During the early stages of clinical drug development, transient transfection methods can serve as a rational alternative to stable cell line derivation for prompt biomolecule production and clinical advancement. However, creating a high-yielding and reproducible transient process can present many challenges due to the lack of understanding crucial aspects of the process. Thus, in this work we aimed to develop a method to comprehensively track the transient expression of a vaccine candidate molecule, Chikungunya virus-like particles (VLP), in suspension HEK293 cells for enhanced process understanding and control. Our analysis method focused on monitoring cell population responses during transfection, understanding how process changes can affect these responses, and determining patterns in cell performance over the culture duration. For enhanced process visualization, plasmid labeling and VLP staining were employed to evaluate cells via flow cytometry and to draw correlations between plasmid DNA uptake and the resulting VLP expression. 

Antibodies were produced using AstraZeneca’s proprietary CHO-based transient expression system at different scales to explore the potential for using transient expression processes to generate clinical-grade material. The presentation will highlight the scalability and productivity of the process up to 200L along with analytical data from independent batches. The benefits and risks of using transient platforms to rapidly generate clinical material will be discussed.

Innovative cell line and early process development are critical to generate high producing cell lines alongside a USP strategy to meet high quality standards. Improving productivity is often done by increasing VCD during USP, however this results in arduous clarification and elevated HCPs. Therefore, Polpharma Biologics developed SPOT™ technology to increase the cell line specific productivity (Qp), which facilitates high volumetric productivity at low VCD accompanied with a simple and efficient DSP process.

Cell-free protein expression is emerging as a viable alternate to traditional in vivo expression of proteins. The field is rapidly advancing to reduce barriers to entry for new users, improve yields, cost, and throughput. This presentation will outline some of these advances, as well as limitations that new users should be aware of.

Different biopharmaceuticals require different glycosylation to unfold their optimal pharmaceutical activity. Optimization can occur with regard to e.g. serum half life, activity, cellular uptake or immunogenicity. With our GlycoExpress^® (GEX^®) toolbox we have established a set of glycoengineered cell lines for the high yield production of biopharmaceuticals. We will provide case studies on how this technology adds benefit to different glycoprotein projects.

Site-specific integration has emerged as a promising strategy for precise CHO cell line engineering and predictable cell line development. CRISPR/Cas9 with the homology-directed repair pathway enables precise integration of transgenes into target genomic sites, but the low targeting efficiency hinders multiplexed gene knock-ins, as well as high-throughput screening. Here, I will present a simple optimized strategy that allows efficient CRISPR/Cas9-mediated knock-in of transgenes in a transient setup in CHO cells.

We present a high-throughput (HT) peptide mapping workflow, which can be applied at early stages of cell line selection, for testing of dozens of clones to report critical information, such as sequence variants and post-translational modifications, and enable biopharmaceutical development (CF Xu, Adv Exp Med Biol. 2019, 1140:225-236). We will also demonstrate the use of native mass spectrometry to exclude potentially problematic clones containing unprocessed leader sequences.

With most top blockbuster drugs therapeutics being glycoproteins, there is a growing interest in engineering their glycan structures for improved safety, efficacy, and manufacturing. Using our systems biology approaches, we can predict the modifications needed to effectively glycoengineer proteins. We further have explored the more global impact glycoengineering has on the host cell, thus helping to define the design space of CHO produced glycoproteins.

With increasing complexity of cell line selection and development, the need for a fit-for-purpose data management platform is greater than ever. This talk will focus on the key considerations in selecting a modern lab informatics platform and how such a platform can transform cell line development.

Mass spectrometry is a powerful technique in the identification and characterization of biologics. New engineering design of sequences have resulted in the immergence of unexpected product quality attributes. These unexpected modifications or “unusual suspects” are analytically more challenging yet important considerations for cell-line development. In this presentation, we show several case studies, highlighting the techniques used to decipher unusual post-translation modifications, sequence extensions, and isomeric sequence substitutions in biologics.

Development of biologic-producing CHO clones in accelerated timelines is encumbered by demonstrating production stability in candidate cell lines.  Screening out unstable lines earlier in development could mitigate the risk of advancing unstable candidates.  Our work reveals significant phenotypic heterogeneity in clonal populations by characterizing subclones from stable and unstable clones.  This highlights the prevalence of phenotypic drift in clonal cell lines providing a basis for investigating gene expression patterns.