Solving the solubility and stability challenges of L-cystine in culture media

Successful biologics manufacturing requires streamlined, intensified cell culture processes that maximize productivity. Key to this goal is optimized media formulation, using high quality supplements at ideal concentrations. Amino acids are critical additions because they boost productivity and offer a host of other benefits. However, some of the inherent properties of these amino acids, create challenges when adding them to culture and can hinder intensification strategies and efforts to streamline bioprocesses.

Low stability and solubility of some amino acids, particularly when added to cell culture media, can negatively impact media formulation, storage stability, process intensification and ultimately overall productivity. For example, L-cystine provides many benefits to cell culture, but exhibits low solubility at neutral pH and is at risk of precipitation at higher concentrations. Thus, these challenges with solubility and stability can limit amino acid bioavailability and result in suboptimal concentrations in bioreactor feeds or perfusion media, resulting in low-performing bioprocesses. To further complicate matters, existing mitigation strategies for amino acids increase cost, add extra processing steps, and extend culture time.

To address these challenges, Evonik has developed a portfolio of chemically defined and highly pure peptides that enable more concentrated and stable media formulations and more efficient cell culture processes. They are already used in many pipeline and commercial programs. The latest addition to this portfolio of peptides is cQrex® KC.

In this Ask the Expert session, we spoke with Martin Schilling, Business Director, Cell Culture Ingredients, Evonik to discuss next generation peptide and how it is improving cell culture processes and increasing productivity.

What is L-cystine and why is it important to cell culture?

L-cystine is an amino acid and an essential component in chemically defined cell culture media. It is critical to supply sufficient L-cystine to obtain high growth and bioprocess productivity, which can ultimately lead to reduced production cost for therapeutic modalities, such as monoclonal antibodies.

How does your new product cQrex® KC help to address the challenge of L-cystine supply in cell culture?

One of the biggest challenges of L-cystine is that it has low solubility in cell culture media at neutral pH. Since, most cell culture It is very difficult to dissolve enough of the nutrient in culture and this can severely limit the productivity and output of the bioprocess. At Evonik, we have developed cQrex® KC to address this challenge and help the industry optimize media formulations and simplfy processing. cQrex® KC is a highly pure, chemically defined peptide that is a highly soluble source of L-cystine efficiently metabolized by cells. It is about 1,000 times more soluble than L-cystine and it also dissolves rapidly in cell culture media, which makes it easy to use.

How does cQrex KC work in bioreactor settings, for instance in small- and large-scale manufacturing?

cQrex® KC can be used to supply enough L-cystine in a pH neutral feed medium. Reduction in the number of nutrient feeds required simplifies the already complex manufacturing process. Since cQrex® peptides are already being used in many manufacturing programs, at clinical scale up through large scale commercial production in the biopharmaceutical industry, we know that they are easily scalable.

In fact, some benefits of the product are expected to be more pronounced at large scale. One example of this is eliminating the separate and highly alkaline amino acid feed that people still often use offers an even bigger benefit at large scale. We have received very positive feedback from several partners and customers testing pre-commercial products with the cQrex® KC over the past one to two years and it scales up nicely.

Does the use of cQrex® KC need to be optimized for each culture?

In general, cQrex® KC can be used as an additive to existing media systems. Evonik provides guidelines and technical support to help customers, so implementing the product is very straightforward. As usual, some parameters might need adjustment. For users who want to fundamentally work on their media platforms to continue to develop these platforms, there is certainly more optimization and experimentation opportunities available. In these cases, we are happy to support customers and have considerable application data to pull from.

What types of cell lines have been tested with cQrex® KC?

We have specifically looked at CHO cells for monoclonal antibody production including several cell lines and feed system process variants. We continue to work with the product to identify benefits and other applications, such as use with viral vectors and cell therapies where we have initial positive data.

For now, the core application is monoclonal antibody production in CHO cells where the media systems and the processes are already highly evolved and platformed. There is still an opportunity to continue to improve these platforms to reduce the cost and improve efficiency of large scale production.

How does cQrex® KC compare to currently available solutions?

Current solutions are not ideal. The industry has expressed a desire for better solutions in terms of solubility and supply and this is why we developed cQrex® KC. We experienced these same challenges when developing cQrex® KC. The challenge is quite fundamental and is related to the chemical properties of this important amino acid. One way to work around this is to use a separate feed for L-cystine, but that presents many challenges. It makes the process much more complex and riskier.  A chemical modification is a good way to increase the solubility of L-cystine at neutral pH, but not every chemical modification makes a good nutrient for cells. The chemically defined peptides  are very natural substrates for cells compared to other chemical derivatives that are also sometimes used.

How do you produce the product and at which quantities can you supply?

In general, cQrex® KC peptides are produced using chemical synthesis from amino acids followed by a purification process. The final product is a highly pure, chemically defined product that meets very tight specifications. Evonik has two sites in Germany that produce the peptides via chemical synthesis and then the final purification and downstream processing happens at their site in France.

Evonik also uses a similar technology to product their injectable grade amino acids for the pharmaceutical industry. These peptides have been produced on several lines at this site at large scale for a long time and Evonik continues to invest in our manufacturing capabilities. This ensures supply for our biopharmaceutical and pharmaceutical customers. Evonik already has the capacity in place to supply commercial quantities for biopharma cell culture applications. This is a product that is available globally and customer support is provided globally.

For more information, please see Biopharma Cell Culture Solutions

About the Expert

Dr. Martin Schilling, Director Cell Culture Ingredients at Evonik

Dr. Martin Schilling is responsible for Evonik’s product portfolio of high-quality cell culture ingredients that are used to improve biopharma cell culture processes. He also manages Evonik’s innovation program that leverages existing core competencies in biotechnology, chemistry and formulation development to address other emerging needs within the cell culture industry. Martin has a strong technical background in cell culture and industrial biotechnology and earned his Ph.D. in biochemistry from the Technical University of Munich in Germany in 2008.


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