Antibodies missing core fucose show enhanced ADCC effector function and anti-tumor activity. Expression of an anti-alpha-(1,6)-fucosyltransferase (FUT8) intrabody engineered to reside in the cell can efficiently reduce FUT8 activity and therefore the core-fucosylation of an antibody. Cell engineering to inhibit directly and specifically FUT8 activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected.

In this presentation, we will introduce the efforts at REGENXBIO to adapt our HEK293 host cell lines from adherent to suspension and further improve AAV productivity of cell lines by multiple cloning efforts. Furthermore, to improve AAV titers, we made a sequential modification of our helper plasmid. By combining the new cell lines and new helper plasmids, we increased our overall transient yield >20-fold.

There is an ever-growing need to make the CLD process more efficient in order to keep up with the demand for better therapies. I will present the benefits of the UP.SIGHT, CYTENA’s new single-cell cloning and plate imaging instrument that achieves a probability of clonality >99.99% using 3D Full Well Imagining. I will also introduce a new, automated work station for screening hundreds of clones without user interaction. 

N-linked glycosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs). There exists naturally occurring heterogeneity in mAb glycosylation produced by Chinese hamster ovary cell cultures. Here, I will describe Glycosylation Flux Analysis, a model-driven data analysis for estimating intracellular glycosylation fluxes, and its application to fed-batch and perfusion CHO cell culture production of immunoglobulin-G. Insights into the controlling factors of N-glycosylation from the analysis will also be discussed.

Biologics production has a need for faster development cycles and intensified processes. Running multiple microbioreactors in parallel enables expedited optimization, and assays are required to achieve desired critical quality attributes and high productivity. While automated microbioreactors have enabled better scale-down models for bioprocess, low microbioreactor volumes limit the media available for daily extractions for metabolite and nutrient analysis. In addition, CQAs such as glycosylation and protein charge variants analysis can be time-consuming and require specialized skill set. We discuss a data-driven and accelerated process optimization approach leveraging tandem automated at-line microfluidic capillary electrophoresis mass spectrometry (CE-MS) analyzer

We developed a novel cell line selection technology, PTSelect, that we used to rapidly generate clones producing the monoclonal antibody, adalimumab. In addition to evaluating productivity and stability, we characterized the glycan profiles of the antibodies produced. Lower productivity clones had glycan profiles that more closely resembled the innovator drug. Medium and bioprocess variations were applied to improve the glycosylation profiles in higher productivity clones.

Root cause analysis for out-of-trend processes can be a challenge when there are confounding factors, multiparameter interactions, and missing data. In silico cell culture modeling by first principles is a promising methodology to identify root causes and reduce the evaluation time. Here, we present an example using digital twin modeling to investigate variation in metabolism and glycosylation across manufacturing sites, elucidate potential causes, and propose corrective actions.

Here, we describe a novel rapid HTP transposon-based CHOZN CLD platform that produces high-titer stable pools and clones with desired product quality attributes. This new platform enables us to: (1) design and screen as many vectors and vector combinations as necessary for selecting the lead cell lines with high productivity and quality; (2) shorten expression timelines to 7 to 10 weeks (1 month shorter than conventional electroporation process).

Using stable CHO pools, high expression levels up to 1 g/L of SARS-CoV-2 trimeric Spike Proteins can be obtained in fed-batch culture within 6 weeks post-transfection. The scalability and stability of stable pools expressing variants of concern (VOCs) have been assessed in 1L bioreactors. Our data also demonstrate process reproducibility, robustness, and versatility.

Current methods for stably expressing biotherapeutics in CHO cells often rely on random or semi-random integration events which result in widely heterogeneous cell populations. In this study, we developed a targeted integration system that expresses recombinant proteins using transposon-mediated landing pads. By targeting predefined genomic locations that support high expression of exogenous proteins, several cell lines expressing different biotherapeutics can be established with a high degree of specificity and reproducibility.

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.

Mammalian protein expression often relies on a limited collection of natural promoters, which offer discrete expression levels and could be challenging to predict their yields in distinct cell types. Additionally, conventional strategies using transient expression or random genome integration with gene(s) of interest, followed by high-throughput clonal screening and expansion, are laborious and time-consuming. To address those issues, we have developed versatile and scalable synthetic biology toolkits to transform mammalian protein expression and cell engineering. Our robust platform technologies can be adapted to a variety of mammalian cell types and biomanufacturing settings to optimize complex therapeutic protein productions or cell behaviors.

Microbial contamination is one of the most universal challenges in the R&D and manufacturing of cell culture-based biologics such as cell and gene therapy, and monoclonal antibodies. This costs a substantial amount of time, efforts and  exorbitant amount of money. Researchers have estimated that mycoplasma had the potential to affect hundreds of millions of dollars of NIH-funded research (Tanabe et. al., 2020). The common methods to overcome microbial contamination such as radiation, elevated heating, the addition of antibiotics in cell media, and manual cleaning with antimicrobial agents all come with their challenges and are not a guaranteed way of preventing contamination.

This is where Caron’s owned antimicrobial technology based on the hydrogen peroxide (H₂O₂) module can be a game changer. This patented technology covers several unique features, including rapid cycle management, a highly repeatable sensor-driven process, and a compact & effective sterilant catalyst system.  Our research has shown a 12-log reduction in 2 hours at 37 degrees C resulting in water and oxygen as the end products avoiding any need for cleanup post the sterilization cycle. As a result, Caron’s patented H₂O₂ technology has been an integral part of cell culture labs in the world’s most reputed biotech and biopharma companies.

Cas-CLOVER is a proprietary dimeric nuclease system comprising restriction endonuclease, Clo051. Using this enzyme for genome cutting makes it fully dimeric, giving it precise site-specificity. However, there is an unmet need to improve the efficiency of the Cas-CLOVER system, and reduce its cellular toxicity, which are discussed in this talk. Case studies in suspension CHO cells targeting key genes for yield selection and enhanced ADCC will be covered.  

Achieving a high-yielding process requires a combination of robust technologies and a masterful process design. In this presentation we are proud to present Cell Insights by Umetrics® Studio, the first self-service analytics application that combines data-driven information and mechanistic knowledge to capture the complexity of cell growth, metabolism, and productivity in-silico.

The potential for mRNA-based products to treat previously untreatable diseases has created significant interest in how these products can be manufactured consistently and scalably, and the feasibility for a platform-based approach for manufacture of different mRNA-LNP products. This talk will discuss the different strategies being taken for development of manufacturing platforms for mRNA-based products and associated challenges, including data and reflections based on our own work at CPI.

T cell engagers are protein therapeutics that tether T cells to surface antigens on tumor cells, leading to activation of those T cells and destruction of the tumor. The TriTAC (Tri-specific T cell Activating Construct) technology was designed to optimize therapeutic window by addressing half-life and stability limitations of pioneering bispecific T cell engagers. This presentation will highlight process development strategies influencing TriTAC product quality.

The Genomic Medicine Unit (GMU) CMC group at Sanofi is dedicated to the establishment of best-in-class manufacturing platforms to support development of life-changing advanced cell and gene therapy products. This presentation will touch upon the technologies available to produce recombinant adeno-associated virus (AAV)-based viral vectors towards treatment of various diseases. With transient transfection mainly being used with HEK-293 cells, the scientific field is advancing the options of generating a stable cell line known platform using HeLa cells. The talk will feature upon Sanofi’s PCL-based platform for production of gene therapy vectors.

Adenovirus-associated virus (AAV) production could pose specific requirements for nutrients or function compounds to meet the need of viral production in host cells. In this study, we use RNA-Seq-based study and spent medium analysis to drive understanding of pathways involved in AAV production. We conducted experiments to optimize culture medium with the supplements chosen to potentially affect AAV production metabolisms.

By using a de novo serum-free adaptation and single-cell cloning method, we successfully developed a HEK293 suspension cell line X-RAP for rAAV production. Compared with other commonly used HEK293 suspension cells, it demonstrates no cell aggregation and robust cell growth. By using half of the nutrients of the Thermo VPC’s need in the bioreactor, X-RAP showed 1.8-fold higher genome titer and 4.5-fold higher packaging efficiency in purified rAAV.