of elevated cell population in the G0/G1 phase, which is generally considered as the most productive state of the cell cycle. In order to integrate the cell growth control strategy described herein, a two stage perfusion concept is designed where the first stage focuses on rapid accumulation of cells to reach the target cell density, and the second stage switches to a slow growth, yet highly productive and viable perfusion culture

Marching toward implementation of an ultra-high density dynamic perfusion process

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Product sieving challenges in TFF perfusion cell culture

Integrated continuous biomanufacturing has gained significant interest because of its potential to streamline production by integrating upstream and downstream processes. Combined with an intensified perfusion bioreactor, continuous processing can greatly reduce cost, space requirements, and handling steps, while improving production efficiency.1,2 During perfusion operation, product and spent media are removed while cells are retained within the bioreactor with a cell separation device. In particular hollow fiber membranes, attached externally to the bioreactor, permit tangential or alternating flow filtration (TFF, ATF), as cells are recirculated through the unit and permeate is harvested for downstream processing.3,4 However, membrane fouling and issues with product sieving, especially associated with a TFF setup, have a direct impact on total product yield from the process, and can cause hollow fiber reliability issues which in some cases can result in premature termination of a bioreactor run. It is hypothesized that membrane fouling from host cell proteins, cell debris, or additives such as antifoam can result in decreased product sieving as product transmission through the membrane decreases over time.5,6 Toward addressing issues with product sieving, we aim to identify the underlying causes of membrane fouling, associated with host cell proteins and antifoam, and to develop new methods to lengthen their lifetime during perfusion operation. This presentation will focus on some tools and experiments that we have conducted to address this issue with the goal to identify factors within the bioreactor that lead to reduced product sieving and implementation of new strategies to mitigate the effects of these factors during operation. 1. Konstantinov, K.B., Cooney C.L “White Paper on Continuous Bioprocessing” Journal of Pharmaceutical Sciences 104 (3), 2015, 813-820. 2. Warikoo, V., et al. “Integrated Continuous Production of Recombinant Therapeutic Proteins” Biotechnology and Bioengineering 109 (12), 2012, 3018-3029. 3. Clinke, M.F., et al. “Very High Density of CHO Cells in Perfusion by ATF or TFF in WAVE Bioreactor. Part I.” Biotechnology Progress 29 (3), 2013, 754-767. 4. Karst, D.J., et al. “Characterization and Comparison of ATF and TFF in Stirred Bioreactors for Continuous Mammalian Cell Culture Processes” Biochemical Engineering Journal 110, 2016, 17-26. 5. Van Reis, R., Zydney, A. “Bioprocess Membrane Technology” Journal of Membrane Science 297, 2007, 16-50. 6. Wang, S., et al. “Shear Contributions to Cell Culture Performance and Product Recovery in ATF and TFF Perfusion Systems” Journal of Biotechnology 246, 2017, 52-60

iSKID: From integrated pilot scale runs to GMP implementation approach

One of the most compelling business reasons for integrated processing is the ability to de-risk capital investment due to a significantly more productive process that takes less space and fewer campaigns to generate clinical and commercial material. Boehringer Ingelheim and Pfizer developed the iSKID, a fully integrated and automated system that hydraulically links the perfusion bioreactor with several downstream unit operations (2xProtein A columns, continuous viral inactivation, AEX in flow through mode, and SPTFF). The Protein A elution cycles are discrete and separated by \u3e2hrs, allowing the ability to discard cycles that do not meet process specifications. The discreteness between product cycles and hydraulic linkage enables the sanitization between cycles for a robust bioburden control strategy. Each cycle is captured in a single use mixer (SUM), where the product is pooled in stable conditions until viral filtration, ultrafiltration/diafiltration and final filtration are performed in batch mode. Identical iSKID prototypes at 100L scale were used at three different sites to generate product quality, process, and bioburden data from three different molecules. The data has been used to understand implementation gaps in GMP facilities and process platforms (CMC1/CMC2). In addition, the team identified specific items to present to the FDA’s Emerging Technology Team (ETT). These items include our strategies for batch definition, microbial control, and process control. In this talk, we will use the data generated from the consistency runs to elaborate on the robustness of the process and touch upon the strategies to be presented to the ETT

The effects of climate change on the Acropora aspera holobiont

Corals host a community of microbes, comprised of endosymbiotic algae, Symbiodinium, bacteria, Archaea, and viruses. This coral/microbe assemblage is known as the coral holobiont. The mutualistic nutrient exchange between coral host and Symbiodinium is arguably the most critical and therefore most studied of these associations; energy supplied by the algae contributes the majority of corals' daily energy requirements. The loss of Symbiodinium during coral bleaching can potentially be fatal to the host. Unfortunately, mass bleaching episodes have become more frequent with intensifying global climate change. Anthropogenic CO₂ emissions are increasing the concentration of atmospheric CO₂, which causes a greenhouse effect resulting in elevated sea surface temperature. These anomalously high temperatures cause the holobiont to become stressed and triggers expulsion of Symbiodinium. Concomitant with temperature rise is the acidification of the surface ocean, caused by uptake of atmospheric CO₂. The interactive effects of these two factors on coral holobiont physiology and community dynamics are largely unknown.\ud \ud Symbiodinium in vitro and in hospite exhibit significant photosynthetic dysfunction above 34°C, which coincides with the bleaching temperature of Acropora aspera. Expression of targeted key photosynthetic and stress response genes of Symbiodinium (measured using quantitative PCR) remains unchanged by heat stress, irrespective of symbiosis status (isolated or in hospite) and pCO₂. The stress response of Symbiodinium, therefore, likely relies on some other mechanism, possibly post-translational modification of transcripts or shifts in the metabolome. Conversely, transcript levels of key carbon metabolism genes of A. aspera are significantly altered by a synergistic effect of pCO₂ and bleaching stress, despite relatively small increases in treatment pCO₂ compared to the diurnal fluctuations experienced by these corals in the reef flat environment. This suggests the physiological effects of ocean acidification will be felt by mid-century, much sooner than predictions made for calcifying processes. Although 34°C bleaching stress resulted in mass expulsion of Symbiodinium in both ambient pCO₂ and elevated pCO₂ treatments, there was no apparent change in the bacterial community. This contradicts many studies that have found an increase in Vibrio in bleached corals. These results suggest a relatively static nature of the A. aspera bacterial community, implicating that shifts in the bacterial assemblage do not contribute to the environmental response of this holobiont. The ability of corals to acclimate to changing temperature regimes was demonstrated in A. aspera. Exposure to sub-lethal temperature stress prior to a bleaching-level stress conferred an acclimatory response in Symbiodinium non-photochemical quenching of chlorophyll fluorescence and A. aspera antioxidant and heat shock protein gene expression, which was not observed in repeated bleaching-level stresses.\ud \ud The results of this thesis suggest Acropora aspera transcriptional regulation and Symbiodinium photophysiology are the key factors in environmental response and Symbiodinium transcriptional regulation and bacterial community dynamics play a limited role in temperature and acidification response