Chromatography refuses to be overwhelmed by the processing demands and cost pressures associated with next-generation medicines. Instead, chromatography is strengthening its commitment to innovation.

The ability to meet new challenges is a highly refined trait in chromatography. Indeed, chromatography has been improving ever since it began separating pigments and dyes in the textile industry. After becoming a vibrant success in textiles, chromatography adapted to drug manufacturing, contributing to tasks ranging from material preparation to molecular characterization. And then, most recently, chromatography entered the warp and weft of commercial biopharmaceutical manufacturing.

The history of chromatography in biopharmaceutical manufacturing is celebrated by Lisa Sapp, director of strategy and market development, Applied Genomics, PerkinElmer. “Humulin was introduced in 1982,” she points out. “It was the first biopharmaceutical product approved by the U.S. Food and Drug Administration. High-performance liquid chromatography (HPLC) was initially used to get pure Humulin from a mixture of proteins. Since then, chromatography has been used in the purification of the hundreds of biopharmaceuticals that have come to market in the last 40 years.

“In addition, chromatographic separations have evolved and are an essential part of the raw material testing and critical quality attribute testing that is done to ensure safety and efficacy of new biopharmaceuticals.”

Resins, modality-specific ligands, and membranes

Chromatography’s ubiquity means that any chromatography improvements are bound to have wide-ranging implications, from overall process efficiency to product quality. To substantiate this point, improvements in chromatography media are emphasized by Christopher Pohl, vice president of chromatography chemistry, Thermo Fisher Scientific.

“The earliest media used in biopharmaceutical chromatography involved relatively soft gels,” Pohl points out. “Due to the compressibility of these gels, their use was restricted to rather large particles. Given the properties of large particle size gel media, mass transport, especially for biomolecules, is relatively slow, necessitating relatively low linear velocities during the separation process.

“The trend is toward using media with smaller particle sizes and improved physical properties, allowing modern media to withstand higher operating pressures without deformation of the particles. At the same time, there has been a trend toward wide-pore media, which enables more rapid mass transport of biomolecules in and out of particles. Thus, the separation power of newer materials is substantially improved over the materials initially used in the industry.”

Another view of chromatography evolution is offered by Priyanka Gupta, head of external collaborations, separations technology, Sartorius Stedim Biotech. She suggests that the evolution of chromatography is exemplified by the range of chemistries that have become available.

“Initially, the chemistry and ligands used were very limited, and use of chromatography technologies like size exclusion were most prevalent,” she explains. “As the industry evolved, additional chemistries with different modes of operation, such as those in affinity, ion-exchange, and hydrophobic interaction chromatography, became increasingly prevalent.

“Even within these modes, the ligand chemistry has been improved. Consequently, more and more modality-specific ligands are being used to increase both the yield and purity.”

Another area of innovation is in ready-to-use chromatography technologies—particularly membrane-based systems. “Membrane chromatography was introduced approximately 20 years ago,” Gupta relates, “but now with the advancements in its performance and better recognition of its importance—especially in terms of having ready-to-use, small-scale, high-flux solutions—membrane chromatography use has increased, and it is replacing traditional resin-based chromatography.”

Traditional and emerging modalities

“Initially, the development of chromatography products and processes was very much R&D driven by vendors,” says Henrik Ihre, PhD, director of global strategic technologies, Cytiva. “Today, the business is more mature, and new chromatography products and processes are often developed in close collaboration between customers and vendors to ensure that the right critical quality attributes are considered from the very beginning.”

The emergence of new therapeutic modalities is another key dynamic, says Pohl, who reinforces his point by citing the example of monoclonal antibody (mAb)-based therapies. “Many areas are challenging for bioprocess chromatography,” he notes. “This includes the general problem of nonspecific binding, which can compromise the purity of the product and product yield. The industry continues to push for higher capacity and higher performance products.

“At the same time, the challenges are different for different classes of analytes. For example, in the case of mAbs, the primary separation mode involves Protein A affinity chromatography. Since the same affinity mode works for a wide range of mAbs, one chemistry can be applied to many different mAbs.

“In contrast, protein drugs each require a specific high-affinity ligand in order to use affinity chromatography. This lack of a general solution to the affinity separation problem relegates affinity chromatography to an infrequently used method for protein drugs.” Instead, Pohl says, protein drug makers tend to use reverse-phase and size-exclusion chromatography.

Sapp also sees the protein drug sector as a major innovation driver, particularly in contrast with the small-molecule medicines industry. “Some of the challenges scientists face when working with protein drugs are in the complexity and size of the molecules,” she remarks. “As an example, the size of a common small-molecule pharmaceutical drug like Lipitor is around 559 Da. Herceptin, a common protein therapeutic used in the treatment of breast cancer, has a molecular weight of 185,000 Da.

“There is much more opportunity for variability in these drugs. Any variability in protein attributes or behaviors, including post-translational modifications, peptide sequences, glycans, charge states, or aggregation, can affect the overall quality of the drug, so these must be characterized and monitored during the manufacturing and release of the product.”

Another challenge for chromatography is posed by the development of antibody-drug conjugates (ADCs). Each ADC consists of a targeting antibody melded to a therapeutic “warhead.” “Many ADCs are highly polydisperse with respect to the points of attachment of the conjugate,” Pohl says. “This makes it extremely difficult to separate all the different ADC variants in many cases.

“Newer methods of synthesizing ADCs provide better synthetic precision and overcome some of these barriers. The other problem, though, is that the ADCs fundamentally change the physical properties of the biomolecules to which they are attached. Thus, it isn’t easy to find a good compromise between chromatographic conditions that are compatible with the biomolecules while allowing for the separation of the ADCs. Hydrophobic interaction chromatography is often useful in overcoming this problem.”

Cell and gene therapies

Chromatography is as vital in the production of cell and gene therapies as it is in the production of traditional biopharmaceuticals. However, for cell and gene therapies, chromatography tends to be applied during the earlier stages of manufacture.

Ihre notes that cell and gene therapy production relies on transfection of cell lines. “Here, different viral vectors and plasmids are required,” he explains. “Both are manufactured from traditional up- and downstream protocols comprising traditional chromatography steps.

“For truly large molecules, such as plasmids, viral vectors, and mRNA, there are sometimes limitations with the bead-based purification since these molecules cannot access the pore structure of the bead resulting in lower capacities. For such large targets, chromatography based on fibers or membranes may offer a more high-capacity alternative.”

Cytiva facility
To facilitate the manufacture of therapeutics, Cytiva designs and develops new purification products at its facilities in Cardiff, Wales, and Uppsala, Sweden. Cytiva indicates that drug companies are playing a greater role in the creation of chromatography technologies.

The appeal of membrane-based chromatography systems for cell and gene therapy manufacturers is also recognized by Amélie Boulais Raveneau, manager of external collaborations at Sartorius. She notes that cell and gene therapy agents “are usually larger and more fragile than proteins, which is leading to low yields of the downstream process.”

“The overall capacity of classical resin matrix is lowered, as these molecules are not diffusing into the pores of the beads,” she adds. “Therefore, we see the growing adoption of convective matrix in the clinical and even commercial production of gene therapy products, such as chromatography membranes or monoliths.”

The pull of industry demand

“I expect the biopharma industry to continue to move toward higher-performance media,” Pohl says. “Newer biopharmaceuticals tend to use higher performance media since they have improved separation performance, which is often critical for high-purity products.”

Ihre agrees that emerging therapeutic modalities will continue to shape the development of chromatography systems: “As new modalities mature, the request for highly productive and scalable chromatography platform solutions will increase. Another potential game changer is personalized medicine—once it becomes a reality. Small cGMP processing runs and a competitive process economy would be required to manage costs of personalized therapies.”

According to Sapp, chromatography technologies, in line with other bioprocessing systems, will evolve to incorporate elements of automation. She points out that pharma and biopharma companies are moving more toward continuous processing, and that regulatory agencies encourage real-time monitoring through process analytical technology (PAT). “With PAT,” she notes, “the manufacturing process continually analyzes pharmaceutical samples to determine critical quality attributes that may fluctuate during production.”

In addition, Sapp predicts that we will see chromatography evolve “to have more usability features and automation capabilities, and to be more connected to other systems in the laboratory.”

The shift to single-use technologies

The move from larger, single-product facilities toward smaller, multiproduct plants will also impact the development of chromatography systems. “Most biopharma players are looking to build multimodality portfolios, which means they cannot establish a platform approach or a universal solution,” Gupta says. “There will be a greater need for flexibility.”

To gain flexibility, industry players will favor single-use technologies to sustain disposable flow paths and eliminate cleaning and sterilization processes. Single-use technologies can also enable a shift to more efficient chromatography devices. Currently, the chromatography devices used in the preparation of material for studies rarely reach their lifetime potential. Accordingly, scientists are gravitating toward a multicolumn chromatography approach where they can pack small columns, reducing costs and facility footprints. This approach is facilitated by single-use technology.

“The desire to have more single-use solutions is also leading to a greater understanding of the advantages of technologies like membrane absorbers and monoliths,” Gupta continues. “More research is focused on finding good affinity solutions using these two platforms, which can really revolutionize the chromatography industry.”

Ihre also believes that the industry needs a range of chromatography solutions—including multi- and single-use technologies. “The molecules that have a high-volume need in the market may still benefit from more fixed installations and stainless-steel solutions,” he argues. “On the other hand, smaller volume and niche drugs may benefit from processes more based on single-use solutions enabling a lower capital expenditure, shorter time to market, and greater flexibility to run true multiproduct facilities with a smaller footprint.”

Whether single-use chromatography technologies come to dominate is ultimately a question of economics. “There is a limited trend toward single-use chromatographic media, but chromatographic materials’ high costs limit this approach to high-value products,” Pohl states. “It’s unlikely that the cost of chromatographic materials will significantly decrease, which will restrict single-use chromatographic applications.”