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

Cynthia A. Challener

The global market for highly potent active pharmaceutical ingredients (HPAPIs) has been growing rapidly in recent years due to the rising prevalence of cancer, diabetes, and cardiovascular diseases, among others. The overall market was valued at $12.6 billion in 2014 and expanding at an estimated compound annual growth rate of 7.8% from 2015 to 2023, to reach $25.1 billion by 2023, according to Transparency Market Research (1).

	The main driver of HPAPI market growth is the development of novel, targeted therapies, particularly antibody-drug conjugates (ADCs), which enable treatment with highly potent compounds that, if delivered systemically, would cause significant damage to healthy cells and tissues. ADCs consist of an antibody linked to a highly potent small-molecule “payload” that is released only once the antibody binds to the targeted cell.

	Two ADCs have been approved by FDA: Adcetris (brentuximab vedotin from Seattle Genetics) and Kadcyla (ado-trastuzumab emtansine from Genentech). Approximately 40 additional ADCs were in various phases of clinical development as of early 2015 (2).

	The production of ADCs requires expertise in both biopharmaceutical and chemical manufacturing, as well as the ability to conjugate highly potent small-molecule APIs to biologic components. This last step can be particularly challenging, given that it involves the manufacture of highly toxic compounds in a biopharmaceutical environment, according to Laurent Ducry, group leader, R&D bioconjugates with Lonza.


	A number of different linker chemistries have been explored to connect the highly potent small-molecule drug to the antibody. The linker chemistry determines the release profile of the payload, and thus is crucial to the safety and efficacy of ADCs. The complete ADC must be stable during storage and throughout the time during which the antibody circulates in the bloodstream so that the drug is only released once the antibody binds to the target cell.

	The types of conjugation chemistry that can be used are largely determined by the available functional groups that are present on the surface of the antibody and sufficiently distant from the binding site so as not to interfere with the function of the antibody. Most commonly, cysteines and lysines have been reacted with hydrazones, disulfides, peptides, and thioethers. Of the ADCs in development, more than half link to the thiol groups of cysteine residues, and at least a quarter are conjugated through surface lysines (2). The locations and quantities of cysteine and lysine residues vary from one antibody molecule to another, however, resulting in heterogeneous conjugation.

	Several new conjugation technologies for site-selective reactions that result in homogeneous conjugation for ADC production are emerging to address this issue, according to Ducry. “These techniques have the potential to provide ADCs with improved therapeutic properties,” he notes.

	Compared to traditional lysine and cysteine conjugation chemistries that produce a heterogeneous mixture of products, these novel conjugation techniques based on engineered antibodies or enzymatic reactions result in site-directed conjugation. 

	Some examples include selective N-terminal conjugation based on the differences in the reactivity of internal and terminal amino acids; site-specific functionalization of glutamines through selective enzymatic conjugation of specific residues; and protein engineering to incorporate at specific sites within the antibody unnatural amino acids with reactive moieties other than thiol or amine groups, such as aldehydes, ketones, azides, or alkynes (2). These new approaches are enabling the use of new conjugation chemistries, such as click reactions and enzymatic ligation (3).


	The design of antibody-drug conjugates, which makes them effective, targeted therapies, also makes them challenging to produce. Expertise in bioprocessing (cell-culture and/or fermentation), chemical synthesis, linker technologies, and conjugation chemistry are all necessary, as is the ability to perform much of the manufacturing process under high-containment conditions due to the high potency of the small-molecule payload.

	Different technologies and plant set-ups are required to synthesize, purify, and isolate small-molecule HPAPIs and biological targeting agents such as monoclonal antibodies. The bioconjugation step, which takes place in mostly aqueous media under aseptic conditions, requires a third set of production capabilities, according to Ducry. 

	In addition, while the conjugation of biologic compounds to non-potent chemical APIs is used for the manufacture of a number of different drug products, conjugation of highly potent APIs is a relatively new technology. “One of the key challenges for ADC manufacturing is addressing the need for high containment when working in a biopharmaceutical environment,” Ducry states. He adds that robust safety processes that ensure occupational safety but also enable effective cGMP manufacturing are required to address these challenges.

	In biomanufacturing, it is important to prevent contamination of the large-molecule API by biologic material originating from operators or the environment. As a result, a cleanroom environment with positive pressure is used. When working with small-molecule HPAPIs, however, protecting operators and the environment from exposure to the API is the main concern. Specialized containment, handling, and personnel protective equipment, highly complex operating procedures, and the use of negative air pressure are employed to ensure a high level of safety.

	The needs for bioconjugation of HPAPIs are therefore complex, because conflicting measures (positive and negative air pressure) are typically taken to prevent contamination and protect operators. Developing manufacturing conditions that ensure both operator safety and product purity is challenging. “Very few contract manufacturers have the unique set of capabilities necessary to achieve the balanced approach required for facilitating bioconjugation with HPAPIs,” observes Ducry.

	Other challenges when performing conjugations with antibodies and highly potent payloads include the need for rigorous equipment cleaning and waste management. Cleaning to necessary levels can be difficult because cleaning limits are calculated based on the toxicity of the material. The cleaning limits for the ADC as well as the small-molecule API component are often quite low as a result, according to Ducry.

	Waste treatment is also complicated by the presence of highly potent compounds. Again, protection of workers, the public, and the environment are the prime concern. Lonza has addressed this issue by incinerating all toxic waste on site at its Visp, Switzerland facility where toxin synthesis and conjugation are performed. “This solution provides optimal control, improved safety, and higher flexibility,” Ducry says.


	“Full-length antibodies remain the primary targeting agent used to selectively deliver cytotoxic agents to cancer cells because they have long circulation times,” Ducry says. He also notes that this approach has been validated by approved drugs such as Adcetris and Kadcyla, both of which Lonza manufactures.

	Nonetheless, smaller scaffolds such as antibody fragments, bispecific antibodies, proteins, and synthetic peptides are being explored with the goal of improving penetration rates into tumors. Additionally, conjugated polymers and nanoparticles are emerging as new drug candidates, according to Ducry. “Based on the R&D activity occurring at many pharmaceutical/biopharmaceutical companies, we expect that the number of APIs being developed with targeted drug-delivery mechanisms will continue to grow.”


	The challenges posed by cleaning permanent stainless-steel equipment and managing the toxic waste generated in these processes can be minimized through the implementation of single-use technologies. “Use of disposable systems eliminates equipment cleaning and the need to develop sensitive analytical assays. The change-over time is shorter, and compared to dedicated equipment, there are no storage or start-up costs,” Ducry explains. He does add, however, that single-use equipment lacks the robustness of stainless steel, and therefore appropriate risk-management strategies must be employed to ensure that the necessary level of containment can be maintained.

	Lonza began manufacturing ADCs in fully disposable systems in 2015 in order to simplify change over and cleaning processes, and has focused on managing the safety risk associated with highly potent cytotoxic compounds in accordance with its safety program. To date, the company has successfully produced five of its ADC products in fully disposable systems without any safety issues, according to Ducry.

Global High Potency Active Pharmaceutical Ingredients Market: Growing Demand for Targeted Therapeutics to Boost Market Growth at 7.8% CAGR

,” Jan. 6, 2016,
	2. N. Jain, S.W. Smith, S. Ghone, and B. Tomczuk, 

. 32(11), 3526-40 (2015).
	3. J.S. McCombs and S.C. Owen, 


	When referring to this article, please cite it as C. Challener, “Conjugation Chemistry with Highly Potent Compounds," 

Articles

Manufacturing highly toxic compounds in a biopharmaceutical environment tests equipment and systems.

Conjugation Chemistry with Highly Potent Compounds

Solid-dosage drugs remain the preferred dosage form due to ease of administration and typically lower manufacturing costs compared to parenteral and other dosage forms. Many of the properties of a chemical relate directly to its physical form, and solid compounds can adopt numerous different crystalline forms (e.g., polymorphs, solvates, and hydrates) and non-crystalline (e.g., mesophases or amorphous) forms. Extensive screening must be completed to identify the different potential stable and meta-stable forms that an API may adopt during manufacturing of the drug substance and drug product, packaging, storage, and within the body. Potentially stable salts and cocrystals of the API may also be evaluated. In addition to the skills and knowledge required to conduct and evaluate the large quantities of structure data generated during polymorph screening projects, the ability to design appropriate screening studies has a direct impact on their success.

Toxicology, efficacy, and stability are important criteria when selecting an appropriate candidate for the development of a solid-dosage drug. “These properties can vary depending on the physical structure of the API. It is necessary to understand the physical properties of each potential solid form and the relationship between these different forms in order to identify the candidates with the greatest likelihood of success,” says Brett Cowans, director of materials science for SSCI (Solid State Chemical Information), a division of AMRI.

	The chances of selecting the best candidate improve considerably the earlier these factors are known. “The challenge for companies is to balance the cost of development and risk of selecting the wrong solid form. Ideally, this balance will include understanding the true physical properties of the selected candidate compound, including polymorphism, and how these properties will affect the API manufacturing and drug product efficacy and stability for each form, as well as the intellectual property position for the formulated drug,” Cowans observes.

From a technical point of view, the main challenge of polymorph screening is to identify all crystalline forms relevant and potentially crucial to development. Solid forms may, for example, be observed in the presence or absence of certain impurities in the final isolation step of the API, upon storage of its intended dosage form, or 

 when administered orally, according to Patricia Andres, director of particle engineering for SSCI. “The screening of such forms typically requires a rational design of experiments often not amenable to high-throughput screening, and the unambiguous identification of new phases necessitates the preparation of pure phases in sufficient quantities for characterization,” she notes.

	Andres cautions, however, that while thorough and targeted screening will considerably reduce the chances that a new critical form is discovered later in development, it is not possible to know without a doubt that all relevant forms have been found. She cites ritonavir as a classic example of a crystalline form not discovered during development. The product was developed using a form designated as Form I, which was later found to be metastable with respect to a new form exhibiting considerably lower solubility and rendering the initially developed drug product bio-unavailable.

“The ability to gauge the level and type of screening that is adequate based on considerations of development phase, dosage form, physicochemical and biological properties of the compound, and regulatory requirements is paramount,” Andres asserts. Risks associated with the discovery of a new crystalline form at a later phase of development must be weighed against the possibility of the compound failing in development. This risk assessment requires both experience of solid-state issues that can affect the performance of the drug substance and drug product and intimate knowledge of the drug substance and drug-product processes.

	Another practical challenge in polymorph screening, according to Marco Gil, a general manager with Hovione, is to identify processes for isolation of different forms that can be scaled in a robust manner to yield the selected polymorphs (or cocrystals).

Polymorph screening is a complicated process that involves many individual experiments and significant data analysis. Significant resources have been invested in efforts to accelerate the process. The dream of displacing rational experimental design in pharmaceutical form screening (e.g., salts, cocrystals, and polymorphs) with high-throughput screening was pursued for about a decade by mid-sized and large pharmaceutical companies, as well as material science contract research organizations (CRO), according to Jon Selbo, director of preformulation with SSCI. “Significant internal resources were dedicated to developing home-built screening instruments and/or purchasing equipment and software to implement this new strategy,” he says.

	The justification for high-throughput screening was that significant improvement in identification of otherwise unknown forms could be more easily obtained by conducting large arrays of experiments in parallel. In most cases, however, this strategy failed for both identification of new forms and desired forms for development. “In general, crystallization quality was poor, there was a lack of experimental diversity (regardless of significant increases in solvents used in crystallization attempts), the analytical results were inadequate, and analyzing the large quantities of poor quality data generated in the experiments often proved difficult,” Selbo observes. In addition, polymorphs identified using high-throughput screening must be considered as general lead compounds and must be thoroughly assessed, including evaluation of their pharmacokinetics, solubility, and other properties, according to Gil.

	The driver for large-scale screening in early development has, for the most part, also disappeared with the new molecular entities failing more often today due to efficacy and toxicity issues rather than biopharmaceutics properties. “Only a few larger pharmaceutical companies and small CROs continue to employ high-throughput screening as a first pass strategy,” says Selbo.

	What is important, according to Cowans, is how the results of high-throughput screening can contribute to better understanding of the properties of potential solid-form candidates. “As a screening tool, high-throughput technology provides a platform to evaluate the potential for crystallizing new solid forms under certain conditions, albeit with some limitations. The information obtained solely from high-throughput technology is not sufficient to select a solid-form candidate for development, however,” he says. Consequently, rational experimental design with fit-for-purpose screening strategies based on rational screening or mixed screening approaches have become the norm for the industry, according to Selbo.

	Large-scale screening is still employed as a part of intellectual property (IP) strategies for later-stage programs where there is more certainty of compound survival. “High-throughput screening is today an essential technology for mapping polymorphic forms, generating IP, and broadening the protected space in terms of polymorphs, and helping to avoid surprises as much as possible in advanced development phases,” Gil comments. For these programs, mixed screening services with rational, mid-, and high-throughput screens may be employed in an attempt to cover all bases, according to Cowans.

With respect to advances in technology for polymorph screening, Cowans points to improvements in x-ray powder diffraction (XRPD), which he refers to as the “quintessential tool for distinguishing different crystalline forms.” Most importantly, current-generation instruments are capable of providing the sharp, high-quality patterns that are necessary for identifying whether a material exists as a single crystalline phase and are amenable to indexing algorithms.

	The combination of indexing software and advanced XRPD technology improves the ability to identify crystalline phases or mixtures, increases screening efficiency, and provides critical information about the various crystalline forms obtained, according to Cowans. SSCI has developed and patented its own indexing software specifically targeting organic molecules and integrated this tool into its routine screening activities.

	Another important advance for polymorph screening is the reduction in the quantity of API required for analytical techniques (e.g., XRPD), according to Gil. Automation has also increased the efficiency of these analyses and accelerated sample pre-characterization.


	When referring to this article, please cite it as C. Challener, " Polymorph Screening for Identification of Relevant Crystalline Forms," 

Gauging the adequate level and type of screening is the challenge when identifying relevant crystalline forms.

Polymorph Screening for Identification of Relevant Crystalline Forms

Biopharmaceutical manufacturing involves a series of complex unit operations linked together to provide high-purity, biologic actives with specified physicochemical and pharmacokinetic properties. The development of commercial-scale processes that consistently provide high-quality product that meets those specifications requires extensive examination of numerous process variables and potential process variations. Conducting the required number of studies on the commercial scale is not practical; scale-down models are therefore employed to determine optimum conditions for downstream separation processes, including chromatography and viral clearance steps. Qualification that such models provide results truly representative of commercial-scale processes becomes necessary as molecules move closer to commercialization.

A scale-down model is a bench-scale process designed to predict the results that will be obtained when the process is run at commercial scale. Unlike initial processes that are used during the discovery and early development phases to produce material for characterization, efficacy, safety, and other studies, scale-down models are intended to aid in optimization and characterization of the process that will be implemented for manufacture of large quantities of a biologic once the product has been approved.

	While process simulations are an ideal surrogate, given the complexity and inaccuracy of many theoretical models, there often remains no substitute for a physical scaled-down model, according to Adam J. Meizinger, a process engineer in Genzyme’s Purification, Manufacturing Science, and Technology Laboratory.

	Scale-down models are used in the development of nearly every unit operation in the overall biopharmaceutical manufacturing process, including both upstream and downstream steps, and separation and filtrations steps, such as chromatography and viral filtration processes. A scaled-down chromatography process, for instance, is designed considering the residence time, flow rate, and column bed height of the large-scale process. Additional parameters that are considered include buffer preparation and feed stream. “The ideal model behaves identically to the scaled-up process such that the results obtained in the lab exactly predict the results that will be obtained in the manufacturing plant,” says Paul Jorjorian, director of global technology transfer for Patheon. Because they are models, however, such a situation does not occur. The goal, therefore, is to mimic the commercial-scale process as closely as possible.

The quality-by-design (QbD) approach to process development requires characterization of the impacts of different process parameters on product critical quality attributes (CQAs), which in turn necessitates considerable experimentation, according to Meizinger. “While the consideration of up-front risk assessments, access to general scientific understanding, and the use of design-of-experiment (DoE) methodology minimizes the required number of runs and maximizes the informational output, it is impractical to conduct such characterization experiments at full scale. Qualified scaled-down models are instrumental for conducting such characterization work, as well as for viral clearance studies, continuous process improvement, and for conducting impromptu investigations in support of large-scale operations, such as when deviations occur,” he adds.


	While ideal scaled-down models correlate exactly with their large-scale counterpart processes, generally a perfect match is not achieved between the bench and commercial scales, and therefore, model processes are not perfectly scalable. The causes, according to Jorjorian, may not relate directly to the process, but can be attributed to small, nuanced differences that still have an impact on the results at different scales. “The issues are often unavoidable and thus must be addressed by developing correction factors and/or transfer functions that enable the accurate prediction of the behavior of manufacturing processes,” explains Jorjorian. “It is important, however, to minimize the need for correction factors and transfer functions, and for those that are required, provide thorough justification and clearly demonstrate their suitability,” he adds.

	Another aspect of scale-down models that can create issues when predicting large-scale behavior is the fact that models are qualified at the center point of the process design space. “This approach can lead to discrepancies in predicted and actual results when the model is applied to the entire design space,” Jorjorian observes. He adds that timing and planning of modeling runs is also important and can negatively influence results if not done effectively. For example, it is important to use the same feed used for production runs in scale-down modeling runs; therefore, modeling runs must be scheduled when the feed is available. In many cases, that isn’t possible, and the feed may need to be frozen, which introduces a new variable that can impact the reliability of the model.

	Specifically for chromatography operations, some key considerations in developing scaled-down models are minimizing the differences in the extra-column volume, which varies considerably with scale, contending with increasing wall effects inherent in small-diameter chromatography columns, and accounting for differences in detector-specific path lengths and the associated non-linearity of detector output, according to Meizinger. He also notes that some steps (e.g., gas overlays for intermediate holds) may be challenging to implement as part of scaled-down model development, and mixing dynamics and heat transfer often remain different between scales. “The impact of these differences must be carefully assessed for their resulting impact on scaled-down model performance,” he says.

	Scale-down modeling of membrane chromatography processes can also be difficult, according to Jorjorian. The reason often relates to the designs used for small- and large-scale membranes; they are often different, with large-scale membranes having added flutes or pleats or other features that affect their performance such that it is difficult to develop bench-scale processes that can reliably predict production-scale outcomes.

There is nothing new about the use of bench-scale process models during the development of biopharmaceutical manufacturing processes. What has become an important trend in process development is the need to demonstrate that scaled-down models truly represent their large-scale counterparts. “It has only recently become standard practice to demonstrate that scaled-down models provide results that are statistically equivalent to those obtained from commercial-scale versions of the same processes,” states Meizinger.

	Often during earlier stages of development, even through Phase I clinical trials, scale-down models of downstream biopharmaceutical processes are generally not formal, qualified models. “For these models, the basis for use of each model and the rationale for why it is expected to be representative of the process at large scale are documented,” notes Jorjorian. As a molecule progresses toward commercialization, however, formal, qualified models are required. The ability of the model to predict results at large-scale must be demonstrated by performing at least triplicate runs. The use of any correction factors and/or transfer functions must also be justified and their application demonstrated to provide robust results.

	Qualification of most models for chromatographic separations is fairly straightforward, according to Jorjorian. “There is a lot of documentation and analyses to complete, so in essence, qualification of scale-down models is often an exercise in coordination, rather than presenting a substantial technical challenge,” he observes. Meizinger agrees that the use of statistical equivalence for testing quality attributes and process performance indicators between scales is generally not an issue. “Demonstration of both individual unit and linked unit output (recovery, product quality attributes, product purity, chromatographic profiles/transition analysis, etc.) comparability between small and large scales remains both practical and accepted,” he states.

Qualification of scale-down models can be challenging, however, when very small column sizes are involved. There is significant interest in using high-throughput approaches for scale-down modeling to speed up development times. There are, however, technical issues that arise due to the different behaviors observed in very large and very small columns, such as the wall effects mentioned previously. 

	“It is definitely more challenging to establish robust scale-down models using high-throughput methodologies because typically transfer functions and correction factors are required. As a result, it is more difficult to adequately demonstrate the scalability of the models to the level required for qualification,” Jorjorian says. He adds that there is a lot of effort being devoted to overcoming these issues to enable high-throughput modeling of downstream bioprocesses.

	A few groups are also investigating the application of first principles calculations to the scale-down modeling of both upstream and downstream unit operations, and particularly for chromatography, for which there is significant understanding of separation behaviors and existing mathematical models. “This approach to scale-down modeling is in the earliest phases and largely limited to large pharmaceutical companies and university research groups with the resources and computing power required,” Jorjorian says.


	“Despite the limitations of existing scale-down modeling methods, the use of scaled-down models is recommended wherever possible, including for process characterization, viral clearance studies, continuous improvement, manufacturing-related investigations, evaluation of changes in raw materials, and so on, barring only validation efforts and clinical trial/commercial manufacturing requirements for performance of large-scale runs,” asserts Meizinger. “Today, scaled-down models serve as efficient and cost-effective representations of full-scale processes. While the inability to demonstrate equivalence between scaled-down and large-scale operations is often inevitable for some metrics, further understanding of these scale-related differences, particularly through the development/refinement of associated engineering models, promises to continuously improve the accuracy and utility of scaled-down models,” he states.

Scale-down modeling accelerates the development of downstream biopharma manufacturing processes.

Small-Scale Modeling Boosts Downstream Development

The ultimate goal in pharmaceutical manufacturing is to achieve complex syntheses in high yields with high selectivities in as few steps as possible, and to meet quality requirements. Many reagents considered hazardous due to their extremely high reactivity and/or toxicity can often be used in processes that meet these criteria. In the past, however, the safe handling of such materials and the design of safe processes using them have been a challenge. Advances in engineering control and reactor/process technologies combined with greater understanding of the underlying fundamental physical and chemical behaviors of hazardous reagents are leading to their increasing use for pharmaceutical production.


	Reagents are typically classified as hazardous if they are highly unstable and/or react violently under specific conditions, such as exposure to oxygen or moisture in the air or when combined with other reactive species. Explosive compounds and substances that are sensitive to shock, friction, and heat are examples of unstable, hazardous reagents. Reactions that produce or use these compounds as raw materials, as well as highly energetic processes, require precise and careful control of the process conditions, including the use of inert blanketing gases, selection of the appropriate order and rate of addition, and maintenance of suitable reaction temperatures. Some toxic reagents also fall into the hazardous reagent category, such as extremely strong acids and bases and other corrosive materials.

	Key examples of hazardous processes that have applicability for the synthesis of pharmaceutical intermediates and APIs include:

	Of these technologies, halogenations, nitrations, and azide-based reactions are the most commonly performed hazardous reaction processes, according to Thierry Durand, R&D director at Novasep Synthesis. “These reactions are the most prevalent because they provide access to a large variety of functionalized aromatics and heterocycles that are present in many APIs,” he notes.


	Although the natural reaction to the concept of hazardous reagent chemistry is to assume that such processes will be more complex, costly, and lead to complex mixtures of products, the opposite is often true. Hazardous reagents often allow the synthesis of desired compounds in fewer steps than more conventional reagents. In addition, the reactions often proceed more rapidly with higher yields and greater selectivities than can be achieved using multi-step routes with less reactive materials. “Overall, the use of hazardous reagents often leads to more cost-effective processes that provide purer products, and in fact as many as 10 of the 12 principles of green chemistry (1) can be fulfilled for these transformations,” Durand asserts. He adds that careful evaluation can lead to the development of short and elegant synthetic routes using hazardous chemistry that have reduced environmental impacts.

	Realization of these benefits within the pharmaceutical industry is leading to greater interest in hazardous chemistry routes, particularly for the production of non-regulated raw materials and intermediates. Durand does note, however, that cGMP requirements are becoming more common for starting materials and earlier intermediates in overall synthetic schemes for APIs than even a few years ago, which can be a challenge to using hazardous chemistry for some pharmaceutical companies.

	“Regulatory and supply chain risk management by pharmaceutical manufacturers requires alignment of the technologies used by multiple partners in their supply networks, often to the most standard technologies. Because specialized technical expertise is necessary for safe operations using hazardous reagents, many contract manufacturers do not have extensive capabilities in this chemistry. Some pharmaceutical companies therefore choose not to take advantage of the benefits offered by this technology,” he explains. On a positive note, however, there are many examples of the use of azide chemistry in the final steps of API syntheses. In addition, recently developed azide building blocks are now used to perform click chemistry to modify a large variety of biomolecules.


	Novasep has been using hazardous reagents for more than 135 years and in that time uncovered many examples where hazardous chemistry offers significant advantages over more conventional synthetic methods. For instance, a recent process for the reduction of a hydroxy ester using a standard ester-reducing agent that originally involved five steps--including protection and deprotection of the hydroxyl group and provided the desired product in 59% yield--was replaced with a two-step route that proceeded via direct amide formation and gaseous diborane reduction (no protection/deprotection required) in 81% yield. Production costs were reduced by 60%, and the reaction using hazardous reagents proceeded with much higher atom efficiency, a lower E-factor, and the use of less solvent.

	In a second recent example, a multistep Grignard/carboxylation process was replaced with a one-step carbonylation reaction. The customer required conversion of an aryl chloride bearing a keto substituent to an aromatic ester. The original process involved three steps, including protection of the keto functionality. The new process developed by Novasep involved Pd-mediated carbonylation and provided the desired product in quantitative yield without any need for a protection step. In this case, raw material consumption was reduced by 50% due to the high atom efficiency of the transformation and reduced solvent use.


	Realization of such dramatic benefits does require extensive experience with hazardous reagent chemistry. Not all processes that at first glance may appear to be suitable for replacement with hazardous chemistry are appropriate. “It is necessary to consider not only the chemistry involved, but also the process conditions and controls required to ensure process safety and performance. A process must be feasible, safe, and economically advantageous,” observes Durand.

	Novasep proposals are based on a careful evaluation of different options, including hazardous chemistry, because it is considered a standard tool in the company’s synthetic tool box and therefore not a negative criterion. “Alignment with customer timelines and cost expectations are the key drivers when determining the optimum synthetic route. Hazardous processes, even after taking into account necessary process safety measures, are often highly competitive solutions,” Durand says.

	Equally important has been an increase in customer awareness with respect to the advantages of hazardous chemistry. They also have a greater understanding of the risks associated with hazardous processes and the advanced safety measures that are available today, according to Durand. “Novasep actively participates in symposia and webinars about hazardous chemistry in order to educate pharmaceutical companies about the state-of-the-art technologies that are being implemented. We believe that sharing information with our customers and the hazardous chemistry community is critical to increasing the perception that hazardous chemistry is safe when properly conducted,” he comments.

	Durand also stresses that there is a significant internal commitment at Novasep surrounding its hazardous chemistry operations. Procedures for all operations are detailed, and extensive training of both employees and contractors is performed to ensure a high level of expertise within the company. Continuity of that expertise is also maintained by identifying future internal experts in anticipation of their career development.


	Hazardous chemistry can only be conducted safely if the process can be effectively monitored and controlled. Reactor and other engineering safety control technologies continue to advance and help address some of these concerns. The most exciting developments, however, have been related to process analytical technology (PAT), according to Durand. He points to ReactIR (Mettler Toledo), a real-time, in situ mid-infrared-based system designed for monitoring the progression of solution-phase reactions and determining the gas-phase concentrations of key reagents (e.g., HN

 concentration). The system also provides specific information about a reaction’s initiation, conversion, intermediates, and endpoint, and is therefore an important new PAT tool. “The development of MRT (micro reaction technology) has contributed to further reductions in the level of risk associated with hazardous processes (extremely efficient control of exotherms and minimization of reaction volumes/quantities) and also helped to decrease somewhat the psychological barriers to using these highly beneficial chemical technologies,” Durand states.

, Oxford University Press (New York, 1998) p.30.

Hazardous reagents can prove to be faster, simpler, cheaper, and greener.

Hazardous Reagents for Higher Yields

Materials science, while often associated with industries other than bio/pharmaceuticals, also plays a significant role in the production of drug substances and formulated products, from reactor and purification equipment design to catalyst technology, the selection of polymorphs, the development of new drug-delivery technologies, and the choice of materials for medical devices. A selection of developments in nanocatalysis for API synthesis and nanoparticle technologies for more effective API delivery is discussed here.


	Many types of nanocatalysts have been developed and investigated as mediators of complex synthetic organic transformations. They are attractive because nanoparticles have different properties from their bulk counterparts, including high surface areas. Their electronic, optical, and magnetic properties are often vastly different from bulk materials, and consequently their reactivities are different on many levels. Synthetic chemists have sought to use these different behaviors to prepare highly stable nanocatalysts with high activities and selectivities. Magnetic nanocatalysts are of particular interest in organic synthesis because they can often be readily recovered and reused, while the separation of non-magnetic nanocatalysts that are also non-supported can be problematic.

	Audrey H. Moores at McGill University in Canada focuses on the synthesis of novel magnetic, reusable metal nanocatalysts for coupling, hydrogenation, C-H activation, and oxidation. Robust, inexpensive, amphiphilic polymer-stabilized Fe(0) nanoparticle (Fe NP) catalysts developed by Moores are highly efficient for the hydrogenation of alkenes, alkynes, aromatic imines, and in ethanol or water in a flow reactor. Aliphatic amines and aldehydes, ketones, esters, arenes, and nitro and aryl halide groups are not affected (1). Nanocatalysts comprising Fe

 particles surrounded by nitrogen-doped graphene layers have also been shown by Matthias Beller of the Liebniz Institute for Catalysis and the University of Rostock in Germany to mediate the hydrogenation of structurally diverse and functionalized nitroarenes to anilines and the one-pot reductive amination of carbonyl compounds with nitroarenes (2). His group prepared more than 40 amines useful as building blocks for pharmaceuticals and other fine/specialty chemicals.

	Moores has also shown that patches of palladium nanoparticles deposited on colloidal cellulose nanocrystallite (CNC) nanowhiskers catalyze the enantioselective hydrogenation of prochiral ketones in water at room temperature and 4 bar H

, achieving an enantiomeric excess of 65% with 100% conversion (3). Meanwhile, researchers at Birjand University and Payame Noor University in Iran deposited ionic liquids (ILs) on magnetic nanoparticles to overcome their limited recyclability (4). This approach led to increased activity of the ILs and their reduced consumption, and the heterogeneous nanoparticles were recoverable. The researchers suggest that the nanoparticle-stabilized ILs can be used as solvents, catalysts, or for the extraction of specific compounds that form bonds with the ILs. 


	A large number of small-molecule drug candidates under development today have poor solubility, and thus reduced bioavailability. In many cases, formulation strategies and drug delivery technologies can overcome this issue. Nanoparticles in particular can increase the stability of APIs, protect them against degradation, and enhance their bioavailability by increasing their solubilization, enhancing their permeability, reducing their rate of metabolism, and/or improving their transport. 

	While biodegradable polymers are often selected for the formation of nanoparticles, some researchers have focused on the use of lipids due to their greater physiologic similarity. Solid lipid nanoparticles (SLNs) also offer site-specific targeting, with high adhesion to the gut wall, which is an ideal location for the absorption of release APIs (5). In addition, both lipophilic and hydrophilic drugs can be incorporated into SLNs without the need to use organic solvents. It is important, however, to select an appropriate lipid for a given API to obtain crystalline SLNs with uniformly dispersed API and suitable lipophilicity for improved drug solubility. In some cases, biodegradable polymers may be used as surfactants to stabilize SLNs or as a surface coating to increase the biodistribution of APIs. Other excipients may also be used in SLN formulations, including non-polymeric nonioninc surfactants, cosolvents, and antioxidants.


	As the potency of many drugs-particularly oncology APIs-increases, there is significant need for technologies that enable their targeted delivery to the site of action to minimize the unwanted side effects common with systemic delivery. Many researchers are turning to nanotechnology to achieve that goal. Careful design of nanoparticle properties, typically through modification of their surface properties, can enable their binding to specific components within the body and influence their API release behaviors.

	Two examples of the use of nanotechnology for targeted drug delivery come from Southern California-based universities. In the first example, scientists from the California NanoSystems Institute at UCLA developed a nanoparticle delivery system for the antibiotic moxifloxacin that vastly improves the drug’s effectiveness against pneumonic tularemia, a type of pneumonia caused by inhalation of the bacterium 

, which is highly infectious and designated as a top-tier bioterrorism agent (6), survives and multiplies within macrophages, particularly those in the liver, spleen, and lung. Jeffrey Zink and colleagues designed mesoporous silica nanoparticles that are readily taken up by macrophages. The nanoparticles are soaked overnight in a solution of the API, which is adsorbed in the pores. The pores are then blocked with nanovalves that are designed to degrade in the acidic environment of the macrophages. As a result, the drug is not distributed systemically, but only once it enters the macrophages. The approach allows for significantly more API to reach the site of infection without the side effects typically observed when moxifloxacin is administered freely into the blood stream (6). The researchers found that in mice given a lethal dose of 

, the nanoparticle-delivered moxifloxacin caused few side effects and was more effective at reducing the number of bacteria in the lungs than a dose of freely injected moxifloxacin two to four times greater.

	Researchers at the University of California, San Diego Jacobs School of Engineering, led by Liangfang Zhang, have taken a different approach to targeted delivery using nanotechnology. In their case, nanoparticles formed of a biodegradable polymer were coated with fragments of human platelet membranes, which preferentially bind to damaged blood vessels and certain pathogens, such as Methicillin-resistant 

 (MRSA) bacteria, and protect the nanoparticles from attack by the immune system (7). Unlike synthetic platelet mimics, the platelet membrane fragments possess the complete set of surface receptors, antigens, and proteins naturally present on platelet membranes. Once the platelet fragments bind to the desired site, the polymeric core degrades and is safely metabolized by the body, releasing the drug at the site of action. When nanoparticles loaded with docetaxel, a drug used to prevent scar tissue formation in the lining of damaged blood vessels, were administered to rats afflicted with injured arteries, the loaded nanoparticles were observed to selectively bind to the damaged sites of arteries and heal them (7). 

	Nanoparticles loaded with vancomycin at one-sixth the clinical dose and injected into mice systemically infected with MRSA bacteria resulted in up to a one-thousand-fold reduction of the bacterial counts than those in mice treated with the clinical dose of vancomycin alone (7).


	A third approach to nanoparticle-based delivery of APIs relies on the use of nanoparticles formed from natural biomolecules. Silk fibroin (SF) has attracted particular attention due to its excellent biocompatibility, biodegradability, and low immunogenicity combined with a high binding capacity for many types of drugs, good controlled release properties, and ease of preparation (8). SF is an alternative to biodegradable polymers, such as poly (lactic acid) (PLA), poly (ε-caprolactone) (PCL), and poly (glycolic acid) (PGA) because protein-based nanoparticles bear functional groups that are naturally designed to interact with cells and cause biological responses. SF in particular is widely used as a biomaterial and is well characterized and understood. Studies of SF nanoparticle systems have shown that its biodegradation rate is influenced by its crystallinity, molecular weight, and degree of crosslinking. However, while numerous methods for the fabrication of nanoparticles have been developed, there is significant variation in the structure of SF obtained from different species and even individuals, and thus interest in genetically recombinant SF-based nanoparticles is increasing. Surface modifications are also required to improve the delivery specificity (8).

	A nanoparticle-based formulation of the drug ormeloxifene (ORM) for the treatment of pancreatic cancer is under development by researchers at the Department of Pharmaceutical Sciences and the Center for Cancer Research at the University of Tennessee Health Science Center in order to enable tumor-specific targeted delivery (9). The ORM was readily encapsulated in poly(lactic-co-glycolic acid) nanoparticles that were then subjected to antibody/aptamer conjugation to achieve tumor-specific targeting. Cellular uptake and internalization studies with the 100-nm-diameter particles confirmed that they did not undergo lysosomal degradation and survived to allow efficient endosomal release. Treatment of a xenograft mice model resulted in increased animal survival and suppressed pancreatic tumor growth (9).

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Nanoscale catalysts and engineered nanoparticles show promise for API synthesis and delivery.

Nanoparticles Demonstrate Potential for Small-Molecule Pharmaceuticals


	When referring to this article, please cite it as C. Challener, "Going Small to Achieve Success on the Commercial Scale," 

Scale-down modeling is instrumental in supporting the development of downstream biopharma manufacturing processes.