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Top Trends in Biopharmaceutical Manufacturing: 2015

New cell-culture techniques, biomanufacturing formats, biological products, and the expansion of single-use applications are driving rapid change in the biopharmaceutical market. Pharmaceutical Technology spoke to industry experts in the field of bioprocessing to identify the key trends impacting the industry in 2015 and beyond.

Novel expression systems and cellular platforms Alternative platforms for industrial development may prove to be more cost-effective than prevailing cell models. While Chinese hamster ovary (CHO) cells are commonly used for the production of recombinant protein therapeutics, alternative expression systems are gaining popularity, according to William Whitford, senior manager, cell culture, at GE Healthcare Life Sciences. “Avian lines (e.g., duck embryo quail sarcoma and chick embryo fibroblasts) have been reported to transfect well, have promoters that work with mammalian genes, and grow (i.e., culture expand) faster. [These lines also] promise higher levels of cell density and specific expression, reduced generation of ammonium and lactate, and reduced product cell-surface fucose, resulting in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity,” Whitford toldPharmaceutical Technology. EB66 is an example of such an avian origin line; these cells were shown to reach high cell densities at short population doubling times and are believed to offer enhanced biological activity as a result of their natural ability to produce glycoproteins with low fucose, a feature that is correlated with improved receptor binding (1). Baculoviral insect cell systems have also been gaining popularity as a substitute for commonly used production schemes for recombinant protein production and have been effective vectors for large-scale production of human monoclonal antibodies (mAbs) (2).

New formats Transient transfection. Instead of introducing a DNA into a cell’s host genome, genetic information can also be introduced into a cell (though not integrated into the cell’s genome) via pores in a cell membrane—a process known as transient transfection. Recent advances in cell culture and transient transfection have allowed cell lines to be transiently transfected to produce large amounts of recombinant proteins before the genetic material is degraded and/or diluted. Whitford says that transient transformation is easier and cheaper than the “standard engineering of stable transformants.” Using retroviruses to genetically modify T cells can also be a concern because of “their propensity to integrate near start sites of genes, which could lead to gene dysregulation, cell transformation, and oncogenesis” (3). The use of nonviral transposon systems or direct RNA electroporation could, therefore, be effective alternative transduction options for T cells and in other applications as well.

According to Life Technologies, the benefits of transient transfection include creating large quantities of post-translationally modified and active mammalian protein in 3–7 days, the ability to express proteins in mammalian cell culture facilities with shake flasks and a platform shaker, and the easy purification of secreted proteins from serum-free cell-culture media (4). Transient expression also eliminates the cell-expansion step required for standard approaches, thereby helping to reduce manufacturing time and potentially, manufacturing costs.

Continuous biomanufacturing. Increasing titers as a result of advances in process efficiencies means that there is more pressure on downstream processing. Though continuous processes such as perfusion are widely adopted upstream, downstream continuous methods are slowly but surely catching up to upstream processes. Chromatography techniques are gradually becoming continuous. “For example, a series of small columns have been demonstrated to mimic one single large column with a diameter and a bed height equal to the total bed height of the smaller columns,” explains Whitford. “Multicolumn setups have been characterized in bind-and-elute (B/E) mAb capture steps. There are even valve-and-column arrangements that lengthen the stationary phase to allow high-solute loadings to the process,” he adds. “From a features and benefits point of view, quasi-, pseudo-, or even partially-continuous capture chromatography can provide the benefits of a more continuous [setup].”

Christel Fenge, vice-president of marketing and product management fermentation technology at Sartorius Stedim Biotech, says that while a few teams are working on establishing end-to-end continuous processes, she predicts it will take the industry at least a decade or more before it achieves robust commercial continuous processes that are completely sequential and closed in an end-to-end loop. “Managing process deviations in a GMP context is not trivial, and the old topic of batch definition needs to be revisited,” Fenge states. “There are also still [performance] gaps with regard to single-use sensor tools, valves, and pumps.” Indeed, the experts agree that industry pilot evaluations of end-to-end processes will have to continue to properly weigh the advantages and disadvantages. “Success at larger scale and under GMP and commercial manufacturing environments will be the next hurdle for end-to-end continuous manufacturing,” Parrish Galliher, chief technology officer, upstream, at GE Healthcare Life Sciences asserted. “We expect that the next five years will reveal the ultimate place and role of continuous manufacturing.”

Progress in perfusion: New cell-culture techniques High-density perfusion/intensified perfusion. With manufacturers constantly trying to reduce material costs and produce more drug in a shorter timeframe, they have looked to new methods to achieve those goals. Alternative culture methods are growing in popularity, according to Whitford. “Intensified perfusion is growing in popularity for [the] production of protein biopharmaceuticals,” Whitford notes. He says that various publications have described intensified perfusion in applications such as cell banking, seed expansion, and cell cultivation for production. Perfusion with intensification is a continuous bioreactor process characterized by a cell-recycle system. Intensification allows titers to increase, with cell mass concentrations reaching more than 100 million cells/mL at laboratory scale and mAb products reaching concentrations of more than 20 g/L, Galliher states.

Typically, intensified perfusion is applied to mAb production, says Fenge, as these molecules are relatively stable. A key benefit of intensified perfusion is the “space-time yield,” says Fenge, as a 2000-L single-use bioreactor can produce as much antibody as a bioreactor that is five to 10 times larger, and does so with a smaller footprint. “Another benefit is speed to market, as the scale used for Phase III trials is the same as commercial manufacture,” meaning that “no further scale-up is necessary.” Compared with fed-batch operations, cost benefits of intensified perfusion vary, says Fenge. “Bottom line, the cost really depends on the individual case, framework, and constraints—but there are clearly scenarios where there is a tangible overall cost benefit.” Galliher notes that a disadvantage of intensified perfusion, however, is that high cell mass overloads conventional cell-removal systems, bottlenecking downstream purification operations.

Hollow-fiber perfusion, packed-bed bioreactors, and bioreactors with microcarriers. In some types of perfusion, cells are bound or grown on a membrane. Other types of perfusion require filtration or centrifugation to retain cells floating around in the bioreactor. Whitford notes that solid substrate systems, used for attachment-dependent cells, can in some cases produce lower apoptosis rates and produce fewer contaminating cell metabolites (5). “In perfusion systems with cells bound to a solid substrate, cells grow more naturally and with less traumatic mixing/agitation and shear,” Whitford writes. Furthermore, all perfusion systems can in some applications provide “recombinant proteins/antibodies that are purer, more like native proteins, and more consistent in their biological activities than fed-batch bioreactors, such as having fewer variations in glycosylation” (5).

In particular, animal cells are increasingly being seeded within cartridges of hollow-fiber perfusion bioreactors. Hollow fibers allow for 3D cell culture. The 3D fibers are biomimetic of actual human tissue, allowing cell interaction via numerous contact points (6). Nutrients and waste are exchanged through capillaries, with fresh media diffusing outside of the fibers into the cells in the intercapillary space and spent culture media flowing back into the fibers for eventual removal. Hollow-fiber perfusion has been shown to create a more constant culture environment in terms of nutrients and metabolites, and products can be harvested continuously at higher concentrations than they can from suspension cultures over longer periods of time (5). This closed platform can be applied to various cell types, including vaccines, monoclonal antibodies, stem cells, and cell therapies (5).

Conversely, Fenge says she believes that hollow-fiber bioreactors are outdated, and are “only used for making antibodies for diagnostic or research purposes, if at all.” Packed-bed bioreactors, on the other hand, have gained traction in emerging economies for the production of vaccines, although Fenge acknowledges it is difficult to ensure homogeneity inside of a packed bed. “Essentially, it is very difficult or impossible to monitor pH, DO [dissolved oxygen] or cell density in a reliable way inside a packed bed. Also, scalability is a challenge, as these systems are difficult to scale in a linear way and typically, users scale-out, i.e., they use multiple parallel bioreactors to produce the amounts needed,” Fenge explicates. “This [scale-out method], in turn, increases the operational and analytical costs compared with an approach that can be scaled in volume.”

Microcarriers are also an attractive option for the production of vaccines, as “they can be used in a classic stirred-tank bioreactor in a homogenous cell-culture mode.” While Fenge recognizes that not all vaccines can be produced in suspension culture, she says that this process is easiest because no “complex seed expansion is necessary, where cells need to be detached from the carriers and transferred to the next larger bioreactor, no bead-to-bead approaches [need to be] validated, and no tedious preparation of the carriers is required.” Galliher says that while vaccines are being produced more often with suspension cells in conventional stirred-tank reactors, in developing markets, “vaccines that still require attachment-dependent cells will expand in those new territories.”

Cell therapies are expanding, and many require attachment to a surface, says Galliher. Microcarriers and packed-bed bioreactors may be most promising for the mass production of stem cells in the manufacture of regenerative medicines, notes Fenge, but the challenges that exist with vaccines also exist with stem cells. Harvesting the stem cells requires releasing them from the attachment surface using enzymes, which need to be washed away in the final dosage form, Galliher asserts. “Additionally, the morphology of these cells may have an impact on their differentiation, leading to consistency issues, or worse, [the production of] nonfunctional cells,” adds Fenge. As a result of these challenges, and because there is an interest in reducing the cost and complexity associated with the processing of attachment-dependent cells, there is a big demand for suspension-adapted stem cells. “We expect that suspension-adapted stem cells will become more widespread in the cell-therapy space in the future,” Galliher emphasizes.

Retrofitting for perfusion. According to Whitford, existing equipment and legacy systems are increasingly being retrofitted to enhance operational efficiencies. While he says retrofitting for single-use bioreactors is going on, retrofits of existing GMP processes “tend to be substantially more difficult.” Despite this fact, “the selection of perfusion technologies available for interface with stainless-steel bioreactors is supporting retrofit of an increasing number of established stainless steel-based (and hybrid) facilities.” Indeed, existing research on the productivity of spin-filter perfusion and alternating tangential-flow perfusion demonstrates that these perfusion culture processes offer cost of goods savings when compared with fed-batch processes (7).

Although retrofitting existing stainless-steel facilities for perfusion is an option, Fenge thinks that this procedure is “not at all straightforward,” and it is much easier to just “rip the old stainless-steel upstream equipment out, retrofit the cleanroom space with new, single-use bioreactors, media preparation, and storage solutions.” She also says that increased efficiencies can come from exploring hybrid solutions with buffer mixing, storage, and intermediate storage in single-use bags.

The process mode of perfusion as a whole needs improvement, says Fenge, especially when it comes to the creation of more robust cell-retention devices, single-use pumps, and sensor technologies. Fenge notes there is also a clear gap in large-scale single-use connectors, “as better cell-culture results are achieved at large scale if the recirculation loop is wide enough to avoid high shear forces on the cells.”

Single-use technologies Formal standards will drive increased expansion of single use. The focus on single-use is correlated with an increased interest in modular facilities, smaller bioreactors (from 10,000–20,000-L sizes to medium-sized, 2000-L bioreactors), the need for facilities to produce multiple products in parallel, and the “need for risk mitigation to better manage strong attrition rates of products coming through the pipeline,” observes Miriam Monge, marketing director, integrated solutions at Sartorius Stedim Biotech. Single use can now be seen in laboratory, pilot, clinical, and commercial manufacturing operations. Galliher says that customer reports demonstrate that after 10 years of active use, single-use products were shown to reduce capital cost by 40–50%, reduce operating costs by 20–30%, and reduced the time-to-build by 30% when compared with traditional stainless-steel technology. “Over the past decade, the question of whether single-use technologies are feasible has dissipated, and they have become an industry standard for the manufacturing of clinical batches in biopharmaceutical production,” says Helene Pora, vice-president of single-use technologies at Pall Life Sciences. She adds, “Many opportunities exist around bringing more control through automation, and the industry continues to focus on robust and reproducible processes with recording systems that create more of a standard mode of operation.”

Despite the process efficiencies of single use, problems still exist, namely, the assurance of product quality, product integrity, vendor supply-chain security, and the need for “change control with timely notification,” according to Monge. She says that even more difficulties arise when an end user attempts to audit a single-use supplier, because there are no true regulatory standards in place, only guidelines.

There is now a trend, driven by end users and suppliers, to facilitate the adoption of enforceable standards for single-use systems, Monge asserts. “One of the greatest challenges that end users currently face in their selection and qualification of single-use technologies is the fact that very rarely are the vendors working with the same testing methodologies, whether we are talking about the way in which they determine and characterize extractables from materials used in single-use applications, characterize leachables released from materials, evaluate integrity testing methods, or characterize particulate burden from single-use systems,” she says. In addition to the validation gaps mentioned, the fact that tube sizes remain small and that there are a myriad of nonstandardized incompatible sterile connectors on the market also present problems, adds Galliher. Single-use systems also have a limited capacity in downstream applications in general, he concludes.

Monge points out that while the The Bio-Process Systems Alliance (BPSA) and BioPhorum Operations Group (BPOG) have written recommendations and guidance documents on single-use systems, and organizations such as ASTM International and US Pharmacopeial Convention (USP) are gaining traction when it comes to the regulatory control of extractables and leachables, integrity testing, and particulates, there will still be performance gaps in the absence of published standards by regulatory bodies. “Standards will significantly facilitate the adoption of single-use systems, as end users will be able to directly compare like with like, and if these standards receive endorsement from the regulatory authorities, the end users will be able to have a much higher level of confidence when widely implementing single-use systems into commercial manufacturing,” Monge stresses. The first approved standards for single-use technologies are expected in late 2015 or early 2016, she says.

Single-use for the conjugation of ADCs. An antibody-drug conjugate (ADC) combines the specificity of a mAb with the efficacy of a cytotoxic small-molecule compound. Christian Manzke, director, marketing and sales for integrated solutions at Sartorius Stedim Biotech points out that the original company that makes a mAb can be a different company than the one that performs the conjugation of the product. Furthermore, a separate company altogether may perform the formulation or filling duties associated with said product. While frozen mAbs or conjugates typically travel to all of these different processing locations in single-use bags, says Manzke, it is not until recently that single-use technologies have been used for the conjugation reaction itself. The biological, aqueous, and large-molecule world of ADC manufacturing is merging with the chemical, solvent-based, small-molecule pharma world, notes Manzke. “Where single use is already an accepted technology in the biopharmaceutical industry, it was not obvious that this [technology] would become a success for the solvent-based chemical linking process as well. The fear about noncompatibility of the plastics used with the solvent-containing reaction liquids or increased leachable profiles led to a slow acceptance in the market,” Manzke says. “Now we see more and more companies weighing the benefits over the challenges and using single-use bags for the reaction process and single-use crossflow systems for the diafiltration and concentration steps.” Because single-use options offer a closed system, they are ideal to use for conjugation process steps. Closed systems protect the operator, environment, and the drug itself, and ensure that residual cytotoxic payloads or conjugate inside the disposable assembly are contained, Manzke adds. Single-use processing equipment also facilitates equipment sharing for multiple different ADCs.

New protein biologicals Allogeneic and autologous therapies for adoptive immunity. “Due to recent heightened investment from large pharmaceutical companies through acquisitions and partnerships with academia and [subject matter experts], the regenerative medicine industry is gaining momentum, poised to confer patient benefits for unmet medical need and new profit areas to prop up ailing conventional drug pipelines,” asserts Kim Bure, director, regenerative medicine at Sartorius Stedim Biotech. Although initially, off-the-shelf allogeneic therapies, such as those exploiting mesenchymal stem cells, were thought to serve a universal population, Bure says that the clinical success of these types of therapies has been limited, and many of the allogeneic products in development have failed in late-stage Phase III trials. As a result, many researchers have turned to autologous immunotherapies, specifically in the form of chimeric antigen receptor T-cell therapies (CART or CAR-T). In these therapies (which can be allogeneic or autologous), T cells are harvested from patients and genetically engineered to recognize cancer antigens.

So far, CAR-T products have been shown to be dramatically effective for blood cancers, but the therapies still face challenges when it comes to eliminating solid tumors. In fact, some companies, such as MaxCyte, in collaboration with the Johns Hopkins Kimmel Cancer Center, are now doing research on the introduction of the CAR construct as a transiently expressing messenger RNA (mRNA) for the treatment of solid tumors. This approach is being investigated as a method to control the “on-target, off-tumor toxicity” of most of the CAR-T therapies being developed for blood cancer indications (8). Other developers are investigating a CAR-T “safety switch” to address severe toxicity concerns within the body due to cytokine storm.

The burgeoning interest in genetically engineered T cells has the potential to further drive the adoption of single-use systems and novel production paradigms, says Bure, given that the safety of the final cellular product in these instances is imperative. A mounting concern, however, is that the cost of autologous therapies will prove unsustainable. “Unique, small-scale lots that still require full-scale quality control and release testing increase the cost of goods and have the potential to make these therapies not commercially viable, so efforts to create a historical design space informed by extensive process analytical technology data could allow for reductions in testing and movement towards real-time release testing,” Bure explains. “Additionally, with the advent of the Falsified Medicines Directive, these blood-derived therapeutics will possibly be deemed as APIs from the initial production stages by regulators, forcing unique identifier techniques to be implemented, such as 2D-data matrices with 21 Code of Federal Regulations-compliant tracking abilities.”

Cell-based vaccines.

There is also an interest among vaccine manufacturers in producing vaccines in humanized cell systems as opposed to what Bure says are “antiquated egg techniques.” Bure notes that researchers are currently investigating dendritic vaccines for hard-to-treat cancers, such as glioblastomas.

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