Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

Perfusion technology was first introduced in the late 1980s to boost the low product concentrations obtained from the early cell lines used for biopharmaceutical manufacturing. Dramatic increases in titers for batch and fed-batch cell-culture processes over the next two decades largely eliminated the need for perfusion and interest waned. A key driver today is the production of unstable proteins that require low residence times in the bioreactor. Recently, however, rising cost pressures have been driving the need to achieve greater efficiency and productivity and reduce costs. Continuous bioprocessing is seen as a possible solution and, consequently, perfusion is once again attracting significant attention.

	In perfusion mode, high cell numbers are sustained for much longer periods by constantly feeding fresh media and removing spent media while the cells remain in culture. With this approach, optimum conditions for growth and production are maintained by supplying the appropriate nutrients and removing toxic waste products. Because the product is also regularly removed and separated from the waste products that can cause degradation, perfusion is highly beneficial for biologic APIs that are unstable under production conditions.

	Different methods are used to remove the spent cells, including centrifugation, in which the cells

	are separated and then returned to the reactor; alternating tangential-flow (ATF) filtration, in which the cells remain in the reactor and the spent media and product are collected in the filtrate; and the use of adherent cells that bind to capillary fibers or membranes in the bioreactor, which allows easy separation from the spent media.

	Introduction of the ATF technology (by Refine Technology; now owned by Repligen) was quite significant for perfusion processes, according to Parrish Galliher, CTO of Xcellerex, which is part of GE Healthcare’s Life Sciences business. “ATF and other filtration perfusion processes allow for complete clarification of the product, whereas with centrifugation, approximately 90% of the cells are recycled back to the bioreactor, and an additional filtration step is required to remove the remaining 10% of the cells from the product.” He also notes that advances in single-use technologies for perfusion have greatly simplified the process because sterilization and cleaning systems are no longer required, which in turn has made it much easier to set up and evaluate perfusion as a viable option for cell culture.


	As indicated above, perfusion cell culture has been used in the industry to produce biologics clinically and commercially for more than 25 years. Currently, a handful of companies manufacture biopharmaceutical drug substances using perfusion technology, largely because these products are not stable in the bioreactor and must be removed quickly to minimize degradation/modification, according to Galliher.

	Many more companies are looking at perfusion at the process development and clinical scales, however. This interest goes beyond the desire to improve the product quality of sensitive biologics; perfusion has the potential to reduce the scale at which reactions can be performed and increase productivity through better equipment utilization. “With the recent growing interest in continuous bioprocessing, there is renewed interest from manufacturers, equipment vendors, and academics in evaluating and pursuing perfusion technology,” says Chris Hwang, senior director of late-stage process development at Genzyme.


	Properly designed perfusion processes can significantly increase volumetric productivities (grams/L of bioreactor working volume per day) such that the bioreactor scale can be significantly reduced, which facilitates adoption of disposable technologies. The result is an increase in operational and capacity flexibility, reduced capital and operational costs, and increased speed (facility readiness and manufacturing operation), according to Hwang.

	In addition, due to the measurable decrease in production scale, it is possible to develop and scale up processes to production scale in development labs using the same control/automation systems and eliminate process scale-up in manufacturing, which significantly decreases technology-transfer risks. Site-to-site transfer risks can also be reduced because perfusion processes can be used to produce any protein (stable or non-stable), which facilitates standardization.

	Product quality is also important. In addition to minimizing the risks associated with product degradation/modification due to the reduced product residence time in the bioreactor compared with what occurs in a fed-batch system, perfusion processes can achieve steady-state product quality and simplify downstream operations, which all result in decreased heterogeneity and greater product consistency.

	The jury is still out, however, on whether perfusion and continuous biomanufacturing can truly increase development speeds and decrease costs for products that do not have stability/degradation issues, according to Galliher. “Perfusion is very easy to achieve on the lab scale, but there are many reports of additional challenges to its use on the commercial scale.

	Opinions vary widely among those who have been involved in implementing larger commercial perfusion processes, with some eager to use the technology again and others who are not,” Galliher says. The potential advantages are sufficiently significant, though, and many people are evaluating perfusion to determine its applicability for their biopharmaceutical processes.

Integration with downstream continuous processes


	Integration of a perfusion bioreactor process with continuous downstream processes (integrated continuous biomanufacturing, or ICB) increases the advantages of perfusion, according to Hwang. “If all of the unit operations between the bioreactor and downstream purification are performed continuously, one can minimize hold times and eliminate many unit operations, and all of the downstream equipment can also be miniaturized and single-use technologies can be utilized. The application of appropriate process analytical technology (PAT) enables automation of the entire production process without operator intervention, except to ensure uninterrupted medium and buffer supply,” he explains.

	Genzyme/Sanofi has successfully demonstrated at production scale the integration of a perfusion bioreactor with continuous capture for model enzymes and monoclonal antibodies with the products Cerezyme (imiglucerase for injection), Fabrazyme (agalsidase beta), and Myozyme (alglucosidase alfa). In addition, proof-of-concept of a fully continuous process (end-to-end) has also been demonstrated at the lab scale, according to Hwang.


	Despite changing perceptions of perfusion and continuous processing, significant challenges do remain. Some are very practical issues. A lack of adequate sensor technology for the on-line direct evaluation of product quality parameters-protein folding, aggregation, glycosylation, oxidation, contamination, etc.-is a risk, according to Galliher. “Product quality at present can only be monitored indirectly on-line through determination of solution temperature, pH, conductivity, etc., and the product quality cannot be determined until it is sampled and analyzed.

	As a result, more business risk is accumulating the longer a process is run, and that risk is compounded even further if perfusion is directly integrated with multiple continuous downstream processes that occur without product sampling and analysis,” he explains. Robust in-line product quality sensors will reduce this operational risk.

	Process quality by design (QbD) and design-space studies can also be more complex, because the ability to maintain product quality over an extended period of time must be verified for a full production run, which can go on for weeks or months. These longer run times can extend development times, according to Parrish, because QbD screening and optimization experiments take longer. It is possible to reduce the number of process parameters that are investigated to keep the development time similar to that of a batch/batch-fed process, but that may lead to the development of a less robust process. The positive view that FDA has of continuous manufacturing presents an opportunity to evolve the guidelines for QbD studies when applied to continuous processing.

	Unexpected costs can also reduce the positive impacts of perfusion. As an example, Galliher notes the benefit that a perfusion bioreactor can be scaled down by as much as 10-fold compared to a conventional bioreactor, enabling adoption of single-use technology with a smaller footprint and lower costs. Because the process is continuous, additional sterile-feed and sterile-harvest collection vessels are required to keep the process operating. “These additional vessels take up room and carry additional costs that must be considered when determining the overall benefits of continuous biomanufacturing,” Galliher observes. Concentrates of cell-growth media and buffers coupled with in-line dilution can reduce these additional costs. Hwang also notes that in an ICB process, many of these tanks would be eliminated.

	Other challenges have more to do with perceptions and company culture. “For companies that have historically relied on batch upstream and downstream processes for protein production, moving to perfusion/continuous processes can be difficult due to management concern about implementing what are considered to be new technologies for the company, particularly with respect to the potential for increased risk and development times,” Hwang explains. There is also often a desire to fully utilize existing production capacity, and it can be very challenging to overcome the deeply entrenched batch technology mindset throughout an organization. For existing products, there are additional concerns about implementing continuous processes as part of lifecycle-management strategies with respect to product comparability.

	To overcome these challenges, Hwang recommends that, to gain experience, companies invest resources and capital for the testing of continuous platforms that is de-coupled from product development. That way, risks can be minimized because a new platform won’t be considered ready for manufacturing until the expected process benefits can indeed be achieved.


	Given the heightened interest in perfusion and continuous bioprocessing, it is not surprising that equipment and device manufacturers are working to develop improved solutions for perfusion processes. “Key goals are to make perfusion more robust, scalable, and easier to operate,” says Galliher. GE is looking at all aspects of the system, such as in-line dilution systems and improvements of the filtration process (prevention of clogging) to increase flow rates for larger-scale, single-use filtration systems.

	Both Galliher and Hwang agree that the development of novel, robust PAT sensors for in-line monitoring of process performance and product attributes in real time will be crucial for the more widespread adoption of perfusion and other continuous bioprocesses on a commercial scale. For full integration into disposable platforms for protein production, pre-sterilized production-scale perfusion systems must also be developed, according to Hwang.

	“FDA has been encouraging the industry to transition to continuous manufacturing (CM) since 2011 because the agency sees CM as a key enabler for modernizing pharmaceutical manufacturing. I anticipate that with the continued improvement of bioreactor cell densities and further increases in volumetric productivities, there will be growing interest in the use of perfusion technology for protein production,” says Hwang. “Coupled with the potential significant strategic advantages of ICB, I suspect that industry adoption will continue to increase and further fuel advancement of the intensification of perfusion and continuous bioprocesses,” he concludes.

:
	When referring to this article, please cite it as C. Challener. “The Potential of Perfusion,” 

Articles

Potential for improved product quality and cost/time savings is reviving interest in perfusion technology.

Pursuing Perfusion

Despite a few significant investments in continuous manufacturing facilities by pharmaceutical companies, including Vertex Pharmaceuticals, Johnson & Johnson, GlaxoSmithKline, and Novartis (1), the adoption of flow chemistry for commercial production of APIs generally remains in the early stages. FDA has encouraged the adoption of continuous manufacturing since 2004, but specific guidelines are lacking from the agency and other regulators around the globe. Both former FDA Commissioner Margaret Hamburg (1) and Center for Drug Evaluation and Research Director Janet Woodcock (2) have been more vocal about the issue, particularly in relation to the proposed 21st Century Cures Act. This legislation requires FDA to support the development and implementation of continuous manufacturing for drugs and biologicals as one of several approaches to speeding up drug development and commercialization (3). In addition to a lack of comprehensive regulatory guidance, however, a dearth of industry personnel with expertise in flow chemistry is a hindrance to rapid adoption of continuous technologies.

Early adopters and fast followers drive change


	“While the industry as a whole is in the early stages of adopting flow chemistry for small-molecule API manufacturing, the early adopters and fast followers not only recognize that flow chemistry is the future of manufacturing, but also believe that they can implement it effectively,” asserts Tim Jamison, professor of chemistry and incoming department head at the Massachusetts Institute of Technology (MIT) and CEO of Snapdragon Chemistry, a new company dedicated to catalyzing the adoption of continuous flow synthesis. “Ultimately, when these companies realize the many benefits of flow chemistry, including reduced operating costs, footprint, and capital expenditures combined with improved process efficiencies, control, and product quality, investors likely will modify their expectations and demand this increased value from the industry as a whole. The rest of the industry will then have to scramble to catch up, and the early adopters and fast followers should reap the rewards of their forward-thinking actions. At that point, the entire industry--out of necessity to remain competitive--likely would shift its view on flow technology,” he explains.

	Most large pharmaceutical companies, according to Peter Poechlauer, innovation manager with Patheon, have at least installed advocate groups with a mission to showcase successful applications of flow processes. Dominique Roberge, head of chemical technologies with Lonza Pharma & Biotech agrees that flow chemistry has become an accepted technology for small-molecule manufacturing, largely for the development of new chemical reactions that have not been feasible in batch operations, to reduce the cost of goods, and to decrease capital expenditures (CAPEX) via process intensification. “These projects are significantly more focused and give a better understanding of what is achievable via flow chemistry. As a result, we have moved past focusing on feasibility studies only and are now evaluating projects that are more mature for tech transfer and scale-up,” he notes.

	“The decision to develop a certain step as a flow process is, however, opportunistic and may be motivated by the speed provided in early development, the need for smooth scale-up of hazardous reactions, and/or the savings in investment for a new drug whose future is still uncertain,” Poechlauer observes. In addition, he notes that these criteria apply to single steps of multi-step pharmaceutical syntheses, and therefore hybrid approaches that combine continuous and batch operations are most common; few companies have developed end-to-end continuous syntheses of pharmaceuticals that combine drug-substance manufacturing and drug-product formulation.

	“Continuous flow manufacturing occupies a similar position to where a technology like spray drying was 10-15 years ago. It shows great promise but the ‘mainstream’ commercial viability within the pharmaceutical industry has yet to mature,” says Patrick Kaiser, a principal scientist in the process development business of SAFC. He believes that, like spray drying, a clear commercial pathway and, more importantly, a clear regulatory pathway will drive more entrants as developers embrace the technology’s promise. Even so, Kaiser expects there will be limitations to its full embrace, because certain systems lend themselves to be more relevant to continuous processing, while others make less sense to perform via continuous means, and this dissonance complicates the pathway forward. “The reality is that CMOs must embrace such disruptive technologies moving forward to ensure long-term competitiveness in the marketplace,” he concludes.


	A positive indication for the future of flow chemistry is its increasing use for different types of reactions and downstream separation/purification operations. With respect to chemical reactions, using flow processes allows better control of yield and selectivity, which have a direct influence on purification and separation steps, according to Roberge. “The best approach is to develop new processes that can and will lead to a significant improvement in the synthetic route, but will typically not work in a traditional batch process,” he comments. His examples include various oxidation reactions (with molecular oxygen or hydrogen peroxide), azide chemistry, and high-temperature/pressure reactions developed via microwave chemistry.

	Jamison adds that the use of flow chemistry for photochemical and electrochemical reactions is also exciting, because these reaction classes are typically difficult to carry out and control and also challenging to translate from laboratory scale to pilot or manufacturing scale, but afford significant opportunities to access completely novel chemical scaffolds and greatly streamline current synthetic routes. “By using flow chemistry to gain better control, predictability, and scalability for these reaction classes, chemists can increase their utilization, which will ultimately result in significant advances and broader adoption in the pharma industry,” Jamison states.

	Integration of chemical synthesis reactions in flow with an increasing diversity of work-up operations in flow, such as continuous extraction, membrane processes, and the crystallization and separation of solids, is also an important development, according to Poechlauer. He further notes that the application of parallel, analytical instruments with sufficiently short response times have been developed, allowing efficient control of these processes.

	The development of a complete toolbox of flow reactors that can be used for all types of reaction rates and phases (e.g., liquid-liquid, solid-liquid, etc.) is also necessary for the broad application of flow chemistry to be achieved, according to Roberge. To address some of this need, Lonza has developed the pulsating coil reactor for liquid-liquid phase reactions and an efficient de-plugging system based on ultrasound technology. “Ultimately, true innovation in this field will come from a few key players in the market who have the variety of experience and infrastructure to optimize multiple reaction platforms,” asserts Roberge.


	In fact, the inadequate supply of scientific talent and expertise necessary to implement continuous flow technology at scale is perhaps the largest factor hindering more rapid adoption of flow chemistry for small-molecule API synthesis, according to Jamison. “It’s not as simple as asking current chemists to start working with continuous flow technology. That is like asking a saxophonist to play oboe. While both instruments are woodwinds, a saxophone uses one reed, while the oboe uses two. Thus, saxophonists can certainly become oboists, but it is not automatic; there will be a learning curve in most, if not all cases. Currently, flow chemistry is generally not in most university chemistry curricula. Thus, there will continue to be a lack of expertise in this area until this situation is changed,” he explains. “In the long run, industry demand will accelerate such changes; as the industry requires more expertise in this area, education/training standards will shift to meet this demand. This shift will not occur immediately, however, and there will likely be a short- to mid-term lack of human resource supply,” Jamison says.

	Rhony Aufdenblatten, manager of small-molecule business development with Lonza Pharma & Biotech, agrees that flow chemistry remains a specialized technology, because it requires specific technical know-how that can only be developed over years of manufacturing different chemical products. “The key challenge for any small-molecule development program is management of scale-up of the lab process for industrialization. Moving this type of scale-up into a new platform like flow chemistry can only be handled by the few players who have experience working with a variety of chemistries and processes,” he observes.

	Poechlauer is not convinced that continuous processing is “experts only” territory any longer, but he also believes that this perception certainly affects decisions regarding adoption of the technology. As a result, he does believe that CMOs with a proven track record in continuous processing may be favored as demand for this capability increases.


	Two other factors that are influencing the rate of adoption of flow chemistry for API synthesis include a lack of clear regulatory guidelines and the existing batch-based manufacturing infrastructure. Although FDA representatives recently made a number of public comments in support of continuous manufacturing in the pharmaceutical industry, from a regulatory standpoint, there remains a need to develop clear, harmonized guidelines accepted across the various regulatory authorities that will facilitate the development of continuous manufacturing routes in a manner that guarantees a consistent way to monitor/regulate their output, according to Jamison. In addition, while there are a growing number of flow processes being filed with auditors despite this lack of clarity, Poechlauer notes that there is little experience with respect to the auditing of continuous process steps.

	The International Conference on Harmonization (ICH) may be an effective body for achieving international guidelines. The organization may in fact be making continuous flow manufacturing a focus issue over the next year to 18 months.

	The impact of existing batch plants is less straightforward. For companies with unused batch capacity, Aufdenblatten notes that additional motivation for further investment into large-scale flow chemistry will be needed to overcome the additional CAPEX for flow infrastructure. “Typically, a gain in yield of 2-3% will not be sufficient; reduced cost of goods, safer processes, and breakthroughs in process platforms must also be considered,” he explains.

	Jamison points out, though, that existing infrastructure is in various stages of maturity, and new capacity (whether batch or continuous) could be established naturally, with continuous plants being built in place of new batch plants, as appropriate. “In addition,” he says, “continuous manufacturing plants could have a 10- to 100-fold smaller footprint than a batch plant of comparable output. Therefore, the CAPEX investment is smaller, which might sway the economic analysis to favor the business case of mothballing a significant number of existing batch-mode plants.”

	The mentality of the process development function must also be considered, according to Kaiser. The use of flow chemistry earlier in development requires both the innovator and CMO to be open to using continuous systems as an option in their process development efforts. Doing so potentially requires developing an initial batch process backed up by a second-generation flow process, perhaps simultaneously if the drug is on an accelerated approval pathway, which is not a small shift in today’s “fast-fail” drug development business. “Development companies are generally averse to investing too much in the manufacturing process until there is good clinical data to show manufacturing on a larger scale is necessary. Unfortunately, waiting too long to develop a continuous process also complicates a company’s regulatory strategy and potentially a challenge with different impurity profiles from different manufacturing processes,” he comments. The best strategy to combat this challenge, according to Kaiser, is to have experienced flow chemistry experts recognize where continuous systems provide the greatest opportunity for results early on in the process development effort.

End-to-end implementation of flow chemistry


	Snapdragon Chemistry-a new company launched in early May 2015 by CEO Tim Jamison, a professor of chemistry and incoming department head at the Massachusetts Institute of Technology (MIT), with Boston University assistant professor of chemistry Aaron Beeler-intends to provide drug companies with assistance in developing flow chemistry strategies and systems. 

	“Snapdragon Chemistry has the capability to help a drug-maker from end-to-end in implementing continuous flow manufacturing,” Jamison says. Backed by a scientific advisory board that includes Steve Buchwald and Klavs Jensen, well-respected MIT chemistry and chemical engineering professors, respectively, the company is offering solutions services that include evaluation of entire portfolios of molecules in development to identify the greatest opportunities for implementation of continuous flow chemistry. The company also offers the translation of desired organic syntheses end-to-end into a continuous-flow format, including the selection and/or design and development of laboratory- to commercial-scale continuous reactors.

	“Our goal is to help adapt flow technology solutions to the context of a specific company. We have the deep technical expertise across chemical synthesis and engineering to help companies make the appropriate decisions on where to invest in flow chemistry and increase their probability of success in implementing the technology,” he adds. Snapdragon Chemistry has already formed partnerships with companies, such as drug-discovery service provider Paraza Pharma and equipment manufacturer Zaiput Flow Technologies, to bring the most innovative and effective continuous flow solutions to clients, according to Jamison.


	SAFC has the capability to manufacture products using continuous systems on a 1-50 kg/day basis and currently is producing monomers, reagents, and building blocks for pharmaceutical and high-technology applications. The company is also investigating the addition of cGMP capacity that will include continuous manufacturing technologies.

	Lonza has produced many intermediates on kilogram and hundreds of kilogram scales for APIs in clinical trials. In 2013, for instance, the company completed a technology transfer to a large-scale manufacturing process under high-temperature and pressure conditions that were not possible in a batch process, according to Rhony Aufdenblatten, manager of small-molecule business development with Lonza Pharma & Biotech. Lonza has also used flow chemistry for the multi-ton production of a niacin product under GMP conditions.


	1. J.D. Rockoff, “Drug Making Breaks Away From Its Old Ways,” 

, February 8, 2015, www.wsj.com/articles/drug-making-breaks-away-from-its-old-ways-1423444049, accessed June 2, 2015.
	2. J. Wechsler, “

Congress Encourages Modern Drug Manufacturing

 website, May 1, 2015, , accessed June 2, 2015.
	3. Energy & Commerce Committee, United States House of Representatives, “

Full Committee Vote on the 21st Century Cures Act

,” May 19, 2015, , accessed June 2, 2015.


	When referring to this article, please cite it as C. Challener. “Lack of Expertise Hinders Adoption of Continuous API Synthesis,” 

FDA, Congress, and early adopters look to speed up the use of continuous API manufacturing.

Lack of Expertise Hinders Adoption of Continuous API Synthesis

Many transformations that afford important categories of organic building blocks useful for the synthesis of pharmaceutical intermediates and APIs cannot be performed due to hazardous conditions. Reactions that require the use of unstable reagents lead to the formation of highly reactive intermediates, and those that are highly exothermic typically are not suitable for implementation on a large scale because of the hazards they pose.

	Research is ongoing in both the industrial and academic setting to develop alternative approaches that will enable these transformations to be performed safely on a commercial scale. Such approaches include the development of new forms of hazardous reagents that are stable and can be readily manipulated in the plant setting; identification of new reaction conditions, such as the use of flow chemistry, to reduce the quantities of hazardous reagents and/or provide highly controlled conditions for energetic reactions; and the identification of entirely new routes that do not have any of these issues.

	Examples of recent approaches to overcome hazardous transformations are described in the following sections.
	
	


	In the Vilsmeier (or Vilsmeier-Hack) reaction, aryl aldehydes and ketones are obtained from substituted amides via their reaction with phosphorous oxychloride and electron-rich aromatic compounds. The substituted chloroiminium ion formed from the reaction of the substituted amide with phosphorous oxychloride is referred to as the Vilsmeier reagent (VR). While this reaction can provide a wide range of interesting carbonyl compounds, its use on a large scale can be difficult, because VRs are often irritants and also hazardous reagents due to their high thermal energies of decomposition.

	Researchers at Novartis Pharma employed flow chemistry to address this issue (1). They achieved the in-line formation and immediate consumption of VRs using a combination of batch and flow technologies and applied the new process to the synthesis of the anti-diabetic drug vildagliptin.

	Researchers at AMRI also performed a potentially hazardous cyclopropane ring-opening reaction in a continuous-flow reactor to increase the safety of the process (2). Subsequent copper-mediated Diels-Alder and palladium-catalyzed Negishi coupling reactions were also used in the optimized synthesis of taxadienone on a decagram scale.

	At Bristol-Myers Squibb, a hazardous analysis of a reaction involving the conversion of a tertiary alcohol to a hydroxypyrrolotriazine intermediate identified the potential for thermal runaway (3). To mitigate this potential, a continuous oxidative rearrangement process was developed that involves the mixing of three different, stable feed streams in a specific order using in-line static mixers. A “plug flow” design achieved using heat exchangers ensures sufficient residence times for completion of the reaction. The continuous process has been employed on the pilot plant and commercial manufacturing scales for the production of the investigational liver cancer drug brivanib alaninate.


	Advances in process analytical technology (PAT) not only help speed up process development and optimization, they can also facilitate the control, and thus increase the safety, of synthetic organic reactions. Researchers at Merck Sharp & Dohme recently reported the successful application of PAT for determining the appropriate timing of reagent addition and reaction quenching (4). The technologies discussed included process video microscopy (PVM), a new focused-beam reflectance measurement (FBRM) method, miniature process infrared (IR) spectroscopy, and a flow IR sensor. PVM was used as a nondestructive method for analyzing particle morphologies in real time, while the new FBRM technique has improved reliability due to its greater resistance to probe fouling. The miniaturized IR system was found to be easier to use at scale-up and more robust, and the miniature IR flow sensor was a more cost-effective tool with a faster response, and thus useful for continuous flow processes.

	The IQ Consortium, an organization of pharmaceutical and biotechnology companies with the mission of advancing science-based and scientifically-driven standards and regulations for pharmaceutical and biotechnology products worldwide, also recently reported on the current state of PAT use in the development of APIs (5). In the paper, the authors point out that PAT can minimize the hazards posed to operators when sampling hazardous materials and also provide more reliable data on processes that involve the use of hazardous materials. Through greater process understanding, increased process control-often through the use of simplified monitoring and control systems-is possible at the commercial production scale.


	One of the most effective methods for eliminating the concerns associated with hazardous reagents is to develop stable derivatives or totally new alternatives that accomplish the desired transformation without the safety concerns of the original compounds. Several examples have appeared in literature recently.

	Michael Willis at the University of Oxford developed a method for the synthesis of sulfonamides via 

 formation of intermediate sulfinates from magnesium-, lithium-, and zinc-based reagents and DABSO, a surrogate for sulfur dioxide (6). The sulfinates were reacted with N-chloroamines, which were also generated in situ by reacting amines with aqueous bleach. While sulfonamide groups are present in biologically relevant compounds, they are often accessed through the corresponding sulfonyl chlorides, which can be unstable. The new method provides a route that avoids sulfanyl chlorides. Primary and secondary amines, anilines, and amino acids were all suitable for the reaction, which proceeded most efficiently with Grignard reagents.

	Sodium metal is often used effectively for Birch reductions and the reduction of esters (Bouveault-Blanc reduction), but does have associated safety concerns at larger scale given the pyrophoric nature of sodium metal and the volatility of ethanol. Researchers at the University of Manchester and Pentagon Fine Chemicals reported that a dispersion of sodium metal particles with diameters ranging from 5-15 μm in a mineral oil is a nonpyrophoric, free-flowing powder alternative that can be handled in air and provides results similar to sodium metal (7). In particular, the Bouveault-Blanc reduction was found to proceed in high yields at 0 °C in hexane for a wide range of aliphatic ester substrates with 2.5 equivalents of isopropanol rather than ethanol and just 4.5 equivalents of the sodium dispersion. Notably, alkenes, aryl amines and fluorides, acidic protons, and sterically hindered carbon centers were tolerated

	Direct aryl C-H chlorination is another desirable reaction for the preparation of aromatic chlorides, but the most reactive methods typically require the use of hazardous reagents such as chlorine (Cl

). Milder reagents are available, including N-chlorosuccinimide (NCS) and 1,3-dichloro-5,5-dimethylhydantoin (DCDMH), but their applicability is limited due to their reduced reactivity.

	Other reagents that have been employed also suffer from unattractive features such as toxicity, hydroscopicity, light/heat sensitivity, and explosive properties among others. Phil S. Baran and colleagues at The Scripps Research Institute and Bristol-Myers Squibb have developed the guanidine-based chlorinating reagent 2-chloro-1,3-bis(methoxycarbonyl)guanidine (CBMG), which they refer to as Palau’chlor, as an alternative for the mild and safe direct chlorination of nitrogen-containing heterocycles and certain arenes, conjugated π-systems, sulfonamides, and silyl enol ethers (8). The new reagent is an air-stable solid that is thermally stable below 100 °C, yet achieves transformations that have only been possible in the past with highly reactive, hazardous chlorinating reagents. Importantly, the cost of the reagent and its functional group tolerability are similar to that of NCS.

, 19 (4), 551-554 (2015).
	2. S. G. Krasutsky, et al., 

. 19 (1), 284-289 2015.
	3. Thomas L. LaPorte, et al., 

. 18 (11), 1492-1502 (2014).
	4. George Zhou, et al., 

. 19 (1), 227-235 (2015).
	5. John D. Orr ,et al., 

. 19 (1), 63-83 (2015).
	6. M.C. Willis, et al., Angew. 

. 54 (4), 1168-1171 (2015)
	7. D.J. Proctor, et al., 

. 79 (14), 6743-6747 (2014).
	8. Phil S. Baran, et al., 

Safer reagents and reaction conditions are making many hazardous transformations possible.

New Ways Around Hazardous Reagent Chemistry

:
	When referring to this article, please cite it as C. Challener, "New Ways Around Hazardous Reagent Chemistry," 

Continuous processing is attracting attention in the manufacture of both small-molecule drugs and biopharmaceuticals. In small-molecule manufacturing, flow chemistry has many advantages, including increased development speeds, improved product and process consistency and thus product quality, greater process safety when employing hazardous chemistries, the opportunity to perform reactions that cannot be run under batch conditions, and reduced capital investment and operating costs. 

	Continuous downstream processing operations offer many similar advantages, and there has been more focus on integrating continuous downstream processes with continuous flow synthesis. While many downstream processes can be readily adapted to run continuously, such as extraction, phase separation, and distillation, others-particularly those related to solids handling-present greater challenges.

	Several research groups supported by industry have focused on continuous crystallization. The Center for Innovative Manufacturing in Continuous Manufacturing and Crystallization (CMAC), for instance, has support from founding members such as GlaxoSmithKline, Novartis, and AstraZeneca and is developing continuous processes for all stages of crystallization that are efficient, cost-effective, scalable, sustainable, and precisely control the form, size, shape, purity, surface structure, and functionality of crystallized drug intermediates and APIs. Researchers at the Novartis-MIT Center for Continuous Manufacturing have investigated methods for the direct crystallization of APIs on polymer excipient surfaces and the incorporation of continuous crystallization processes within an integrated continuous manufacturing pilot plant, which requires careful selection of appropriate process analytical technology (PAT).

One of the advantages that can be realized by implementing integrated continuous processing is reduction of the number of processing steps typically required for completion of the same process in batch equipment. For crystallization processes, direct crystallization of a pharmaceutical intermediate or API on the surface of a polymeric excipient can lead to the simultaneous separation of impurities and formation of a drug-excipient composite. This result may cause the elimination of several solid-handling steps that are typically required for the downstream processing of solid products, according to Bernhardt L. Trout, the Raymond F. Baddour professor of chemical engineering at MIT and director of the Novartis-MIT Center for Continuous Manufacturing. “If crystallization can provide a product with the appropriate physical properties, it is possible to eliminate the need for milling, granulation, and other processes,” he says.

Importance of heterogeneous nucleation
	

Heteronucleation involves the formation of crystal nuclei at a solid-liquid interface and can be employed to control nucleation and, potentially, polymorphism, which is important for the production of APIs with stable forms (1). There is some evidence, according to Trout, that the surface morphology of the solid affects the crystallization process. “In particular, the use of nano-patterned substrates can affect not only the nucleation kinetics, but phase stabilization, polymorphism, and the orientation of the resultant crystals,” he adds.

Heteronucelation of a single compound using different polymeric substrates can provide access to a wide range of polymorphs for that compound, because the different surface morphologies of the polymers result in different interactions at the polymer-crystal interface (1). Nanoimprinting of polymers provides a way to impart specific surface geometries that can be used to influence nuclei formation and ultimately polymorphism, according to Trout. “Our supposition was, therefore, that nanoimprinting of polymers that already provide favorable surface-solid interactions to create surface geometries that lead to nanoconfinement should allow for control of crystallization behavior,” he comments. It should be noted that the functionality of the polymer and whether it is more hydrophilic or hydrophobic determines the interactions between the solute and the polymer surface, and thus the crystallization process.

	Trout and his colleagues examined a range of polymer types to determine their effects on this templated crystallization process. To create the engineered polymer surfaces, a rigid, flat silicon mold with nanometer-sized and mono-dispersed pillars of varying angles was fabricated using interference lithography. Negatives of the mold were then transferred to polymer films using nanoimprint lithography. “With this method, it is possible to create both hydrophobic and hydrophilic polymer surfaces with nanopores of many different shapes and sizes. By considering the structure of the API, it may therefore be possible to choose a specific polymer and nanopore structure that will result in crystallization of a specific polymorph of the API, even metastable forms,” Trout notes. For example, favorable interactions can be observed by matching the surface geometry angles with the angles between dominant faces of the desired polymorph of a compound, according to Trout. Enhanced nucleation rates and alternation of the polymorph distribution can result.

	Initially the biocompatibility of the polymers was not considered; eventually the researchers focused solely on polymers that have generally-recognized-as-safe (GRAS) status given that these polymers can be used as excipients in final drug formulations, says Trout. The processability and solubilities of the polymers were also considered. Pharmaceutically acceptable organic solvents were also screened to find an appropriate crystallization solvent.

Effective continuous crystallization in an integrated process
	

Continuous crystallization techniques in the pharmaceutical industry are typically developed as stand-alone processes, but in many cases, the goal will be to develop a fully integrated continuous process involving flow chemistry, purification, and final formulation. In such a setting, the crystallization process can be affected by events that occur upstream, and careful control of the crystallization conditions is required to ensure that all critical material attributes and critical process parameters are met on a continual basis.

	Trout and several colleagues at MIT have investigated the implementation of continuous crystallization in the pilot plant developed at the Novartis-MIT Center, which is designed for integrated continuous manufacturing (ICM) of pharmaceutical compounds (2). “The most significant result of this study, which involved the two-stage continuous crystallization of aliskiren hemifumarate as part of an ICM pilot plant, was the determination that the use of a combination of different PAT tools and automated control loops was sufficient to maintain operational performance within specifications for extended periods of time,” Trout states. In fact, the continuous reactive crystallization was performed for more than 100 hours in the integrated process, providing product with a purity >99% and a yield of 91.4% (2).

1. V. Lopez-Mejías, A. S. Myerson, and B. L. Trout, 

, 13 (8) 3835−3841 (2013).
	2. H. Zhang et al., 


	When referring to this article, please cite as C. Challener, “Considering Continuous Crystallization,” 

Research groups supported by industry tackle the challenges of continuous crystallization and process analytical technology.

Advances in Continuous Crystallization

	In September 2011, the US Government Accounting Office (GAO) published a report on the challenges that FDA faced in assuring the safety of the API supply chain given that reliance on APIs produced by foreign manufacturers has risen dramatically and the complexity of the global pharmaceutical supply chain has increased significantly over the past two decades (1). The report found that while FDA had increased inspections of foreign API manufacturers (up 27% from 2007 to 2009, for example), its rate of inspection for foreign establishments was still far below that for domestic manufacturers. In addition, most foreign inspections were scheduled far in advance, conducted under controlled circumstances, and typically occurred during much shorter time frames. GAO also determined that FDA still lacked important information about foreign API manufacturers (1).

	On a positive note, GAO recognized that FDA had begun to implement initiatives designed to improve its oversight of the drug supply chain, including increased training by overseas offices, the development of programs for control of APIs and other drug products entering the United States, and a push for risk-based inspections rather than a set schedule (1). The GAO concluded, however, that FDA needed to implement changes more rapidly to better assure the safety of drugs on the US market (1).

	Since that report, new legislation has affected FDA ‘s ability to schedule and conduct inspections of API manufacturers. As a result, inspections of foreign manufacturers have increased dramatically--along with warning letters--while inspections of domestic manufacturers have decreased.


	The most important piece of legislation affecting FDA ‘s ability to improve the safety of the pharmaceutical supply chain is the Food and Drug Administration Safety and Innovation Act (FDASIA), signed into law on July 9, 2012. The Generic Drug User Fee Amendments (GDUFA), included as part of FDASIA, instituted a Generic Drug User Fee Program that was agreed to by FDA and the generic-drug industry, specifically the Bulk Pharmaceutical Task Force (BPTF) of the Society of Chemical Manufacturers and Affiliates (SOCMA), the European Fine Chemicals Group (EFCG), and the Generic Pharmaceutical Association (GPhA).

	The user fees are intended to provide additional funding for FDA ‘s drug approval and inspection efforts with the goal of increasing the safety of generic drugs and the ingredients used to produce them, increase the speed of the drug approval process, and the transparency of the industry. One of the main goals of GDUFA is to ensure that foreign facilities are inspected at a rate equal to that of domestic facilities. The fees raised through GDUFA have enabled FDA to hire approximately 1000 additional employees, making it possible for the agency to complete more inspections and speed up the approval process for abbreviated new drug applications (ANDAs) and the review of drug master files (DMFs).


	Several other aspects of the FDASIA legislation have also impacted the API and formulated-drug supply chain. For instance, FDASIA requires FDA to identify facilities involved in the manufacture of generic drugs and associated APIs. “Prior to FDASIA, FDA didn ‘t know how many facilities were producing formulated drugs or APIs in the US, let alone how many companies around the world were manufacturing APIs and formulated drugs and exporting them to the US,” says John DiLoreto, executive director of BPTF. Before the self-registration process began, the industry estimated that there were 1700-2000 manufacturing sites around the world. After the registration process was complete, that number was reduced to approximately 1300. DiLoreto believes that some consolidation of manufacturing plants occurred as companies looked to reduce the GDUFA fees they would have to pay by consolidating operations.

	In the most recent report on foreign and domestic drug establishments issued by FDA (2), the agency identified a total of 12,949 registered drug establishments in 2014, including 9330 domestic and 3619 foreign (slightly up from 12,613 in 2013. Of those 12,949 facilities, 4383 were registered as finished drug product (FDP) establishments, and 1495 were registered as API establishments. (If a site produces both finished drug products and APIs, it is placed in the FDP category.) The remaining 7071 establishments fell into the “other” category, which includes facilities that produce, compound, or process medical gases, medicated feed, and some biologic drugs.

	FDASIA also grants FDA the authority to confiscate and destroy unsafe APIs and drug products being imported into the US. With its increased ability to track the manufacturing activities and inspection histories of API and drug manufacturing sites, the agency can determine if APIs or finished drug products were produced at a facility that has not been FDA-inspected or is not in compliance. The agency has also published guidance on what conduct it considers as delaying, denying, limiting, or refusing inspection, actions that can result in determination of a drug to be adulterated. In addition, under GDUFA, all drugs produced in an unregistered facility or in a facility for which the GDUFA fees have not paid are considered “misbranded.”

	“Together, these new capabilities of the agency make it possible for potentially unsafe APIs and drug products to be removed from the marketplace completely,” DiLoreto says.

	FDA is also using the information on establishments to prioritize them according to the level of risk each represents. Rather than inspect facilities on a set schedule as was the case in the past-typically once every two and one-half years on average for domestic facilities and once every 10 years or more for foreign manufacturing sites-the agency now determines which facilities to inspect based on the overall level of risk they pose, which is determined using a model that takes into account inherent risk, outbreaks, recalls, adverse events, and compliance history (3). This move has led to a dramatic increase in foreign inspections and a concomitant decline in domestic inspections.

	In fiscal year (FY) 2014, the total number of foreign and domestic high-risk human drug inspections by FDA was 918, which exceeded the agency ‘s target of 750 (3). In FY 2013, 443 domestic and 365 foreign high-risk establishments were inspected (total of 808); 43 GMP-based warning letters were issued as a result of those inspections (4). Overall in FY 2013, FDA conducted 967 domestic and 604 foreign GMP inspections (3). In FY 2014, those numbers were 780 and 757.  FDA estimates that in both FY 2015 and 2016, there will be 591 domestic and 843 foreign inspections.

	Both the agency and the industry are adjusting to the risk-based inspection approach, according to DiLoreto. “When FDA first indicated that it would be decreasing domestic inspections so significantly, BPTF was initially concerned that domestic facilities would not be receiving inspections often enough, particularly those exporting to Europe, because the European Medicines Agency (EMA) requires pharmaceutical manufacturers importing products into the European Union to have had an inspection within the previous three years,” notes DiLoreto. The agency has addressed those concerns and established an agreement with EMA. “One of the primary goals of GDUFA was to achieve inspection parity between foreign and domestic facilities, but the change to a risk-based program allows FDA to better utilize resources,” he adds.


	An additional step in the right direction was made in December 2014 when FDA Commissioner Margaret Hamburg signed an agreement with Chinese officials to collaborate on inspections in China that builds on an initial agreement signed in 2007 (5). China also finally agreed to provide additional visas for more FDA inspectors, which will allow the agency to boost its number of employees from 13 to 33.

	FDA is working with EMA on joint inspections and trying to establish mechanisms for sharing of inspection data, according to DiLoreto. BPTF would like to eventually see EMA and FDA inspections results considered to be equivalent. There are, however, concerns on the part of some manufacturers about how confidential business information can be adequately protected under such a scenario.


	Despite the numerous advances that FDA has made in addressing concerns about APIs and formulated drugs manufactured overseas, there are still many challenges facing the agency. Some foreign governments still do not welcome the agency, and inspections of foreign facilities still suffer from many restrictions. DiLoreto does believe, however, that the situation is improving in many countries, as indicated by the recent agreement in China.

	There is also the issue of the dramatic increase in warning letters issued to foreign manufacturers that has occurred along with the rise in foreign inspections. DiLoreto expects these problems to be resolved once these manufacturers have been educated about GMP requirements and become familiar with the expectations of FDA. “It is not surprising that issues are being found at facilities that are being inspected for the first time. These facilities will implement the required improvements, and the number of citations will decline as more effective quality programs are put in place,” he observes.

	It is also important to remember, according to DiLoreto, that many of the FDA inspectors now on the job are still quite new. “It takes at least two years for an FDA inspector to be fully trained, because it take time for him/her to gain the practical experience needed for the job,” he says. “A large percentage of current FDA inspectors don ‘t have that two years of experience yet, and while new inspectors are on the learning curve, issues can arise,” DiLoreto continues. These difficulties, however, should also be resolved in the next few years, he notes.

, “Drug Safety: FDA Faces Challenges Overseeing the Foreign Drug Manufacturing Supply Chain,” Sept. 14, 2011 (Washington, DC).
	2. FDA, 

2015 Annual Report on Inspections of Establishments in FY 2014

, January 2015 (Rockville, MD).
	3. Department of Health and Human Services, 

Fiscal Year 2015 Food and Drug Administration Justification of Estimates for Appropriations Committees

, February 2014 (Washington, DC).
	4. Department of Health and Human Services, 

Fiscal Year 2016 Food and Drug Administration Justification of Estimates for Appropriations Committees

, Jan. 30, 2015 (Washington, DC).
	5. M. Mansour et al., “United States: FDA And China Agree To Collaborate On Inspections,” 


	When referring to this article, please cite as C. Challener, “FDA Steps Up Foreign Inspections,” 

FDA steps up inspections of foreign API manufacturers, thanks to new legislation and changes in policy.