In the downstream portion of any bioprocess, one must pick through the dross before one can seize the gold—the biotherapeutic that the bioprocess was always meant to generate. Unfortunately, the dross is both voluminous and various. And the biotherapeutic gold, unlike real gold, is corruptible. That is, it can suffer structural damage and activity loss.
When discarding the dross and collecting the gold, bioprocessors must be efficient and gentle. They must, to the extent possible, eliminate contaminants and organic debris while ensuring that biotherapeutics avoid aggregation-inducing stresses and retain their integrity during purification and recovery. Anything less compromises purity and reduces yield.
To purify and recover biotherapeutics efficiently and gently, bioprocessors must avail themselves of the most appropriate tools and techniques. Here, we talk with several experts about which tools and techniques can help bioprocessors overcome persistent challenges. Some of these experts also touch on new approaches that can help bioprocessors address emerging challenges.
Optimizing affinity purification
Most efficient purification processes start with an affinity chromatography capture step that “removes the majority of the impurities and provides the desired concentration factor,” says Karol Lacki, PhD, vice president of technology development at Avitide. Subsequent purification steps are used to remove traces of remaining process- and product-related impurities.
Lacki and his colleagues develop custom affinity resins for purification of difficult-to-purify biotherapeutics. Avitide’s “affinity resins are characterized by high specificity toward the target, high binding capacities, and high alkaline stability—up to 0.5 molar sodium hydroxide,” Lacki explains. The resins are developed against specifications from the client, and Avitide will make a custom resin in 12 weeks.
“This would be a resin for process development, testing, and even for toxicology studies,” Lacki asserts. A cGMP-ready resin can be developed in an additional five months. Besides developing custom resins, Avitide is expanding its catalog of affinity resins for the purification of adeno-associated virus (AAV) gene therapies and SARS-CoV-2 spike protein vaccines. “Since these resins are also alkali-stable, you can use a column several times, significantly reducing purification costs,” Lacki explains.
Lacki says that no matter how bioprocessing evolves, separation technologies won’t undergo any major changes in the foreseeable future. “Affinity-based purification is here to stay,” he insists. “Purification or separation happens on the surface, and surface chemistry will remain the heart and soul of chromatography.” He adds that scientists will also continue to face challenges in purification.
Addressing antibody aggregation
Aggregation that occurs in an antibody-based therapeutic can reduce the product’s shelf life. Worse, it can endanger patients. An aggregated antibody-based therapeutic can trigger an unwanted immune response. If aggregates get big enough, they can even block blood vessels.
Aggregation can occur in the bioprocessing of most antibodies—at least under some conditions. It is no surprise, then, that common bioprocessing challenges include the preservation of protein stability and the prevention of protein aggregation.
“Antibody aggregates are formed during bioprocessing if antibodies are exposed to stress conditions such as excessive pH and temperature levels, effects related to the use of low-quality raw materials, the presence of trace elements, shear forces, and the presence of micro- or nanoparticulate impurities,” says Nandu Deorkar, PhD, vice president of R&D for biopharma production solutions at Avantor. “Native protein monomers can also aggregate by adhering to preexisting protein oligomers, contaminants, or vessel surfaces during bioprocessing.”
With aggregation being so prevalent and problematic, bioprocessors take various approaches to reducing or eliminating it. “Process materials and excipients such as buffers, salts, surfactants, amino acids, and sugars can help prevent aggregate formation and ensure adequate stability throughout a product’s shelf life,” Deorkar details. “The key is to choose raw materials of the appropriate quality, and excipients that consistently control impurities such as free fatty acids, trace elements, and reactive and nanoparticulate impurities.”
Bioprocessors can also reduce aggregation by taking advantage of new technologies. For example, as Deorkar explains, there are “certain new Protein A affinity ligand–based resins that can remove aggregates and minimize aggregates by utilizing additives in load or elution buffers.” He adds that Avantor has recently demonstrated that its Protein A resin, J.T.Baker Bakerbond PROchievA, can be used to “remove more than 90% of high-molecular-weight impurities by optimizing conditions and fractionating elution.”
In the future, antibody aggregation could be an even bigger challenge. “A trend in biotherapeutics is the development of subcutaneous products,” Deorkar points out. “These products, which offer administration convenience and a reduced number of hospital visits, typically require a high-concentration formulation.” Producing a high-concentration formulation, though, requires overcoming three crucial challenges: poor solubility, high viscosity, and increased aggregation. So, Avantor develops excipients that address these challenges. “These excipients,” Deorkar asserts, “are suitable for use in a wide range of concentrations.”
Like antibodies, antibody-drug conjugates (ADCs) need special handling in bioprocessing. An ADC is a targeted drug that, as the name suggests, consists of an antibody conjugated to a drug or even multiple drugs. The antibody in an ADC binds to specific antigens, and so it can guide a conjugated drug or drugs to cells in the body that express those antigens.
Today’s ADCs keep getting more complicated. For instance, Mersana Therapeutics has an ADC platform called Dolaflexin that can attach more than the usual number of drugs to each antibody. “[It can attach] 10 warheads to an antibody—compared with the industry standard of 2, 3, or 4,” asserts Michael Kaufman, PhD, Mersana’s chief manufacturing officer.
Despite the potential clinical benefits of creating a 10-warhead ADC, some complications come with it. “The Dolaflexin platform is complex and produces a very complicated mixture of materials,” Kaufman points out. “That in itself is not bad—a lot of highly established medicines are mixtures of things. (Heparin comes to mind as a very complex thing.) But complex mixtures put extra pressure on your processes. For example, complex mixtures can make it difficult for you to run your purification reproducibly, lot after lot after lot.”
To cope with complex mixtures, Mersana uses a purification method that incorporates strong cation exchange chromatography. Although this is an established form of chromatography, using it to purify products generated by the Dolaflexin platform requires what Kaufman describes as “a very complex optimization.”
“We need to make sure that we get the parts of the molecule that we truly want to develop as a therapy,” he explains. “We exclude the other parts of the molecule that really add nothing.”
Complex methods of purification can reduce the yield from a bioprocess, and the purification method that is used with the Dolaflexin platform is certainly complex. So, unsurprisingly, the platform posed a challenge with respect to yields. Initially, the platform plus strong cation exchange chromatography produced yields of just 40%. However, Mersana scientists managed to increase yields to 65%.
In addition, the scientists reduced the purification time by optimizing the amount of drug that the resin could capture. “We’ve got that from about two days to roughly a half a day,” Kaufman details. “So, let’s call it a fourfold increase in productivity.”
Improving the amount of drug recovered is not all about better chromatography. “In general, if you could recover your product and do separations and recovery by filtration, as opposed to chromatography, that’s a huge efficiency improvement for any process, because these filtrations are much faster and much simpler than chromatographic separations,” Kaufman explains. “In the Dolaflexin platform, we replaced three chromatography steps with tangential flow filtration, which runs quite quickly and efficiently.”
Getting to the genes
Beyond antibody-related therapeutics, bioprocessors also make gene therapies, and these “products are increasing in popularity and scope,” says Baley Reeves, PhD, a research scientist at the National Center for Therapeutics Manufacturing (NCTM). “There are over 80 products in Phase III clinical trials at the moment—representing about 12% of the biopharmaceutical pipeline—and these numbers just continue to climb.”
At the NCTM, Reeves and her colleagues are studying baculovirus-expressed AAV-2 vectors for gene therapy. As Reeves observes, “Multiple expression hosts and different vector serotypes can be used.” All gene therapy products, it should be noted, consist of a gene of interest inside a viral capsid or protein shell.
“Unfortunately, during the upstream process, not all capsids produced end up with the gene of interest inside,” Reeves continues. “So, a critical part of the downstream process is to separate the full capsids—the desired product—from the empty capsids—a product-related impurity.”
To address this purification challenge, vendors have developed new affinity resins for AAV purification. One such resin, Reeves notes, is Thermo Fisher Scientific’s POROS CaptureSelect AAVX affinity resin. “It is not the only affinity resin available for AAV capsids, but it has been demonstrated to be superior to the other brands we have tried for the capture chromatography step,” she remarks. “Additional steps—typically either ion exchange chromatography or ultracentrifugation—would still be required to separate empty from full capsids.”
The high level of empty capsids also creates a recovery challenge. “In the gene therapy space, overall process recoveries tend to be lower than they would be for a traditional biopharmaceutical product,” Reeves says. “We typically see process recoveries less than 25%, and our collaborators report similar findings.”
Reeves sees those low levels of recovery as the biggest hurdle for gene therapies. So far, a solution to that problem remains elusive. “It will probably require a multipronged approach involving the use of higher production titers upstream, as well as the development of more robust platform processes downstream,” Reeves suggests. “These will be developed over time as more and more studies are published and best practices are established. As we learn more about how to manufacture viral vectors, process recoveries will undoubtedly rise.”
Focusing on the future
Tomorrow’s bioprocessing methods will surely make purification and recovery even more efficient. The process should also run faster and maybe even cost less. Still, those metrics—efficiency, speed, and cost—will not be all that the industry will consider.
Even today, bioprocessing faces pressure to increase its sustainability. Some companies already take that seriously. At Mersana, for example, Kaufman made sure to mention that tangential flow filtration is greener than purifying therapeutics with chromatography alone. “This filtration generates far less waste and doesn’t generate any organic solvent waste at all,” he says. “I feel good about that from an environmental standpoint. It’s just the right thing to do for the environment.”