We hear a great deal lately about the maturation of the biopharmaceutical industry — and much advancement over the past decade or so has been in business models, financing, and product pipelines. Meanwhile, regulators around the world have become more well versed in the subject matter and have adjusted their approaches to and expectations from the industry. However, the practical side of developing, characterizing, and manufacturing biotherapeutic products cannot be overlooked — nor its importance overstated. Many technological advancements in recent years have enabled companies to shorten time to market, to better understand their manufacturing processes, and to characterize their products well. In fact, the technical aspects have driven much of the increasing sophistication on both the regulatory and business sides of this drug-industry sector.
In August 2013, BPI informally surveyed readers about those technologies. In addition to the usual demographics, we asked what upstream, downstream, analytical, formulation/fill–finish, facilities, and discovery technologies have most improved (and/or are most improving) bioprocessing. The answers helped guide me in preparing this special report.
And they’ve brought me to some interesting conclusions — in some cases confirming my sense of things from all my reading, editing, and conference attendance; and in others bringing to light some trends of which I was yet to be fully aware. What follows is a celebration of sorts — highlighting and acknowledging some of the main technical advancements that have taken the biopharmaceutical industry from being big pharma’s little sister to become a force to be reckoned with. As major drug companies begin restructuring their organizations away from the old blockbuster-pill mentality toward more personalized approaches and biologics, it’s clear that all these technologies have a bright future ahead.
I begin where bioprocessing always begins: upstream with production methods and equipment. The past couple decades have seen great advancement in cell lines and genetic engineering as well as feeding strategies, culture media, and bioreactor technologies. New expression systems have appeared on the scene to raise the expression bar: the PER.C6 human cell line from Crucell (now owned by Johnson & Johnson) and Pseudomonas fluorescens from Pfenex (spun out from Dow Chemical Company), just to name a couple. Serum-free media and chemically defined supplements became the norm for cultures producing human therapeutics. Special promoters and other genetic engineering elements have helped companies develop high-expressing cell lines, and a plethora of hardware improvements and new gadgets have flooded the market (1,2,3,4,5,6,7,8,9,10,11).
The results of our survey reflected all the above. Over a third of respondents pointed to new bioreactor technologies as the most influential advancement, with about a fifth choosing new culture media and another fifth divided between cell-line engineering and new cell lines. Of the 22% who answered “other,” most elaborated with, “single-use technologies,” echoing the commentary of those who offered more detail with their “new bioreactor technologies” responses. Clearly, hardware improvements and inventions have had great impact.
Mats Inganäs (senior director of R&D science and applications at Gyros AB in Sweden) highlighted the importance of analytics in modern upstream process development. He pointed out that product quantification (e.g., IgG titers) and characterization (e.g., glycan profiling of therapeutic antibody in cell culture media) provide valuable information for modern process developers.
I asked editorial advisor Bill Whitford (bioprocessing market senior manager at Thermo Fisher Scientific) for his thoughts on these results. “Improvements in biopharmaceutical production can be categorized into a few rather distinct arenas,” he said, pointing to cells and genetic engineering, media, and hardware.
“From the producer cell-line perspective, we see entirely new cell lines, such as EB66 cell line from Vivalis and SAFC. Derived from duck embryonic stem cells, it maintains genetic stability in serum-free media while supporting virus production in suspension culture.” Serum-free, animal-product–free, protein-free, and chemically defined media haven’t been enabling technologies as much as they have presented new challenges. In seeking to remove potential contamination sources and variability from their upstream processes, biopharmaceutical companies have had to find ways to maintain product quality in an increasingly unnatural culture environment. “But the newest products from premier media suppliers are now providing robust consistency with unprecedented performance.”
When it comes to cell engineering, Whitford said, “we see sponsors cloning in (or knocking out) particular glycosylation pathways in established cell lines. We even see more advanced control, such as of the cell cycle itself using CDI Bioscience’s RP Shift technology. The emerging fields of systems biology and metabolomics are just beginning to yield practical results in practically controlling the aggregate effects of individual biochemical pathways in producing cells.”
From a process perspective, Whitford explained, the flexibility and support of modular processes afforded by single-use systems have changed manufacturing processes forever. “Beyond that, the marriage of QbD and PAT principles with improved process analytics has significantly increased process understanding and control. This all has led to the emerging revolution of continuous biomanufacturing, which has already established its potential in such individual operations as the intensified perfusion mode of culture and SMB [simulated moving bed] chromatography. Combine that with developments in process control from fuzzy logic in algorithms to SCADA (supervisory control and data acquisition) systems more enterprise control systems. The results have inspired the expectation of truly integrated, closed, and continuous bioproduction in the near future.”
Tim Ward heads the cell culture team at TAP Biosystems. “One of the most revolutionary developments in recent years,” he told me, “are in the tools available to improve the efficiency of cell line selection and process development. Historically, tools for early clone evaluation have been poor predictors of how cell lines would behave at scale.” That led to repeated process development activities when originally selected cell lines did not perform well in large bioreactors. “Early stage screening tools have improved with the use of clone selection technologies that better assess antibody productivity,” Ward said, “as well as improved shaken-plate–based culture systems that make it easier to make an initial selection.” More important, he said, is the availability of microscale bioreactors that better mimic large-performance than ever before. They have enabled clone selection under process conditions, dramatically improving success rates in cell line development. “The best systems are fully automated,” he pointed out, “with single-use, fully disposable microbioreactors (complete with impeller and sparge gas), that can mimic the pH, DO [dissolved oxygen], and feeding control of full production-scale platforms. These are widely reported to be very predictive of performance in 5-L and 10-L benchtop bioreactors.”
Such microscale systems perform parallel processing with 24–48 separate reactors. In combination with automated sampling, said Ward, they
can be used for large design of experiment (DoE) testing, which can be a challenge when carried out using conventional benchtop systems — and expensive in terms of operator time and needed resources. “The software on these systems makes it easy to import designs,” said Ward, “and (in automated systems) to set up media and feed combinations.” That makes such systems “truly enabling” for understanding an upstream design space. “Fortunately, analytical tools have been developing in parallel,” Ward pointed out, “to enable the use of smaller samples so that the 10- to 15-mL volumes of microbioreactor systems are not too depleted by the need for frequent sampling.”
Looking to the future, Ward said, “a similar approach has been applied to larger-scale arrays (100–250 mL) in single-use, fully disposable bioreactors that can incorporate more precise control and continuous feeding. They can be used for enhanced process development and scale-down models of ≤150-L systems, potentially removing the need for laboratory-scale bench-top bioreactors. This could provide a further step change in productivity for process development laboratories and consequent reductions in development time and cost.”
Chromatography is the cornerstone of downstream processing. Protein A affinity has anchored the antibody-purification platform since its introduction. Ion-exchange is often part of that platform — and an essential element of separation and purification for most antibody proteins as well. Size-exclusion, reverse-phase, and hydrophobic-interaction chemistries all have their place in bioprocessing. Other technologies may fit between chromatography steps, provide viral filtration, harvest supernatant and clarify process streams, and so on — but none have yet knocked the heavyweight champion from the ring.
A Downstream Technology Provider Weighs In
One example of a new downstream technololgy that’s beginning to make an impact is advective chromatography from Natrix Separations, Inc. While analyzing our survey results, I spoke with James Stout, PhD, the company’s vice president of process sciences.
BPI: The top responses in our survey for innovations that have enabled companies to improve their downstream processing were new chromatography media, technology, and membrane adsorbers. Where does advective chromatography fall into that mix?
JS: Natrix HD membranes fall into all three categories. This new chromatographic technology combines the protein binding capacity of resin-based chromatography with the speed of membrane adsorbers. A patented three-dimensional, macroporous fiber structure is polymerized with a hydrogel made of ≥95% functional monomer, making the hydrogel structure itself functional. So the membranes are uniformly and densely saturated with binding sites. We can engineer Natrix HD membranes with traditional functional chemistries, and we are also investigating and developing new advanced chemistries (e.g., mixed-mode and affinity ligands).
HD membranes offer superior binding capacity, high resolution and selectivity, and unparalleled flow kinetics with residence time in seconds. Advective technology will allow faster separations using less chromatographic material in a simple, flexible, and cost-effective plug-and-play format.
BPI: Many companies are seeking alternatives to protein A affinity as that crucial primary unit operation for antibody purification. Do you think a new sorbent will be the answer — a new chromatogographic approach — or some other technology entirely? Can protein A find its way into advective media?
JS: Natrix is exploring various affinity ligands, including protein A, to design into HD membranes as a future separation modality. Our focus is on providing a simple, disposable chromatography approach to meet the most demanding process challenges for monoclonal antibodies (MAbs) and other biomolecules. We want to provide a full downstream processing tool-set for for critical unit operations and all process needs now and in the future.
HD membranes are designed for rapid processing and can be cycled in minutes, so the technology is suited for continuous processing either using conventional systems or in emerging simulated moving-bed (SMB) platforms. SMB techniques are starting to mature and being combined with advanced clarification techniques, such as precipitation, tangential filtration, and centrifugation strategies. Natrix membranes fit well with all three and can help close the gap in processing bottleneck challenges.
BPI: Nonantibody biopharmaceuticals are a diverse collection of protein classes. What are the chances for protein-A type platforms to establish themselves for (some of) these products? What nonantibody protein types might have the most promise for such a platform approach? And where would advective chromatography fit in?
JS: This is an enormous challenge for chromatographic processing. Presently, the ProA ligand is the best affinity chromatography step for antibodies and Fc-fusion proteins due to its selectivity, reproducibility, cycle life, and history in downstream processing and with the regulatory agencies. A similar type of platform for other biomolecules without a specific universal binding site as a target for affinity ligands will be challenging to design. However, with deeper understanding into biomolecule properties and limited to specific classes of molecules — e.g., viruses, glycoproteins, or vaccines — it may be possible to design affinity ligands that are biomolecule class specific. Such affinity ligands could be developed into a wide range of downstream processing matrices, and advective chromatography would be well-suited for such designs.
For now, traditional chemistry ligands and mixed-mode functional groups are being used for their orthogonal binding properties with nonantibody and non–Fc-fusion molecules. They usually require more chromatography steps to achieve the required throughput at the expense of overall yield — and sometimes activity. Natrix is currently working with a number of ligand partners who have unique tools for these applications, and we see great promise for HD membranes using these ligands in the future.
Our survey results made that clear. Nearly three-quarters of respondents pointed to something chromatographic in nature as the most significant enabling technology for downstream processing: from new media (28%) to membrane adsorbers (23%) and new chromatographic technologies (21%) such as monoliths and SMB. For all the talk we’ve heard over the past decade about “anything but chromatography” (ABC) (12), only 14% of respondents pointed us in that direction, just as many as are lumped together under “other.” However, if you consider that most “other” responses — e.g., centrifugation and improved filter membranes — would also fall under the ABC umbrella, that moves it into second place behind new chromatography media with 26%. So even as the champ continues to reign (13,14,15,16,17,18,19), the challengers may be ganging up to topple him from his place (20,21,22,23). Is chromatography the downstream processing equivalent of petroleum-based energy — a monopolistic dinosaur that will yield to many challenging solutions (with filtration, partition, and other separation methods filling the role of solar, wind, and wave power)? Or will it settle alongside them in a company of equals? So far, the latter seems to be the case. And taken as a whole, chromatographic technology is still king.
I asked Nanying Bian, PhD (a principal
scientist at EMD Millipore Corporation) about these results. She wrote, “Process development scientists and engineers in the 21st century have many more choices of chromatography resins than their counterparts had 20 years ago. Innovations from resin suppliers have generated new chromatography resins with higher capacity and better resolution for purification of more challenging biological feed streams.”
Specific to protein A affinity chromatography resins, Bian added, “The first generation of protein A resins are generally characterized with low capacities, slower flow rate, and susceptibility to alkaline cleaning solutions. Over the years, the combination of resin base-matrix invention and protein ligand development have provided higher capacities and faster-flow resins that can be cleaned by alkaline and/or acid solutions (e.g., EMD Millipore’s Eshmuno A brand). This type of advancement offers customers significantly higher productivities, operational flexibility, and regulatory compliance, especially for modern high-titer expression systems.”
Another trend Bian pointed out is in disposable chromatography, especially for purification of early to mid-clinical phase materials. “Prepacked chromatography columns have been gaining popularity with process development scientists and engineers. These prepacked columns reduce non–value-added time (column packing) and increase throughput while freeing technical staff for more important tasks. In addition, scientists and engineers are no longer content to use the heavy plastic cylinders of early disposable chromatography solutions. In this heavily regulated industry, they want a column that is sanitized and ready to use, ideally designed with operator ergonomics in mind.” Bian cited her company’s Chromabolt family of columns as an example.
“As time-to-market and productivity continue to drive many biopharmaceutical companies,” she concluded, “prepacked columns definitely offer something that process development scientists and engineers from the past century would have envied.”
Part 2 of this special report (February 2014) will examine the results for analytical, formulation/fill–finish, and facilities technologies, as well as touch on drug discovery.
Cheryl Scott is cofounder and senior technical editor of BioProcess International, 1574 Coburg Road #242, Eugene, OR 97401; 1-646-957-8879; firstname.lastname@example.org.