CHO Media Development for Therapeutic Protein Production

Introduction

Cell culture medium plays a crucial role in industrial biopharmaceutical processes since it provides essential nutrients and the proper environment for high viable cell density and efficient therapeutic protein expression. Besides growth and productivity, other media performance aspects should be considered, such as product quality and medium powder manufacturability. In the past few decades, industrial cell culture media have evolved from formulations containing animal-derived or plant-derived components to chemically defined (CD) formulations to reduce potential for adventitious agent introduction and to reduce raw material variability. This review focuses on media development for Chinese Hamster Ovary (CHO) cell cultures, including a brief history of mammalian cell culture medium, general media development strategies and considerations, as well as some more recent progress in CHO media development.

History of CHO Culture Media

Multiple mammalian host cell lines have been used to manufacture therapeutic proteins, including CHO, NS0, BHK, HEK-293 and PER-C6.¹ However, CHO is used as the predominant host in the biologics industry due to its well-characterized genomic background and its relatively fast growth and high protein production in suspension culture. In the 1950s, cell culture pioneers developed basic formulations such as EMEM and DMEM, which required supplementation with serum to support mammalian cell growth. Ham developed the first CD, serum-free formulation F-12 to support the clonal growth of CHO in 1965.²

Insulin, transferrin, ethanolamine, and selenium were then successfully used to replace serum in CD media in other early research.³ Enabled by emerging technologies, CD media development for CHO has progressed dramatically in the past few decades, with recombinant protein titers exceeding 10 g/L in some cases.⁴

Media Design Strategies

One key advantage of custom media over off-the-shelf options is the knowledge of the formulation, which enables efficient process optimization, troubleshooting, and better understanding of media component impact to product quality. It is a commonly used strategy for biopharmaceutical companies to develop a platform process for early-stage clinical projects with the same CHO host cell line in order to quickly move the projects from research to clinical trials.⁵ Platform medium, as part of the platform process, could be developed from in-house media using multiple CHO cell lines for evaluations. If required, further media optimization could be conducted at a later stage for individual cell lines.

Nutrient Requirements

Typical CHO basal medium consists of 50-100 components⁵ and the concentration of each should be in its optimal range as depletion or excess may harm growth, productivity, or product quality. Cell culture medium is a mixture of component groups, such as carbohydrates, amino acids, vitamins, trace metals, salts, lipids, polyamines, protectants, and buffers. Media development efforts have typically focused on optimizing the concentrations of carbohydrates, amino acids, vitamins, and trace metals. Glucose is the predominant carbohydrate used to provide energy to CHO cells via glycolysis and oxidative phosphorylation. Lactate is produced from pyruvate via glycolysis and its accumulation inhibits cell growth. Strategies have been developed to control lactate through glucose limitation or supplementing other carbohydrates.^6,7 Amino acids are the building blocks of cells and therapeutic proteins, and optimizing their concentrations has been a major focus of media development research. Xing et al. identified four limiting amino acids and four in excess amino acids in continuous CHO culture using metabolic flux analysis. After the concentrations of those eight amino acids were adjusted, peak cell density and protein titer were increased by 55% and 27%, respectively.⁸ In another example, Carrillo-Cocom et al. used HPLC to monitor amino acid concentrations in fed-batch cultivation, and identified depleted, steady-state, and accumulating amino acids, providing information for rational media design.⁹ Certain amino acids, such as asparagine and serine, play a key role for specific CHO cell lines, and their metabolism was elucidated by a metabolic profiling study.¹⁰ Water-soluble B-vitamins are essential in cell culture media due to their roles as enzyme cofactors, although they are supplemented at small quantities. Vitamins A, D, and E have been evaluated for their physiological functions in cell culture media, but they are less commonly used due to their low solubility in water. Trace metals are another indispensable nutrient group in serum-free media, and their optimal concentrations are generally even lower than the vitamin concentrations. Similar to vitamins, trace metals function as the cofactors of various enzymes and significantly impact cell growth, productivity, and product quality. As the cofactor of oxidative phosphorylation enzymes, copper is commonly supplemented to cultures to shift lactate from production to consumption.¹¹ Iron has been shown to boost monoclonal antibody (mAb) production, but it has to be properly chelated to prevent the toxic effects of its free form (transferrinbound in serum-free medium or citrate-chelated in protein-free medium).^12,13 Besides copper and iron, zinc and selenium are essential trace metals, which have been reported to enhance mAb production and reduce oxidative stress, respectively.^14,15

In addition to traditional nutrient components discussed above, various growth factors including peptides, low molecular weight proteins, and hormones have been demonstrated to enhance product titer. The Long®R3 IGF-I is an insulin-like growth factor-1 (IGF-1) analog engineered for improved stability, and it has been shown to increase fusion protein titer by increasing viable cell density.¹⁶ Dipeptide or tripeptide containing cysteine and tyrosine have been used to improve mAb titer, cell density, and metabolic profiles.¹⁷ Hormones are known to modulate various cellular activities. Hydrocortisone, a steroid hormone, was reported to extend cell viability and boost fusion protein titer in a dose- and time-dependent manner.¹⁸ Moreover, recent studies have identified additional titer enhancers with different mechanisms of action, including pyrimidine nucleosides, histone deacetylase inhibitors, and non-proteinogenic amino acids.^19,20,21 However, the following aspects may need to be considered when working with growth factors and titer enhancers: cost, solubility, stability, material sourcing, impact on product quality attributes, and their clearance during product manufacturing.

Experimental Approaches

The traditional one-factor-at-a-time approach only allows altering the concentration of a single component at a time. This method becomes exceedingly labor-intensive for the large number of components in CHO media. Also, since this approach cannot investigate the interactions of components, it is more likely to only achieve a local optimum instead of the global optimum in the range of experimental conditions. On the other hand, a statistical approach called Design of Experiments (DoE) evaluates component concentrations and component interactions simultaneously, while greatly reducing the number of experimental conditions. In general, screening designs such as factorial design, Plackett-Burman design, and definitive screening design are first used to select components with significant effects or interactions, followed by response surface methodology, such as central composite design or Box-Behnken design, to identify their optimal concentrations and interactions of each component.²² For example, Huang et al. used Plackett-Burman design to screen eleven components with only twelve conditions, generating a CD medium to support CHO cell growth.²³ In another case study, the optimal concentrations of five groups of components were determined using central composite design, with viable cell density and titer increased by 35% and 50%, respectively.²⁴ Media blending has also been applied to optimize medium formulation. This approach can generate and evaluate a large number of media formulations simultaneously, predict the optimal formulation, and identify individual critical components for further investigation. As an example, a 40% titer improvement was achieved by blending 16 CD formulations, and seven components were found to be titer enhancers.²⁵

Although DoE and media blending methods are efficient for media design, the total number of conditions can still be substantial, especially when exploring many components with multiple ranges and experimental replicates. Therefore, high-throughput culture systems with high repeatability and robustness are desired. Shaken vessels such as deep-well plate, conical tubes, and shake flasks are most commonly used since they are easy to set up and cost-effective. With the recent introduction of ambr®15 and ambr®250 bioreactor systems, it is feasible to optimize media with high-throughput in miniature bioreactors with pH and dissolved oxygen control, which has been demonstrated to be representative of large-scale bioreactors.^26,27

Media development is an iterative process with rounds of performance testing, spent medium analysis, and formulation refinement, with incremental improvement after each round.⁵ In order to optimize existing components in media formulation, it is vital to profile them during the culture time course accurately. Various analytical techniques have been utilized to quantify nutrients and metabolites, including high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), gas chromatography (GC), inductively coupled plasma mass spectrometry (ICP-MS), liquid chromatography or GCcoupled with mass spectrometry (LC-MS or GC-MS).²⁸ For example, NMR and HPLC have been combined to measure all 20 amino acids, followed by the supplementation of identified five depleted amino acids, resulting in improved cell culture performance.²⁹

Product Quality Considerations

Although cell growth and productivity have been the focus of media development research, particular attention should be paid to product quality throughout the optimization process since it is closely associated with efficacy, potency, and safety of therapeutic proteins. Media components have been reported to impact many product quality attributes, including glycosylation, charge variants, size variants, and sequence variants.³⁰ The effects of selected media components on these product quality attributes are exemplified in some case studies as follows. Copper has been widely reported to increase basic charge variants and affect mAb aggregation and fragmentation.^31-33 Manganese is a well-known factor to modulate glycosylation pattern, such as high mannose species, galactosylation, sialylation, and glycation.³⁰ Cystine, asparagine, and glutamine have been found to increase sialylation while cystine was also described to decrease acidic charge variant and low molecular weight species.^30,34,35 In addition to their positive effect on titer, hydrocortisone and Long®R3 IGF-1 were shown to increase the sialylation level of an Fc-fusion protein significantly.^16,18 Non-nutritional components, such as acetylated N-acetylmannosamine and glycerol, were also indicated to improve protein sialylation.^36,37 In terms of amino acid misincorporation, an efficient preventative step is to provide sufficient amino acids to keep culture from exhausting those nutrients.³⁸

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Manufacturing Considerations

Although liquid media and stock solutions are extensively used during media development to generate and optimize formulations, it is highly preferable to use powder media for manufacturing because they are more stable, and easier to store and transport.⁵ When evaluating a formulation for powder media manufacture, the proper chemical form of each component should be selected based on solubility, stability, and cost. For example, cystine and tyrosine have very low solubility, and are often supplemented to cultures in a separate feed with high pH. The chemically modified amino acids s-sulfocysteine and phosphotyrosine have significantly higher solubility, allowing them to be combined with a single feed to greatly simplify the cell culture process.^39,40 As for the powder milling device, the pill mill has established its predominance over the ball mill for powder media manufacture due to its easier maintenance, higher component homogeneity, and less heat generation.⁵ Because of the composite nature of CD media powder, it is critical to characterize and control its variability to ensure consistent performance in large-scale manufacturing. Media powder variability sources include component degradation, impurities or contaminants of raw materials, milling and blending process, as well as raw material sourcing.⁴¹ Chemometrics in combination with fluorescence, nearinfrared (NIR), and Raman spectroscopy have been demonstrated to assess cell culture media consistency.⁴²

Recent Progress in CHO Media Development

Media Development for Perfusion Processes

Perfusion technology has gained increasing popularity in the past decade due to its potential to increase bioreactor usage efficiency, lower manufacturing cost, and improve product quality.⁴³ A new medium must be developed to support high cell density and high productivity of perfusion culture, when switching from fed-batch process to perfusion process. In one case study, existing fed-batch media were leveraged to develop perfusion medium, achieving a productivity of 1.2 g/L-day at steady state by blending and concentrating basal and feed media, removing unnecessary components and enriching with amino acids, vitamins, and lipids.⁴⁴ Similarly, perfusion media were derived from fed-batch basal and feed with DoE approaches in another study, achieving high productivities and desirable product quality.⁴⁵

Omics Technologies

Transcriptomics, proteomics, and metabolomics are currently being used to improve bioproduction processes. Metabolomics profiling of metabolite flux in fed-batch process could guide the design of media and feed supplement schedules.⁴⁶ Data generated from transcriptomics analysis could be used to understand metabolic pathways and optimize cell culture media and the feeding pattern. Schaub and colleagues exemplified the use of transcriptomics for targeted media optimization.

Lipid concentration was increased threefold based on the differential gene expression data of high-titer and low-titer processes, leading to a 20% titer increase.⁴⁷ By using LC-MS based metabolomics approach, some nucleotides/nucleosides and amino acid derivatives were found to be apoptosis inducers, which could help them avoid pitfalls of media design.⁴⁸ Moreover, proteomics and metabolomics have been combined to identify cell stresses associated with four metabolic systems for CHO, enabling the targeted nutrient supplementation with a 75% increase of peak cell density.⁴⁹

Summary and Future Perspectives

This review provided an overview of CHO cell culture media development, covering the nutrient and product quality requirements, media design elements and experimental approaches, as well as media manufacture considerations. While empirical approaches will continue to be used, mechanistic modeling can be helpful in accelerating media development in the future. As two widely used approaches, stoichiometric models and kinetic models can potentially be leveraged to determine all key media components and perform media optimization in silico, significantly reducing experiment workload and resources. Combined with emerging omics, high-throughput, and analytical technologies, mechanistic modeling has a great potential to achieve the optimal cellular performance and desired product quality attributes with incredible efficiency and robustness.

Acknowledgements

The author would like to thank Cary Opel, Gayle Derfus, and Yas Saotome from Gilead Sciences for their review and advice.

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