CHO in biomanufacturing: Past, present and future

Mario Pereira, field application scientist for bioproduction at Horizon Discovery, a PerkinElmer company, comments on the advantages of using CHO cells for biomanufacturing and explains how gene editing technologies are increasing their utility.

Since the approval in 1986 of the first mammalian-expressed recombinant therapeutic—human tissue plasminogen activator produced in Chinese hamster ovary (CHO) cells—CHO cell lines have dominated therapeutic protein production. The capacity of CHO cells to grow in suspension culture at very high cell densities, combined with their unparalleled adaptability to different growth conditions, has led them to become a mammalian “workhorse” for producing proteins at scale. However, as biologic drugs increase in complexity, the cellular systems used in their production must evolve to address various biomanufacturing needs.

Intricacies of the biomanufacturing workflow

Manufacturing a biologic drug in a mammalian system at commercial scale is inherently time-consuming and complex. It begins with cell line development, whereby a DNA vector encoding the therapeutic protein is transfected into the chosen host cell line. Following selection of expressing cells and the screening of hundreds of individual clones, a small number of candidates is identified for further evaluation and characterisation.

Candidate identification involves assessment of key cell culture characteristics such as growth, productivity, and clone stability. It also typically includes evaluation of critical product quality attributes such as the intact protein sequence, glycosylation profile, and aggregate levels of the biologic drug.

Once the candidate clones have been identified, they are cultured in small-scale bioreactors where further optimisation takes place. This involves establishing the impact of physical (e.g., temperature, agitation speed, gas flow rate) and chemical (e.g., pH, dissolved oxygen and CO2 levels, substrate concentration) parameters on cellular growth attributes. Most importantly, it determines each clone’s performance as a producing cell.

Often, during this period, an appropriate growth medium and feed strategy is developed in parallel. For therapeutic protein production, fed-batch culture is a tried and trusted method and can routinely achieve cell specific productivity of over 20 pg/cell/day. Continuous culture, although more technically difficult to implement and validate, provides the advantage of minimising by-products since it allows key nutrients to be maintained at lower concentrations.

Only when the best-performing clones and pilot scale growth conditions have been confirmed is it possible to transfer manufacturing to a cGMP facility. To both expedite and de-risk the biomanufacturing development process, it can be prudent to use a well-characterised system, such as a commercially available, GMP-grade CHO cell line.

Advantages of CHO cells in biomanufacturing

Several important features of CHO cells make them an attractive choice for biomanufacturing. First, they can grow in suspension culture and in chemically defined (serum-free) media, facilitating the development of precise, controlled, and reproducible manufacturing processes. CHO cells also benefit from a short doubling time (16–22 hours) that rapidly provides the critical cell mass needed to produce a recombinant therapeutic at scale. However, a main advantage of CHO cells is their safety profile, which has been established over a period of several decades and helps ensure compliance with regulatory safety and quality requirements.

A further benefit of CHO cells is their ability to replicate high quality post-translational modifications (PTMs), which far surpasses that of commonly used microbial, insect or plant-based platforms. Unlike non-mammalian systems, CHO cells can produce almost all human-like PTMs, significantly reducing the potential immunogenic response when the final therapeutic product is dosed in man. Moreover, compared with other mammalian cell lines, CHO cells have been shown to be less susceptible to certain viral infections, which minimises the possibility of contaminating adventitious agents being transferred to humans.

Inherent challenges of cell line development

Various types of biomolecules are manufactured in mammalian cells, with well-known examples being therapeutic proteins such as hormones or blood factors, and antibody-based drug products including monoclonal antibodies, Fc-fusion proteins, and bispecific antibodies.

To function correctly, these large, complex structures must be properly folded and, in the case of many antibody-based drugs, assembled appropriately from their constituent parts. They must also undergo multiple PTMs following synthesis and must avoid degradation.

A further challenge associated with cell line development for biomanufacturing is ensuring the cell line is fully traceable. For CHO cells, this means knowing their history, including any manipulations (e.g., genetic modification or treatment with selective agents) that may have taken place.

This issue is easily overcome by selecting a CHO cell line from a reliable commercial source. Such a cell line will be backed by comprehensive documentation and will often be provided with optimised cell culture media and detailed technical protocols describing its use.

Overcoming biomanufacturing challenges with engineered CHO

Since the turn of the century, emerging technologies for gene editing have enabled the development of new CHO variants. These include zinc finger nucleases, systems based in recombinant adeno-associated virus, and, most recently, CRISPR-Cas9, all of which have been exploited to overcome diverse biomanufacturing challenges.

Using these novel platforms, CHO cells have been engineered to incorporate missing human-like PTMs or remove unwanted modifications, resulting in better quality biomolecules demonstrating superior product performance. Additionally, in certain cases, the half-life of protein therapeutics has been extended. Other gene editing strategies have increased the consistency and speed of CHO cell line generation, enhanced process robustness and productivity, or helped reduce manufacturing costs.

Horizon Discovery has attempted to ensure the benefits of gene editing are accessible to those engaged in CHO-based bioproduction with its CHOSOURCE CHO-K1 GS knockout expression platform. The platform has been designed to enable glutamine synthetase (GS) selection to support the rapid identification of high-expressing clones.