From its initial development back in the 1950s, cell culture media development has been plagued by impurities, often present in small quantities, difficult to characterize, and trace back to their origins. Classically, culture media have required the addition of animal serum, which serves a nutritional function, but also may absorb and bind up trace impurities that may be inhibitory to cell growth.
However, with higher purity reagents and better characterization it has been possible to concoct a large range of different serum-free media, of special value when proteins synthesized by engineered cells need to be purified from their resident media. This means that it is even more critical to trace minor impurities in culture media.
GEN recently interviewed several experts about issues related to the management of these challenges. The panel included:
Trissa Borgschulte, PhD, senior director and head of bioprocessing upstream R&D, MilliporeSigma
Vince Nguyen, senior upstream process development engineer, Precision Biosciences
Atherly Pennybaker, product applications scientist, InVitria
GEN: Because of its complexity, impurities in cell culture media may be difficult to detect and measure. Trace metals can frequently serve as contaminants and may have significant effects on the quality and quantity of protein production. Does this constitute a problem for you in monitoring your media? And if so, how do you monitor it?
Borgschulte: There are many raw materials in a cell culture media formulation. Although the supply chain for these raw materials remains relatively unchanged over the last 40 years, the way we view the supply chain and the potential for variability of the bioprocess has changed.
The role of trace elements and their impact on protein and product quality is well documented. As such, trace element introduction by formulation addition is critical. Variation in these same critical components has been linked to unintended impurities in the prevailing cell culture raw material supply chains. The first step in understanding and managing variability is acquiring reliable data on these critical trace elements.
MilliporeSigma is committed to providing scientists and engineers with best-in-class lab materials, technologies, and services, so we have been studying the impact of trace elements. We now offer testing on finished products for trace elements, as well as the monitoring of our products and high-risk raw materials within our supply chains.
Nguyen: From a quality perspective, the concern with trace metals is related to the Fenton reaction, in which metal is a catalyst resulting in the formation of free radicals. Counteracting this is the fact that as long as the cells are alive, the cell culture environment is a reducing environment. The result is that for the majority of the time, trace metals are not a problem. Of course, it is never “one size fits all.” Some processes are sensitive or simply not robust and the cells can lose the balance between the oxidative and reduced states in the present of excess trace metal.
Of course, there are many potential sources of trace metals, including lot-to-lot variations in disposables. When there is a deviation in IEX profiles for a process, the source of disposables is one of the first places to where I would look.
Conversely, low levels of certain trace metals can also be an issue. There are publications describing situations where low copper levels can cause lactate production. In that case, the author solved this problem by actually adding trace amounts of copper to shift the environment to a slightly more oxidative state.
The views expressed in this forum are the opinions of the interviewee, and do not represent the policies or beliefs of the company at which he is employed.
GEN: Trace metals are also vital for optimal growth and performance. What approaches do you use in adjusting levels of trace elements, including copper, manganese, zinc, and selenium?
Borgschulte: The source of trace elements in cell culture applications comes from two primary sources: from intended trace metals by formulation addition and from unintended trace metal impurities from raw materials in the prevailing cell culture supply chain. For intended trace metals we have developed controlled processes for adding trace elements at the ppb levels in cell culture media. And for unintended trace metal impurities, we have established monitoring programs for high-risk raw materials to include key sources such as iron salts, as changes to the prevailing supply chains aren’t always possible.
GEN: Contaminants in culture media may arise from varied sources, including water, added serum, and various chemical components. What has been your experience concerning the most frequent sources of contaminated components?
Borgschulte: MilliporeSigma has always aligned with the needs of the industry and tackles the same tough challenges. For cell culture media, there have been no significant changes in the prevailing supply chains. Any observed impurities have always been present. The movement to chemically defined formulations and the sophistication of measuring protein quality attributes have uncovered the supply chain variability as there are no set rules for impurities such as trace elements. What is acceptable as a trace element for one company and its protein may be harmful to another company and its protein.
The best proactive tool is to measure the impurity and then work collaboratively with a customer to develop strategies to manage the variability, noting that changes to a prevailing supply chain may not be possible. Molecule management should include limits of tolerance to the inherent variability of the bioprocess.
Pennybaker: Contamination is not a new problem in this industry. Most frequently we see it coming from the variability of fetal bovine serum (FBS) or human serum which introduces a higher risk of adventitious infectious impurities entering the culture media. It’s well known that lot-to-lot inconsistency of serum and serum-derived proteins exists, but its extent is not fully understood. In some cases, lots of FBS can exhibit greater than a 10-fold difference in known components on top of the potential presence of other undefined substances.
Further, human serum albumin manufacturing guidelines only require 96% purity, opening the door for unknown and potentially biologically active substances and factors to be present, including that of viral contaminants. There have even been outbreaks associated with more complex serum-based products such as plasma protein fraction (PPF) that indicate a differential safety profile in this class of serum-derived components. To reduce contamination risks and high variability, supplementation of recombinant human serum proteins that not only demonstrates more uniform RP-UPLC peaks between lots but also eliminates the need for virus-specific testing poses as a very attractive solution.
GEN: Interestingly enough, water can actually be overly purified, in which case it can be reactive, and drive the leaching of toxic chemicals from the processing vessels used in media preparation. Have you ever encountered this unusual situation, and what steps did you take to avoid it or deal with it?
Borgschulte: Water in its purest form is ionic in nature and can be reactive. MilliporeSigma follows the same global compendial standards for industrial water usage as either water for injection for liquid preparations and/or purified water for dry powder cleaning rinse standards. All liquid formulations are built with buffer systems, which, in our experience, have not been an issue in terms of reacting with process vessels. Liquid solutions have quality standards for pH and osmolarity and shifts in these key attributes would indicate a problem.
Equally, all bioprocess organizations, including MilliporeSigma, establish internal standards for passivation/re-passivation, select top-grade cleaning agents which can reduce the need for re-passivation, and establish the appropriate standards for qualification/re-qualification of equipment and processes. Overall, this topic has not been an area where we see concerns.
GEN: Endotoxins, products of gram negative bacteria, are also a frequent issue in culture media preparation and monitoring. Can you discuss your experiences with endotoxins: detection, treatment, troubleshooting?
Borgschulte: Endotoxin monitoring and management are key to quality cell culture manufacturing. Our company follows the same global compendial testing for endotoxin measurement and establishes the appropriate monitoring and control practices of incoming high-risk raw materials, key process steps, and outgoing finished products.
Pennybaker: This is a great example of the benefit of testing individual cell culture components for endotoxin in order to have control of their level in the complete cell culture media. For our final recombinant protein products, we utilize a limulus amebocyte lysate (LAL) kinetic chromogenic endotoxin assay. We have found this method to be best in its ability to not only determine whether endotoxins are present but to also reveal how much is present. This level of quantitative data helps significantly with QC and QA control and can also be advantageous for troubleshooting when endotoxin issues arise in culture media that can be traced back to a single component.
From a manufacturing standpoint, having this level of control when formulating a completely chemically-defined media results in a final cell culture media product of unrivaled quality for the end user.
GEN: Free radicals can be generated from certain types of media exposed to fluorescent lights. How do you deal with this problem?
Pennybaker: The most important point in the problem of free radical generation is that prevention is key. Simple steps such as placing reminders around the lab or directly on the media bottles to keep out of light and aliquoting smaller working volumes can go a long way in successfully reducing the amount of light exposure. Serum-free media are even more fragile in this respect and so it becomes crucial for the operator to follow good cell culture practices to alleviate this problem. Although proactive steps are important, oxidative stress and the generation of free radicals is an imminent threat in cell culture technology. These free radicals can impede cellular antioxidant defenses making it critical to supplement the media with other sources of antioxidants that can support those functions. Common oxygen scavengers such as ascorbic acid (Vitamin C), glutathione, uric acid, or even albumin are often used for this purpose.
Chemically-defined media become a great tool in this case because it is known exactly what components are contributing to prevent damage from free radicals and how those affect the overall performance of the culture.
Borgschulte: Where necessary, we label finished goods and in-process materials with a “Protect from Light” and note established processes and procedures to support the requirement.