Exploring the molecular content of CHO exosomes during bioprocessing

Abstract

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. 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.

Extracellular Vesicles Facilitate Large-Scale, Homogenizing Dynamic Exchange of Proteins and RNA Among Cultured Chinese Hamster Ovary (CHO) and Human Cells

“…The measured EV diameters in the TEM samples were representative of the diameter size distribution measured by NTA. Additionally, EV morphology observed with TEM was similar to other reported CHO EV samples isolated via differential ultracentrifugation (Keysberg et al, 2021). SEM of CHO cells (Fig.…”

“…2A and 2B) had high expression of the surface markers Annexin A1 and CD81, while CD63 and TSG101 were detected on the exosomes at a lower abundance as determined by the differences in MFI. CHO EVs have been reported in other studies to be high in CD81 expression and lower in TSG101 expression (Han & Rhee, 2018; Keysberg et al, 2021). MPs (Fig.…”

“…Chinese hamster ovary (CHO) cells are the most widely used cell host in the biopharmaceutical industry for production of therapeutic proteins (Han & Rhee, 2018; Keysberg et al, 2021). CHO cells are grown in suspension cultures in large scale bioreactors at high cell densities and CHO cell lines are optimized to extend culture lifespan, increase protein production, and limit the accumulation of toxic by-products (Han & Rhee, 2018).…”

“…Yet very little is known about the biology and impact of EVs on CHO cells in culture. In a recent study (Keysberg et al, 2021), proteomic and small RNA sequencing of CHO EVs harvested from different phases of growth identified differentially expressed proteins and small RNAs. Additionally, the protein and RNA content of the exosomes were compared to the parent cell, identifying proteins and RNA highly enriched in exosomes, contributing to early understanding of the regulatory effect CHO EVs may have in culture.…”

“…Fluorescent studies of CHO EVs from these overexpressing cell lines were observed in vitro to be taken up by primary mouse aortic endothelial cells (Seras-Franzoso et al, 2021). Current literature provides evidence and support for exploring and detailing the dynamic release and uptake of native EVs of CHO and other cells in culture (Han & Rhee, 2018; Keysberg et al, 2021; Seras-Franzoso et al, 2021). Such detailing would shed light to the potential impact of EV exchange between cells on cell fate both in vitro and, by extrapolation, in vivo .…”