April 1, 2014 (Vol. 34, No. 7)

During the early 2000s, biomanufacturers believed that the industry was on the verge of a shortage in manufacturing capacity. As companies planned for purchases of 10 and 20 kL stainless-steel bioreactors, process developers were improving titers by factors of 2, 5, and 10, to the point where the “capacity crunch” never materialized.

Experts such as Dr. Florian Wurm at Switzerland’s Polytechnic University of Lausanne attributed the bulk of volumetric productivity improvements to the emergence of advanced media products and feeding strategies.

As production-worthy cell culture media continue to improve, bioprocessors are enjoying process intensification, even higher titers, and access to diverse upstream single-use production vessels.

Cell culture-based processes have seen a shift in focus, from maximizing individual unit operation to an emphasis on overall process efficiency. “This represents a distinct trend upstream,” says Morgan Norris, general manager for upstream products at GE Healthcare. “Five years ago, we were still worried about titer. Today, the concern is on overall process yield and quality, as well as the culture media constituents that affect the efficiency of downstream steps.”

For GE Healthcare and other media vendors, this concern translates to the need to design upstream processes to make purification more efficient.

A flip-side challenge, as the industry moves toward flexible manufacturing, is that processes occur at relatively modest volumetric scales. This difference is offset, to a significant extent, by higher titers. With single-use bioreactors stalled at around 2,000 L, bioprocessors exploiting flexible manufacturing strategies must retain economic viability when scaling single-use systems. “We must also assure that we’re not simply pushing up the yield and kicking problems down the road to downstream operations,” Norris adds.

Understanding a culture medium’s raw material ingredients has become a major goal for large-scale biomanufacturing. A team at SAFC headed by Kevin Kayser, Ph.D., characterizes media ingredients to satisfy the market’s regulatory, safety, and sourcing concerns. “As an industry, we need to get a lot better at deciding where we source raw materials and knowing how they’re made,” Dr. Kayser says. More interestingly, his group investigates how media ingredients affect product quality. “As we learn more about CHO biology, we’re discovering how a medium’s raw materials affect design specifications for cell culture processes.”

Simplifying media has been one way to achieve tighter quality control. At one time, culture media routinely consisted of 90 or more components. SAFC has reduced that number to about 65 with no impact on performance. “We’ve gotten good at knowing what we can take out,” Dr. Kayser adds. This work has, in turn, revealed which components are responsible for quality attributes such as glycosylation.

An obvious outlet for this insight is biosimilars, where process details for the originator molecule are sketchy at best. Dr. Kayser asserts his group can now control a protein’s quality attributes by manipulating raw materials in the media formula. This has involved “lots of science” that resulted in fine-tuning, through control of post-translational modifications, of therapeutic half-life, efficacy, and dosing. “Bioprocessors used to believe that these attributes arose from clone selection and specific parental cell lines, but we’ve learned that a good deal of control is available through media composition. And these benefits do not affect yield,” Dr. Kayser concludes.


SAFC strives to ensure that all media and feeds are readily scalable to bioreactors.

Process Simplification

For Joerg von Hagen, Ph.D., R&D director for upstream cell culture media at Merck Group, the major issues in cell culture media development are developing robust large-scale bioprocesses by simplification, achieving similarity to originator molecules for biosimilars, and dealing with the variability of raw materials like poloxamer (known under several trade names).

Poloxamers are nonionic, amphiphilic, surfactant-like triblock copolymers used as shear stress-relieving agents in many fed-batch processes. In addition to preventing cell disruption from the agitation mechanism, poloxamers are believed to improve oxygen transfer and help cells avoid being swept up by gas bubbles into the reactor headspace, where they may be subjected to reactive oxygen species.

The additives work as advertised in liquid media, which are principally the domain of R&D and basic research. They also do their magic in large-scale processes based on dry-formulated media, but with unacceptable variability, which seems to stem from variability in the raw material itself.

“The disconnect between small- and large-scale processes affects bioprocess scale up,” Dr. von Hagen says, “but nobody knows the root cause.” It remains unclear, he notes, why different batches have such divergent susceptibility to shear stresses and likely oxygen transfer characteristics.

BASF is the main supplier of branded poloxamer products, but company prepares these materials for use outside biotechnology. Thus, BASF’s product specifications are not directly meaningful for discriminating nonperforming batches from lots that perform at large scale.

With poloxamer concentrations in reconstituted media around 1g/L, and other solid ingredients in the 15–25 g/L range, the additives represent a significant fraction of a medium’s ingredients. Poloxamer variability is manifest in loss of viable cell density. “The level of unpredictability is not acceptable,” Dr. von Hagen explains. “Bioprocessors talk about quality by design and process analytics, but how can you achieve them with so much variability from one media ingredient?”

With respect to process simplification, Dr. von Hagen points to feed addition as a significant area for improvement. Fed-batch CHO processes undergo multiple, separate feeds for glucose; amino acids and vitamins; and two specific amino acids, tyrosine and cysteine. The latter do not easily solubilize at process-friendly pH, so processors must add them at pH 11.5. Even with mixing times of a minute or less, pH “hot spots” form to the detriment of pockets of cells. Although initial cell death is relatively unremarkable, this event sets off a cascade of events that may seriously affect the batch.

For example, dead CHO cells release proteins that promote cell-cell aggregation, which in turn affects the rheologic behavior of the batch. Adjusting the tip speed upward to accommodate the higher overall “viscosity” kills additional cells through shear forces, leading to a vicious cycle.

Ideally, one would be able to solubilize tyrosine and cysteine at close to neutral pH. Gaining this ability requires the development (as Merck Group is attempting) of modified amino acids with enhanced solubility.

1 + 1 + 1 + 1 = 2: Media, Equipment Companies Rearrange Assets

As you read this, GE Healthcare is in the process of acquiring the Hyclone cell culture media and sera business from Thermo Fisher Scientific, which will retain Hyclone’s single-use bioprocessing business.

GE Healthcare’s Morgan Norris sees the Hyclone deal as “a really good fit for GE.” With its earlier acquisition of PAA Laboratories, GE gained its first experience with cell culture and a greater appreciation for cell culture within the context of GE’s downstream product lines. Then, through the merger with Xcellerex, the “big picture” for GE expanded further, encompassing single-use, stirred-tank bioreactors and related products and process knowledge. GE currently maintains a small-scale manufacturing facility at Malborough, where it conducts process development and early-stage clinical manufacturing for customers.

“These acquisitions have brought us considerable additional expertise across the biomanufacturing landscape, but in particular on how the individual pieces—cell lines, bioreactors, and media—fit together,” Norris says. “Hyclone’s reputation as a top media company helps us to provide content and context for our hardware business, not to mention greater scale and depth of understanding.”

On February 3, Thermo completed its acquisition of Life Technologies for cash and debt assumption totaling $15.1 billion. Over the years, Life Technologies had expanded well beyond culture media to include next-generation sequencing, instrumentation, reagents, diagnostics, antibodies, and consumables through such well-known brands as Invitrogen, Applied Biosystems, Molecular Probes, Ion Torrent, and Novex.

The two deals are related in an interesting way. Thermo had apparently planned to retain the media and serum businesses of both Hyclone and Life Technologies’ Gibco brand. But regulators saw that as anticompetitive. Thermo Fisher was forced to reinstate the balance of power by divesting itself of Hyclone while retaining Gibco. It will be interesting to see how the two megacompanies handle the growing “verticalization” of their cell culture and media businesses.

Serum Shortages?

For years, production-level cell culture has increasingly relied on serum-free, reduced-serum, and chemically defined media. Initially, bioprocessors were concerned with the risk of infectious agents from animal-derived media components ending up in their processes. More recently, manufacturers came to value the consistency that came with precisely formulated media.

Now, notes Jason Walsh, business director for media at Corning Life Sciences, an unexpected factor is coming into play that makes serum-based media even less attractive.

“Serum prices have gone up substantially due to global shortfalls,” Walsh explains. One would assume the issue would be loss of serum component manufacturing capacity, but that is not the primary driver. Walsh notes that drought has caused U.S. and global cattle herds to decline in number to near-historic lows. “Media formulators are the first to feel that price pressure. They have no choice but to pass the added costs onto their customers.” As a result, Walsh predicts demand for alternatives to serum-based products will accelerate.

Since its 2011 acquisition of MediaTech, Corning, which was better known for cell culture-related labware, has invested steadily in providing new serum-free media, which Walsh describes as the future of cell culture media. Examples of the company’s efforts are the Corning® stemgro® hMSC Medium (for mesenchymal stem cells derived from cord blood, bone marrow, or adipose tissue) and Corning® hybrigro™ SF Medium (for monoclonal antibody production from hybridoma cells).


Media, packaged in single-use bags, can be used to fill Corning® HYPERStack® vessels

Keeping Primary Cells “Happy”

With the economic focus of cell culture media development residing in biomanufacturing, it becomes easy to overlook volumetrically smaller (but nevertheless lucrative) cell therapy markets for media.

Primary cell cultures have a limited lifespan, but their potential utility in cell-based therapy and regenerative medicine has revived interest in primary cell culture media. According to Hermann Bohnenkamp, Ph.D., business area manager for Miltenyi Biotec, his company hopes to provide defined media that will make primary cell therapy a reality. “To make this possible, you have to keep primary cells happy ex vivo, which is why primary medium is so important.”

Given the diversity of primary cells, this appears to be a daunting task. Miltenyi takes a modular approach that consists of a limited number of basal media recipes, to which specific supplements are added. This strategy is familiar to individuals engaged in large-scale bioprocessing, where vendors have simplified media recipes over the years while becoming increasingly reliant on basal media plus supplementation.

“This creates a platform for serving the great diversity of primary cells with a limited number of media,” Dr. Bohnenkamp tells GEN.

Miltenyi’s strategy is similar for primary cells used in drug screening and for cell-based therapy, although the latter appears to be in the company’s crosshairs. Another target is creation of high-performing primary media for research efforts, which easily translate to a clinical or translational medicine setting. And under GMP, these media will pose less regulatory uncertainty and be easier to characterize.

High-quality media is a must at every level, Dr. Bohnenkamp explains, yet traditionally research scientists have not been overly concerned with quality. “We’d like to supply media of similar quality for research and clinical use—products that are easy to scale from benchtop to bedside.”

FBS and Traceability

Fetal bovine serum (FBS) is the media additive for many mammalian cells grown in vitro for a wide range of biomedical applications, each of which carries a different potential for adverse effects stemming from the use of FBS. To effectively manage this issue, classic risk-management strategies, such as quality testing, must be expanded to include geographic source verification, or traceability.

“Traceability is the sole quality parameter that cannot be determined by testing.  Global geographic variation in prevalent bovine diseases, or adventitious agents, allows for risk reduction by restricting purchases to those geographic areas with the most limited disease/agent profiles,” says Rosie Versteegen, CEO of the International Serum Industry Association (ISIA).

For this reason, FBS from New Zealand and Australia is priced higher when compared with U.S. and Canadian origins. Further, serum from most South American countries costs significantly less than these four countries. Various pharmaceutical applications consume serum in large volumes resulting in a price stratification due to their demand for FBS from preferred geographies. “These significant price differentials reinforce the need for vigilance in assessing accuracy of origin,” adds Versteegen.

To that end, ISIA recently implemented traceability standards and an auditing system to verify the authenticity of the country-of-origin claims made by any serum supplier.

“This program sets industry standards for the documentation trail and product preparation from the abattoir to the buyer, based on a rigid program of third-party audits that verifies standards compliance,” continues Versteegen.

Although most of the procedures used in routine supplier audits will work with serum suppliers, a thorough audit of the traceability system is not routine. Information on audit procedures that are customized for serum suppliers is available on the ISIA website.

“ISIA Traceability Certified status is awarded to suppliers who adhere to ISIA guidelines and are the subject of a successful audit,” explains Versteegen. “Hence, additional measures of safety and supply-chain confidence are obtained by requiring that suppliers are ISIA Traceability Certified.

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