Quality and productivity in the manufacture of therapeutic proteins is a function of both nature and nurture: Cellular expression systems and all aspects of the process, primarily but not exclusively cell culture media. Thus optimization of media and feed go a long way toward achieving product in good yield and of outstanding quality.
Andreas Wiesner, global senior product manager for protein manufacturing media at Sartorius Stedim Biotech, believes that media and feed strategies have been most responsible for improvements in yield, titers, and cell density over the past 15 years, especially for CHO cultures, but that the big picture is more complex.
“It’s due to culture media and feed strategies that manufacturing processes are expected to generate at least three grams per liter whereas we were satisfied with protein titers of 500 milligrams per liter fifteen years ago,” he explains.
Next in terms of influence is cell-line development. Much has improved in tweaking CHO cells by developing and selecting high producers and combining them with advanced promoter technology to drive protein expression. Wiesner refers to the innate ability of CHO to produce and modify proteins, and improvements thereto. But cells become self-limiting, even within the context of media/feed improvements, because in the end “a CHO cell is a CHO cell is a CHO cell, and there’s only so much you can do with them.”
Process control, third on Wiesner’s list, finetunes the benefits of the first two factors. “Process control helps to manage reproducibility and process consistency, which establishes the best manufacturing process possible. If the understanding and control of the process is sub-optimal it won’t be reproducible, irrespective of cell or medium.”
Consistency also feeds into quality… particularly for emerging biopharmaceuticals that demand far more stringent characterization than conventional mAbs.
Consistency also feeds into quality, which is all the rage today, particularly for emerging biopharmaceuticals that demand far more stringent characterization than conventional mAbs.
According to Wiesner the key to improving media is to take a holistic view, and regard media optimization as part of cell-line and process development. “You can’t just take an isolated part of the process, optimize it, and expect the pieces to flow together. Companies offering media development services often take a customer’s cells into their own labs, develop a medium in isolation of the process, and claim to return the ‘perfect’ product. That doesn’t always work.”
The most successful media development services are conducted on site where the real process development is happening: with the equipment, along with the protocols, and under the same control and monitoring systems as the entire process development. This way everything occurs within the proper context, and is established in cross-communication between the media and the process. This can be achieved with Sartorius’ CHOptimizer.
Image: At Sartorius-Stedim, integrated process/media optimization combines state-of-the art technology, expertise and media support.
Ancillary benefits
While agreeing that media optimization may be responsible for two- to five-fold titer improvements over the past ten years or so, Peggy Lio, global cell culture process science leader at GE Healthcare, notes broader benefits.
“Media free of animal-derived components alleviate regulatory safety concerns, implementation of chemically defined (CD) formulations benefits process robustness, and fine-tuning of certain media components helps to modulate a product’s critical quality attributes.”
The success of media optimization, Lio adds, is often defined as achieving optimal performance within a short timeline. Media optimization thus benefits from several inputs: the existing knowledge base on cell culture processes and media, application of advanced design of experiment (DOE) methodology based on high-throughput cell culture systems, and modern analytical assays for spent media analysis. On the latter point GE HyClone has established assays for quantification of metabolites, amino acids, vitamins, lipids, and trace elements that reveal critical details of cellular metabolism and guide media optimization in high-throughput formats.
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Platform media strategies are applicable toward the majority of monoclonal antibodies, Lio says. “One exception is biosimilar development where platform media may be incapable of delivering quality attributes that are comparable to the innovator molecule. Those quality attributes differ massively between molecules. In this case, it is desired to screen multiple media formulations and identify promising candidates followed by finetuning cell culture media and process settings to match the innovator molecule.”
Computational methods
In addition to their contributions toward rising titers, media and feeds have significantly and positively influenced critical quality attributes for therapeutic proteins. “Raw media components can be manipulated to achieve biosimilarity or alter specific critical quality attributes of originator molecules,” says Andrew Bulpin, Ph.D., head of process solutions at MilliporeSigma. “Media and feeds have not been the sole drivers, however. Process development, new cell lines, clone isolation and selection, harmonization of media and feeds, and innovative expression systems have also contributed to these advances.”
MilliporeSigma’s recommended protocol for media/feed optimization utilizes a design strategy that integrates a data- and mechanistic-driven model.
“Since each clone may have different metabolic routes toward achieving high cell growth and productivity, we carefully design sets of media that ‘ask’ the cell what it likes best to provide the best environment for growth,” Bulpin explains. Each cell culture media in this diversity set contains 70-plus components, which are then tested using high-throughput automation systems. This process generates a significant amount of data about each clone, which is then used for optimization.
MilliporeSigma claims great success using this data—and computation-driven approach for enhancing enhance product quality—to the point where mathematicians are key R&D team members.
Niche markets
In November this year, InVitria launched a new media formulation service that utilizes the company’s ExpressTec plant-based protein expression and production system. Among the proteins manufactured through ExpressTec are albumin and transferrin, ingredients used in mammalian cell culture at concentrations of up to one gram per liter.
“When you view the global media market it’s difficult to reach that scale through other means,” says Randall Alfano, vice president for product development at Invitria.
Since ExpressTec products are inherently animal component-free, they are far less likely than animal-derived ingredients to trigger regulatory red flags.
While addition to large-scale cell cultures of recombinant or serum-derived albumin and transferrin often results in yield improvements, that is not InVitria’s target market. CHO cultures can be productive without these additives, and in fact many bioprocessors are satisfied with today’s yields.
Recombinant media additives happen to be effective in facilitating single-cell cloning for CHO cell-line selection, and seed train expansion. They also aid recovery during single-cell cloning to enhance the number of clones to analyze for mAB production. By playing this role in one cell culture niche, Invitria’s recombinant ingredients expede scaleup from small to large cultures, particularly for therapeutic cells, while eliminating risk to patients from animal-derived pathogens.