Bioproduction

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Chinese Hamster Ovary (CHO) Cells

Biological therapeutics such as monoclonal antibodies are an increasingly prominent part of many drug development pipelines. Horizon Discovery believes that to maintain pace with industry needs in this area, access to Chinese Hamster Ovary or CHO cells must be improved to make them available to companies of all sizes. High entry costs and restrictive licensing conditions are prohibitive for access to CHO cells for the majority of biomanufacturing groups.

CHO GS vs. DHFR Metabolic Selection

CHO cells have become the expression system of choice for the manufacturing of biological therapeutics. They have been shown to have the capacity to express a variety of proteins such as therapeutic enzymes or monoclonal antibodies at multi-gram per litre titres. As expression technologies have developed, focus on increasing titre has mainly been achieved through improvements to media and feed, while the ability to identify high productivity clones has been streamlined through the use of different selection systems.

Antibiotic selection has been used for a number of years, but the requirement to maintain cells in antibiotics is costly and requires removal of the antibiotic from the production media during downstream steps, which means that metabolic selection has become the preferred industry method. Metabolic selection can broadly be split into those that use Glutamine Synthetase (GS) or Dihydrofolate reductase (DHFR) systems.

Glutamine Synthetase (GS) Null

Glutamine Synthetase is an enzyme that catalyses the conversion of glutamate to the amino acid glutamine and is the only mechanism for cells to generate their own glutamine. If the expression of Glutamine Synthetase is reduced through chemical or genetic means, then the cells are not viable unless they are either cultured in media containing additional glutamine or have an alternative Glutamine Synthetase exogenously expressed. This mechanism has been exploited for over ten years to generate a metabolic selection system that links the expression of an exogenous GS gene to the expression of a protein of interest (for example a monoclonal antibody). This means that when the cassette stably integrates into the genome, expression of the monoclonal antibody is proportional to the amount of Glutamine Synthetase expressed. Cells can then be placed into media that lacks glutamine, and those expressing insufficient GS (and by extension a low level of monoclonal antibody) are unable to survive.

Originally, the mouse cell line NS0 exploited this mechanism as it is naturally deficient in Glutamine Synthetase. To adapt this system for use in CHO cell expression, GS was inhibited by the chemical inhibitor Methionine Sulphoximine (MSX). However, this led to high levels of background due to the cell line increasing the expression of its endogenous GS gene, and MSX needed to be included in production culture to maintain the selection. As a highly toxic compound, this needs to be removed from the production media during downstream processing, leading to increased costs and time.

More recently, CHO K1 cells have been engineered to be null for Glutamine Synthetase. Horizon Discovery has engineered a GS null CHO cell line using its proprietary rAAV technology, while Lonza used Meganucleases and Sigma Aldrich used Zinc Fingers (ZFNs). This GS null CHO K1 selection system is now considered to be the industry standard method of selecting high expressing clones following transfection with a vector expressing the biotherapeutic.

DHFR

An alternative metabolic selection system exists that utilises the DHFR gene. DHFR reduces dihydrofolic acid to tetrahydrofolic acid and in its absence, cells require supplementation of the media with glycine, hypoxanthine, and thymidine for viability. To reduce the level of DHFR in the cells, increasing levels of the chemical inhibitor Methotrexate (MTX) was used, or there are also cells lines (such as DG44 cells) that are null for the DHFR gene. Similar to the GS system, vectors have been designed that express the protein of interest (for example a monoclonal antibody) as well as a separate expression of an exogenous DHFR gene. However, due to decreased timelines associated with the GS system, this has become the system of choice for most companies.

Furthermore, metabolic selection using MSX or MTX is often performed using a number of amplification steps to increase copy number and expression levels of clones, which has led to some concerns over the long-term stability of the final producer clone. Together with the increased downstream processing burden, null cell lines are a preferable technology for utilszing the metabolic selection through DHFR or GS.



Custom Bioproduction

Horizon provide a service that enables custom optimisation of CHO expression platforms. This allows you to direct the manipulation of the CHO genome to suit your specific requirements. They have successfully engineered thousands of cell lines, including direct experience of CHO. Not only has Horizon generated their own knockout cell line, but they have extensive experience of handling proprietary cell lines. With dedicated lab space and enhanced SOPs to ensure the highest standards, Horizon have become a trusted partner for large pharma and for biotechs wishing to engineer their CHO cells.

Horizon are uniquely placed to provide a range of engineering possibilities. They are truly technology agnostic, and with their wealth of experience in rAAV and nuclease genome editing technologies including CRISPR, Horizon have the flexibility to make almost any modification you may require, ranging from point mutations to large scale translocations. Projects can be based on a cell line provided to them or on one of their commercially available lines, and can be structured in different ways depending on budgetary requirements.



CHO Expression Data

Horizon's GS Null CHO K1 cells are in the process of being independently evaluated in over 15 organisations globally. Some of these evaluations have given Horizon permission to share their cell line development data relating to the expression of recombinant proteins, including monoclonal antibodies and bispecifics. Although at this stage considerable optimisation is still being performed on the culture process, Horizon are consistently receiving reports of expression exceeding expectations. While some of the data provided below utilises MSX to improve the stringency of selection with individual vectors, the CHO SOURCE platform combining Horizon's GS null CHO K1 cell line with an expression vector from ProteoNic does not require MSX during selection, nor does it require any amplification steps commonly associated with alternative metabolic selection systems such as the Methotrexate / DHFR selection approaches.

Comparison with msx based gs system

After being transfected with a GS selection vector containing a monoclonal antibody and placed under metabolic selection in glutamine free media, the cells expressed significantly higher titres of recombinant protein than the reference system comprising MSX treated wild type CHO K1 cells. Data shown is generated in a 5L Bioreactor and represents titre from stable pools of cells.

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Stable pool expression

As part of an evaluation of different vector technologies, the cells were transfected with a construct expressing a monoclonal antibody in a vector containing UnicTM elements from ProteoNic.

Titres in excess of 2g/L of a biosimilar IgG were achieved at stable pool stage.

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Clonal expression

Four hundred clones from the stable pool above were screened and assessed for productivity. Three clones expressed at >5g/L.

This is currently an un-optimised process, with further advances expected.

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Stable pool expression

As part of an evaluation of HD-BIOP3, a Biopharmaceutical company in China developed stable pools using a vector provided by DNA 2.0. They assessed the impact of using bulk selection compared with generating minipools through applying selection to 500 cells. Together with this, they evaluated the impact of using MSX with this vector.

Data shown is after 5 days of shake culture.

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Stable Pool expression

As part of an evaluation of HD-BIOP3, a Biopharmaceutical company in the USA developed stable pools using a vector provided by DNA 2.0, together with increasing concentrations of MSX.

Data shown is after Protein A purification.

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