Formulation/Fill–Finish: BPI Theater @ BIO 2015

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Matt Feldman (senior manager of strategic communications, Aeras) 10:00–10:25 am

Vaccines, Global Health, and the Future of Fighting Tuberculosis

Aeras is a nonprofit biotechnology company located in Rockville, MD, with other locations in Beijing, China, and in Cape Town, South Africa. Its mission is to advance tuberculosis (TB) vaccines worldwide. The fully integrated biotech company has in-house capabilities in finance, portfolio management, and manufacturing, taking products from preclinical through phase 2b clinical trials. Aeras is governed by a board of directors, external and internal advisory groups, and a leadership team with experience in public- and private-sector vaccine development — all in all, employing about 160 people with an annual budget of about $55 million. Financing primarily comes from donors such as the Gates Foundation and from governments around the world.

Feldman began with a short history of infectious diseases and how they have affected civilizations over thousands of years. He observed that TB is “Mother Nature’s number-one killer over the past 200 years,” and it is likely to have been responsible for more than a billion deaths throughout human history. It is spread like a common cold, transmitted by coughing, sneezing, and talking. This year alone will see nine million new cases — and a million and a half deaths (more than 4,000 deaths a day, which Feldman compared with the recent Ebola crisis, which killed about 22 people a day). TB is responsible for the deaths of about one in four people with HIV and is responsible for what he called “a remarkable amount of documented drug resistance.” One in four new TB cases coming out of Eastern Europe is drug resistant, and the implications of that on treatment costs make TB “an incredibly expensive disease. In the United states, a case of drug-sensitive TB costs about US$17,000; multidrug resistant TB, a quarter of a million dollars; and extensively drug-resistant TB (referred to as XDR), more than a half a million dollars.

Feldman explained more about XDR, calling it “Ebola with wings.” It has a similar mortality rate to that of Ebola but with airborne transmission and is found in more than 100 countries. He emphasized the mortality rates, the expensive and lengthy nature of treatments, and the impossibility of containment in these days of international travel.

The 90-year-old bacille Calmette-Guerin (BCG) vaccine reduces the risk of severe pediatric disease and meningococcal TB, but it offers unreliable protection against adult pulmonary TB — which accounts for the bulk of the world’s TB transmission in adults. According to the World Health Organization (WHO), a new vaccine, introduced around 2025, should be critical to ending the threat of TB and accelerate the decline of cases per year. Several stages of the disease offer different potential breakpoints to interrupt its transmission. He described some programs under way toward developing vaccines, preventing latent infections from progressing, and shortening treatment times. A number of viral vector vaccines are in phase 1, protein adjuvant vaccines are in phase 2, and a product called M72, currently in a multicountry phase 2b trial, is one for which Aeras is enrolling subjects in southern Africa.

Partnerships Are Key: Aeras built its own vaccine manufacturing facility in 2008 to perform end-to-end manufacturing, going from master cell line creation to finished vials. The facility was then made available for contract manufacturing services to other organizations, notably for an initial partnership with Crucel on an adenovirus platform. That provided an opportunity in 2014 for Aeras to become involved with producing a vaccine for Ebola — a different disease from TB, but one that shares the adenovirus platform. The company worked with Janssen to help support its accelerated time line in 2014, getting the vaccine produced and into clinical trials. In November and December of 2014, Aeras filled more than 11,000 vials of the candidate Ebola vaccine.

Through partnerships, Feldman said, “we can achieve more, we can achieve it better, and we can probably achieve it faster.” Whether to fight TB, Ebola, or any other urgent infectious disease threat, new tools are essential, but “no one organization is going to do that on its own. Worldwide collaborations are essential to this process. Novel approaches help us accelerate the learning curve. We can’t just take one approach and beat it to death. We have to look at TB from a holistic perspective and take new strategies to interrupt this disease. Only through looking at and exhausting all options are we going to find the option that works and works in a way that is accessible, affordable, and effective for the world.”

He elaborated on a comprehensive view of vaccine development that maximizes key resources. Efforts to define and differentiate vaccine platforms are critical to this fight, and diversity is needed in the pipeline, approaching TB from several different platforms. “Different pieces of the puzzle are going to get filled in by those different candidates, informing the models, informing the adjuvants, informing how to get there. Better data get us to better decision making. Success is going to be based on transparency, trust, coordination around the world and among various stakeholders with different resources and different priorities, but working together. And together, these partnerships are going to make progress a virtuous cycle, not only for TB and for individuals and organizations, but for the world.”

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Greg Adams (director of analytical and formulation development, Fujifilm DioSynth) 10:30–10:55 am

Biopharmaceutical Formulation: A Journey from Expression to Patient

Adams described a holistic approach in which “formulation development shouldn’t be thought of as just drug-substance or drug-product development. It’s part of the protein’s journey from expression and recovery through purification and fill–finish, then ultimately administration to patients. Processing and handling affect protein stability. Integrating preformulation and biophysical characterization at all stages enables companies to better understand the conditions their drug molecules need to remain stable. Early stress testing helps in downstream process development. “People tend to think of protein structure as frozen, like a computer-generated model locked in space and time.” But in solution, a protein twists and turns about, and depending on the conditions and concentration, it may also lose structure. Adams showed three stages a protein can go through: from denatured to an intermediate, partly folded state, and then its native state. That behavior, he said, depends on the aqueous environment.

“Protein purification is a stress-inducing process,” Adams explained, from recovery to capture to chromatography and postcolumn processing, concentration, and different filling processes. At all those stages, a protein is susceptible to shear, agitation, and degradation. “We want to monitor what’s happening to a protein as it goes through that process,” he said, listing several instruments in the “biophysical toolbox” on a slide. Then he highlighted two methods: differential scanning calorimetry (DSC) and isothermal chemical denaturation.

thermal chemical denaturation. DSC provides information on thermal stability as a function of solution conditions. Adams showed the transition that occurs as a sample is slowly heated, causing proteins to unfold. Depending on conditions, the temperature at which that happens generally indicates protein stability. At different points in a typical monoclonal antibody (MAb) downstream process — protein A capture, viral inactivation, two-column chromatography, and ultrafiltration/diafiltration (UF/DF) concentration — it is subjected to stressful conditions and potential instability.

Adams showed a MAb coming off protein A at a fairly low pH, at which it is held for viral inactivation before pH is brought up for ion exchange. DSC screening elucidates how those pH changes affect the product. He showed a DSC profile that remained fairly stable with acetate rather than citrate used as a buffer. The higher the denaturation temperature, the more stable a protein is in solution. And the acetate–sodium-phosphate condition was found to maximize stability for cation exchange.

Adams’ second case study described a protein refolding process. This is often needed with microbial proteins expressed as inclusion bodies (IBs) in Escherichia coli. IBs are basically clumps of aggregated/denatured protein that must be solubilized and refolded, what Adams called a “black- box” process, typically involving very high concentrations of detergents. Yields can be low, with many inactive, misfolded, and aggregated proteins as impurities and very little soluble, fully folded product. He described how the new technique of isothermal chemical denaturation can help companies “peer into this black box” to monitor refold processing. Analysts subject a sample to high detergent concentrations and then drop those levels stepwise with guanidine and dithiothreitol (DTT), noting at each stage (through fluorescence monitoring) the mix of isoforms. Ideally, he explained, they want to see free-state transitions.

Case study results showed one graph tracking solubilization (increasing guanidine concentration) and one the refolding. Comparing with reference material, analysts look for conditions at which refolding and formulation buffers show the same results. They assay both a pure drug substance and solubilized inclusion bodies. Having expected interference from other components found in inclusion bodies (e.g., other host proteins and DNA), his company was pleased to find the comparison to be relatively easy. Using design of experiments (DoE) to further compare different DTT concentrations, the team optimized and doubled the refolding yield.

Adams concluded by describing “a new paradigm in formulation development.” The traditional stepwise approach includes biophysical characterization and screening, then testing different buffers at various temperatures over time. It typically takes nine to 12 months. With today’s increasing emphasis on speed to market, formulators need ways to make this a faster process. Prior knowledge and literature awareness help, especially for MAbs. New biophysical tools and high- throughput technologies do too, shortening by at least half.

“We really want a middle ground,” Adams said. Formulators need both speed and deliberation in their work. He offered more examples of a combination approach, pointing first to the lack of correlation found between MAb pI and final pH of that MAb’s formulation. Some people have tried to develop a platform formulation approach that works around a protein’s pH, but it rarely works out. However, Adams said, a formulation platform approach to excipients may be possible.

Only a few excipients have been shown to work in MAb formulations. Most are formulated in some combination of polysorbate and salt. In a case study involving DoE screening based on that knowledge, the team found its high- concentration formulation in three months rather than a year with accelerated stability testing. Further testing extended that stability window to 25 weeks.

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Anthony Cannon (head of drug product manufacturing technology, Samsung Biologics) 11:30–11:55 am

Anatomy of the Lyophilization Process: Considerations for Successful Tech Transfer

Beginning with ICH Q10’s definition of technology transfer, Cannon emphasized ensuring sufficient control of variability in a commercial production environment. The ultimate goal is documented evidence that a manufacturing process is robust and effective in producing good manufacturing practice (GMP)-compliant products.

Freeze-drying (lyophilization) removes a solvent (e.g., water) to convert a solution to a solid state. This prolongs shelf life for products that are sensitive to reactions in liquid state. Some proteins are stable in liquid for less than a day, some up to a year — neither of which is an acceptable commercial shelf life. Some products are stored frozen, particularly those in early clinical testing without fully validated lyophilization processes in place. Most lyophilized products can be stored at room temperature. After a brief overview of the freeze-drying process — lowered temperature, primary drying below the vapor pressure of ice, and secondary drying to remove residual water — Cannon went on to discuss associated tech- transfer considerations.

Companies must evaluate the influence of process- and equipment-related differences. Performance differences of laboratory, pilot-scale, and manufacturing freeze-dryers need to be characterized early on. Cannon said the product itself — not the equipment — should be the only limiting factor in optimizing process conditions. He also said that freezing is the most important step to focus on in both process development and tech transfer. Shelf temperature and pressure controls are also important. Types and locations of gauges and control mechanisms make a difference.

In a manufacturing setting, loading and stoppering of numerous vials further complicate the basic freeze–dry process. Both take time, depending on batch size and level of automation. The time lag itself can affect stability and behavior of some products, as can related temperature differences. It’s better to load product at 5 °C (or colder) than at 20 °C. Hold time should be minimized before the lyophilization process begins. Everything should be at the same temperature before the cooling phase begins.

Once product vials are loaded, cooling rates are critical. Shelf uniformity within the freeze-dryer makes a difference. If temperature is too high in some locations, then product solutions may not completely solidify there before drying begins. With a case study, Cannon showed how a difference in process controls between the original laboratory-scale equipment and the commercial lyophilizers affected freezing rates. All product temperatures must be the same before cooling, with the temperature lowered from, e.g., –5 °C to –45 °C. Newer large-scale units provide very good control for a straight-line decrease. But with a product transferring from an older system with less precise control, his company had the challenge of reproducing the exact parameters of the original freezing step. Other freezing scenarios include cooling that is too fast or too slow. When transferring up in scale, equipment size will be an issue. Larger units require more time to cool down.

In primary drying, shelf temperature and chamber pressure are important to the rate of sublimation, which will occur only at low pressure with slight heating. “Shelf spacing is one consideration you wouldn’t necessarily think of,” said Cannon. It will be different in different lyophilizers and can affect sublimation rates through radiant heating. Product temperature must be maintained below the threshold at which glass transition, collapse, and eutectic melt occur. The unit condenser must be cold enough to condense the water vapor that comes off as products dry out.

Pressure control is extremely important, and smaller- scale units are easier to control more precisely than commercial units. Companies should consider edge effects and equipment design as well. For example, the port between the chamber and condenser in some units may allow backflow during aggressive sublimation processes. Chamber pressure that is too high directly influences product temperature; chamber pressure that is too low slows down the sublimation rate.

In secondary drying, shelf temperature most directly influences the amount of moisture that remains. Excessively low shelf temperatures allow moisture to remain; high temperatures can detrimentally affect the protein product. Product temperature is still an issue even with all the ice removed. Now the threshold temperature isn’t necessarily below freezing, but biotherapeutics can be sensitive to some temperature above room temperature. Secondary drying up to 50 °C would damage most such products. Again, condenser temperature is important to pull off the vapor coming from products in the unit.

The last step of a commercial lyophilization process is stoppering. Shelf temperature may affect the smoothness of this process; stoppers may stick at higher temperatures. And for safety reasons, stoppering and unloading shouldn’t occur at temperatures that are too high. Some products may still need to be stored at 5 °C. Chamber pressure during stoppering directly determines vial pressure inside the stoppered vials. Significantly low pressure can pull atmosphere inside when the vial stopper is punctured. Higher pressures may complicate reconstitution efforts.

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