Evaluation of cell culture with a simulated continuous manufacturing (sCM) process in 50mL tubespins for clone selection

Continuous Manufacturing (CM) is a process where perfusion cell culture for \u3e30 days is performed with a pre-defined constant biomass set point achieved by bleeding extra cells from the bioreactor (BR). Requirements for cell lines cultured in CM include: (1) good growth to achieve biomass set-point and maintain viability \u3e90%; (2) constant cell-specific productivity as function of culture time (and consequently, volumetric productivity); and (3) constant product quality as function of culture time. In comparison to traditional batch or fed-batch cultures, early screening of numerous clones for a CM process may need to include further evaluation of these three additional attributes to better choose the top performing clones in a CM-like culture. With this purpose, we evaluated a small-scale simulated CM process (sCM) in 50mL Tubespins to screen up to 20 different clones simultaneously. This sCM small-scale model mimics a BR CM process with a simulated perfusion via daily medium exchange. Additionally, sCM can match the cell-specific perfusion rate (CSPR) in the CM BR and includes a discrete daily manual bleed to maintain a target cell density. We performed two sets of experiments to determine sCM performance including (1) evaluation of 16 cell lines expressing a model molecule and cultured in both sCM and small-scale fed-batch process, and (2) evaluation of 5 clones in both sCM and 2L CM BR. Our results indicate clone ranking accordingly to product quality is comparable between small-scale fed-batch and sCM, but ranking accordingly to viability and growth could differ between the two formats. Comparing to BR CM results, sCM predicts well daily volumetric productivity and overall growth performance, but final viability is lower in sCM for some clones. Overall product quality trends as function of culture time were similar between BR CM and sCM. In summary, we established a small-scale Tubespin model for CM that could be used as an additional tool during clone screening

Increasing diversity of production cell lines through miniaturization, automation, and high-throughput analytics

The development of a successful biologic therapeutic manufacturing process begins with the creation of a stable clonal cell line. Since attributes of the production cell line will significantly impact upstream and downstream processes, researchers must find ways to generate several candidate lines with diverse properties. However, a wide diversity is difficult to achieve since cultures are commonly selected, maintained, and screened as populations. In these populations, robust sub-populations can overtake the overall culture and reduce diversity. To combat this, sub-populations must be physically separated by splitting or subcloning, and maintained in individual vessels requiring intensive labor and infrastructure. As a result, researchers must balance between either increasing diversity vs. increasing resources need to maintain and screen hundreds of cultures. In order to shift this balance towards greater diversity, we have developed systems that combines miniaturization of culture vessels, targeted use of automation, and single cell analysis to allow for hundreds of cell lines to be isolated, maintained, and analyzed. We demonstrate cell lines can be easily maintained in simple low volume formats with no impact on cells. We show that we can significantly improve and maintain diversity through separation and isolation of hundreds of cultures. Additionally, higher throughputs allows to assess cell line phenotypes of multiple candidate lines early in development. Benefits achieved through this approach did not increase resources or timelines. Moving towards miniaturization combined with single cell analysis will also enable future possibilities for more precise cell engineering and gene editing

Maintaining productivity over extended durations for perfusion processes

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Modulation of half antibody and aggregate formation in a CHO cell line expressing a bispecific antibody

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two heavy chains that fold and correctly pair via a diversity of engineered interchain interactions (e.g., orthogonal interface, domain crossover, charged mutations, sterically complementary mutations [1-3]). As a consequence of these complex mechanisms that mediate chain assembly, product-related impurities (e.g., half antibodies, homodimers, mispaired light chain species) can be prevalent when expressing bispecific antibodies in cell culture, requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable CHO cell line X expressing a bispecific antibody formed by light chains LC1 and LC2 and heavy chains HC1 and HC2. In particular, this cell line responded to cell culture temperature by decreasing half antibody formation from ~14% to less than 3% when temperature changed from 36°C to 32.5°C. However, the decrease in half antibody also correlated with increased protein aggregates from ~4% to ~10%. We established that half antibody and aggregate formation correlated to intracellular events and not to extracellular degradation mechanism (studies included Western blots of cell lysates and extended supernatant incubation). Analytical characterization showed that protein A-purified pools from cell line X cultured at lower temperatures were enriched in LC1-contaning species, whereas pools from cultures at 36°C were enriched in LC2-containing species. When comparing the LC1 to LC2 ratio in antibodies secreted by cell line X to the ratio in another different 30 cell lines expressing the same bispecific antibody, it revealed a pattern with half antibody formation only present in ratios lower than one, and with enhanced aggregation in ratios larger than one. These results suggested the imbalance of expressed light chains led to one of the two main impurities being preferentially formed. Further studies for cell line X showed that cell culture temperature also modulated mRNA levels of the four expressed chains, which possibly led to misassembled species that contributed to either increased half antibody levels at 36°C (enriched with LC2-containing species), or increased aggregate formation at 32.5°C (enriched with LC1-containing species). Overall, we identified culture conditions that could alter the overall process yield by adjusting impurity amounts and types and consequently, the impurity separation in subsequent purification steps such as cation exchange chromatography. References Lewis SM et al. Nature Biotechnology (2014), 32:191 Gunasekaran K et al. The Journal of Biological Chemistry (2010), 25:19637 Merchant AM et al. Nature Biotechnology (1998), 16:67

Engineered transposon for improved cell line development

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Limitations of subcloning as a tool to characterize homogeneity of a cell population

Cloning, or the derivation of a cell line from a single cell is a critical step in the generation of a manufacturing cell line. The expectation is that the process of cloning will result in a uniform and homogeneous cell line that will ensure robust product quality over the lifetime of the product. Regulatory guidelines require the sponsors provide assurance of clonality of the production cell line and when such evidence is not available, additional studies are required to further ensure consistent long-term manufacturing of the product. One approach to characterize homogeneity of a cell line is subclone analysis where clones are generated from the original cell line and an evaluation of their similarity is performed lines. To study the suitability of subclone analysis to provide additional assurance that a production cell line is clonally derived, an antibody producing CHO Master Cell Bank (MCB), which was cloned by a validated FACS method and with a clear documented day 0 image was characterized. Specifically, this MCB was subcloned and imaged to assure each of the subclones were derived from a single cell. A total of 46 subclones were analyzed for growth, productivity, product quality, as well as copy number and integration site analysis. Despite demonstration of clonality for both the MCB and the subclones, significant diversity in cell growth, protein productivity, and product quality attributes was observed between the 46 subclones. The diversity in protein productivity and quality were reproduced across bioreactor scales, suggesting that albeit different, the subclones were stable populations that varied from the parental clonal cell line. Additionally, while ~2-fold shifts in copy number were seen, no significant integration site changes were observed. Our data suggest subcloning induces changes (genetic or epigenetic) outside the region of the transgene which result in the subclones exhibiting a wide diversity in cell growth protein productivity, and product quality. Transcriptomic and genomic characterization studies are underway to further characterize the differences between subclones and the MCB. Importantly, the subclones do keep their individual characteristics as they mature and stabilize, suggesting that the resulting population that grows out of a single cell is stable but with unique properties. Overall, this work adds to the growing body of work on CHO cell plasticity and suggests that subcloning is not an effective approach to demonstrate homogeneity of a cell bank

Designing a highly productive next generation CHO fed batch platform

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Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single-cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub-cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank