Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

Amann, Thomas; Schmieder, Valerie; Kildegaard, Helene Faustrup; Borth, Nicole; Andersen, Mikael Rørdam

doi:10.1002/bit.27101

Cited by 41 publications

(27 citation statements)

References 227 publications

(327 reference statements)

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“…In recent years, mammalian cells have become the main host systems for recombinant protein production (Walsh, 2014). This is due to their innate membranous organelles allowing post-translational modification (PTM) of the expressed proteins, which is critical for certain proteins to fold properly and/or exert its biological activity (Amann et al, 2019). In particular, given their ease of in vitro cell culture and ability to adapt to selection pressure, Chinese hamster ovary (CHO) cells are established as the workhorse for industrial protein production (Berger et al, 2020).…”

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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“…Therapeutic protein immunogenicity risk assessment, with attention to assays and in vivo models, has been described as a way to mitigate this risk in a recent study (Tourdot and Hickling, 2019). The use of gene knockout/knockdown and overexpression to develop meaningful approaches to improve the PTMs of biopharmaceuticals in different production platforms and their applicability were well-described in a recent study (Amann et al, 2019). Recent developments in metabolic engineering also include the use of gene-editing tools for successful clone and product development.…”

Section: Introductionmentioning

confidence: 99%

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

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

397

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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“…While these cells have proven to have a cellular machinery well fit for production of monoclonal antibodies, i.e. IgGs, and several other human proteins for therapeutic usage, recent interests in more difficult-to-express human and artificial protein-fusions, have highlighted product yield and quality bottlenecks in CHO ( 5–7 ). Studies using transient expression in CHO cells have shown that product yield can be enhanced by regulating the expression of protein subunits and helper proteins ( 8 , 9 ).…”

Section: Introductionmentioning

confidence: 99%

Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

Eisenhut

Mebrahtu

Barzadd

et al. 2020

Nucleic Acids Research

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Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

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Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

Cited by 41 publications

References 227 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

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

Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

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