The Medium Is the Message: Considering the Effects of Trace Metals on Cell-Culture Performance

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Since the 1990s, Chinese hamster ovary (CHO) cell lines have served as the biopharmaceutical industry’s “workhorse” for producing recombinant proteins, especially monoclonal antibodies (mAbs) and derivative formats. Process and product knowledge accumulated over the past 30 years has enabled vast improvements in expression titers and yields. However, development scientists still have much to discover about optimizing cell culture, especially regarding maintenance of product-quality targets for processes involving high-producing cell lines. Aside from selecting clonal cell lines with particularly advantageous characteristics for both quality and yield, upstream teams can experiment with different culture-media compositions and supplementation strategies to control levels of cell growth, protein expression, and metabolite buildup.

While moving through the conference circuit, I have heard many presentations about culture-media development. Often, presenters focus on their teams’ efforts to optimize glucose and amino-acid levels. Less often do scientists discuss trace metals. Sometimes metals enter culture processes as impurities from equipment and media components (1). But upstream scientists also can add metals into growth media to modify culture conditions (1).

At the March 2024 BioProcess International West Conference and Exhibition (BPI West) in San Diego, CA, Ryan Graham (a principal scientist in technical development at Genentech) explored that approach in “Achieving Product Quality Targets While Maintaining High Titer in CHO Cell-Culture Processes.” The talk chronicled a Genentech team’s optimization of three CHO-based culture processes. One of those cases required optimization of trace-metal concentrations in a culture medium.

From that presentation and subsequent correspondence with Graham, I learned about how significantly a medium’s trace-metal composition can influence a target protein’s critical quality attributes (CQAs) (1, 2). Moreover, because trace metals are present in extremely low (submillimolar) concentrations in culture media, they are susceptible to variability — and even slight variations in their levels can manifest as inconsistencies in product quality (1, 2). In our discussions, Graham highlighted the importance of developing a holistic understanding of growth-medium components considering that a trace metal can enhance or mitigate the effects of other metals and raw materials in a formulation (3).

Before joining Genentech in 2020, Graham was an Oak Ridge Institute for Science and Education (ORISE) research fellow in the Division of Product Quality Research at the US Food and Drug Administration (FDA) in Silver Spring, MD (2018–2019). Under the direction of Seongkyu Yoon, he earned a PhD in chemical engineering from the University of Massachusetts at Lowell, where his research focused on the impacts of trace-metal variability on CHO-culture performance.

Metals Matter

Your BPI West presentation included a case study on optimization of trace metals in culture media. What concern was your team trying to address, and how did you go about that work? Our primary focus was to reduce the percentage of acidic charge variants to maintain our therapeutic protein’s charge profile within a target range. Glycation, oxidation, and other posttranslational modifications (PTMs) can yield acidic charge variants. Generally, those modifications involve reduction–oxidation (redox)–active residues and raw materials. Certain metals such as iron and copper are redox active and thus can create culture environments that promote PTMs (4, 5).

With that in mind, we knew that optimizing trace-metal content in our media would be helpful. Antioxidants can alleviate oxidative stress, so coupling metal optimization with antioxidants gave us a unique strategy that helped us achieve our goal. At the same time, we wanted to keep expression titers high. It was important to ensure that optimizing our media for product quality did not compromise a high-titer process. We developed a media-optimization strategy that enabled us to meet our product-quality and titer metrics.

How does your team generally approach media development? One of our most important considerations during media development is to understand a cell line’s specific needs. All CHO cell lines require some level of amino acids, metals, vitamins, and so on to perform well in culture. But those needs can change across cell lines, especially when those lines host molecules that are more difficult to express than standard mAbs are.

We leverage laboratory work, data analysis, and colleagues’ historical knowledge about our processes to learn as much as we can about our cells. And by getting a clear sense of how a cell line is expected to perform, we can make better and more informed decisions regarding which components to include in a media system, what concentrations to supply, and what times would be optimal to feed our culture. Getting our cells to reach their full potential hinges on understanding their specific needs.

What role do trace metals and related components play in cell culture? Metals play a significant role in culture performance. In one way or another, trace metals strongly influence cell growth, productivity, glycosylation patterns, charge profiles, and energy metabolism — just to name a few parameters from a comprehensive list (1–8). As far as culture processes, all trace metals have a similar story: They bring both benefits and limitations depending on what concentrations are supplied and what cell lines are applied. The key to landing on optimal trace-metal concentrations is understanding what purposes those metals serve.

What trace metals are of greatest concern during media optimization? What trace metals merit more attention than they usually receive? In literature and conference presentations on the topic, you will find a special focus on iron, zinc, copper, and manganese because they have well-understood effects on cell growth, expression titer, and other product-quality metrics during CHO cell culture. Other metals that are commonly included in cell-culture media also have important roles to play. For example, calcium and magnesium are cofactors for various enzymes in the tricarboxylic acid (TCA) cycle (9, 10). Potassium is known to influence membrane integrity and to help regulate calcium transport (11). Selenium’s antioxidant properties also can be favorable for cells (12). A handful of other metals serve as cofactors for enzymes related to processes from energy metabolism to glycosylation.

Although metals that directly manifest in key performance indicators (such as titer and product quality) tend to get more exposure in the literature, it is important to recognize all of the ways that the different metals we supply to culture media are helpful for CHO cells.

In recent articles, you and your coauthors note that scientists generally have reported about the effects of individual trace metals on cell culture but not about those components’ synergistic effects (2, 3). Why should trace metals be examined in tandem, and what kinds of investigations still need to happen in that area? Different metals can participate in similar functions. When you change a particular metal’s concentration in a culture medium, cellular responses can depend on concentrations of other metals present. Oxidative stress is a good example of that phenomenon. Both copper and iron can yield reactive oxygen species (ROS) via the Fenton reaction. If improperly balanced with antioxidants, ROS can cause oxidative stress and damage organelles, lipids, and DNA. Reducing iron concentrations might have little effect on Fenton-induced ROS generation if copper concentrations are already high — and vice versa. On the other hand, zinc is an antioxidant that is known to stabilize sulfhydryl residues that are prone to oxidation. So we already have three different metals to consider.

It can be helpful to understand how such metals relate to one another, especially during titrations. I noticed that available literature sometimes has left that part out of the discussion. In my 2019 review, I tried to highlight some of those gaps (3).

Collaborative Culture

What kinds of concerns arise during optimization regarding media quality and consistency? We expect to run high-producing cell cultures consistently and reliably across our network by designing robust processes. Media development is no different. The trace-metal composition in our media systems is designed to maximize the performance of our cell lines. But a media system is not really optimized if it is not robust. So we ensure that robustness is a key focus during all of our media-development efforts.

My impression is that some bioprocess suppliers provide proprietary and customized media formulations. In your experience, how greatly does that factor influence how end users can test and optimize their culture media? Yes, some suppliers provide proprietary and custom media formulations. However, off-the-shelf media also have intellectual property (IP) restrictions that can complicate media-development efforts. That is why many companies have established their own media-development teams. There are situations in which acquiring vendor formulations is appropriate, and in those cases, it is important for vendors and end users to establish collaborative relationships so that they can customize a media formulation to a cell line’s particular needs. Transparency from both parties is mutually beneficial here.

What bottlenecks remain in media-optimization workflows, and how might those concerns be reduced or eliminated? Thankfully, useful resources are available to scientists who work in media development. We have instruments that are well suited for at-line, in-line, and off-line analyses of cell-culture samples. New modeling techniques and data-analysis tools are helping us to understand our cell lines and our processes. So not many bottlenecks remain in terms of acquiring and analyzing data for media development.

I greatly appreciate working with colleagues who can share their experiences with media and process development. There is so much information to glean from the collective experiences and expertise of colleagues who have worked with different cell lines, media systems, and production processes. That kind of knowledge can be instrumental for media-development work. Of course, we only have so much bandwidth; I suppose that is a bottleneck. But so much value can come from leveraging diverse perspectives, and Genentech fosters a collaborative environment. That is a unique part of our culture and yet another reason why I enjoy working here.

What else should scientists know about culture media and processes for their testing and optimization? As with trace metals, upstream scientists need to consider the relationships among all culture-media components. CHO cells can interconvert certain amino acids, some of which can increase or decrease the stability of other media components. A vitamin might be more or less sensitive in the presence of other raw materials. Those examples serve as a reminder that media development should consider not only direct relationships between a given raw material and titer or product quality, but also relationships that your raw materials share and how they might work together to maximize culture performance.

I also would like to underscore some of my earlier points, especially that taking the time to understand a cell line’s specific needs will lead to a high-performing cell culture. Robustness should always be a primary focus during media development. And although bioreactor runs, sample analysis, and data reviews are critical for media-development efforts, leveraging diverse insights from colleagues enables us to have rewarding experiences along the way.

References

1 Prabhu A, Gadgil M. Trace Metals in Cellular Metabolism and Their Impact on Recombinant Protein Production. Process Biochem. 110, 2021: 251–262; https://doi.org/10.1016/j.procbio.2021.08.006.

2 Polanco A, et al. Trace Metal Optimization in CHO Cell Culture Through Statistical Design of Experiments. Biotechnol. Prog. 39(6) 2023: e3368; https://doi.org/10.1002/btpr.3368.

3 Graham RJ, Bhatia H, Yoon S. Consequences of Trace Metal Variability and Supplementation on Chinese Hamster Ovary (CHO) Cell Culture Performance: A Review of Key Mechanisms and Considerations. Biotechnol. Bioeng. 116(12) 2019: 3446–3456; https://doi.org/10.1002/bit.27140.

4 Yuk IH, et al. Effects of Copper on CHO Cells: Cellular Requirements and Product Quality Considerations. Biotechnol. Prog. 31(1) 2015: 226–238; https://doi.org/10.1002/btpr.2004.

5 Weiss CH, Merkel C, Zimmer A. Impact of Iron Raw Materials and Their Impurities on CHO Metabolism and Recombinant Protein Product Quality. Biotechnol. Prog. 37(4) 2021: e3148; https://doi.org/10.1002/btpr.3148.

6 Brühlmann D, et al. Tailoring Recombinant Protein Quality by Rational Media Design. Biotechnol. Prog. 31(3) 2015: 615–629; https://doi.org/10.1002/btpr.2089.

7 Graham RJ, et al. Zinc Supplementation Improves the Harvest Purity of β-Glucuronidase from CHO Cell Culture by Suppressing Apoptosis. Appl. Microbiol. Biotechnol. 104(3) 2020: 1097–1108; https://doi.org/10.1007/s00253-019-10296-1.

8 Kim BG, Park HW. High Zinc Ion Supplementation of More Than 30 μM Can Increase Monoclonal Antibody Production in Recombinant Chinese Hamster Ovary DG44 Cell Culture. Appl. Microbiol. Biotechnol. 100(5) 2016: 2163–2170; https://doi.org/10.1007/s00253-015-7096-x.

9 Pérez-Rodriguez S, et al. Nutrient Supplementation Strategy Improves Cell Concentration and Longevity, Monoclonal Antibody Production and Lactate Metabolism of Chinese Hamster Ovary Cells. Bioengineered 11(1) 2020: 463–471; https://doi.org/10.1080/21655979.2020.1744266.

10 Kim WH, et al. Effect of Ca2+ and Mg2+ Concentration in Culture Medium on the Activation of Recombinant Factor IX Produced in Chinese Hamster Ovary Cells. J. Biotechnol. 142(3–4) 2009: 275–278; https://doi.org/10.1016/j.jbiotec.2009.06.001.

11 Wang SB, et al. Manipulation of the Sodium-Potassium Ratio as a Lever for Controlling Cell Growth and Improving Cell Specific Productivity in Perfusion CHO Cell Cultures. Biotechnol. Bioeng. 115(4) 2018: 921–931; https://doi.org/10.1002/bit.26527.

12 Zhang J, Robinson D, Salmon P. A Novel Function for Selenium in Biological System: Selenite as a Highly Effective Iron Carrier for Chinese Hamster Ovary Cell Growth and Monoclonal Antibody Production. Biotechnol. Bioeng. 95(6) 2006: 1188–1197; https://doi.org/10.1002/bit.21081.

Brian Gazaille, PhD, is managing editor for BioProcess International (part of Informa Connect Life Sciences); [email protected]; 1-212-600-3594. Ryan Graham, PhD, is a principal scientist in technical development at Genentech, a member of the Roche group, in South San Francisco, CA.