Cell Line Development Platform Research Articles

A deep-well plate enabled automated high-throughput cell line development platform.

Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource-demanding process. Here we have developed a deep-well plate (DWP) enabled high throughput (DEHT) CLD platform using 24-well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed-batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well-to-well and plate-to-plate variability and the absence of well-to-well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Assuring Clonality on the Beacon Digital Cell Line Development Platform.

During biomanufacturing cell lines development, the generation and screening for single-cell derived subclones using methods that enable assurance of clonal derivation can be resource- and time-intensive. High-throughput miniaturization, automation, and analytic strategies are often employed to reduce such bottlenecks. The Beacon platform from Berkeley Lights offers a strategy to eliminate these limitations through culturing, manipulating, and characterizing cells on custom nanofluidic chips via software-controlled operations. However, explicit demonstration of this technology to provide high assurance of a single cell progenitor has not been reported. Here, a methodology that utilizes the Beacon instrument to ensure high levels of clonality is described. It is demonstrated that the Beacon platform can efficiently generate production cell lines with a superior clonality data package, detailed tracking, and minimal resources. A stringent in-process quality control strategy is established to enable rapid verification of clonal origin, and the workflow is validated using representative Chinese hamster ovary-derived cell lines stably expressing either green or red fluorescence protein. Under these conditions, a >99% assurance of clonal origin is achieved, which is comparable to existing imaging-coupled fluorescence-activated cell sorting seeding methods.

A novel mammalian cell line development platform utilizing nanofluidics and optoelectro positioning technology

Generating a highly productive cell line is resource intensive and typically involves long timelines because of the need to screen large numbers of candidates in protein production studies. This has led to miniaturization and automation strategies to allow for reductions in resources and higher throughput. Current approaches rely on the use of standard cell culture vessels and bulky liquid handling equipment. New nanofludic technologies offer novel solutions to surpass these limits, further miniaturizing cell culture volumes (105 times smaller) by growing cells on custom nanofluidic chips. Berkeley Lights’ OptoElectro Positioning technology projects light patterns to activate photoconductors that gently repel cells to manipulate single cells on nanofluidic culturing chips. Using a fully integrated technology platform (Beacon), common cell culture tasks can be programmed through software, allowing maintenance and analysis of thousands of cell lines in parallel on a single chip. Here, we describe the ability to perform key cell line development work on the Beacon platform. We demonstrate that commercial production Chinese hamster ovary cell lines can be isolated, cultured, screened, and exported at high efficiency. We compare this process head to head with a FACS‐enabled microtiter plate‐based workflow and demonstrate generation of comparable clonal cell lines with reduced resources. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1438–1446, 2018

Sustaining an efficient and effective CHO cell line development platform by incorporation of 24-deep well plate screening and multivariate analysis.

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24-deep well plate (24-DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24-DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24-DWP achieved the highest productivity improvement upon scaling-up to shake flasks. Product quality of the protein purified from shake flasks and 24-DWP were also compared. The 24-DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N-glycan species, and increase the high-mannose N-glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled-up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24-DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175-186, 2018.

CHO-DHFR cell line development platform: Application of Clonepix and Automated Mini Bioreactor (AMBR) technologies to meet accelerated timelines

Background The Holy Grail sought by all Bioprocess Cell Line Development (CLD) groups is achieving high yields from easilycultured, robustly-growing cells in timelines measured in weeks rather than months. As the first bottleneck in process development, CLD must first birth its product for upstream and downstream groups to initiate their own reproductive cycles. To facilitate shortened CLD timelines, scientists have turned to new technologies and automation platforms. Emerging high-throughput instrumentation such as Clonepix and Automated MicroBioreactors (AMBR) have been enthusiastically integrated into stable cell line generation platforms; however, application of these methodologies among users is divergent. In this study, we describe method development that is part of an ongoing effort to create a vigorous, adaptable, high-throughput cell line development platform, incorporating in-house VRC-DG44-CHO cells, expression vectors with high-activity regulatory elements, and efficient selection and evaluation methods that result in early generation of optimal-yielding, vigorous, stable cell lines. This study was aimed toward a) evaluating early incorporation of Clonepix technology to maximize transfectant pool heterogeneity, b) identifying Clonepix fluorescence attributes that increase the probability of high-productivity clone isolation, c) evaluating and optimizing AMBR operating conditions to achieve process attributes comparable to and predictive of medium-scale bioreactors, and implementation of these practices for maximal results with shortened timelines.

High performance CHO cell line development platform for enhanced production of recombinant proteins including difficult-to-express proteins

Background In an effort to improve product yield of mammalian cell lines, we have previously demonstrated that our proprietary DNA elements, Selexis Genetic Elements (SGEs), increase the transcription of a given transgene, thus boosting the overall expression of a therapeutic protein drug in mammalian cells [1]. However, there are additional cellular bottlenecks, notably in the molecular machineries of the secretory pathways. Most importantly, mammalian cells have some limitations in their intrinsic capacity to manage high level of protein synthesis as well as folding recombinant proteins. These bottlenecks often lead to increased cellular stress and, therefore, low production rates.

Combination of the 2A/furin technology with an animal component free cell line development platform process

The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.