Despite the definition of critical quality attributes early in the development process having long been the standard in the industry, the sudden identification of novel risk factors can lead to a need for major redevelopment at mid- or even late-stage process development. We show how the redevelopment of a production cell line to remove an HCP can result in suddenly having to focus on completely different attributes.

Epigenetic modifications to the nucleotides in RNA species have been generating considerable interest in recent years. The role of methylation in particular, including characterising the proteins that add (writers), remove (erasers), and interpret (readers) this epigenetic mark, will be discussed. This talk will consider the potential of targeted methylation as an enhancer of mRNA translation and how this mechanism might be applied to improving biologics production.

Macromolecular complexes are cornerstones of most, if not all, biological processes in cells. We will illustrate how the CRISPR/Cas9 editing technology can be used for gene tagging in order to introduce affinity tags and facilitate the purification of proteins/macromolecular assemblies expressed in physiological conditions. We will also discuss tagging proteins with fluorescent reporters in view of imaging and functional proteomics applications.

• Current trends
• When to implement in the development process? Is it possible to address and/or initiate this as part of the QbD process?
• Removal of protein from expression cell line vs. introduction of GOI into dedicated knockdown/knockout cell line?
• IP, especially in the context of long development timelines – potential advantage of late filing?​

We present CLD4, a four-step method for autonomous lead clone selection for biopharmaceutical processes. Step 1 digitises and stores data in a structured data lake. Step 2 calculates the cell line manufacturability index (MICL). Step 3 deploys machine learning for process understanding. Step 4 uses NLG to generate automated reports. CLD4 revealed hidden issues in a CHO cell line, showcasing the power of industry 4.0 principles for informed decision-making.

Insect cells are a high-yield and low-cost expression system with mammalian-like post-translational modifications. In contrast to the commonly used baculovirus expression vector system (BEVS), the plasmid-based system enables faster production in high yield and quality, especially of secreted proteins and Virus-Like Particles (VLP). We here present the production of different proteins as well as of Hanta, influenza, entero-, and noro-VLPs, and their applications for antibody development.

Cell-line development typically starts with pool generation, and is followed by single-cell cloning, starting with thousands or hundreds of clones and several rounds of ranking and selection, until the final clone can be nominated. Selection and ranking is usually based on cell growth, productivity, monoclonality, stability, and product quality attributes. The latter is gaining more and more importance—highlighting the need to look at product quality attributes as early as possible.

Protein production using recombinant DNA technology is time-consuming and labor-intensive. We have established a protein production platform based on the cell-free technologies that can produce milligram quantities of proteins totally without the use of recombinant DNA technology. By using this platform, time-consuming and labor-intensive protein expression/purification processes are dramatically accelerated, especially when combined with automated equipment. This platform is therefore very useful for upstream processing.

Viral vaccine antigens produced in mammalian cells often hide from the immune system using glycan shields. S2 cell-produced antigens can reduce glycan shielding due to paucimannosidic glycosylation. Conversely, glyco-engineering S2 cells enhances vaccine effectiveness. Suitable for large-scale production, S2-produced antigens have reached Phase III clinical trials. Utilizing image-based confirmation of monoclonality and micro-scale cultivation accelerates monoclonal S2 cell-line generation, improving efficiency and throughput in cell-line development.

Immunoglobulin G (IgG) glycans regulate inflammation and as such participate in both disease and aging. To unravel the unknown mechanisms responsible for changes in IgG glycome composition, we utilize a dual approach: large-scale genome-wide association studies (GWAS) to identify genes that associate with IgG glycosylation, and CRISPR/dCas9-based in vitro systems for gene validation. We have discovered that the regulatory networks of IgG glycosylation extend far beyond the known glycosylation-related genes.

Stable cell lines are widely considered to be the stepping stone to simplify manufacturing processes and further increase yields. In this presentation you will be given an overview of the importance of lentiviral vectors for cell and gene therapy and presentation of Orchard’s stable cell-line development platform. Details on steps to an optimised CLD workflow and an overview of GMP requirements when manufacturing LVV with stable cell lines.

In this presentation we will discuss key aspects related to rAAV production systems' scalability plus current challenges and potential directions for product characterisation. Our presentation focus lies in improving the scalable HeLaS3-based production system. Efforts involve optimising the cell line generation process and improving the cell host; relevant genes for rAAV production, identified through CRISPR-Cas9 genetic screens, will be engineered to create a cell host with enhanced production capabilities.