As a high-value biomanufacturing operation, mammalian cell culture has become the focal point of process improvements that have enabled huge productivity improvements.
Ellen McCormick, director, bioprocess R&D at Pfizer, describes cell-culture process development as mostly science, but part art as well. “At the end of the day we’re working with biological systems that are not fully defined,” says McCormick. “We don’t know everything we would like to know about cellular machinery, so some aspects of cell culture—what works and what doesn’t—are based on trial and error.”
Pfizer’s global biologics facility in St. Louis oversees the development and manufacture of a large portfolio of monoclonal antibodies, peptides, and oligonucleotides. Advanced products in development include CP-751, 871 (monoclonal antibody to IGF1r, insulin-like growth factor receptor 1) and tanezumab (a monoclonal antibody to human nerve growth factor).
Today, cutting-edge work in mammalian cell culture involves defining metabolic processes and interdependencies of various factors affecting cell growth, viability, productivity, and, ultimately, product quality. One is reminded of FDA’s recent initiatives on process understanding, quality by design, and process analytics, which while promulgated separately are, in fact, closely related and share the same goal: well-characterized products.
Biotech has made significant strides toward understanding cell lines and expression systems, their capabilities and limitations, across the manufacture of dozens of biotherapeutics products. Other areas of substantial progress have been the understanding of metabolic systems, cellular physiology, process robustness, physical and mechanical effects like shear, and the ability to employ these tools and knowledge sets at the largest scales. Data mining commercial-scale processes over long periods of time also provides additional perspectives and understanding of manufacturing methods.
“The closer we move toward rational process design, the stronger our mammalian cell culture-based processes will be, and the easier they will be to adapt and change as our knowledge increases. In the end, biotech products will be less expensive because their manufacture is optimally controlled and productive,” McCormick notes.
“Biomanufacturers already control processes well, despite the challenge of the inherent heterogeneity of protein products,” she adds, “but we can always do better.”
Despite the good news on rising protein titers and its impact on shrinking bioprocesses, large-scale cell culture is still alive and well. Successful monoclonal antibody drugs have been a major driver.
Productivity, Scale Not Incompatible
Rising titers notwithstanding, large batches carry economies of scale that multiple, volumetric-equivalent batches do not. Cell cultures in the 10,000 liter range and higher pose huge burdens on infrastructure, facilities, equipment, and downstream operations, but biomanufacturers have had a long time to resolve these engineering and facility issues, and for the most part have done so quite well.
Lonza Biologics, a typical large-scale biomanufacturer, operates cell culture vessels of up to 20,000 liters in volume but nevertheless continues to improve its cell lines for greater productivity. Lonza’s U.K. facility, which produces monoclonal antibodies and other biotherapeutics, has developed a variant of CHO cells that grows spontaneously in suspension in chemically defined media, with growth characteristics that support high productivity as well.
“A lot of effort goes into finding ways, through genetic engineering, to improve growth and productivity in CHO cells,” notes John Birch, Ph.D., CSO for biopharmaceuticals.
Lonza’s protein-expression system uses glutamine synthetase as the selectable marker, which routinely leads to productivity levels of several grams per liter.
“We expect those productivities will continue to increase,” says Dr. Birch, adding that yields of 10 to 20 grams per liter should be attainable within the next few years.
Biotech’s successes have not just caused processes and corporate cell-culture capacities to grow, they have also led to vastly improved processes, particularly with respect to cell lines and protein expression.
When asked to choose the area of most significant progress in cell culture, Dr. Birch immediately points to media and feed strategies as primarily responsible for the 100-fold rise in volumetric productivity over the last 20 years. Many experts, such as Florian Wurm, Ph.D., at the Ecole Polytechnique Fédérale, agree. Lonza has put considerable effort into designing and optimizing media and feeds for fed-batch processes.
The ability to engineer cell lines for product quality as well as productivity has been one of biotech’s great successes. In addition to programming-in high titers, one can now modify a CHO cell’s glycosylation pathways to produce the desired post-translational modifications for specific protein therapeutics.
More than Media
Laurie Donahue-Hjelle, Ph.D., director of cell line development at Invitrogen, agrees that media and feed strategies have been instrumental in raising protein titers, but only to a point.
“If you want the largest improvement possible, you can’t ignore the cell line itself. Cell-line optimization gives you the best chance of success because getting the most out of inherently low-producing cells is going to be tough,” she adds.
Cell-culture improvements occur in cycles, says Dr. Donahue-Hjelle, with some disciplines more productive than others at any given time. “We have just gone through a cycle that relied heavily on improvements in base media and feeds. While there were certainly significant efforts in cell-line engineering going on, the translation of breakthrough academic research has been slower to translate into our space,” Dr. Donahue-Hjelle says.
“I think we are now entering an era where we can begin to make more mechanistically driven advances in our cell-line engineering.”
Significantly improving productivity will require raising the bar in vector and host cell systems, she says, by a better understanding of the cell biology underlying processes such as protein folding and secretion. “We will need to make larger advances in these areas to reach the twenty to thirty gram per liter titers people are talking about. The only way to make that leap is to re-engineer the cells,” she notes.
Re-engineering based on subtle mechanisms such as folding and secretion addresses the issue of quality as well as productivity. Enzymes that carry out transcription, translation, post-translational modifications, folding, and other critical quality-related operations have an optimal catalytic rate but an innate error rate as well. Simply upregulating the appropriate genes may in fact introduce more mistakes that cause the product to be less homogeneous, and perhaps less valuable as well.
Cell-engineering and feed/media strategies are inter-related rather than independent variables. The enzymes discussed require cofactors like vitamins and salts and, in many cases, feedstock such as sugars and amino acids. Thus media/feeds fuel not just the cell’s metabolic activity, but support specific pathways of bioengineered protein production as well.
Lean, Mean, Productivity Machine
Biomanufacturing is growing leaner from an operational standpoint, says Bruce Lehr, director of marketing at SAFC Biosciences. The lean imperative has led to streamlining at every stage and level of manufacturing processes.
“Customers increasingly ask for applied solutions. For example, reconstituting media and buffers into concentrates, supplying them in disposable packaging, and integrating into the customer’s process to save time, labor, and cost,” says Lehr. SAFC performs significant work for customers on simplifying feeds, combining multiple feeds into one product, and providing highly concentrated feed or media solutions from standard formulations.
SAFC’s imMEDIAte ADVANTAGE™ service, for example, creates made-to-order cell culture media, buffers, concentrates, and supplements, and supplies them at smaller minimum volumes than is typical for large-scale manufacturing. Most imMEDIAte ADVANTAGE orders ship within 10 working days of receipt.
The push from 2–3 grams per liter protein titers to 5 and 10 g/L and beyond will require a confluence of high-producing cells, optimized media and feed strategies, and advanced bioreactor technologies. “All of these must work together to enable the massive titers expected over the next few years,” Lehr notes.
For now, collaborations between SAFC and bioreactor companies are limited to the disposables arena. SAFC has been working with Advanced Scientifics on prefilled bags containing concentrates, buffers, and media, for pilot-scale processes.
The trend toward animal-derived component-free (ADCF) ingredients in mammalian cell culture continues, as does usage of chemically defined media. With these advances comes greater concerns over raw material purity, where ingredients are sourced, and an interest in supply chain integrity that is usually reserved for finished pharmaceutical products.
One of SAFC’s recent service offerings is a CHO media and feed library consisting of more than 20 unique animal component-free, chemically defined formulations that cover nutrient requirements for a wide range of CHO cell lines. Customers can screen cells against each of the media and use the products as-is, or ask SAFC to optimize them for their specific cells.
Lehr does not claim that the library will optimally cover every CHO cell’s requirements for maximum growth and productivity. “Individual clones can vary quite a bit, even among those from the same parental cell, depending on what proteins are expressed,” he says.
SAFC has been marketing ADCF long R3-IGF (insulin-like growth factor), which replaces insulin as a growth factor in mammalian cell culture. Insulin manufacturers, says Lehr, typically focus on therapeutic markets, relegating insulin for cell culture as an afterthought. This product addresses the particular need for growth factors in cell culture and is produced independently of the much larger therapeutic market. Novozymes manufactures the IGF product for SAFC.
Shorter time-to-market drives a good deal of cell-culture work these days, according to Stephen Gorfien, Ph.D., director of bioproduction media products and services at Invitrogen.
Invitrogen serves cell-line development and media/process optimization through its PD-Direct® bioservices division, of which PD-Direct Revolution™ is a key component. PD-Direct Revolution is an advanced cell-engineering technology that accelerates natural genetic evolution for enhancing productivity and optimizing critical cell characteristics such as resistance to high osmolarity or ammonia.
For this service, Invitrogen relies in part on ClonePix FL from Genetix Systems, an instrument that screens thousands of clones, identifies the best producers with good growth characteristics, and robotically picks the most desired clones in just a few hours, according to the company. ClonePix quantifies the cells’ protein secretion (or relevant cell surface markers) through fluorescent probes at up to five wavelengths. ClonePix is compatible with a range of suspension and adherent cells, including hybridoma fusions, myelomas, CHO, HEK293 and mouse embryonic stem cells. Dr. Gorfien describes the instrument as an automated “robotic plating and picking machine.”
Invitrogen is well known for its GIBCO® media and feed products, which is a significant area of the company’s research and cell culture-related services.
Media and Feed Products
Many processes use media and feed strategies that were formulated years ago for the approximate cell line in use but perhaps not for the specific clone in use, or even for the same product. Legacy media/feeds are typically complex, and often not optimal for protein-producing cells. Invitrogen can, at customer requests, deconvolute the media and reconstruct it using components ideal for the particular cell line. For this service Invitrogen uses the StarPLUS automated fluid handler from Hamilton. This instrument can be used to break down formulations into their components, and recombine them to create hundreds of new formulations in a short period.
Another tool Invitrogen employs to select cells and optimize media and process conditions is the SimCell™ high-throughput cell-culture platform from BioProcessors. SimCell is an integrated cell-culture platform consisting of a microbioreactor array, automated cell-culture robotics, and data handling. The system runs hundreds of cell-culture experiments simultaneously at 750 microliters each. Users can control dissolved oxygen, pH, and temperature. “We use SimCell for both PD-Direct client projects and for new product development” says Dr. Gorfien.