Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi, Nagesh K.; Shrivastava, Ambuj

doi:10.3389/fbioe.2019.00420

Cited by 369 publications

(282 citation statements)

References 435 publications

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“…The advent of recombinant DNA technology has enabled the production of these proteins in industrial-scale bioreactors. The process involves upstream genetic engineering of host systems, such as bacterial, yeast, insect, or transgenic plant and mammalian cells, along with downstream purification of extracted proteins (Tripathi and Shrivastava, 2019). In recent years, mammalian cells have become the main host systems for recombinant protein production (Walsh, 2014).…”

Section: Introductionmentioning

confidence: 99%

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression

Chua

Zhang³

et al. 2020

Front. Bioeng. Biotechnol.

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The global cell culture market is experiencing significant growth due to the rapid advancement in antibody-based and cell-based therapies. Both rely on the capacity of different living factories, namely prokaryotic and eukaryotic cells, plants or animals for reliable and mass production. The ability to improve production yield is of important concern. Among many strategies pursued, optimizing the complex nutritional requirements for cell growth and protein production has been frequently performed via culture media component titration and serum replacement. The addition of specific ingredients into culture media to modulate host cells' metabolism has also recently been explored. In this study, we examined the use of extracted bioactive components of the microalgae Chlorella vulgaris, termed chlorella growth factor (CGF), as a cell culture additive for serum replacement and protein expression induction. We first established a chemical fingerprint of CGF using ultraviolet-visible spectroscopy and liquid chromatography-mass spectrometry and evaluated its ability to enhance cell proliferation in mammalian host cells. CGF successfully promoted the growth of Chinese hamster ovary (CHO) and mesenchymal stem cells (MSC), in both 2D and 3D cell cultures under reduced serum conditions for up to 21 days. In addition, CGF preserved cell functions as evident by an increase in protein expression in CHO cells and the maintenance of stem cell phenotype in MSC. Taken together, our results suggest that CGF is a viable culture media additive and growth matrix component, with wide ranging applications in biotechnology and tissue engineering.

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Section: Introductionmentioning

confidence: 99%

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression

Chua

Zhang³

et al. 2020

Front. Bioeng. Biotechnol.

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“…Escherichia coli is a commonly used host for the production of biotherapeutic and other highvalue proteins. A favored expression strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation (Karyolaimos et al, 2019;Tripathi and Shrivastava, 2019). Yields of several g/L of active human growth hormone, containing two disulfide bonds and exported to the periplasm using the Tat pathway, were recently demonstrated in E. coli "TatExpress" strains (Guerrero et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli

Zhu

Ruan

et al. 2020

Front. Bioeng. Biotechnol.

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Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.

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“…Importantly, associated procedures should be equally amenable to production of other Cas9 effectors, including dCas9, Cas12a, and variants thereof. Pharmaceutical companies have been highly incentivized to optimize scaled protein production environments, 94 including moving to continuous flow rather than batch setups, 95 due to lucrative demand for protein therapies, especially monoclonal antibodies. Process advancements brought about by this economic landscape will translate well to CRISPR-based protein drugs.…”

Section: Administrationmentioning

confidence: 99%

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

et al. 2020

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CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities. Here, we review how CRISPR-Cas therapeutics are translated from technological systems to therapeutic modalities, paying particular attention to the therapeutic cascade from cargo to delivery vector, manufacturing, administration, pipelines, safety, and therapeutic target profiles. We also explore potential solutions to some of the obstacles facing successful CRISPR-Cas translation. We hope to illuminate how CRISPR-Cas is brought from the academic bench toward use in the clinic.

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Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Cited by 369 publications

References 435 publications

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

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