Adoption of perfusion in N-1 bioreactors yields a reduction in process time and/or increase in manufacturing capacity without increasing volume capacity. Due to recent advances in cell retention technologies, cell line and media development, perfusion becomes a more viable alternative to expand performance beyond what traditional platforms can attain.  This presentation explores the benefits of a pre-sterilized, high-throughput perfusion filtration systems in an N-1 perfusion application and demonstrates scalability from 3 L to 50 L.

Using our high-throughput cell-line engineering platform, we have engineered a panel of CHO cells able to produce proteins with specifically designed glycoprofiles. We have used these cells to produce therapeutic proteins that have previously not been possible to produce in CHO cells. This approach can be used to produce therapeutic proteins with better biological properties, such as increased half-life, improved activity, etc.

To enable large-scale recombinant antibody production, a high-producer cell line is essential. Selecting such a cell line is, however, time-consuming and labor-intensive. By combining the design of a tricistronic vector expressing GFP and both antibody chains, separated by a GT2A sequence, with single-cell sorting and automated image analysis, a CHO cell line was rapidly selected producing high amounts of recombinant antibodies, which showed minimal degradation.

Cell Counting is often overlooked, but is essential for reproducibility in experiments, assays and manufacturing processes. Cell counting is a challenging technique, with many pitfalls, that can delay entire projects. The NucleoCounter NC-202, solve all of these challenges, and standardize cell counting across organizations.

Epigenetic modifications to the nucleotides in RNA species have generated considerable interest recently. The role of methylation in particular, including characterising the proteins that add (writers), remove (erasers), and interpret (readers) this epigenetic mark, is considered. This talk will consider its potential as an enhancer of mRNA translation and how this mechanism might be applied to improving recombinant protein expression systems.

Although technologies for production of complexes have been tremendously improved, sample preparation is still a major bottleneck for structural biology and functional analysis both in academia and industry. Using the 10 subunits transcription/DNA repair factor TFIIH, I will illustrate reconstitution of multisubunit complexes using the baculovirus expression system and characterization of endogenous assemblies from CRISPR/Cas9-edited cell lines.

Bio-pharma development is shifting rapidly from classical IgG molecules to more challenging complex biopharmaceuticals like bispecifics or non-IgG molecules. With CHO being a good production cell line for IgG molecules, they might fail to produce more challenging candidates. The GlycoExpress® (GEX®) system represents an ideal alternative for the production these difficult to express protein molecules.

By taking advantage of CRISPR-Cas9 genome editing, we have developed a novel mammalian cell system for the expression of full-length antibodies. The Plug-and-(Dis)play (PnP) workflow included the initial generation of a platform cell line in which a novel specificity is introduced by means of a synthetic antibody. Subsequently, we optimized HDR efficiency to render the system amenable to antibody discovery and engineering; specifically, we were able to display libraries for the discovery of antibodies from immunized animals, and to affinity mature a known antibody by screening a library of random mutagenesis variants.

In many cases, the cell-line development (CLD) process is on the critical path of projects towards IND filings. In order to shorten the time required to start clinical trials, there is a strong interest in speeding up the duration of the CLD process. Ichnos Sciences has evaluated the implementation of a single-cell deposition device in the CLD process. This presentation will provide a critical review of this technology with regards to the desired shortening of timelines, as well as clonal derivation (“monoclonality”), productivity, product quality, and expression stability of the resulting cell populations.

An increasing number of new CLD labs are being established around the World. A major challenge is how they decide for purchasing the right platform which must ensure rapid implementation, cost-effectiveness, regulatory assurance, and deliver on the corporate cell line project commitments. We provide solutions in this presentation.

Stable cell-line generation is a time-consuming and labour-intensive process. We report the impact of implementing systems such as a Verified In-situ Plate Seeding system (VIPS™) linked with the Cell Metric imager. This resulted in an increase in throughput, reduced timelines, and provided clonality assurance for our stable cell-line processes.

Vaccines based on virus-like particles (VLPs) have gained interest over the last years due to their high immunogenic capacity, the advantages they present compared to traditional vaccines, and their versatility when combining different antigens. Cell specific perfusion rate (CSPR), concentration of DNA and PEI, and time of multiple transfections were optimized using design of experiments in a HEK 293 suspension culture perfusion process. The VLPs obtained were quantified and characterized using a new SRFM-based method in crude supernatants.

In order to increase the bioavailability of coagulation Factor VII, the N-glycosylation machinery of the human embryonic kidney, FreeStyle 293-F (HEK 293 F), cell line was engineered using CRISPR/Cas9. N-acetylgalactosamine transferase knock-outs led to the replacement of N-acetylgalactosamine with galactose and increased sialylation. Knock-in of the sialic acid transferase, ST6Gal1, further increased sialylation of Factor VII and greatly reduced glycosylation-related heterogeneity of Factor VII molecules. Factor VII produced in the engineered cell lines showed reduced asialoglycoprotein receptor binding in vitro and increased bioavailability in vivo.