Elemental Impurities in Cell Culture Media: A Case Study

Elemental Impurities in Cell Culture Media: A Case Study

Why are cell culture media important?

A cell culture is developed when cells, tissues, or organs (whether animal or plant in origin) are placed into artificial environments conducive to their survival and/or proliferation.1 Cell culture technology is used in the manufacture of biologic drugs (or “biopharmaceuticals”) and vaccines as well as in assessment of drug efficacy and toxicity. When manufacturing biopharmaceuticals, it is absolutely crucial to minimize variables, maximize yield, simplify purification, and meet regulatory requirements. The industry has been growing more aware of the impact of cell culture media selection on these considerations.

In order for viable and optimal in vitro cultivation of cells, an appropriate growth medium must be selected. Cell culture media are absolutely vital to the productivity, reproducibility, and quality of these bioproduction processes. The projected market for cell culture media and reagents is expected to reach $7 billion in the next few years.2 With the greater variety of culture media available commercially each day, and with heightened competition in biopharmaceutical manufacturing across the world, it is increasingly crucial that we closely evaluate these media.

What is cell culture media?

A typical culture medium is composed of amino acids, vitamins, inorganic salts, carbohydrates (such as glucose), lipids (cholesterol, steroids, fatty acids, etc.), polyamines, reductants (such as 2-mercaptoethanol), protective agents (carboxymethyl cellulose, polyvinyl pyrrolidone, Tween 80, etc.), detergents/surfactants, and serum as a source of growth factors, hormones, and attachment/adhesion factors (fibronectin, laminin, etc).3 There are an extensive number of medium formulations for every application.

Elemental Impurities in Cell Culture Media: A Case Study

Metals in Cell Culture Media

Inorganic salts in the media help maintain pH, retain the osmotic balance, and regulate membrane potential by providing sodium, potassium, and calcium ions.1 The transition metals Co, Cu, Cr, Fe, I, Mn, Mo, Se, and Zn function in the active centers of enzymes and drive protein quality.3 Cu, Fe, Se, and Zn, in particular, must be present at least in micro amounts in most cell culture media to assist with proper cell growth.1,3 Because of the importance of many metals for proteins, trace elements contaminating the water or raw materials have led to some positive developments in cell culture media, such that many trace elements may now be intentionally added to media used for specific purposes.

However, addition of metals must be considered carefully as many promote the production of reactive oxygen species. Trace elements Al, Ag, Ba, Br, Cd, Co, Cr, F, Ge, I, Mn, Mo, Ni, Rb, Se, Si, Sn, Ti, V, and Zr have all been shown to negatively affect the efficiency of cell culture media via the production of reactive oxygen species.3 As such, development work to improve quality or yield is often wrapped up in understanding trace metal impact, which can be costly and time-wasting.

It is generally understood that elemental impurities are ubiquitous in biologically-derived cell culture media. Serum and hydrolysates have undefined compositions, with high variation in contaminants. Many proteins carry trace elements such as nickel, cobalt, or magnesium.

Genetic Engineering & Biotechnology News has a thorough illustration of the complexity of this issue: “For example, iron is a standard medium component, but various grades of ferrous sulfate contain differing quantities of trace metals, among them manganese, which CHO cells require for glycosylation. But that need falls within a narrow range: too little manganese is as deleterious as too much. Combined contributions from impurities present in iron sources and other ingredients may alone supply more than the desired level of manganese”.2

CD Media

The introduction of serum-free, chemically defined (CD) media in the 1970s looked to assist researchers and manufacturers in achieving consistent results with the production of uniform media. CD media has a defined composition, with known components at controlled concentrations. CD media generally does not contain proteins or hydrolysates, and is animal-origin free. Any protein additives (growth factors) in CD media are produced in bacteria or yeast by genetic engineering.

As such, chemical definition can allow for validation of the cultivation process.3 There are many advantages of using CD media, including reduced variability/improved reproducibility between lots and reduction of contamination in upstream biologic drug production. This in turn saves time and effort in purification and downstream processing.

Metals in CD Media

While chemically defined media has many advantages and dodges issues pertaining to complex animal and plant derived proteins, CD media still has problems with trace metals that affect protein quality. As a recent article in Pharmaceutical Technology put it, “Chemically defined does not mean chemically pure.”4 Without the inconsistencies of serum, it has become apparent that other ingredients of CD media, such as vitamin stocks and salts, may introduce concerning elemental impurities. And chemically defined cell culture media may contain more than 100 ingredients, each with the potential to introduce impurities that affect cell culture and/or protein quality.2 In fact, within a serum-free medium, these impurities may have stronger effects on the cultured cells because of a lack of toxin-neutralizing activity.3

Case Study

In order to investigate elemental impurities in chemically defined cell culture media, EKG developed a digestion procedure and inductively coupled plasma-mass spectrometry (ICP-MS) method. The team chose to analyze four similar formulations of Dulbecco’s Modified Eagle’s Medium (DMEM).

DMEM is a basal medium, meaning it contains no proteins and requires serum supplementation to be a “complete” medium. DMEM is prepared with higher concentrations of inorganic salts and vitamins than other media and thus is at greater risk for trace metal contamination.

EKG acquired DMEM from two large and well-recognized suppliers (the two most common suppliers of DMEM referenced in literature, according to a survey performed by Labome1) as well as from two smaller and lesser-known suppliers. EKG was able to obtain precise formulations for the media analyzed/acquired and calculated known quantities of metals present from salts and other metal-containing compounds for comparison against what elements were observed by ICP-MS.

EKG evaluated a number of different types of digestions, generally utilizing various concentrations of nitric acid, hydrochloric acid, and hydrogen peroxide under applications of heat between 4 hours and 24 hours. By evaluating preparations of each DMEM sample spiked with known concentrations of approximately 60 elements, EKG was able to demonstrate consistent recoveries between 95% and 105% for most elements. The ICP-MS method had in-sample limits of detection between 0.15 µg/mL and 0.35 µg/mL in DMEM.

Barium (Ba) was the only elemental impurity observed with a limited daily exposure regulated by the FDA in USP 232/233, and was present in all four formulations. Na, K, Ca, Mg, and Fe were observed at expected levels, according to inorganic salts added in the DMEM formulations. Though not intentionally added to the formulations, Al was distinctly and quantitatively observed in all four formulations. Zn, quantitatively observed in a DMEM purchased from one of the smaller suppliers, was the only element that was significantly present in only one formulation. Rb, Ti, and Zr were observed around the limit of detection in all four formulations. Trace presence of Al, Ba, Rb, Ti, and Zr have all been shown to negatively affect the efficiency of cell culture media (see above).

Element ObservedIntentionally Added to Media?Of Concern?
BaNo (Detected in every sample)USP 232/233 regulated exposure
NaYes (Found at expected levels)No
KYes (Found at expected levels)No
CaYes (Found at expected levels)No
MgYes (Found at expected levels)No
FeYes (Found at expected levels)No
AlNo (Quantifiable in every sample)Could negatively affect the efficiency of cell culture media
ZnNo (Quantifiable in one sample)No
RbNo (Quantifiable in every sample)Could negatively affect the efficiency of cell culture media
TiNo (Quantifiable in every sample)Could negatively affect the efficiency of cell culture media
ZrNo (Quantifiable in every sample)Could negatively affect the efficiency of cell culture media
Additional Work at EKG

This case study was an effective proof of concept that ICP-MS can be used to evaluate cell culture media. If approached by a client, EKG would assist the client in performing/carrying out a risk-based assessment to identify potential elemental impurities of concern. EKG would then tailor this method development to those metals of concern, to continually improve recoveries and instrumental limits. EKG could also expand the scope of a study to include quantification of impurities in both starting raw materials and/or final formulations.

E&L Considerations

In addition to elemental impurities, plasticizers and other leachables have been found in a wide variety of cell culture media.3 Any plastic substances that come in contact with the formulation (including manufacturing components, filters used in sterilization, packaging, etc.) should be evaluated from a risk perspective. In many ways, cell culture media can pose a higher risk for leachables, since supplements such as serum albumin can bind to potential toxins, such as phthalates.3 EKG Labs is quickly becoming a leading name in extractables and leachable studies, and could assist with risk assessments and these types of investigations.

About EKG Labs

EKG Labs is an analytical service provider for the medical device and pharmaceutical industries. Our analytical services support regulatory filings, product development, and analytical chemistry investigations. Specialty analytical services include: extractables, leachables, biocompatibility, chemical characterization, impurity identification, method development, method validation, and other investigational activities. EKG Labs operates out of Innovative Technology Enterprises (ITE) at the University of Missouri, St. Louis (UMSL).

If you are experiencing a challenge with impurities or need analytical services to support your product development, please contact us at info@ekglabs.com or at 810-354-5229.

References:
1Cell Culture Media: A Review (University of Pittsburgh’s Materials and Methods, updated 2019-08-20)
2Cell Culture Cocktails with Fewer Hangovers (Genetic Engineering and Biotechnology News; Vol. 37, No. 3; February 2017)
3Animal‐cell culture media: History, characteristics, and current issues (Reproductive Medicine and Biology; Vol. 16, Iss. 2; April 2017)
4Optimizing Cell Culture Media (Pharmaceutical Technology; Vol. 41, Iss. 8; August 2017)