Single Use bioreactors: Geometry does matter

The first generation of single use bioreactors (SUBs) departed from conventional stirred tank bioreactor (STR) geometry in terms of impeller number, and orientation and sparger hole diameter. Moreover, one marked feature of SUB bioreactors was that they could be operated at lower volumes than conventional STRs, bringing considerable operational flexibility. This practice, however, further negates the principle of geometric similarity. Whilst some processes may be able to remain within a SUB for the whole product life cycle, many products will require scale up to larger-scale vessels as demand for the product increases. This poster considers the implications of changing reactor geometry on scale up of mammalian cell culture processes using multivariate data analysis to compare different geometries and different fill volumes. This approach uncovered a surprising result when working at half volume, which may not have been spotted using conventional data analysis methods. The first generation of SUBs challenged two of the industry’s key principles of scale up: geometric similarity and maintenance of KLa. There is now a wider variety of SUBs on the market, including vessels that display a higher degree of geometrical similarity to conventional STR geometry. As a result a study was performed to evaluate similarity of process performance between systems with different geometries in order to support Lonza’s expansion of single use upstream capacity. In this study we have compared performance of two SUB systems; one with a conventional STR geometry (SUB 1) and one with a non‑conventional geometry (SUB 2). Mass transfer studies performed with both systems using the gassing-out approach demonstrated that empirical models built to describe KLa performance in Lonza’s conventional STRs (10 to 20,000 L) were better able to predict KLa’s in SUB 1 than in SUB 2, as would be expected given the geometries. Cell culture evaluations were performed with a model cell line in both SUB systems. Multivariate analysis of the data showed that the behavior of the cultures performed in the SUB 1 was closer to behavior of cultures performed in Lonza’s conventional scale-down model than those performed in SUB 2. However, Hoteling’s T2 and Q residuals analysis suggested that difference in behavior in SUB 2 was not extreme. The impact of operating SUB 1 at half volume was investigated for two different vessel volumes. Multivariate data analysis showed that there was considerable difference in behavior of the cultures performed at half volume when compared to cultures performed in the conventional scale-down model. At several time points towards the end of the cultures, Q residual values were outside the 95% confidence interval, indicating significantly different culture behavior. Furthermore, the analysis indicated that there was also a difference in behavior of the half-volume cultures in different size vessels. This indicated a lack of scalability between half-volume cultures performed in different scale vessels of SUB 1, which was not apparent when the same vessels were run at full volume. It was concluded that SUB geometry does matter when scaling processes up and should be a key consideration in a quality by design approach to minimizing differences in culture behavior during cell culture process scale up. Moreover, multivariate data analysis can provide useful supplemental insight in bioreactor process performance comparison

Scale-up in the single use age: Does geometry matter?

Singe use bioreactors (SUBs) are becoming standard work horses in the biopharmaceutical industry. These SUBs are supplied by vendors as off the shelf designs limiting the cell culture engineer’s ability to match the geometry of the SUB to the geometry of their existing stirred tank reactor (STR) capacity. The first generation of SUBs departed from conventional stirred tank bioreactor (STR) geometry in terms of impeller number, and orientation and sparger hole diameter. Moreover, one marked feature of SUB bioreactors was that they could be operated at lower volumes than conventional STRs, bringing considerable operational flexibility. This practice, however, further negated the principle of geometric similarity. This presentation considers the implications of changing reactor geometry on scale up of mammalian cell culture processes using multivariate data analysis to compare different geometries and different fill volumes. This approach uncovered a surprising result when working at half volume, which may not have been spotted using conventional data analysis methods. The first generation of SUBs challenged two of the industry’s key principles of scale up: geometric similarity and maintenance of KLa. As an early adopter of SUBs Lonza had to overcome these challenges. This was done by following an approach advocated by the SUB manufactures which departs from a conventional scale up strategy. Conditions were found empirically that matched the oxygen mass transfer in a conventional STR as closely as possible. There is now however a wider variety of SUBs on the market, including vessels that display a higher degree of geometrical similarity to conventional STR geometry. As a result a study was performed to evaluate similarity of process performance between systems with different geometries in order to support Lonza’s expansion of single use upstream capacity. In this study we have compared performance of two SUB systems; one with a conventional STR geometry (SUB 1) and one with a non‑conventional geometry (SUB 2). Mass transfer studies were performed with both systems using the gassing-out approach. Results demonstrated that empirical models built to describe KLa performance in Lonza’s conventional STRs (10 to 20,000 L) were better able to predict KLa’s in SUB 1 than in SUB 2, as would be expected given the geometries. Cell culture evaluations were performed with a model cell line in both SUB systems. Multivariate analysis of the data showed that the behavior of the cultures performed in the SUB 1 was closer to behavior of cultures performed in Lonza’s conventional scale-down model than those performed in SUB 2. However, Hoteling’s T2 and Q residuals analysis suggested that difference in behavior in SUB 2 was not extreme. The impact of operating SUB 1 at half volume was investigated for two different vessel volumes. Multivariate data analysis showed that there was considerable difference in behavior of the cultures performed at half volume when compared to cultures performed in the conventional scale-down model. At several time points towards the end of the cultures, Q residual values were outside the 95% confidence interval, indicating significantly different culture behavior. Furthermore, the analysis indicated that there was also a difference in behavior of the half-volume cultures in different size vessels. This indicated a lack of scalability between half-volume cultures performed in different scale vessels of SUB 1, which was not apparent when the same vessels were run at full volume. It was concluded that SUB geometry does matter when scaling processes up and should be a key consideration in a quality by design approach to minimizing differences in culture behavior during cell culture process scale up. Moreover, multivariate data analysis can provide useful supplemental insight in bioreactor process performance comparisons

Proteasome-based selection systems for generation of recombinant CHOK1SV GS- KO™ cell lines with enhanced productivity

Chinese hamster ovary (CHO) cells are widely used industrially for the production of biotherapeutic proteins. In order to generate recombinant CHO cell lines expressing the target biotherapeutic gene(s) of interest, metabolic markers are used to select for those cells that have stably incorporated the gene(s) of interest. Such selection systems work very efficiently but do not directly select cells based upon secreted biotherapeutic recombinant protein productivity characteristics. When such biotherapeutic proteins are synthesised in eukaryotic cells, typically the polypeptide is co-translationally fed into the endoplasmic reticulum where it is folded, and if required, assembled with other polypeptides/domains, as in the case of antibodies. An overload of the capacity of the ER to fold and assemble recombinant proteins can result in upregulation of ER-associated degradation (ERAD) where unfolded or incorrectly folded or assembled material is retro-translocated out of the ER to the proteasome for degradation and recycling of amino acids. We have therefore investigated whether the susceptibility of cells to proteasome inhibitors during cell line construction when a recombinant load is placed upon the cell can be used to select for cells with a greater capacity for producing recombinant biotherapeutic proteins whilst maintaining or enhancing the quality of the material secreted. A number of proteasome inhibitors were therefore investigated, epoxomicin, MG-132 and bortezomib, at different concentrations to identify concentrations that would provide selection but not result in complete cell death. A range of concentrations was then added to CHO cells, along with MSX, during cell pool construction using Lonza’s CHOK1SV GS-KO™ proprietary host cell line to investigate whether this resulted in the generation of cell pools with enhanced productivity characteristics as compared to selection using MSX alone. Using this approach, and a number of different recombinant biotherapeutic molecules, we have found that CHO pools giving enhanced product concentrations can be generated (see Figure 1 for example) and validated this in ambr15 miniature bioreactor experiments. We have thus shown that stable transfectants derived from pools that had been cultured with proteasome inhibitors were more productive than pools generated without proteasome inhibitors. Further, stable transfectants generated using proteasome inhibitors retained their higher productivity characteristics even when the proteasome inhibitors were no longer added at subculture, meaning that proteasome inhibitors are only required in the initial stages of cell line construction. Please click Additional Files below to see the full abstract

The challenges of performing high density perfusion processes in single use bioreactors

Single use technologies (SUT) have become embedded in the biopharmaceutical industry over the last 20 years. An important recent trend in the industry is a pivot towards continuous processing. SUT are being deployed for continuous perfusion cultures where expected viable cell concentrations are higher than 1 x 108 /mL. Although SUT provides many advantages, there are also some challenges associated with its application to intensified perfusion processes. In particular, the single use bioreactor has to provide enough oxygen to the culture to maintain aerobic respiration. The main challenges of adapting single use systems for perfusion culture are limited power dissipation (P/V) and limited gas flow rates. These problems arise because plastic is a weak material of construction limiting torque on the simpler shaft and back pressure on the gas outlet filters. These problems are compounded at larger scales due to unfavourable cross-sectional area to volume ratios. Lonza have characterised a number of single use bioreactor systems for their ability to achieve sufficient mass transfer to support perfusion culture. In this poster we will present results from one such system. Oxygen mass transfer was characterised for different sparger and impeller configurations over a range of power dissipations and superficial gas velocities at various levels of oxygen enrichment. A DoE approach was used to characterise the design space for oxygen mass transfer in two different bioreactor scales. Response surface models were used to quantify the contributions of different factors on the overall oxygen mass transfer. Whilst the response of oxygen mass transfer was broadly in line with expectations certain interesting observations were made. For example, an interaction between impeller type and power dissipation was observed. This caused us to look more closely at the impact of impeller design on the distribution of the gas phase. The experiments we have carried out so far enabled us to design a new approach to manipulate the oxygen transfer rate via multiple spargers. It was concluded that the single use bioreactor system under evaluation was capable of supporting between 0.4 and 1 x 108 viable cells/mL depending on the cell specific oxygen consumption rate

Challenges of mass transfer for perfusion cultures in single use bioreactors part 1:Oxygen

Please click Additional Files below to see the full abstract

Controlling pCO2 in high density perfusion cultures

Please click Additional Files below to see the full abstract

Inhibition of protein degradation for improved production

Disclosed herein are methods and compositions useful for evaluating, selecting, identifying, or making a cell or cell line that has improved production capacity for generating higher yields of products and/or improved capacity to produce higher quality products. Products, as described herein, can include a polypeptide that is endogenously expressed by the cell, a recombinant polypeptide that is not endogenously expressed, or a non-naturally occurring recombinant polypeptide. The methods described herein include modulating, e.g., inhibiting, the protein degradation pathway by using a proteasome inhibitor, an ER-associated de-gradation (ERAD) inhibitor, or a ubiquitin pathway inhibitor

Cell Pool Selection of CHO Host and Recombinant Cell Pools by Inhibition of the Proteasome Results in Enhanced Product Yields and Cell Specific Productivity

Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc‐fusion protein produced in CHO cells

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins

Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production

Please click Additional Files below to see the full abstract