CHO cell line selection is a painful bottleneck in biotherapeutic development, particularly for complex molecules like bispecifics. The Opto™ CLD workflow on the Beacon® system accelerates early CLD by integrating high throughput cell sorting, cloning, culture, productivity, growth, and product quality assays into a single, 5-day automated process. Hear about capabilities of on-chip detection that pinpoints best clones early on.  

Genetic vector design underpins both optimal synthesis of an encoded recombinant protein and engineering of the host cell to improve functional performance. Engineering context is paramount.  However, biopharmaceutical proteins made by CHO cells are structurally variable and accordingly require a combination of dynamic cellular processes attuned to their idiosyncratic requirements. To achieve product-specific cell engineering we need new tools and strategies that enable us to design and precisely control the relative rate of synthetic processes encoded on genetic vectors that directly impact product manufacturability.

Understanding of oxidative stress is key to support future development of control strategies. In literature, oxidative stress is defined as the imbalance between oxidant molecules generation and antioxidant defense within the cells. Under bioprocess conditions, reactive oxygen species can be generated by multiple sources. By playing on media composition we modulated oxidative stress to improve product quality and process performance.

Process intensification is broadly discussed to increase biopharmaceutical production output. This encompasses classic elements of process development combined with largely increased cell densities, the latter being usually enabled by cell retention techniques of various kinds. We show a case study to apply such an approach to an existing cell line and analyze consequential requirements for optimal cell lines to start right from the beginning in a highly intensified process setting.

Launched only a few years ago, the Leap-In Transposase platform has rapidly become an industry standard technology for the generation of CHO cells for the manufacturing of antibodies and other biologics.  This presentation will highlight achievements and case studies of the platform including high titer mAb manufacturing, rapid anti-COVID responses, and some novel, next generation, applications.  

Macromolecular complexes are cornerstones of most, if not all, biological processes in cells. We will illustrate how the CrispR/Cas9 editing technology allows to label and isolate native protein assemblies from their natural cellular environment and discuss the potential of the baculovirus expression vector system for reconstitution of multi-subunit complexes. As model systems, we will use transcription regulators such pTefb, nuclear receptors or the 10 subunits transcription factor TFIIH.

Biotherapeutics are mainly produced by Chinese hamster ovary (CHO) cells. However, in nature terminally differentiated plasma cells are responsible for highly efficient antibody production in mammals. In this study we performed a comprehensive multi-omics analysis of CHO and plasma cells to identify crucial differentially expressed genes with beneficial impact on CHO cell characteristics such as productivity, growth or viability and rationally optimized our industrial workhorse by leveraging nature as blueprint.

To maximize the productivity of biotherapeutics, we developed a novel expression system, which allows for the simultaneous integration of numerous independent selective markers. Combining a newly created multi-auxotrophic mutant of CHO-K1 host lineage and transposon-based vector system, a single-step transfection with 8 transgenes resulted in a high frequency of transfectant clones bearing high copy numbers of incorporated vectors and that are capable of pronounced productivity.

The National Biologics Facility has delivered state-of-the-art cell line engineering work since 2012. The obtained knowhow, expertise, infrastructure and optimised CHO cell lines are now being deployed for the delivery of high quality cell line engineering/development and protein production to the benefit of industry and academia. Examples of engineered CHO cell lines resulting in improved product quality, robust bioprocess, predictable gene integration, elimination of clonal variation and tailored N-glycans will be presented.

Application of LC-MS based quantitative proteomics is described for investigating bioprocess behaviour. In the first study, we used proteomics to determine process response to altered conditions, combined with in depth characterisation of the expressed monoclonal antibody to explore product process interplay. In the second study, proteomics was applied to study cells pre and post transfection for AAV viral vector generation followed by functional validation targeting identified pathways to increase yield.

During the live cycle of a biopharmaceutical project the production needs to stay up to date with productivity and quality demands from early pre-clinical to market phase. Optimization can be done at different stages and on different levels with selecting the right cell line, selecting the right clone or optimizing the media/feed combination and / or optimizing process parameters. We provide case studies which address the different possibilities of optimization.

Nature has provided us with mammalian cells that can (and are used) as factories for production of the desirable biological therapeutics. However, yield and capacity to make specific products can be limited by the properties of different cell types. Nature also tells us (although we may not fully understand it) that specific cellular properties will enable us to generate better cell factories, properties that can be engineered into existing cells.

CHO has proven capable to produce a vast array of biological products at high yield and quality during the last couple of decades. Here we report on our attempts to use systems biology to learn from the cases where the secretional, translational and metabolic burden challenges the normal capacity of these cells. We further show how challenging proteins could be rescued in HEK293 cells and our hypothesis around why. Further, we exemplify how OMICS can be used to define limiting factors improving the activity of therapeutic enzymes over 100-fold.

Site Specific Integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for standard mAbs.  However, complex multi-specific modalities can prove challenging for SSI expression systems.  Here we illustrate how an SSI system can be implemented for the production of a bi-specific molecule, and present work towards vector engineering for production of multi-chain, multi-specific molecules.

ApolloX™ is a cell line development platform, incorporating next-generation screening technology, enhanced media selection, and technical expertise. The technology supports aggressive cell line development timelines, whilst ensuring robust and high quality cell lines are obtained. Also described here is a ‘manufacturability assessment’ strategy, which is performed both in-silico using a proprietary bioinformatics tool (Orbit™) together with material generated from transfectant pools, and used to evaluate platform-fit for multiple candidate molecules.

The impact of cell culture media and feeds on the performance of CHO cells has been well established with many groups going so far as to develop specific in-house media for each project/clone. As this approach is generally incompatible with today’s highly compacted timelines in development, we compared the performance of commercially available media and feeds both before and after selection of production clones.

With over 300 candidates in clinical trials and preclinical development, bispecific antibodies are bound to make a difference to human health, and are invaluable drugs due to their unique modes of action. Their intense engineering resulted in a plethora of different formats. Especially in asymmetrical multispecifics, the issue of chain mispairing requires novel solutions; some of them, based on Fab constant domain-exchanged antibody and chemically composed trispecifics, will be presented.

Cell-free protein synthesis (CFPS) is a dynamic field that facilitates rapid protein expression enabling high-throughput screening prior to large scale cell-based expression. The vast majority of protein-based biotherapeutics such as monoclonal antibodies are predominantly produced in mammalian cell-based expression systems, primarily Chinese hamster ovary (CHO). A key feature of CHO cells is the ability to perform human-like glycosylation. This presentation will focus on evaluating the performance of an in-house CHO CFPS for the production of different monoclonal antibody targets whilst investigating their glycosylation pattern. Finally, a novel modular strategy to modify the glycosylation pattern will also be presented.