Tipping the Scales of Therapeutic Antibody Production


Monoclonal antibody (mAb) production was once measured in micrograms per liter. Now, nearly two decades after the first wave of therapeutic mAbs hit the market (with their patents now beginning to expire), it’s routine to see production titers of many grams per liter. Modern cell lines, expression vectors, media formulations, equipment and process parameters enable manufacturers not only to generate more protein, but also, in many instances, to do so to much tighter specifications of glycoforms and other “quality” attributes than was previously possible. Yet turning a laboratory-scaled therapeutic mAb culture into a manufacturing process is still not routine. Here we explore the process of scaling up and optimizing those processes.

How much?

By the time researchers are looking to scale up, oftentimes the antibodies have already been screened for efficacy, with the relevant portions PCR-amplified and cloned into an expression vector, and that vector transfected into a producer cell line such as Chinese hamster ovary (CHO) cells. At this point—with frozen-down master and working-cell-bank cells—begins a second process of analysis and optimization “to make sure the product is still right, and you’re getting the right titers so you can keep your cost of goods in line with where you want it to be,” says Donald Traul, field marketing manager at Sartorius Stedim Biotech.

To simulate process conditions that wouldn’t exist during cloning and early development, this stage of scale-up often takes place in small (two- to five-liter) benchtop bioreactors (often referred to as “scale-down models”). “The scale-down model used depends on the program needs and available resources—a lot of development work can still be accomplished using shake flasks,” notes Serena Fries Smith, process science manager at Thermo Fisher Scientific. Other options include rocking bag bioreactors, which are available in a range of sizes and from several vendors. Some of these are able to handle small working volumes—GE Healthcare Life Sciences’ ReadyToProcess WAVE 25 requires as little as 300 ml, for example, and Sartorius’ BIOSTAT® RM can handle cultures down to 100 ml. Sartorius also offers the ambr® 15 automated microbioreactor which can handle up to 24 or 48 15-ml stirred culture chambers—to simultaneously test different media and conditions, for example, or to compare clones. “You can then go up to two [liters], 10 [liters], 100 [liters], whatever scale you want to, and know that those parameters that you’re using will work at a larger scale,” Traul says.

What that scale ultimately will be depends on the dosage required, the market size and the cell line used, notes Cory Card, principal scientist for GE Healthcare’s cell culture business. “But you want to be able to scale up to that as quickly as possible—that’s time when you’re not making any revenue.”

From growth to production

The challenge is that growth and production conditions are often antagonistic.

“You want cells that grow well during that scale-up but then ideally spend their time and energy making protein rather than wasting them on dividing and growing at that point,” Card continues "Proteins, including antibodies, need sufficient retention time in the endoplasmic reticulum and Golgi apparatus to properly fold and glycosylate, and “if the cells are being pushed into mitosis and growth, that is often sacrificing the amount of protein of interest the cells are expressing, as well as the quality of the protein.”

To address this, a “two-medium” system can be used: Start with medium optimized for growth while the cells are in the seed train and then switch over to a medium optimized for protein production. However, switching basal media during a production process is not easy and makes for a more complex process. Most of Card’s customers want to keep things simple, so they opt to use the same basal medium all through the process, with different feeds and supplements added at various times to modulate growth, productivity and protein quality.

Another tack is to lower the temperature of the culture. “At lower temperatures, the cells are less likely to go through mitosis and more likely to stay in G phase and produce protein,” Card explains. Controlling pH and glucose levels can have a beneficial impact. And some people have seen that raising the osmolality of the culture can help the cells produce more protein, as well. But “there are arguments out there whether that’s leading to secretion of unfinished (vs. properly glycosylated) protein. It’s not a favorite of mine.”


So with the advent of biosimilars (and a marked improvement of the tools to analyze the proteins), “it’s almost like we’ve moved from focusing on yield, to how to adjust process, to the goal of product quality,” says Tom Fletcher, scientific director at Irvine Scientific. “You hear that much more at conferences these days.”

Glycosylation and aggregation—even if they do not affect mAb specificity or affinity, or avidity to the target antigen—can have an enormous influence on efficacy, immunogenicity and clearance from the body.

There is some, but incomplete, understanding of what influences protein quality. Fletcher likes to use an iterative, rational design-of-experiment approach, with statistical software, “to solve a particular challenge, such as increasing galactosylation or reducing fucosylation or whatever,” he notes. “We don’t have a whole knowledge base that can prescribe solutions, but we have an understanding of what to try.” That may include balancing the ratio of amino acids, trace metals concentrations and other key media components, as well as timing of the feeds.

“There are components that can be added to your process to modulate glycosylation. There is also a lot of information in the literature saying which of these components can help you shift your glycosylation patterns,” explains Smith. Most such products, including Thermo Fisher’s GlycanTune™ line, are proprietary. Many vendors also offer custom services to help optimize media and feeds for a particular cell line.

Culture mode

Recombinant antibodies are typically produced in suspension-adapted CHO cell cultures in fed-batch mode (meaning the cultures are periodically “fed” the nutrients that are used up, as determined by spent-media analysis), explains Amber Jones, Lonza’s product manager, media. They also can be grown in a continuously fed perfusion mode. Each has its advantages and disadvantages when it comes to issues such as media consumption, product stability and downstream processing.

Adherent cells that are not, or cannot be, suspension-adapted can be grown at very high densities in apparatus such as ZellWerk’s Z®-RP Cell Culture System or hollow-fiber reactors. Stanley Goldberg, director of Glen Mills, ZellWerk’s North American distributor, says that long-term (up to 200 days) culture in five-liter Z®-RP can grow as many cells as 10,000 roller bottles or a 10,000-liter fermenter: “You put it under a hood, and that’s your construction costs.”

Regardless of the culture mode, when scaling up for production it’s important to begin with the end in mind, notes Fletcher. For example, don’t use materials in the optimization process that can’t be obtained, or would be prohibitively expensive, in production-scale quantities—and certainly don’t use anything (like bovine serum) that may raise eyebrows at the U.S. Food and Drug Administration (FDA). Similarly, if something (like sheer protectant) will be used in production, use it during optimization, as well.

As much as possible, that also goes for how the media and feeds are sourced, prepared and stored. “You can put your formulation from a bottle into a bag and all of a sudden start realizing that lipids are sticking to the bag, and you have to go back and change your formulation,” notes Jones.