Search for: All records

ABSTRACT The implementation of site‐specific integration (SSI) systems in Chinese hamster ovary (CHO) cells for the production of monoclonal antibodies (mAbs) can alleviate concerns associated with production instability and reduce cell line development timelines. SSI cell line performance is driven by the interaction between genomic integration location, clonal background, and the transgene expression cassette, requiring optimization of all three parameters to maximize productivity. Systematic comparison of these parameters has been hindered by SSI platforms involving low‐throughput enrichment strategies, such as cell sorting. This study presents a recombinase‐mediated cassette exchange (RMCE)‐capable SSI system that uses only chemical selection to enrich for transgene‐expressing RMCE pools in less than one month. The system was used to compare eight mAb expression cassettes containing two novel genetic regulatory elements, theAzin1CpG island and the Piggybac transposase 5’ terminal repeat, in various orientations to improve the expression of two therapeutic mAbs from two genomic loci. Similar patterns of productivity and mRNA expression were observed across sites and mAbs, and the best performing cassette universally increased mAb productivity by 7‐ to 11‐fold. This flexible system allows for higher‐throughput comparison of expression cassettes from a consistent clonal and transcriptional background to optimize RMCE‐derived cell lines for industrial production of mAbs. 

Abstract Chinese hamster ovary (CHO) cells release and exchange large quantities of extracellular vesicles (EVs). EVs are highly enriched in microRNAs (miRs, or miRNAs), which are responsible for most of their biological effects. We have recently shown that the miR content of CHO EVs varies significantly under culture stress conditions. Here, we provide a novel stoichiometric (“per‐EV”) quantification of miR and protein levels in large CHO EVs produced under ammonia, lactate, osmotic, and age‐related stress. Each stress resulted in distinct EV miR levels, with selective miR loading by parent cells. Our data provide a proof of concept for the use of CHO EV cargo as a diagnostic tool for identifying culture stress. We also tested the impact of three select miRs (let‐7a, miR‐21, and miR‐92a) on CHO cell growth and viability. Let‐7a—abundant in CHO EVs from stressed cultures—reduced CHO cell viability, while miR‐92a—abundant in CHO EVs from unstressed cultures—promoted cell survival. Overexpression of miR‐21 had a slight detrimental impact on CHO cell growth and viability during late exponential‐phase culture, an unexpected result based on the reported antiapoptotic role of miR‐21 in other mammalian cell lines. These findings provide novel relationships between CHO EV cargo and cell phenotype, suggesting that CHO EVs may exert both pro‐ and antiapoptotic effects on target cells, depending on the conditions under which they were produced. 

Industrial biotechnology and biopharmaceutical manufacturing leverage biology to enable cellular systems to serve as factories to produce molecules of value to humankind. These biotechnological processes utilize diverse host organisms and address applications from biofuel, polymer building blocks, antibiotics, and whole cell therapies. Industrial biotechnology can address environmental and sustainability goals in addition to chemical production. In a similar fashion, the field of biopharmaceutical manufacturing has and continues to produce life-saving medicines. Despite these diverse applications, these fields rely on common biological themes and require similar approaches for genetic and metabolic engineering as discussed in this review. Through advances in synthetic biology, targeted genetic engineering, DNA sequencing, adaptation and high-throughput screening, industrial biotechnology and biopharmaceutical manufacturing utilize the same framework for efficient biochemical production which can be leveraged in current and future collaborations to enable rapid innovation. 

This review describes key milestones related to the production of biopharmaceuticals—therapies manufactured using recombinant DNA technology. The market for biopharmaceuticals has grown significantly since the first biopharmaceutical approval in 1982, and the scientific maturity of the technologies used in their manufacturing processes has grown concomitantly. Early processes relied on established unit operations, with research focused on process scale-up and improved culture productivity. In the early 2000s, changes in regulatory frameworks and the introduction of Quality by Design emphasized the importance of developing manufacturing processes to deliver a desired product quality profile. As a result, companies adopted platform processes and focused on understanding the dynamic interplay between product quality and processing conditions. The consistent and reproducible manufacturing processes of today's biopharmaceutical industry have set high standards for product efficacy, quality, and safety, and as the industry continues to evolve in the coming decade, intensified processing capabilities for an expanded range of therapeutic modalities will likely become routine.