Hyperthermic shift and cell engineering increase small extracellular vesicle production in HEK293F cells.

Although small extracellular vesicles (sEVs) have promising features as an emerging therapeutic format for a broad spectrum of applications, for example, blood-brain-barrier permeability, low immunogenicity, and targeted delivery, economic manufacturability will be a crucial factor for the therapeutic applicability of sEVs. In the past, bioprocess optimization and cell line engineering improved titers of classical biologics multifold. We therefore performed a design of experiments (DoE) screening to identify beneficial bioprocess conditions for sEV production in HEK293F suspension cells. Short-term hyperthermia at 40°C elevated volumetric productivity 5.4-fold while sEVs displayed improved exosomal characteristics and cells retained >90% viability. Investigating the effects of hyperthermia via transcriptomics and proteomics analyses, an expectable, cellular heat-shock response was found together with an upregulation of many exosome biogenesis and vesicle trafficking related molecules, which could cause the productivity boost in tandem with heat shock proteins (HSPs), like HSP90 and HSC70. Because of these findings, a selection of 44 genes associated with exosome biogenesis, vesicle secretion machinery, or heat-shock response was screened for their influence on sEV production. Overexpression of six genes, CHMP1A, CHMP3, CHMP5, VPS28, CD82, and EZR, significantly increased both sEV secretion and titer, making them suitable targets for cell line engineering.

Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform.

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells® (CAP®) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.

The potential of emerging sub-omics technologies for CHO cell engineering.

Chinese hamster ovary (CHO) cells have been the predominant host for recombinant protein production over the past decades with major efforts directed towards cell line engineering to increase amount and quality of biopharmaceutics. Emerging omics approaches and corresponding techniques now allow an extensive characterization of cellular expression systems. Technical improvements in sequencing, mass spectrometry techniques and focusing on defined cellular subsystems unraveled new possibilities for knowledge-based CHO engineering. We found that spotlighting a defined subset of molecules with certain properties or localization, called sub-omics, can provide a better understanding of the respective cellular subsets, enabling the identification of new engineering targets. In this review, we provide an overview of how recent advances from cellular sub-transcriptomes, sub-proteomes and the secretome analyses were and can be utilized for cell line development.

Exploring the molecular content of CHO exosomes during bioprocessing.

In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Graphical Abstract KEY POINTS: • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.