Despite the high efficacy and target specificity of biopharmaceutical drugs, there are still multiple challenges associated with achieving efficient production of therapeutic proteins. These include discovering new approaches for maximum protein expression, developing economical, flexible and robust manufacturing processes to maximize product yields, and solving the complex challenges of refolding proteins into their active state.
One area of growing interest to help solve these challenges is trace elements in cell culture media and upstream processing. Trace elements in cell culture media and supplements may promote or inhibit cell growth and protein expression or quality to different levels during upstream processes.
Dr. Nandu Deorkar, vice president of research and development at Avantor, and Claudia Berrón, vice president of global commercial development for biopharma at Avantor, discuss the impact trace metals can have on cell culture media and upstream processing.
Why is it important to understand the impact of trace metals in biopharma production, especially upstream?
Upstream process (USP) in biopharma manufacturing is typically defined as the stage where therapeutic protein molecules are produced, usually by bacterial or mammalian cell lines, in bioreactors. When they reach the desired density for either batch or fed-batch cultures, the material is taken through the harvest process, most often continuous centrifugation followed by depth filtration, in preparation for downstream processing (DSP).
Levels and, most importantly, lot-to-lot consistency of trace elements are critical variables that may affect cell growth, thus all elemental impurities must be closely monitored for all incoming raw materials. Minimal changes at ppb levels of certain elemental impurities can impact glycosylation patterns, reduce target cell growth and, in certain cases, cease growth or impact therapeutic attributes.
How extensively has this issue been studied?
As a part of cell culture media development, the key role of trace elements on adjusting osmolality and cell function has been studied extensively. However, trace elements and their impact on upstream processing are getting more attention, with the focus on how trace metal levels affect protein expression and bioreactor process yield along with investigation and documentation on the impact of low trace metal levels. Over the last several years, biopharma manufacturers and suppliers of cell culture media and upstream supplements and nutrients have started to improve knowledge around effects of lot-to-lot variability of trace metal impurities in upstream processing. Within the low levels of trace metals, the effects of variability in the low levels need to be further studied, given that variability will impact each molecule differently.
How are trace metals introduced in upstream processing?
They can be introduced in multiple ways: Trace elements can be present in the cell culture media, the carbohydrate energy sources (sugars) like sucrose and galactose, and they can be present in materials like sodium bicarbonate that are used to regulate pH factors in bioreactors. There are also biopharmaceutical producers who have determined that introducing mineral elements as supplements can aid in achieving targeted yields by affecting the glycosylation patterns and by impacting the protein folding/unfolding processes in the target molecule.
The trace metals that most impact upstream processing are zinc, aluminum, manganese, molybdenum and iron. To some extent, a second category includes copper and nickel. Those are the most common elements that impact the glycosylation pattern.
What effects can trace metals have on the upstream process?
There are multiple potential effects. As discussed, changes in the glycosylation pattern can occur. In general, glycosylation is difficult to control precisely in mammalian cells as it is dependent on a variety of factors, such as clonal variations, media, as well as culture conditions. Optimization of cell culture media is cell line dependent due to metabolism and nutrient consumption of specific cell lines. Trace elements can impact glycosylation as they can modulate activities of various enzymes and transporters, such as glycosyltransferases, mannosidases and lysosomal hydrolases.
In addition, if the protein being manufactured has reactive spaces that respond to different trace metals or ratio of certain metals, there can be reactions during protein folding and unfolding, like oxidation. In the case of enzyme replacement therapy production, other reactions can occur. The enzyme can have trace metals as part of their intake, which can be replaced by other trace metals coming from external sources like galactose or sodium bicarbonate, causing different reactions that can have an effect on the manufacturing yield.
What levels of trace metals can produce these effects?
Unfortunately, the best answer to this question is, “it depends.” It involves the target molecule, the enzyme, the genetic sequencing being used and, since each biopharmaceutical company uses these elements in unique ways, the trace metal requirements are extremely custom. It’s also important to be aware that the process conditions can affect how the trace metals themselves interact with the target cell during bioprocessing. The cell environment, the outside media and the media temperature can all affect the uptake of the trace elements and how they metabolize in the cells.
At Avantor, we have conducted studies on the impact of the variability of trace iron levels, and we have studied variations in levels between 100 to 300 ppb. In the molecule used for this study, if iron levels exceeded 300 ppb, a change in glycosylation was observed. So those 100 ppb levels are worth investigating and, we believe, worth controlling because of the impacts observed.
How well understood are these effects?
This is an important area of biologic processing that needs further investigation. It is well understood that trace metals impact biologic upstream production, and lower levels are better, yet some level of trace metals is needed. It is understood that there are a variety of sources of trace metals, and there is a growing understanding that variations in the low levels of trace metals can negatively impact upstream bioprocess yields, as well as the quality of the cells harvested. But exactly how and why these trace metals cause these effects has not been significantly investigated.
A further area of investigation is the interaction of two different trace metals with each other and their combined impact on the process. Some proteins have more complex processes, and we have observed that if the ratio of two elements changes – in this example, nickel and copper – then there are impacts on the way the protein operates. Exactly why this happens is still to be studied and understood.
How do these trace metal interactions in the upstream process affect the overall drug production?
Ultimately, improperly controlled trace metal levels impact upstream process yield. Incorrect or inconsistent levels of trace metals introduced into the process through different sources, there can be an impact on upstream yield. For example, if you produce three grams per liter of an antibody, and, out of that, half a gram per liter is not properly glycosylated, then you’ve significantly reduced your upstream yield.
It also impacts your downstream purification process. In the upstream cell culture production process, the goal is to yield the highest amount of the purest material possible. If the purity levels are not optimum, you may need to perform an additional downstream purification process step to meet your overall process yield goals. This adds time and material costs, so, by exerting better control over all the sources of trace metals in the upstream process and identifying the right low levels of those trace metals and then ensuring that those low levels are consistent, this risk of these negative impacts can be reduced.
It’s also important to note that once you have determined the optimum trace metal levels and the interactions of the different trace metals on the cell culture process, the same levels of trace metals need to be present in the cell banking process. If the trace metal concentration in the cell banking step differs from the cell production media, the banked cells could be “shocked” when transferred from cell banks into production.
How should trace metals be addressed, or controlled?
There are two ways in which we can achieve better control. The first is better understanding of how trace metals are fully impacting yields, depending on the cells and the process. This includes performing DOE studies that use materials with fully characterized trace metal levels that are consistent and produced under cGMP processes. If not, there is the risk that as manufacturers scale up from the studies to production levels, results could significantly vary. Knowing what the trace metal levels were in the DOE study and ensuring that materials used when scaling up have the same consistent, low levels would keep upstream yields on target.
This means having a strategy for treating raw materials used for upstream production and defining the acceptable levels of trace metals in all the raw materials that feed your upstream process. This goes beyond just the initial cell culture medium makeup at the start of the process. It should include carbon sources like galactose or dextrose that may be added in large quantities at multiple feed points in the upstream process.
Avantor, for example, produces high-purity low-endotoxin (HPLE) sugars, including sucrose, galactose and trehalose dihydrate. These sugars have been produced using proprietary purification methods and have been characterized to consistently include less than five ppb of trace metals, including zinc, nickel, copper and cadmium.
A manufacturer may be adding other materials just one time at some point in the process, such as amino acids, growth factors and pH control materials. All are potential sources of trace metals that can accumulate; while there has been more work done to characterize trace metal levels in carbohydrate sources, not as much attention has been paid to these other materials.
What outcomes can biopharma drug manufacturers expect with better trace metal characterization and control?
The potential outcome is a robust upstream process that is much more predictable and under control. That means there is no change in the lot-to-lot protein production from the target cell and at much more pure outputs. This can then impact the downstream processes, enabling purification steps to be as streamlined as possible and potentially eliminating a purification step.
Ultimately, understanding and defining the target levels of trace metals means recognizing that they play a role in processes like protein unfolding and glycosylation. It means recognizing that all the materials used in the processes – carbohydrates, amino acids, pH control materials – can contribute trace metals in ways that can cumulatively affect upstream yield and quality. Identifying the trace metal levels and ensuring all your source materials are fully characterized will provide the foundation for the most rigorous control.