The Central Dogma defines the “what” of biology: genes are transcribed into mRNAs that are translated into proteins. But it says nothing about the “when” or “how much” of expressing thousands of genes in cells. Using convergent technologies, we discovered an information-rich scheduling system for gene expression involving the dozens of chemical modifications of RNA in every cell—the epitranscriptome. Stress reprograms the tRNA epitranscriptome to facilitate selective translation of mRNAs critical to cell survival. We are now leveraging this discovery in a variety of applications, including protein manufacturing and cell line engineering.

Genome engineering technologies allow for nearly limitless edits to tailor genomes for research or manufacturing needs. CRISPR-based systems are currently the most popular choice in genome editing and are typically paired with Cas9 nuclease, but results from alternative nucleases support their use in a wide range of model systems. MAD7 is an engineered class 2 type V-A CRISPR-Cas nuclease (Cas12a/Cpf1) shown to function in bacteria, human cells lines, and mice. In this study, we evaluated its use for genetic editing in CHO cells commonly used for the creation of manufacturing cell lines expressing biologic medicines.  Here we show that MAD7 can effectively modify the CHO genome and facilitate a site-specific genomic knock-in through homology directed repair.

Past efforts to find or engineer improved host cells have met with limited success, owing to natural limitations inherent to those cells. Our novel cell-engineering platform is based partly on directed evolution principles. We create engineered cells with diverse and increased gene copy numbers at the chromosomal level by homotypic cell fusions, followed by screening, or by application of selective pressure, to isolate cells with desired phenotypes. This platform has been used to create: CHO cells with Qp 117 pg/cell·day for mAbs; HEK-293 cells with 9-fold higher AAV productivity, and 2-fold higher percent full; CHO cells with 13.5-hour doubling time.

Phosphoinositide 3-kinase (PI3K) is a signal kinase that affects basic cell functions such as growth, metabolism, and motility. Thus, p110a is an ideal therapeutic target for cancer treatment. Previously, Pfizer optimized production of PIK3. We have further optimized production by incorporation of their constructs into our His6-MBP-tev-target expression vector, our Tni-FNL insect line, and buffer optimization achieving a 40-fold increase in yield for p110a/p85 and a 3-fold increase in p110a.

Long known for their ability to enhance protein folding, the use of chaperones is becoming increasingly important as recombinant protein production projects target more complex proteins and complexes. This talk will highlight some of our labs recent work with, and modifications to, the insect cell/baculovirus platform to enhance the production of several of these challenging target proteins.

Site-specific integration (SSI) via recombinase-mediated cassette exchange has shown advantages for expression of biotherapeutics. We developed a dual-site SSI system having two independent integration sites at different genomic loci, each containing a unique landing pad. The system brings a significant improvement in the efficiency of our cell line development process. The dual landing pad architecture also affords a high degree of flexibility for development of complex protein modalities.

Adjuvanted recombinant proteins remain amongst the most reliable and safest approaches to vaccine development. Using the examples of influenza and SARS-CoV-2 pandemic vaccines produced using the baculovirus insect cell expression system (BEVS), challenges in adapting such systems to large-scale vaccine manufacture will be discussed. Particular issues are lack of analytical tests and presence of adventitious viruses. As demonstrated by the successful licensed SpikoGen COVID-19 vaccine, the challenges of the BEVS system can be overcome to produce high quality recombinant protein vaccines at high yield and low cost of goods.

In addition to producing increased levels of the protein of interest, higher productivity cell lines undergo a range of physiological changes in response to the increased productivity. In this presentation, I will discuss proteomic and transcriptomic differences between parental cell lines and DHFR-amplified progeny including changes in ribosomal proteins, energetic pathways, proteosomal activity, and tRNA biosynthetic enzymes. In addition, transcription factor alterations were seen, potentially altering transcription rates, as were changes in methylation of the CMV promoter, demonstrating a comprehensive response to increased productivity.

DNASU Plasmid Repository receives, grows, houses, and distributes clones across the world at-cost. DNASU hosts one of the largest clone collections in the world, representing bacteria, viruses, and humans. DNASU maintains the largest human collections, with 18000 unique ORFs. With continued commitment to the scientific community, DNASU will have a sustainable resource to help expedite efforts toward scientific discovery and advancement for many years to come.

Challenges exist with the Baculovirus expression system including time and effort to generate, screen, and store large numbers of viruses. To address this we have developed a streamlined process to QC new viral constructs by incorporating 1.) off-the-shelf automation platforms 2.) screening miniaturization techniques, and 3.) data management platforms. This workflow accelerates viral generation through an improved screening funnel and reduces the total number of viral samples that need to be managed.

The insect cell lines widely used as hosts in the baculovirus-insect cell system are contaminated with adventitous viruses. We assessed the infectivity of Sf-rhabdoviruses for mammalian cell lines and immunocompromised mice to determine their impact on the biosafety profile of this biologics manufacturing platform.

There is a current industrial research focus on strategies to engineer the next generation of cell lines with enhanced protein production and tailored performance attributes.  The screening of many random clones that takes place during traditional CHO cell line development offers a wealth of information on the molecular basis of both "good" and "bad" production cell lines.  In this presentation I will review studies using genomics, transcriptomics, and morphological profiling to characterize high performance CHO clones and discuss how these lessons can be applied to cell line engineering and clone selection.

This presentation explores strategies that fast-track the screening process for early candidates, enhancing precision and speed in cell line development. Here we offer key insights into accelerating timelines and optimizing resources, offering a valuable guide for researchers and developers seeking to streamline their bioproduction workflows.

Glycoproteomic analysis of CHO host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings is reported with intracellular and secreted glycoproteins examined. Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, and glycoproteins that are problematic contaminants in recombinant antibody production. Moreover, site-specific glycosylation comparisons of recombinant proteins secreted from CHO and HEK cells are also presented to reveal the importance of cell line choice best suited for a particular bioproduction application.

The work presented here focuses upon the current and emerging technologies available to produce recombinant adeno-associated virus (AAV)-based viral vectors towards treatment of diseases. We will highlight automation enabled serum-free Sanofi’s producer cell lines (PCLs)-based platform for production of gene therapy vectors for delivering safer therapeutics to the patients.