Novel, efficient scale-up of inclined settlers for perfusion bioreactor cultures

Inclined settler has been introduced as a unique cell retention device for perfusion bioreactor cultures over 25 years ago by these authors (Batt et al, 1990; Searles et al. 1994) to selectively remove dead cells and cell debris, while the live and productive cells are recycled back to bioreactor. This device has been scaled up successfully as lamellar settlers and utilized to achieve high cell densities and productivities in production-scale perfusion bioreactors by a number of biotechnology manufacturers, such as Bayer, Biomarin, Eli Lilly, Roche, etc. (Shen and Yanagimachi, 2011; Pohlscheidt, et al., 2013). However, the rectilinear scale up of inclined settlers into lamellar settlers is not very efficient and creates a large footprint. Further, this powerful cell retention technology has not so far been demonstrated or applied successfully for retention of microbial cells to our knowledge. We have recently developed novel more efficiently scaled up compact cell settlers in cylindrical, spiral and conical geometries (patent-pending). These novel settlers use the three-dimensional space more efficiently to maximize the cell settling efficiencies within smaller footprint and can be used to achieve high cell retention in microbial perfusion cultures as well as mammalian cell cultures. We have first demonstrated the increased cell retention capabilities of these novel cell settlers with the smaller (hence more demanding) microbial yeast cell cultures, achieving high cell densities and viabilities through selective removal of dead cells and cell debris. Now we are demonstrating the effectiveness of these more efficiently scalable cell settlers for perfusion cultures of mammalian cells. CHO cells cultured in a 5 liter Celligen bioreactor are pumped into the compact cell settlers, in which the larger live cells settle and are recycled to the bioreactors, while the smaller dead cells and cell debris are selectively removed from the top outlet of the settlers, as demonstrated initially by us over twenty five years ago. However the size of the settlers required for the same bioreactor is significantly smaller with reduced footprint, compared to the results obtained with the traditional rectilinear inclined settlers. Latest results from these on-going high cell density perfusion bioreactors with CHO cells will be presented, along with the scale-up considerations for duplicating these successful results at larger production-scale bioreactor

Single use disposable BioSettler removes the dead cells and cell debris selectively to increase the viability percentage of mammalian cells (e.g., CAR-T) during expansion

Current challenge in a FDA approved cell therapy product for adult B-cell leukemia is reported to be the percentage of viable cells after manufacturing is sometimes out of specified range. As the head of a large contract development and manufacturing organization observed, this fall in viability percentage during CAR-T cell manufacturing is a challenge to the whole industry. We present a simple and powerful off-the-shelf solution to this big challenge in all mammalian cell culture expansion bioreactor system. Our single use disposable BioSettler has been demonstrated to be uniquely capable of removing dead cells and cell debris selectively from the bioreactor and returning or recycling live mammalian cells back to the expansion bioreactor. The mechanism of this very fine separation of dead cells from live cells is the exploitation of their vastly different sedimentation rates during enhanced sedimentation of live cells on inclined surfaces. This inclined sedimentation technology has been proven extensively with Chinese hamster ovary (CHO) cells used in commercial manufacture of therapeutic antibodies. As the size and sedimentation velocity difference between live and dead cells are similar for CHO cells and CAR-T cells, our off-the-shelf BioSettler will be readily useful for removing the dead cells and cell debris from the rocking or Wave cell expansion bioreactor and increasing the percentage of viable CAR-T cells being expanded for adult cell therapy

Integration of low-pH viral inactivation and primary clarification processes in a single use disposable biosettle

Please click Additional Files below to see the full abstract

Novel compact cell settlers for continuous perfusion bioreactor cultures of microbial (and mammalian) cells

About 25 years ago, we first demonstrated the use of inclined settlers for selective removal of dead cells and cell debris and complete recycle of live and productive cells back to perfusion bioreactors with murine hybridoma cells (Batt et al., 1990) and Chinese hamster ovary cells (Searles et al., 1994). These devices have been scaled up as lamellar settlers (Probstein and Yung, 1979) and applied successfully over the last two decades for commercial manufacture of several therapeutic biologics in high cell density continuous perfusion bioreactor cultures of recombinant mammalian cells by different biotech companies. However, the rectilinear scale up of inclined settlers into lamellar settlers is not very efficient and creates a large footprint. Further, this powerful cell retention technology has not so far been demonstrated or applied for successful retention of microbial cells to our knowledge. As microbial secretory expression systems have now become well developed for some larger microbial cells, such as yeast Saccharomyces cerevisiae and Pichia pastoris cells, it is now possible to develop high cell density continuous perfusion cultures of microbial yeast cells. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel patent-pending compact cell settlers that can be coupled more efficiently with microbial perfusion bioreactors. We have constructed several prototypes with minor design variations in stainless steel 316 and attached these compact cell settlers to computer-controlled bioreactors (5 liter and 50 liters) growing the yeast cells. Early results from these devices attached to 5 liter perfusion bioreactors demonstrate complete sedimentation of the larger live yeast cells and their immediate recycle back to the bioreactor, while the harvest stream at the top of these cell settlers is a significantly clarified liquid, containing less than 10% of cell concentration, (i.e. OD) of the bioreactor OD. Further the size of cells and debris in the harvest stream is strikingly smaller under microscopic observation, suggesting that we have now successfully demonstrated for microbial yeast cells the unique advantage of inclined settlers over all other cell retention devices in removing selectively the dead cells and cell debris from the perfusion bioreactor. We are now carrying more quantitative cell size analysis in more extended operation of microbial yeast perfusion bioreactors at 5 liter and 50 liter scale. The latest results from these on-going experiments will be presented

Clarification of cell culture broth using single use disposable BioSettlers

Enhanced sedimentation on inclined surfaces has been exploited successfully in mammalian cell cultures to recycle live and productive mammalian cells into the continuous perfusion bioreactors, while removing selectively the smaller dead cells and cell debris into the harvest stream. We have developed a compact settler design using cylindrical and conical geometries, with about 5-10X more settling surfaces over a given footprint, compared to the traditional rectilinear scale up design. With this improved design, we have demonstrated that these compact settlers can also recycle smaller microbial yeast cells back to the perfusion bioreactors operated continuously over two months. We have fabricated the compact settler as a single use disposable plastic settler at an initial size of 150 mm diameter (“BioSettler150”). Perfusion bioreactors attached with this BioSettler as the selective cell retention device achieve high cell densities and viabilities of CHO cells over extended culture durations by selectively removing smaller dead cells and cell debris. Please click Additional Files below to see the full abstract

Single use plastic settlers for clarifying cell culture broth, selective removal of dead cells and affinity capture of antibodies on protein A beads

We have redesigned the successful “inclined settler” technology used as a selective cell retention device in mammalian cell perfusion bioreactor cultures into a more compact and easily scalable design using cylindrical and conical geometries. Through this novel settler design, we have achieved 6 – 10 x more settling area for the same footprint, compared to the traditional multi-plate or lamellar rectilinear scale up design. Using this compact settler design, we have demonstrated significant clarification of the smaller yeast Pichia pastoris cells in continuous harvest or settler effluent stream and high cell densities (700 – 1000 O.D.) in perfusion bioreactors operated over several months. We have now fabricated this compact settler as a single use disposable plastic settler at 6 inch diameter scale and are planning to fabricate it in two larger sizes: 12” diameter and 24” diameter over the next six to twelve months. Recent experimental data with this compact settler as the selective cell retention device for achieving high cell densities in mammalian perfusion bioreactor cultures (operated for over a month of culture) will be presented. Our industrial collaborators are testing this device as a single use disposable device for clarification of cell culture broth from fed-batch bioreactor, for potential replacement of centrifuge for this operation. Another exciting application of this compact plastic settler is the affinity capture of antibodies from cell culture broth directly onto protein A beads suspended inside the settler, while the cells and unbound host cell proteins are easily washed away in the settler top effluent, followed by elution, cleaning and regeneration steps on the beads suspended inside the settler. This integrated bioprocessing application can potentially replace the current unit operations of centrifugation, depth filtration and affinity column chromatography. Reproducible data from the preliminary experiments on this novel bioprocess application will also be presented

Effect of Deferoxamine on Late Deaths Following CPR in Rats

The iron chelating agent deferoxamine was studied in an animal model as post-resuscitation therapy to prevent late deaths and brain damage following total circulatory arrest and resuscitation. Cardio-respiratory arrest was induced by injection of cold, 1% KC1 into the left ventricles of ketamine anesthetized rats pretreated with succinylcholine chloride, and by discontinuation of positive pressure ventilation. CPR was begun after six minutes, and animals with return of spontaneous circulation were entered into the study. Within five minutes after return of spontaneous circulation, treated animals received deferoxamine (50 mg/kg, IV). At ten days, 16 of 25 (64%) of treated animals had survived without neurologic deficit, compared to nine of 25 (36%) of controls (chi square = 3.92, P \u3c .05). Chelation of intracellular iron by deferoxamine may have prevented free-radical-mediated reactions that led to late deaths in control animals

SUD inclined settling bioreactor for gentle expansion, concentration and harvest of gene and cell therapies

Please click Additional Files below to see the full abstrac

In silico evolution of diauxic growth

The glucose effect is a well known phenomenon whereby cells, when presented with two different nutrients, show a diauxic growth pattern, i.e. an episode of exponential growth followed by a lag phase of reduced growth followed by a second phase of exponential growth. Diauxic growth is usually thought of as a an adaptation to maximise biomass production in an environment offering two or more carbon sources. While diauxic growth has been studied widely both experimentally and theoretically, the hypothesis that diauxic growth is a strategy to increase overall growth has remained an unconfirmed conjecture. Here, we present a minimal mathematical model of a bacterial nutrient uptake system and metabolism. We subject this model to artificial evolution to test under which conditions diauxic growth evolves. As a result, we find that, indeed, sequential uptake of nutrients emerges if there is competition for nutrients and the metabolism/uptake system is capacity limited. However, we also find that diauxic growth is a secondary effect of this system and that the speed-up of nutrient uptake is a much larger effect. Notably, this speed-up of nutrient uptake coincides with an overall reduction of efficiency. Our two main conclusions are: (i) Cells competing for the same nutrients evolve rapid but inefficient growth dynamics. (ii) In the deterministic models we use here no substantial lag-phase evolves. This suggests that the lag-phase is a consequence of stochastic gene expression

Impact of selected plant species on enzymatic activity of soil substratum on post-mining heaps

Published by Polish Society of Ecological Engineering (PTIE). This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.12911/22998993/93867© 2019 Polish Society of Ecological Engineering (PTIE). The natural mineral resources (hard coal, sands, dolomites, lead and zinc ores) found in the Silesia and the excavation of them led to significant transformation or even degradation of the environment. The landscape of Upper Silesia was dominated by heaps created as a result of the accumulation of post-mining coal waste. These post-industrial sites are characterised by difficult conditions for the development of plant communities. Nevertheless, the heaps are spontaneously overgrowing and over time, a separate ecosystem can be observed (for heaps). The article analyzes the enzymatic activity of the substrate in relation to the selected dominant grass (Monocots) and herbaceous (Dicots) plant species. The aim of this study was to compare the activity of particular enzymes in soil substratum of the vegetation patches dominated by grass and herbaceous plants.Published versio