Multiplex secretome engineering enhances recombinant protein production and purity

Kol, Stefan; Ley, Daniel; Wulff, Tune; Decker, Marianne; Hansen, Jens Aage; Schoffelen, Sanne; Hansen, Anders Holmgaard; Jensen, Tanja Lyholm; Gutierrez, Jahir M.; Chiang, Austin W. T.; Masson, Helen O.; Palsson, Bernhard Ø.; Voldborg, Bjørn Gunnar; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

doi:10.1038/s41467-020-15866-w

Cited by 79 publications

(69 citation statements)

References 53 publications

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“…Deleting metabolically expensive host proteins relieves bottlenecks in protein production. Indeed, the Lewis lab has elegantly demonstrated that deleting highly expressed proteins in CHO cells increases the yield of heterologous secreted proteins [25,26]. Modification of the secretory pathway, such as the optimization of signal sequences for protein targeting [71] and reducing the effect of the ERAD system [10], provides varying degrees of success and is contingent on the complexity and optimization of the protein product [72,73].…”

Section: Discussionmentioning

confidence: 99%

“…Per molecule costs can be coupled with measurements of gene expression to identify most expensive host proteins. Deletion of these proteins improves yields of secreted heterologous proteins in mammalian systems [25,26]. However, while these analyses account for demands on global resources, they are limited by insufficient experimental data which links gene products to specific biogenesis subnetworks.…”

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confidence: 99%

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Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

Alva¹,

Riera²,

Chartron³

2020

Preprint

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Background: Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traﬃc, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the ﬂux through various routes into the early secretory pathway, we quantiﬁed the global and membrane-associated translatomes of Komagataella phaﬃi. Results: By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantiﬁed demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway. Conclusion: A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

Alva¹,

Riera²,

Chartron³

2020

Preprint

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“…Saitua et al employed a dynamic GeM of P. pastoris to predict system behaviour across batch and fed-batch cultivation under glucose-limited aerobic conditions, followed by the design of single knock-out genetic engineering strategies that can boost volumetric protein productivity [46] . The CHO GeM has also been used to identify burdensome host cell proteins for deletion to ease pressure on downstream processing [16] , [47] . However, due to the size and complexity of mammalian cell GeMs, this kind of algorithms have not been widely applied to mammalian cell systems yet.…”

Section: Advances In Gem Development and Applicationmentioning

confidence: 99%

The era of big data: Genome-scale modelling meets machine learning

Antonakoudis

Barbosa

Kotidis

et al. 2020

Computational and Structural Biotechnology Journal

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“…ELISA analysis of these samples showed that the total HCP concentrations were greatly reduced from 510,762 ppm in OM, to 1,081 ppm in PrA, 557 ppm in CEX, and finally only 7 ppm in the AEX. In the SWATH-MS analysis, total 1,900 proteins were quantified (at 20% CV cutoff and 1% peptide FDR in OM) from one microgram of each DSP sample, including previously reported difficult-to-remove and problematic HCPs [43][44][45] (Online-only Table 1). Heat map analysis of all the identified proteins showed that majority of the impurities were removed after protein A affinity chromatography (Fig.…”

Section: Robustness Of Protein Identification and Quantification In Cmentioning

confidence: 99%

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins

et al. 2020

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Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) strategy that requires a specific spectral library to generate unbiased and consistent quantitative data matrices of all peptides. SWATH-MS is a promising approach for in-depth proteomic profiling of Chinese hamster Ovary (CHO) cell lines, improving mechanistic understanding of process optimization, and real-time monitoring of process parameters in biologics R&D and manufacturing. However, no spectral library for CHO cells is publicly available. Here we present a comprehensive CHO global spectral library to measure the abundance of more than 10,000 proteins consisting of 199,102 identified peptides from a CHO-K1 cell proteome. The robustness, accuracy and consistency of the spectral library were validated for high confidence in protein identification and reproducible quantification in different CHO-derived cell lines, instrumental setups and downstream processing samples. The availability of a comprehensive SWATH CHO global spectral library will facilitate detailed characterization of upstream and downstream processes, as well as quality by design (QbD) in biomanufacturing. The data have been deposited to ProteomeXchange (PXD016047). Background & Summary Chinese hamster ovary (CHO) cells are widely studied in biomedical research and have been used for the production of nearly 70% recombinant therapeutic proteins, including the blockbuster monoclonal antibodies (mAbs) such as adalimumab (Humira), bevacizumab (Avastin), and trastuzumab (Herceptin) 1-4. As with other biopharmaceuticals, the production of recombinant mAbs using CHO cells is strictly regulated by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) 5. The quality by design (QbD) approach is emphasized in order to ensure all aspects of CHO mAb product development and manufacturing are evaluated and consistent 6-9. Hence, in order to enhance the mAb product quality while minimizing potential adverse effects, a thorough understanding of the recombinant mAbs, the early-stage product development process as well as the production and purification process is critical. Mass spectrometry (MS)-based proteomics techniques have been applied to facilitate the QbD approach for optimal bioprocess design, and enhanced quality and yield of biotherapeutic products 10-13. For example, the data-dependent acquisition (DDA) shotgun proteomics has been demonstrated to complement the traditional immunoassays (western blotting and ELISA) to monitor and analyze known and/or previously unseen host cell proteins (HCPs) in the drug manufacturing processes 14-16. Nevertheless, due to the stochastic nature of peptide sampling by DDA-MS technique, multi-dimensional separation or fractionation steps and lengthy chromatographic gradients are often required to increase the proteome coverage over a large dynamic range of protein concentrations. Additionally, the advanced MS methodologies usually require well-trained professionals to operate t...

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Multiplex secretome engineering enhances recombinant protein production and purity

Cited by 79 publications

References 53 publications

Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

The era of big data: Genome-scale modelling meets machine learning

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins

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