Quantitation of individual cell culture media components before batch release for use in a biopharmaceutical process is critical to ensure media identity and consistency. Because of the complexity of these formulations, comprehensive media identification continues to be a challenge.
Cell culture media (CCM) play a critical role in the upstream production of biologic drugs. Media formulations provide the essential nutrients, growth factors, and environmental conditions needed to support optimal cell growth and productivity. The composition of the media directly impacts cell viability, recombinant protein expression levels, and the quality attributes of the biologic being produced, such as glycosylation. Hence, consistent, well-optimized media formulations are essential for achieving high yields, batch-to-batch reproducibility, and regulatory compliance in biologics manufacturing.
Media used in upstream processes are complex formulations, often containing more than 60 components, including vitamins, amino acids, nucleosides, carbohydrates, trace elements, and salts, many of which are sourced from different suppliers. Even minor variations in the quality or concentration of these components can negatively impact cell growth, productivity, and the quality of the final biologic product. To mitigate this risk, media manufacturers must implement rigorous quality control measures for every formulation.
Risk mitigation and regulatory requirements
Despite significant advancements in CCM over the past two decades, driven by innovations in formulation, manufacturing, and quality control, critical gaps remain. One longstanding challenge is media identification and characterization. As biologics manufacturing grows more complex, the need for robust, high-resolution analytical tools to confirm media identity has become increasingly urgent.
The BioPhorum Operations Group (BPOG) emphasized this risk in their 2022 publication , highlighting the potential consequences of using improperly characterized media:
“If the critical components of a medium are not known, then a sub-optimal source or raw material could be used, which could mean the loss of one or more batches. This could be very expensive and have a significant impact on schedule and market supply.”
In addition to operational risk, regulatory compliance is a key driver. The same report reinforces the following:
“It is a regulatory expectation that raw materials received and used in producing drug products…are qualified using a system to ensure that raw materials are of acceptable identity, quality, and purity before use. This supports that the safety, identity, strength, potency, and quality (SISPQ) of a drug product is maintained throughout its manufacturing process.”
To meet these regulatory expectations and mitigate risk, biologics manufacturers require analytical approaches that can reliably verify the identity and consistency of media formulations.
Limitations of current media identification assays
Common CCM release tests, such as endotoxin, osmolality, appearance, pH, and growth promotion, are not adequate for media identification. Because the specifications of these assays are similar across multiple media, they fail to distinguish one medium from another. While growth promotion testing confirms the suitability of a medium for cell growth, it does not guarantee correct formulation. Improperly formulated media may pass growth promotion specifications, but still lead to out-of-trend quality attributes in the final product. A genuine media identity test should distinguish between different media formulations, even if they contain the same components in varying amounts or differ by just a few components.
Partial testing is sometimes used for media identity verification, with a focus mostly on amino acids and, in some cases, vitamins. However, this limited approach leaves a significant portion of the formulation uncharacterized. In most cases, CCM formulations are released based solely on basic quality control parameters such as pH, appearance, endotoxin, and osmolality. This widespread reliance on minimal testing underscores the need for more comprehensive analytical strategies to ensure media identity and consistency.
Currently, there is no single analytical test that offers a fast and comprehensive approach to identifying and quantifying most individual media components at the time of release. Existing rapid media fingerprinting techniques, such as Raman and IR spectroscopy, fail to provide true identification of CCM and their components because the techniques primarily detect signals from the most abundant substances in the media. Consequently, multiple analytical techniques are required to accurately identify and quantify CCM components. This approach is costly, resource-intensive, and inefficient.
LC-MS fingerprinting
There is a need for advanced analytical tools capable of quantifying a broad range of media components in a single assay. Because CCM contains diverse compounds spanning multiple chemical classes and concentrations, from high-abundance salts to trace-level vitamins, liquid chromatography coupled with mass spectrometry (LC-MS) is particularly well-suited for this task. LC-MS offers exceptional sensitivity, specificity, and a broad linear dynamic range, enabling the simultaneous detection and quantitation of numerous analytes in a single run. Its proven utility in metabolomics further underscores its value for comprehensive media profiling. An LC-MS media fingerprinting assay can quantify 100+ media components.
The application of LC-MS fingerprinting for media analysis greatly improves media component coverage (MCC) the percentage of quantified individual components relative to the total number of components present in the medium.
For instance, for one medium containing 81 components, amino acid testing would achieve less than 30% MCC, while combining amino acid and vitamin testing would increase it to 42%. However, LC-MS fingerprinting alone would significantly improve the component coverage to 74%, representing a 32% improvement over amino acid and vitamin testing. When combined with trace element analysis, LC-MS fingerprinting can further enhance coverage to 90%, a desirable coverage for media identity purposes. This data will be published soon.
For some media, LC-MS fingerprinting and trace element testing can achieve 100% component coverage. Therefore, integrating LC-MS fingerprinting, whether alone or in combination with trace element analysis, effectively captures most of the media components that influence cell culture performance and protein critical quality attributes.
In addition to supporting media identity testing, LC-MS generates a wealth of data and insights relevant to broader applications in cell culture media, including root cause investigations of poor media performance and the strategic design of custom formulations tailored to specific process requirements. Application of LC-MS fingerprinting has proven to be particularly effective in resolving complex media issues that would have otherwise required extensive time, effort, and multiple analytical techniques.
For example, in one investigation involving a liquid medium formulation, LC-MS fingerprinting revealed significant degradation of a specific component during manufacturing. The degradation resulted in the formation of a toxic byproduct that adversely affected cell performance. This finding enabled the implementation of targeted corrective and preventive actions to avoid recurrence. In another application that we were involved in, the method was used to develop a custom medium formulation for a customer, resulting in improved cell growth and increased product titer.
A new standard for media quality
LC-MS fingerprinting significantly enhances CCM characterization, offering detailed insights and enabling proactive quality control. However, because of its cost and complexity, it's more practical for media suppliers to integrate fingerprinting into their quality control processes and provide comprehensive compositional data in the Certificate of Analysis. Biomanufacturers can then verify incoming batches with simplified tests, ensuring consistency without redundant efforts.
As biopharmaceutical manufacturers advance drug pipelines and expand development of increasingly complex biologics in a stringent regulatory environment, engaging with a partner that leverages LC-MS-based CCM fingerprinting could be a strategic advantage.
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