Engineering, expression screening, and production cell line development of hetero Ig molecules using charge pair mutations

In recent years, there has been an increase in therapeutic indications that require bispecific targeting. Bispecific Hetero Ig antibodies that can target two antigens have long been considered as an attractive approach to drive synergistic biologic activity while maintaining the structure and stability of a traditional antibody. However, clinical development of such molecules has been hampered by CMC related challenges relating to product heterogeneity. During the development of a Hetero Ig molecule targeting the Wnt pathway antagonists Dkk-1 and SCL-1, we employed a novel strategy to drive the heterodimerization of IgG antibodies through the addition of charge pair reside mutations (CPM) at both the heavy chain and light chain surface interface. Through electrostatic interactions, these CPMs drive appropriate chain pairing, while minimizing undesired side products. During engineering and expression in transient expression systems, we identified combinations of single residue pair mutations that promoted correct chain pairing. However, the combination of antibody pairs and expression balance is important to enable reduction in undesired side products. These findings extend to stable cell line development, where vector design and appropriate analytics enable the identification of pools and then clones with desired product quality. We have expanded this strategy for the development of a platform approach toward the efficient development of HeteroIg molecules

A mathematical modeling framework for determining the probability of obtaining a clonally-derived mammalian cell line

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Fast predictive expression platform – CHO-K1 with transposase

The Mammalian Expression group in Therapeutics Discovery Research produces panels of therapeutic candidates in high-throughput (HT) for early stage screening in addition to generating larger productions that are used for manufacturability assessment, pre-clinical PK, and efficacy studies. As our molecule types expand from monoclonal antibodies to include various bispecific modalities, the ability to predict how a molecule will behave in our stable manufacturing host becomes more difficult. To more quickly predict characteristics and manufacturability of our candidate therapeutics, we are developing in-house stable expression systems. The transposase is a mobile genetic element that efficiently transposes between vectors and chromosomes via a “cut and paste” mechanism. Because the transposase facilitates non-random, efficient genetic integration, we investigated the possible incorporation of this technology into our current in-house transient expression vector system. We have observed that its stable-like integration properties provide us with a foundation for enabling a short production time, comparable with a transient expression system, while generating proteins with attributes that are predictive of our manufacturing system. This approach has been validated and implemented with our CHO-K1 stable expression system. With this technology, we are able to reliably generate the diverse array of therapeutic candidate modalities in a high-throughput format while achieving higher predictability of material derived from our manufacturing hosts

Enabling next-generation cell line development using continuous perfusion and nanofluidic technologies

The manufacturing process for a biologic begins with establishing a clonally derived, stable production cell line. Generating a highly productive cell line is time and resource intensive and involves screening of a large number of candidates. While miniaturization and automation strategies can reduce resources and increase throughput, they have matured and recent advances have been incremental. With increasing pressure on time to commercialization and the increasing diversity and complexity of therapies in discovery research, there is a need to transform cell line development to accelerate patient access to novel therapies and nanofluidic technology are on potential solution. In this study, we present cell line development data on the Berkeley Lights integrated platform. Cells are manipulated at a single cell level though use of OptoElectronic Positioning (OEP) technology which utilizes projected light patterns to activate photoconductors that gently moves cells. Common cell culture tasks can be programmed though software allowing thousands of cell lines to cultured simultaneously. Cultures can be interrogated for productivity and growth characteristics while on the chip at ~100-fold miniaturization and continuous perfusion of cell culture medium enables effective and robust cell growth and product concentration despite starting from a single cell. Concepts from perfusion culture are also applied to measure productivity and product quality. We demonstrate that commercial production CHO cell lines can be cultured in this nanofluidic environment and show that sub clone isolation, recovery, and selection are achieved with high efficiency. Overall, this technology has the potential to transform cell line development workflows through the replacement of laborious manual processes with nanofluidics and automation, and can ultimately enable the rapid selection of high performing cell lines

Rethinking clonality using modeling approaches

A combination of experimental procedures, imaging, and probability estimation are typically used as evidence of clonality for the manufacture of a biotherapeutic product. In situations where the totality of evidence is unavailable, establishing a high statistical probability for monoclonality can help strengthen the argument for clonality. In this study, the probability of clonality was re-examined for the limiting dilution method using a combination of experimental and modeling approaches. A limiting dilution experiment was performed using a 50:50 mixed population of GFP-and RFP-expressing cells and the plates were imaged over a span of two weeks. The imaged cells were scored for clonality and double checked with fluorescence imager. Among all wells that had single colony-like growth on day 14 and a single cell-like image on day 0, a fraction of the wells were confirmed to have two colors on day 14 by fluorescence imaging, indicating the singe cell-like day 0 images for these wells were false reads. Considering the possibility of having 2 or more cells with the same color in a particular well, we estimated the worst case total possible number of wells with 2 or more cells on day 0. Moreover, assuming a Poisson distribution for limiting dilution, the recovery rate of any single cell that grew into a visible colony by day 14 was estimated. Our modeling analysis indicated that only a fraction of the wells with \u3e2 cells on day 0 could grow into non-monoclonal colonies. If cells from any of the wells with single colony-like growth on day 14 and single cell-like image on day 0 were chosen as the final clone, the probability of monoclonality was estimated to be \u3e 95% with a 95% upper confidence limit

Interrogating cell culture populations for the selection of production cell lines using microfluidic culturing, single cell analysis, and predictive modelling

Cell line development for manufacturing is a lengthy, multi-step, resource intensive, critical path activity. Attempts to perform in silico modelling and prediction of cell culture has been difficult due to complexities around heterogeneous cell culture populations that rapidly shift over generations under changing selective conditions. For example, early populations will often change as response to media and culturing conditions from a static colony culturing in microtiter plates, to small scale suspension culturing, and finally in a controlled bioreactor processes. As a result, it is challenging to make the final cell line selection early, while predicting future bioprocess performance, and ultimately estimate the protein product quality. We address this challenge by drastically increasing the amount of early cell culture population data obtained through use of emerging single cell technologies. Data obtained is combined with modelling approaches to select the best cell lines upfront to reduce timelines and processing steps. To achieve this, we have implemented a platform from Berkeley Lights that effectively digitalizes most aspects of cell culture. Thousands of individual cell lines can be manipulated, cultured and interrogated on a perfusion nanofluidic chip resulting in extensive data on cell behavior on an individual cell level as well as the populations. Through multivariate predictive modeling of this data, we can predict the performance of candidate clonal cell lines in larger scale production runs. Incorporation of additional single cell analysis such as digital droplet RT-PCR and next generation sequencing further predicts product quality, such as heterogeneity of bispecifics and sequence variant detection. Similar approaches can further be used to then study the stability and integrity of a final CHO cell banks. When combined, single cell interrogation of early culture populations allow for the dematerialization of the CLD process, make better predictions of bioprocess performance, and reduce select the final production clone earlier

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

Engineered transposon for improved cell line development

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Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

Tools and methods for providing assurance of clonality for legacy cell lines

Over the last several years demonstration of cell line clonality has been a topic of many industry and regulatory presentations and papers. Guidance has been provided by the regulatory authorities, especially the FDA, on a path forward for providing evidence of clonality with high probability. It has been recommended that two-rounds of limiting dilution cloning (LDC) at sufficiently low seeding densities (≤0.5 cells/well) provides sufficient evidence that a cell line is clonal. Furthermore, one-round of LDC may also suffice if supplemental data from a characterized FACS or plate-imaging workflow are also included in the package. Cell lines generated by methods that do not demonstrate high probability of clonal derivation, including legacy cell lines, may require additional studies to provide assurance and/or process control strategies to satisfy regulatory expectations. Within the Biologics function of the IQ Consortium the “Clonality” Working Group is focusing on methods and tools which could be utilized to provide a high assurance of clonality for legacy cell lines. The presentation will outline a three tier approach to address legacy cell line clonality assurance: standard practices already used in industry to support limit of in vitro cell age studies, enhanced control strategies to ensure process consistency, and emerging technologies that could be used to further support cell line clonality