Biology subjects

Malm, M.

Publications and source records attributed to Malm, M..

5 recordsLinked to original sources

bioRxiv2022

Deciphering the determinants of recombinant protein yield across the human secretome

Mammalian cells are critical hosts for the production of most therapeutic proteins and many proteins for biomedical research. While cell line engineering and bioprocess optimization have yielded high protein titers of some recombinant proteins, many proteins remain difficult to express. Here, we decipher the factors influencing yields in Chinese hamster ovary (CHO) cells as they produce 2165 different proteins from the human secretome. We demonstrate that variation within our panel of proteins cannot be explained by transgene mRNA abundance. Analyzing the expression of the 2165 human proteins with machine learning, we find that protein features account for only 15% of the variability in recombinant protein yield. Meanwhile, transcriptomic signatures account for 75% of the variability across 95 representative samples. In particular, we observe divergent signatures regarding ER stress and metabolism among the panel of cultures expressing different recombinant proteins. Thus, our study unravels the factors underlying the variation on recombinant protein production in CHO and highlights transcriptomics signatures that could guide the rational design of CHO cell systems tailored to specific proteins.

systems biology↗

bioRxiv2021

Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.

systems biology↗

bioRxiv2020

Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

Higher eukaryotic cell lines like HEK293 are the preferred hosts for production of therapeutic proteins requiring human post translational processing. However, recombinant protein production can result in severe stress on the cellular machinery, resulting in limited titre and product quality. To investigate the cellular and metabolic characteristics associated with these limitations, we compared erythropoietin (secretory) and GFP (non-secretory) protein producer HEK293 cell-lines using transcriptomics analysis. Despite the high demand for ATP in all protein producer clones, a significantly higher capacity for ATP production was observed with erythropoietin producers as evidenced by the enrichment of upregulated genes in the oxidative phosphorylation pathway. In addition, ribosomal genes exhibited specific patterns of expression depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased erythropoietin secretion, we detected higher ER stress, including upregulation of the ATF6B gene. Our results are significant in recognizing key pathways for recombinant protein production and identifying potential target genes for further development of secretory power in mammalian cell factories. In BriefAlthough the protein secretion process has been widely studied, the complexity of it leaves many questions with regards to defining bottlenecks for successful protein secretion to be answered. By investigating the transcriptomic profiles of different HEK293 clones with varying translational rates producing either the secreted protein erythropoietin or the intracellular GFP, we reveal that high ATP production and improved capacity of specific post-translational pathways are key factors associated with boosting erythropoietin production. HighlightsO_LITranscriptomics analysis of a panel of HEK293 stable cell lines expressing GFP or erythropoietin (EPO) at varying translational rates C_LIO_LIExpression of mitochondrial ribosomal genes is positively correlated with EPO secretion C_LIO_LIExpression of different cytosolic ribosomal genes are correlated with productivity in a recombinant-protein specific manner C_LIO_LIHigh EPO producing clones have significant upregulation of ATF6B, potentially enabling a beneficial ER stress response to cope with high protein secretion C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/299966v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1e35efforg.highwire.dtl.DTLVardef@71cee2org.highwire.dtl.DTLVardef@1c56d33org.highwire.dtl.DTLVardef@843a6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Evolution from adherent to suspension - systems biology of HEK293 cell line development

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production and is today used for manufacturing of several therapeutic proteins and viral vectors. Despite this increasing popularity there is still limited knowledge of the detailed genetic and composition of derivatives of this strain. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

systems biology↗

bioRxiv2018

The human secretome - the proteins secreted from human cells

The proteins secreted by human tissues (the secretome) are important for the basic understanding of human biology, but also for identification of potential targets for future diagnosis and therapy. Here, we present an annotation of all predicted secreted proteins (n=2,623) with information about their spatial distribution in the human body. A high-throughput mammalian cell factory was established to create a resource of recombinant full-length proteins. This resource was used for phenotypic assays involving {beta}-cell dedifferentiation and for development of targeted proteomics assays. A comparison between host cells, including omics analysis, shows that many of the proteins that failed to be generated in CHO cells could be rescued in human HEK293 cells. In conclusion, the human secretome has been mapped and characterized to facilitate further exploration of the human secretome.

molecular biology↗