Glycosylation in Cell Culture

Spearman, Maureen; Butler, Michael

doi:10.1007/978-3-319-10320-4_9

Cited by 8 publications

(5 citation statements)

References 137 publications

(127 reference statements)

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“…An additional area of focus, apart from improving growth and final titers, is controlling the PTMs, especially glycosylation. Glycosylation patterns are often heterogeneous and they are heavily influenced by the culture status [179,180]. It has been described that glycosylation patterns within CHO platforms can be modulated by varying the culture conditions (e.g., culture medium supplements) [181].…”

Section: Ensuring Product Qualitymentioning

confidence: 99%

Key Challenges in Designing CHO Chassis Platforms

et al. 2020

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Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.

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Section: Ensuring Product Qualitymentioning

confidence: 99%

Key Challenges in Designing CHO Chassis Platforms

et al. 2020

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“…The use of CHO cells for therapeutic proteins production has many advantages such as the easier adaptation to grow in serum-free and/or chemically defined media, the ability to reach high cell densities in suspension culture, high product titer and similar glycosylation patterns to human cell lines (Bandaranayake and Almo 2014 ; Kim et al 2012 ; Kuystermans and Al-Rubeai 2015a ; Spearman and Butler 2015 ). Nowadays, platforms based on CHO cells use cells pre-adapted to grow in chemically defined, serum-free media and suspension culture to decrease time and efforts related to adaptation of the recombinant CHO cell lines (Jostock 2011 ; Kim et al 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

et al. 2021

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The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

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“…Antibodies with reduced glycan heterogeneity or as homogeneous single glycoforms may show significantly higher specific bioactivities . Enzymatic remodeling of an isolated antibody with selected glycosylation enzymes is a simple and versatile approach to generate single glycoforms .…”

Section: Introductionmentioning

confidence: 99%

“…[15,16] Antibodies with reduced glycan heterogeneity or as homogeneous single glycoforms may show significantly higher specific bioactivities. [17] Enzymatic remodeling of an isolated antibody with selected glycosylation enzymes is a simple and versatile approach to generate single glycoforms. [18] However, such modifications may require multiple enzymatic steps that may necessitate intermediate antibody purification resulting in product loss during each step.…”

Section: Introductionmentioning

confidence: 99%

Solid‐Phase Enzymatic Remodeling Produces High Yields of Single Glycoform Antibodies

Tayi

Butler

2017

Biotechnology Journal

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Antibodies are synthesized in mammalian cell culture as heterogeneous mixtures of glycoforms. Production of single glycoforms remains a challenge despite their value as therapeutics. The authors report a method of sequential enzymatic-based changes to antibodies while immobilized on an affinity column. Various antibodies (monoclonal and polyclonal) are isolated on Protein A or G columns and their glycans modified by sequential addition of enzymes for a desired transformation. Galactosylated antibodies (>90% yield) are produced by a one stage reaction process with sialidase to remove any sialic acid residues and addition of galactose with galactosyltransferase and UDP-Gal. Sialylated antibodies (>90%) are produced by a 2 stage conversion involving α(2,3) sialidase and galactosyltransferase followed by treatment with α(2,6) sialyltransferase in the presence of CMP-NANA. By this method, >90% of a disialylated human-llama antibody (EG2-hFc) and equimolar quantities of monosialylated and disialylated forms of human antibodies (αIL8-hFc and human polyclonal) are produced. Such high levels of sialylation are very difficult to obtain by typical cell culture methods. This method of transformation while the antibody is held on a solid-phase column is superior to previous methods because it allows a series of enzymatic steps without the need for intermediate purification. This is an efficient and rapid method to generate therapeutic antibodies with predefined glycosylation profiles. This should also assist in investigating the structure-function relationship of antibody glycans to find the desired glycosylation profile for high functional activity. With further optimization the method can be used to modify antibodies in large-scale manufacturing.

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Glycosylation in Cell Culture

Cited by 8 publications

References 137 publications

Key Challenges in Designing CHO Chassis Platforms

Key Challenges in Designing CHO Chassis Platforms

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Solid‐Phase Enzymatic Remodeling Produces High Yields of Single Glycoform Antibodies

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