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

Cynthia A. Challener

The rise of antibiotic-resistant bacteria is recognized as a significant threat to the future practice of medicine. Continually rising resistance rates have resulted in infections with bacteria resistant to all existing antibiotic treatment options. There is concern that if the current treatment system remains unchanged, the resistance epidemic could push the world into a post-antibiotic era.

	Alternatives are therefore needed to replace current small-molecule antibiotics. Given that the development of resistance is a natural form of evolution for bacteria, the challenge is to find new drugs that kill bacteria in a way that dramatically slows down their ability to counteract them. Biologic drug substances-monoclonal antibodies (mAbs) in particular-may be a key component of the solution.

	Regardless of the antibiotic, resistance will develop, according to MedImmune’s director of microbial sciences Bret Sellman. “Most available antibiotics are related to natural products for which resistance already exists in nature,” he explains. Bacteria also divide rapidly, which increases the likelihood for antibiotic-resistant mutants to evolve.

	In addition, over the past four decades there have been few truly novel antibiotics, according to James Levin, director of preclinical development at Cidara Therapeutics. “We have been targeting the same limited subset of essential proteins, and therefore, bacteria have ample opportunity to evolve and become resistant to entire antibiotic classes over time,” he observes.

	Sellman argues that development of antibiotic resistance has less to do with the structure or chemistry of antibiotics than it does their method of attacking a pathogen and their widespread use in modern medicine and farming. “By killing bacteria directly, antibiotics select for the outgrowth of resistant mutants. In addition, the misuse of antibiotics to treat viral diseases (e.g., the common cold) unnecessarily exposes patients and their bacteria to antibiotics and fails to treat the actual disease being experienced. This ease of access only increases exposure and subsequently the risk of resistance,” he asserts.

	Resistance can arise from chemical modification of the antibiotic by bacterial enzymes or mutations to the antibiotic target, adds Levin. He also notes that bacteria are able to swap genes that impart antibiotic resistance with other bacteria, allowing resistance to spread rapidly.

	Adding to these escape mechanism issues, Levin points out that gram-negative bacteria are intrinsically resistant to many antibiotics because they possess an outer membrane that is impermeable to most drugs-and they can mutate to reduce permeability further when under selective pressure.

	The problem with broad-spectrum antibiotics

	There is an additional problem associated with the use of broad-spectrum antibiotics: they kill not only harmful pathogens, but “good” bacteria that make up the microbiome within humans. Doing so results in the development of resistance in the target pathogen as well as the members of healthy microbiome, which can then transfer resistance to pathogens they encounter, further spreading the problem, according to Sellman.

	Damage to the healthy microbiome can have significant consequences as well. “Killing of the healthy microbiome has been linked not only to the development of 

 diarrhea but also diabetes, obesity, immune defects, and antibiotic resistance spread through gene transfer,” he says.

	While antibiotics will always play an important role in saving and preserving life, the growing antibiotic resistance epidemic and increasing understanding of the adverse effects of broad-spectrum antibiotics on the healthy microbiome necessitate the development of alternatives such as pathogen-specific strategies to prevent or treat bacterial infections, according to Sellman. “We firmly believe that moving away from traditional small molecules is the path forward in anti-infectives research,” Levin agrees.

	Most efforts are focused on new drugs based on mAbs because of their specificity. “Such targeted antibacterials should have reduced toxicity, cause less harm to patients’ beneficial microbiomes, and not promote resistance in bacteria not targeted,” Sellman comments.

	Antibacterial mAbs also directly neutralize bacterial virulence mechanisms and engage the patient’s immune system, according to Sellman. “By boosting the immune system to kill the pathogen rather than killing the bacteria directly, the emergence of resistance might be reduced,” he explains.

	Cidara Therapeutics is developing antimicrobial antibody-drug conjugates (ADCs). “These bispecific molecules capitalize on the potency of antibiotics coupled with the beneficial aspects of an effective and robust immune response and can be designed with a prolonged half-life,” says Levin. He believes that any antimicrobial, including small molecules, that binds to a surface or cell-wall component of the bacterium is a viable candidate for conjugation to an antibody fragment crystallizable (Fc) region.

	In addition to antibody-based drug candidates, Sellman notes that researchers across industry and academia are also exploring phage lysins and viral phage approaches as alternatives to small-molecule antimicrobials.

	Antibacterial biologics require new thinking

	Development of mAb antimicrobial drugs does not come without challenges, but those difficulties are not solely in the scientific arena. “In order to realize the promise of biologics in infectious disease, we need to evolve the way we plan to manufacture and diagnose for these medicines,” Sellman states. Because antibacterial mAbs would likely be most effective in the earlier stages of infections, a move to integrate mAbs into the mainstream infectious disease protocol would require a commitment to more rapid diagnostic methods.

	In addition, he notes that because pathogen-specific mAb treatments must account for bacterial strain diversity and the expression of multiple virulence determinants by the infecting pathogen, mAb combinations may be required for optimal efficacy.

	The higher cost of biologic antibiotic drug substances compared to their small-molecule counterparts could also be an issue, according to Levin. His hope is, though, that the significantly longer half-life that should be achievable for biologic antibiotics, including ADCs, will enable less frequent dosing and thus offset the higher cost.

	Cidara Therapeutics set out to develop ADC antibiotics that exert a direct killing effect on the pathogen; engage the immune system, bringing a second mechanism of killing into play; potentiate standard-of-care antibiotics by attacking the bacterial cell wall and allowing them to penetrate the cell more effectively; and have superior (antibody-like) pharmacokinetic and distribution properties.

	The company conjugates surface-acting antimicrobials (targeting moieties [TMs]) to Fc regions of human antibodies using non-cleavable linkers. The bispecific Cloudbreak ADCs exert direct killing activity on bacteria while targeting the cell for destruction by the immune system, according to Levin. “We believe that by developing drugs with a dual killing mechanism we will reduce the opportunity for the target pathogen to develop resistance. In addition, since our TMs do not have to reach the inside of the cell to kill the bacterium, we avoid the daunting problem of having to breach the bacterial membrane in gram-negative bacteria,” he says. In addition, because antibodies can remain at effective concentrations in plasma for a month or longer, Cidara believes its ADCs can ultimately be engineered to achieve a similar half-life.

	The company recently demonstrated proof of concept with an ADC comprising a peptidic antimicrobial conjugated to a human Fc. “Although not our final drug candidate, this ADC was efficacious in murine 

 pneumonia models. It also demonstrated a much longer half-life than the polypeptide alone,” Levin notes. In-house characterization by Cidara’s immunology team further demonstrated the ability of this conjugate to successfully engage the immune system to enhance bacterial killing. Some of this work was performed in collaboration with Professor Ashraf Ibrahim at UCLA and has yielded important insights into the mechanism of action of ADCs.

	The Cloudbreak ADCs are in preclinical development, but Levin expects a clinical candidate to be nominated in 2019. Current efforts are focused on evaluation of lead candidates in preclinical toxicology studies and exploration of Fc modifications to further extend 

 half-life. The company received a National Institutes of Health grant in 2018 in conjunction with Professor David Perlin at Rutgers that should accelerate the pace of its ADC program, according to Levin. Cidara is also applying its Cloudbreak technology to the development of antivirals.

	Within MedImmune, the global biologics research and development arm of AstraZeneca, two Phase II mAb assets are in clinical testing. MEDI4893 (suvratoxumab) is under investigation for the prevention of Staphylococcus aureus pneumonia in intensive care unit patients, while MEDI3902 is being developed for the prevention of 

 pneumonia in intensive care unit patients.

	“As we continue to explore this field, we are constantly learning about the critical role of the commensal microbiome in maintaining overall health, and even the role it can play in possibly treating certain diseases. With this understanding comes a commitment to exploring new therapeutic options that avoid damaging these beneficial bacteria. The targeting specificity of biologics offers tremendous promise in making this goal a reality,” Sellman concludes. 

Vol. 42, No. 12
	December 2018
	Pages: 36–37

	When referring to this article, please cite it as C. Challener, " Fighting Bacterial Resistance with Biologics," 

Articles

Antibody-based drugs offer new mechanisms of action and greater specificity.

Fighting Bacterial Resistance with Biologics

Highly potent APIs (HPAPIs) are an increasingly important segment of the API market. While there are many contract development and manufacturing organizations (CDMOs) that claim to have potent compound production capabilities, few have the facilities, equipment, and trained personnel needed for the production of highly potent compounds with occupational exposure levels (OELs) of 10 µg/m

/8h. CDMOs with established expertise are investing in expanded capabilities to meet the growing need for HPAPI production capacity.

	Global demand for HPAPIs is estimated by various market research firms to be approximately $17 billion in 2018 and expanding to about $26 billion by 2023, according to Claudio Salvagnini, director of CDMO API and HPAPI activities at Minakem. The rapid growth is driven largely by the global oncology market, as many highly potent compounds are used for the treatment of cancer. This market has already surpassed $100 billion in global sales and is expanding at double-digit rates, according to intelligence reports reviewed by Minakem.

	Maurits Janssen, head of commercial development, API development, and manufacturing with Lonza Pharma & Biotech, adds that the percentage of HPAPIs that are cancer treatments has grown from more than 50% in 2015 to approximately 60% in 2018, with the rest found in hormonal treatments, central nervous system treatments, and all other therapeutic areas. There are also many rare diseases under focus, and many of these conditions are being treated with highly active compounds according to Marko Salo, vice-president of marketing and sales with Fermion.

	Three-quarters of HPAPIs are novel compounds. In particular, the growing focus on precision and personalized medicines such as antibody-drug conjugates (ADCs) that treat specific patient subgroups or conditions is driving development of novel APIs. Innovation is therefore another important factor in the growth of the HPAPI market, according to Salvagnini. He adds that despite the increasing number of biologic drugs in the pharmaceutical pipeline, the HPAPI market remains dominated and is foreseen to continue to be dominated within the next five years by small-molecule, chemical APIs.

	Lonza also expects the generic HPAPI market to see strong growth over the next few years due to patent expirations of some branded HPAPI products, according to Janssen. Another driver for the growth of HPAPI capacity demand is the additional knowledge being obtained for existing compounds that previously were not considered to be HPAPIs. As new toxicity data are generated, some of these compounds are being reclassified as HPAPIs, according to Salo.

	Outsourcing of HPAPI production is also expected to expand faster than the overall HPAPI market, according to Janssen. “This growth is due to the fact that the majority of innovation is taking place in smaller emerging biotech companies with no in-house manufacturing capabilities and therefore the need to outsource,” he explains.

	Limited options for highly potent production

	The number of companies advertising highly potent manufacturing capabilities is quite high, according to a recent survey conducted by Minakem. “In a market analysis, approximately 100 [contract manufacturing organizations (CMOs)]/CDMOs were identified as claiming to focus on high potency and making numerous investments in the past 10 years, so what was once a niche segment appears to be more crowded,” Salvagnini notes.

	Upon closer look, however, Minakem found that classification of highly po.tent manufacturing capability comes down to how each company defines potent vs. highly potent. Many of these companies are capable of processing compounds with OELs down to 10 µg/m

/8h, but only a handful of active CDMOs have the capability of handling highly potent intermediates and APIs with OELs of 0.10–0.01 µg/m

/8h and lower, Salvagnini says. This HPAPI business is highly concentrated in West.ern countries, according to Janssen.

	Salo adds that many of these recent investments have involved smaller capacities. Janssen notes that the majority of HPAPI equipment for commercial manufacturing is mid-scale (4–6m

), where there is the highest de.mand. “Only a few companies, including Lonza, can cover the full range of reactor scales along the drug lifecycle from 2–10m

, allowing customers to access phase-appropriate equipment for their individual needs,” he says.

	“Entry into this segment of the market is limited by the high initial investment required to install containment systems, glove boxes, proper air flow and conditioning systems; develop effective processes; and train operators,” observes Salvagnini. He adds that having a solid reputation for success in HPAPI manufacturing is probably more important than in other segments of the pharma industry, given that the safety of operators, the environment, and patients is a key concern. “We have seen that clients looking for HPAPI services conduct more thorough audits and risk assessments when evaluating potential outsourcing partners,” Salvagnini says.

	There are currently no indications of overcapacity in the market, according to Janssen, but increasing competition may lead to a lower perceived market growth of the segment in the future. Continued investment will still be needed, however; as manufacturing and handling technologies for HPAPIs continue to improve, older facilities will require upgrading from time to time to prevent becoming sub-standard.

	Minakem recently expanded HPAPI capabilities at its Louvain-la-Neuve plant in Belgium, which it acquired in 2015 from Ajinomoto OmniChem. “We acquired high-containment technology with this site and set as a goal to become a leader in high potency development and manufacturing,” notes Salvagnini. With available space for expansion within the building and on the site, it was possible for Minakem to revamp existing facilities and add new equipment and production trains. In addition, the company has access to talent due to its location near universities and pharmaceutical companies.

	“The HPAPI segment is a high-technology market that requires continuous investment to ensure state-of-the art capabilities,” Salvagnini asserts. Not only is the HPAPI market expanding, highly potent compounds are becoming increasingly complex and more potent. “We need to be certain that we have the broadest range of synthetic and containment capabilities to meet the evolving needs of the marketplace,” he adds.

	The new closed-controlled, high containment laboratory facility was opened in August 2018 and extends Minakem’s capacity to develop and manufacture HPAPI compounds from small-scale development to full GMP batch releases. In addition to nine fume hoods, a double-barrier of protection in the form of new-generation glove boxes in a room dedicated to dry powder handling and weighing, air locks, HEPA air filters, and cascading flow, the facility includes a preparative chromatography system for the isolation and purification of target molecules from milligrams to hundreds of grams for analysis and production applications.

	The current production suite enables production of gram, 1–10 Kg, 20–50 Kg, and 100–150 Kg batches of compounds with OELs down to 0.1 µg/m

/8h. Minakem has also installed 5-, 50-, and 300-mm preparative high-performance liquid chromatography (HPLC) columns, according to Salvagnini. The company is also looking to invest in additional hydrogenation equipment under containment.

	Fermion’s three-year project to expand HPAPI production capability at is Hanko, Finland plant reached its conclusion in the summer of 2018, with first batches and regulatory inspection completed. Unit 4 consists of a fully automated, completely closed HPAPI unit of more than 50 m

 capacity for the production of highly potent compounds with occupational exposure bands (OEBs) 4 and 5, according to Salo.

	The facility can be run by just three operators due to a high level of automation. As a result, Fermion is able to produce HPAPIs cost-competitively, Salo adds. A bar-code system is fully integrated into the automation system, preventing charging of the wrong materials and avoidance of paperwork.

	The project was commissioned in response not only to the growing demand for HPAPIs, but also because Fermion was finding that some older generic APIs that were previously categorized as OEB3 are today considered to be category OEB4 or OEB5 compounds. “We want to protect our operators to the highest possible degree and would rather overprotect our employees than take any risks even for lower OEB class products,” asserts Salo. He adds that it is Fermion’s point of view that operator working conditions with modern containment protection systems such as isolators are better than those that rely on old-fashioned personal protective gear.

	One factor contributing to Lonza’s continued investment in HPAPI capacity and capabilities is the growth of oncology as a focus of biopharmaceutical research and development, according to Janssen. “Since these products use increasingly complex and toxic molecules, the companies that develop them more frequently seek external partners like Lonza Pharma & Biotech with the facilities and track record and expertise to properly handle HPAPI formulations,” he says. As an example, he points to ADCs and their use of highly toxic molecules to specifically target and kill cancer cells as representing a step up in oncology treatment that would neither be possible nor effective without extremely potent payload molecules and the capabilities required to handle them.

	The company’s HPAPI investments have focused on providing agile manufacturing solutions for the wide range of product volumes required by biopharma companies’ increasingly specialized and pharmaceutically potent products, according to Janssen. Lonza Pharma & Biotech’s Visp, Switzerland Center of Excellence in HPAPI Development and Manufacturing has multi-purpose facilities designed to efficiently and safely manufacture multiple products simultaneously at various scales. Lonza currently has active HPAPI projects with OELs ranging from 5 to 0.04 µg/m

	In February 2018, the company announced expansion of its HPAPI capabilities in North America, including the addition of two commercial-scale processing suites for the handling and encapsulation of HPAPIs using drum-dosing technology for powder-in-capsule filling at its Tampa, FL site.

	In late 2017, Cambrex announced a $24-million investment in a new facility to manufacture HPAPIs at its Charles City, IA plant in the United States. The 4500-sq.-ft. production area will operate to an OEL of 0.1 µg/m

 and have a total reactor capacity of 2200 gallons and be able to manufacture batches from 50 to 300 kg. The small-scale manufacturing area at the site is also being reconfigured to provide a single high-containment building to support early stage development and manufacturing. Construction and installation of all new equipment is expected to be completed by Q1 2019.

	The investment is in response to the growing number of highly potent, small-molecule APIs in the pharma pipeline, according to Shawn Cavanagh, COO of Cambrex. It is also part of an ongoing strategic campaign to invest in small-molecule API development and manufacturing across Cambrex’s global network of facilities.

	Implementing new HPAPI development and manufacturing capabilities presents multiple challenges that must be addressed within a limited timeline, according to Salvagnini. “Multiple departments are involved, so cross-functional teams with representatives from environmental health and safety, program management, R&D, production, etc., must collaborate with external consultants and project design and construction companies. Having solid internal experience in containment technology and working efficiently together in close collaboration on a daily basis is essential for keeping such types of investment projects on track,” he says.

	Previous experience with the addition of HPAPI units is also highly beneficial for successfully completing new projects on time and without major issues, according to Salo. Janssen agrees: “Considering Lonza’s 20 years of experience in handling HPAPIs and in maintaining the required facilities, the construction of new capacity was supported heavily from past experience, and progress was demonstrated by the short realization timeline and absence of critical challenges,” he observes. He also notes that Lonza has found it extremely valuable to build on a stable team of specialists and to keep everyone pointed in the same direction.

	In addition to the expected healthy growth of the HPAPI market that is expected to continue in the coming years, significant changes in manufacturing and logistics are anticipated in response to changing market dynamics. “The move into personalized and specialized medicines means that large numbers of molecules will be produced at small scale, which is quite a massive change from the way the supply chain operates today,” asserts Salvagnini.

	There is also a movement toward higher containment protection levels, according to Salo. Molecular complexity will increase further as well and present ongoing manufacturing challenges, according to Salvagnini. In addition, many HPAPIs in the pharma pipeline face bioavailability challenges and/or require modified-release technologies, according to Janssen. “As a result, HPAPI production and manufacturing today requires more specialized capabilities than ever before,” he says.

	In addition, many drug formulating companies are closing their in-house production facilities and shifting toward contract outsourcing as a more cost-effective approach to HPAPI manufacturing. “These companies are looking for external CDMOs that offer integrated services and can tailor their enhanced technology platforms and supply-chain solutions to specific companies’ needs to facilitate development and reduce time to market,” Janssen comments. Outsourcing, he adds, is strongly driven by emerging and mid-sized pharmaceutical companies, which account for most of the innovations in the pharma industry today.

	“CDMOs will need both flexibility and agility to accommodate the broader range of chemistries, toxicities, delivery technologies, and product volumes for highly potent intermediates and products that will exist in the near future,” asserts Salvagnini. Responsiveness will also be important, as increasing numbers of projects are designated as accelerated approval programs. “Minakem is preparing by adapting both its facility and internal organization, building dedicated supply-chain and building project teams in order to rapidly proceed through increasingly complex processes and stay on the critical path for shortened timelines,” Salvagnini says. He adds that its ongoing investments are made with this new future in mind, including investments in analytical capabilities and skilled and talented workforce.


	Vol. 42, No. 11
	November 2018
	Pages: 24–26

	When referring to this article, please cite it as C. Challener, "Building Capacity for Potent API Production," 

Demand for highly potent APIs continues to rise.

Building Capacity for Potent API Production

A key focus of the pharmaceutical industry today is increasing efficiency and productivity to reduce cost and time to market. These issues are being addressed across the entire development lifecycle, including in API development labs. From improvement of existing technologies to the introduction of more advanced analytical instruments and modeling software, development labs are focused on increasing speed of optimization and reducing issues during scale up.

	Innovation in API development labs is taking place at all pharmaceutical companies. Adam Kujath, senior director of global manufacturing sciences and technology at Alcami, points out how this innovation is being driven largely by smaller pharma and biotech companies. “Speed is the most important thing for these organizations as they work to get into and through the clinic as quickly as possible. Therefore, most investments are not necessarily for exotic new technologies, but rather expansion and improvement of those that drive more efficient throughput,” he comments. Examples include robotic screening equipment, parallel reactors, and more advanced in-line analytics to support process characterization.

	Flow chemistry for the synthesis of APIs is an important trend in the industry, according to Rui Loureiro, director of R&D process chemistry development for Hovione. “Flow chemistry enables the implementation of chemistries that previously were not possible due to a lack of technology. As a result, chemists are gaining access to new methods for producing new and more complex molecules,” he says. It can also dramatically reduce scale-up times because the same equipment can be used in the lab and for production, just for longer periods of time and/or in multiple copies.

	A side benefit of the interest in flow chemistry is improvements in process analytical technology (PAT)-including nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography (HPLC)-are being developed to allow their use for continuous manufacturing, according to Loureiro.

	Equipment integration and miniaturization

	Not only advances in equipment technology, but the ability to integrate different aspects of API development laboratory initiatives is helping to speed up activities. Access to a growing selection of miniaturized probes with high resolutions allows researchers to more quickly gain a better understanding of how crystals are formed and how polymorphic forms can be controlled, according to Jerod Robertson, a senior process chemist at Hovione.

	 He points to smaller probes for focused- beam reflectance measurements and particle vision and measurement from Mettler Toledo as examples that allow performance of crystallization studies in smaller reactors using smaller quantities of expensive API. “Using less material is important since at the beginning of development there normally aren’t significant amounts of product available, but the shape and size of the obtained crystals should be understood as in-depth as possible because these parameters can significantly impact process development down the road to reaching the commercial phase,” Robertson explains.

	Most notable for Alcami when it comes to equipment advances has been the integration of multiple systems, according to Kujath. “When a piece of equipment capable of performing automated, high-throughput synthesis or crystallization experiments is directly integrated with direct sampling for multiple forms of analysis on the same system, it drives efficiency, such as the Bruker D8 Discover HTS

. Better, more robust data sets can be obtained, making tools such as design of experiments more accessible for earlier development activities and thereby allowing Alcami to create stronger early clinical processes,” he observes.

	Advances in software are equally important as improved equipment and technology. “Software packages are becoming more intuitive, which is important as the databases behind them grow,” Kujath notes. “Scientists today build on the developments of those who came before them, and the software packages that exist today are making that information more accessible for application on a daily basis,” he adds.

	At Hovione, using the simple but effective Dynochem (Scale-Up Systems) and Visimix (VisiMix Ltd.) software packages for optimizing scale up and mixing processes and equipment have been great tools for chemists responsible for the scale-up of API syntheses. “The use of Dynochem has enabled Hovione to achieve faster development of unit operations such as solvent swapping, and it has also been a great tool for understanding reaction mechanisms, including those that lead to impurity formation,” Loureiro says. Such understanding helps the development chemists implement effective control strategies that ensure product quality. The use of tools such as Visimix provides chemists with a greater understanding of effects like mass transfer and mixing and how they can impact product quality, according to Robertson. This information can be used to gain insight into how reactions will run at scale or when they are changed from one piece of equipment to another.

	Hovione is also leveraging software designed for 

 calculations, such as Gaussian calculations. “These types of software are very important because they provide chemists with a better understanding of the possible transition states that can be formed during the different steps in an API synthesis route. This information is helpful for identification of pathways that lead to impurity formation,” says Loureiro.

	The software packages used at Hovione mainly help with modeling. The information that is obtained on process kinetics and impurity formation is used to determine the optimum control strategies, according to Robertson. The company also uses software such as SuperPro Designer (Intelligen) for batch process simulations and computational fluid dynamics software for modeling the scale up of processes when moving from the lab to large-scale production.

	The algorithms used in modeling tools are becoming more accurate and predictive in part because the data behind them continue to grow, according to Kujath. Alcami has seen that they are as a result useful for further refining processes.

	As importantly Kujath notes that while the new predictive synthesis applications being developed in academia are not yet widely used in industry, they hold tremendous future potential in reducing time and materials spent in early screening work. He also expects further development of applications of predictive models like solvent maps, which through principal component analysis enable scientists to make more data-driven decisions in solvent and reagent selection.

	In-line and bench-top analytical advances

	As the pharmaceutical industry moves toward continuous manufacturing, work is also progressing with respect to in-line process analytical technology for use in both the production plant and development labs, according to Kujath. “These new tools not only provide more rapid feedback on experimental results, but are being effectively used to establish proof of concept for scale up at Alcami,” he observes.

	For Hovione, advances in two technologies in particular are speeding of development work: bench-top NRM systems and ultra-high-pressure liquid chromatography (UHPLC).

	Traditional NMR systems were quite large and carried high capital and consumable costs. Newer benchtop systems are much less expensive and do not carry the running costs of older machines because they do not require the use of liquid helium for cooling, according to Robertson. “Although they are much smaller, the new bench-top NMRs still provide high resolution and allow chemists to follow reactions that previously were not analyzed due to lack of immediate access to NMR instruments,” he says.

	Hovione has found that it is possible to more quickly gather information about impurity formation that was possible before. In addition, the bench-top NMR is used in place of gas chromatography to quantify solvents in distillations more quickly and cheaply. Loureiro also notes that the bench-top NMR system can be connected to flow reactors for continuous monitoring of product formation, providing real-time data and enabling faster process development.

	While UHPLC is not new, it is not yet widely used throughout the industry. Many projects that Hovione accepts come with HPLC methods. “We often work with our clients to improve and where possible further convert them using a quality-by-design approach to UHPLC methods,” comments Loureiro.

	Both Kujath and Loureiro expect to see more focus on the development of continuous- flow chemical processes going forward. “New small-molecule entities as a whole are becoming more potent. Chemical synthesis already carries inherent risk with potential high energetics, flammable solvents, and other safety management challenges. Coupling that with the need to continually be more cost effective, it simply makes sense to apply this concept whenever possible,” asserts Kujath.

	Adds Loureiro: “We think that the continuous manufacturing of APIs still has some space to be further improved. Several people are working on the downstream steps, which still require further development before fully continuous processes can be implemented from addition of the starting raw materials to packaging of the final API.”


	Vol. 42, No. 10
	October 2018
	Pages: 22–24

	When referring to this article, please cite it as C. Challener, “Efficiency Demands Drive Advances in API Labs,” 

Integration of new modeling and analytical tools with flow chemistry are notable trends.  

Efficiency Demands Drive Advances in API Labs

The pharmaceutical industry is currently challenged to accelerate the development of new drugs while reducing the cost of these efforts. Drug repositioning is an effective approach for achieving these goals. Evaluating existing compounds that have been proven safe-whether approved, still in clinical trials, or previously found to be ineffective for the diseases they were originally targeting-takes less time and money. The challenge is to identify the right existing compounds to investigate for a given disease.

	Drug repositioning today is currently pursued using one of five key approaches, according to Aris Persidis, co-founder and president of Biovista, which is developing a pipeline of repositioned drug candidates in neurodegenerative diseases, epilepsy, oncology, and orphan diseases. Phenotypic screening is the one method that involves laboratory testing. In this approach, a drug substance is tested in parallel against a small set (10–50) of animal models or cell lines. “This approach is neither comprehensive nor systematic by any means, but if one is lucky and gets a hit, then a potential new use of a drug in a specific new disease has been identified with some initial experimental proof,” Persidis says.

	To achieve systematic or comprehensive repositioning, computer-based approaches must be used. There is no single preferred digital tool or technology for accomplishing drug repositioning because different tools are optimally suited for different strategies, according to Hermann Mucke, CEO of H.M. Pharma Consultancy, a firm that provides drug development services, including support for drug repositioning activities. “Many pharmaceutical companies use their own in-house algorithmic solutions or leverage technology platforms developed by specialists in repositioning, such as Biovista, Excelra, etc.,” he notes.

	In addition to in-silico modeling and target docking using algorithmical solutions beyond medicinal chemistry, Mucke points to data mining of side effects and drug interactions as a way to find valuable clues for APIs formulated into marketed drugs. “Crucial information can also be extracted from the peer-reviewed and patent literature by creative pharmacologists who can think out of the box and ‘connect the dots’,” he adds.

	The other four approaches identified by Persidis overlap somewhat with these strategies and include literature mining, pathway mining, adverse-event matching, and gene-regulation mining. “Pathway mining involves the use of pathway keywords to search the literature and other gene/protein pathway databases where a specific pathway is linked to different diseases, and then match drugs known to hit that pathway across the different diseases,” he explains.

	Similarly, in adverse-event matching, lists of adverse events are used to mine the labels of drugs for the same adverse events. “If different drugs that work in different diseases have the same adverse events, then maybe they can work in the same diseases,” Persidis observes. Gene regulation mining uses data from microarrays to find genes that may be up/down-regulated in a disease microarray, and then the literature is searched to find drugs known to have the opposite effect.

	Unfortunately, there are few data sources that are specifically tuned to the needs of drug repurposers, according to Mucke. In addition, analysis tools are not yet sufficiently advanced to assist without extensive training in pathway and systems medicine.

	The existing approaches have all yielded some successes and failures, adds Persidis. They fall into the “quick/easy/cheap category,” however, and are limited in their applicability. “To do repositioning is easy, to do it well is hard. The key limitation of these existing computational approaches is that they are focused on reducing repositioning to one parameter (gene up/down, common side effects, etc). As a result, they do not take into account the complexity of diseases and their mechanisms of action, or the complexity of patient populations,” he said.

	Going forward, both Persidis and Mucke believe that the application of artificial intelligence (AI) to drug repositioning will take the field much further than it can go today. “AI is emerging as the best tool to do repositioning well. New AI-driven technology shows the best promise, because AI is able to integrate and use many different types of data much more effectively than before,” Persidis explains.

	Adds Mucke: “Advanced AI technologies that can look for connections they have not been specifically trained to identify will revolutionize drug repositioning within the next few years, taking us beyond mere expert systems.”

	It is not just existing compounds that have potential for drug repositioning; their derivatives can also be explored for therapeutic activity. “Once you have a repositioning candidate compound identified for your disease target (or vice versa), you can let loose all the medicinal chemistry and pharmacology tools that are out there,” says Mucke. Derivatives can be generated using known quantitative structure-activity relationship approaches that have been widely employed in drug discovery for many years, according to Persidis.

	Of course, the parent compound must be identified first, which is a repositioning exercise. “The real challenge is not the derivatives, but the starting points. Once you have a drug, then deriving different versions can be done, but you can’t derive a different version unless you know where to start from. The difficulty is finding a good starting point,” says Persidis.

	H.M. Pharma Consultancy maintains the Discontinued Drug and Candidate Database (DDCD), a comprehensive machine-readable database of information on compounds that might be suitable for drug repositioning. The DDCD is regularly updated with information on approved drugs and drug candidates that might be available for repositioning efforts taken from peer-reviewed papers, patent documents, and chemistry, pharmacology, and development information in various public sources, according to Mucke.

	The company uses the DDCD as an internal resource for drug development projects and for repositioning projects that it is completing as part of the European Union Research and Innovation program, Horizon 2020. “The DDCD has been constructed over the past 20 years and goes beyond any single public source in defining the pool of compounds that are potentially available for drug repositioning,” Mucke comments.

	Over the past 10 years, Biovista has been developing an AI solution called Project Prodigy. “A major challenge in drug repositioning is to balance the insights into a new use with equivalent insights into potential side effects,” Persidis says. “Biovista’s AI program is one of very few equally able to identify both potential new uses and the specific subpopulations to include or avoid, based on a full understanding of the benefit/risk balance,” he notes.

	The Project Prodigy AI is also one of few systems that does not limit itself to machine learning (ML), says Persidis. That is important because ML only affords ‘hits’ that are basically already known from training sets, even if hard to find. “Biovista’s Project Prodigy AI is capable of building entirely new clinical scenarios that have never been seen before and can be evaluated as truly novel. No training sets are needed or used. This ability provides a key advantage over typical ML-based approaches,” he states.

	Project Prodigy, according to Persidis, has already been used in a number of projects. Internal efforts at Biovista have led to repositioned drugs with animal model/cell line validation and issued or granted patents in multiple sclerosis, epilepsy, anti-glomerular basement membrane disease, and some rare diseases such as Friedreich’s ataxia and Leber’s hereditary optic neuropathy. The company has also used the AI system in collaborations with major pharmaceutical companies, patient advocacy groups, and FDA.

	Developing a drug is a long, expensive process. Repositioning is the most efficient way to get to a valid starting point. “Drug repositioning should be a standard operating procedure at any stage of development, especially early on. Its ‘right way’ systematic use also reduces development and investor risk across different phases of the portfolio,” said Persidis.

	While none of the current digital tools available for repositioning are capable of providing anything beyond assistance for pharmacologists who know how to use them and who can interpret their results properly, according to Mucke, this situation will change soon with the application of continually improved AI technology.

	“AI has already shown its promise, with actual results achieved much more efficiently than other approaches. Now, it is beginning to be understood as one of the most powerful ways forward for drug development and healthcare cost containment,” Persidis concludes.

Vol. 42, No. 9
	September 2018
	Page: 22Â­–26

	When referring to this article, please cite it as C. Challener, “Can Artificial Intelligence Take the Next Step for Drug Repositioning?" 

Existing software tools cannot take into account the complexity of disease.

Can Artificial Intelligence Take the Next Step for Drug Repositioning?

As the pharmaceutical industry matures, the complexity of new drug candidates is increasing. More complex molecules are more challenging to synthesize, often requiring advanced chemistry techniques to ensure both high yields and high selectivity. Sophisticated chemistry such as exotic catalytic transformations are used more widely as a result. Many of these reactions are highly sensitive to temperature and pressure, with control of one or both of these reaction conditions improving the selectivity and yields of desired products, particularly in cases where undesired impurities have similar structures and physical characteristics. Low-temperature chemistry, and in particular cryogenic chemistry at temperatures down to -80 °C, can facilitate transformations that cannot be achieved at higher temperatures.

	In the past few years, there has been an increase in the number of new API projects being brought to contract development and manufacturing organizations that require low-temperature chemistry. New routes to existing products that are designed to improve efficiency and productivity also more often involve cryogenic processes, according to Jean-Pierre Pilleux, site director at Novasep’s Chasse-sur-Rhône facility in France. “As novel APIs become more and more complex, cryogenic conditions are often mandatory to obtain the required selectivity. For example, processes employing organometallic chemistry can be critical in API synthesis,” adds Jean-Baptiste Guillermin, head of process development at the Chasse-sur-Rhône plant.

	Use of low temperature can influence the reaction pathway, particularly for reactions in which desired and undesired products differ only slightly from an energetic standpoint, whether with respect to stereo-, regio-, or chemoselectivity. Reactions involving unstable intermediates, notably organometallic reagents, that cannot be conducted at or near room temperature are often possible at much lower temperatures. In addition, processing involving gaseous reagents can be easier to implement at low temperature.

	“Cryogenic chemistry is an enabling technology that can allow the limiting of impurities, performance of processes with highly reactive compounds, improvement of reaction selectivity, elimination or reduction of unwanted side reactions, prevention of ice crystal formation, and reduction of the volatility of compounds for greater safety,” notes Ed Price, president and CEO of PCI Synthesis.

	Running large-scale cryogenic processes is an entirely different proposition than performing low-temperature chemistry in the lab, according to Price. “In the lab, glassware can be placed in an acetone/dry ice bath. For commercial production, heat transfer fluids must be pumped through jacketed vessels using sophisticated pump technology (costing five to six times that of conventional pumps) and complex control systems,” he explains.

	In addition, specialized analytical tools are required; measuring cryogenic temperatures cannot be achieved with normal mercury or alcohol thermometers because they freeze. Platinum resistance thermometers that exhibit well-defined electrical resistance behavior as a function of temperature must be employed instead.

	“The need for specific and expensive equipment means that the capex [capital expenditure] for newcomers can be significant,” observes Pilleux. He adds that greater energy consumption and handling of unstable intermediates also must be considered. In addition, while there are always challenges going from small to large scale, scaling cryogenic chemistry and processing is significantly more complicated because extreme temperatures must be delivered and maintained, according to Price.

	“Running processes under such cold conditions is as much an art as a science. These reactions are very touchy and sensitive. They have to be run very specifically, which involves controlling an entire system of pumps and heat exchangers to reach, maintain, and control the temperature as the reaction progresses,” he notes. For that reason and based on 40 years of experience running cGMP cryogenic processes, Pilleux considers process robustness to be key. “Understanding the impact of reaction parameters using a quality-by-design approach during development, coupled with close interactions between chemists and chemical engineers using thermal modeling allows the efficient prediction of scale-up parameters for such highly demanding processes,” he states.

	The need for strict control of the reaction temperature throughout the entire reaction mixture can pose challenges as well. The addition of reactive reagents to such processes may lead to the generation of local exotherms that must be removed via efficient mixing and good heat exchange properties of the reactors, according to Pilleux.

	In addition, the low surface area-to-volume ratio of batch reactors restrains the size of vessels for production. In fact, it is not just the need to achieve and maintain low temperatures that is challenging. It is the fact that batch processing has not changed dramatically from a technology standpoint for decades, according to Price. “Pharmaceutical plants today look similar to those in operation 20–30 years ago,” he says.

	For certain chemistries, there is no other option than to perform them at low temperature. “It goes back to the perennial organic chemistry battle between yield, cycle time, and impurities. Achieving the required purity levels is always the first priority. In early development phases, the goal is to deliver high-purity products, and the economics of the process are not as important. That comes into play if a candidate progresses to later clinical stages. If the process cannot be performed economically at low temperature, another route will need to be identified,” Price comments.

	It is important during development, he notes, to gain an extensive understanding of the process and determine the warmest reaction temperature that won’t cause significant problems and the potential benefits that can be gained if the reaction is performed at the lowest possible temperature.

	In many cases, gains in yield and selectivity can overcome the additional costs of running a process at cryogenic temperatures. For instance, Prices notes that a product that is produced in multiple small batches can cost significantly more and take more time to manufacture than if that product is produced in one or two large-scale cryogenic runs.

									Some reactions that are most often performed under extreme low temperature conditions, according to Ed Price, president and CEO of PCI Synthesis, include:

	As drug candidates continue to become more sophisticated, demand for processing under extreme conditions, including cryogenic temperatures, will continue to grow. “The question then becomes, how do we incorporate novel engineering/processing solutions that really move the needle for drug manufacturing?” according to Price. Manufacturing on a smaller scale in a continuous manner to obtain the same yields and throughputs of larger equipment could be one answer, he notes.

	Guillermin agrees. “Reactors having a higher surface-to-volume ratio and more efficient mixing can be used to increase the productivity of low-temperature processes and avoid the potential for high-temperature hot spots,” he says. “Continuous flow reactors are a breakthrough solution.”

	Novasep is, in fact, focusing on the development of flow-chemistry solutions at production scale in order to offer alternatives to batch processes. “This technology generally enables the use of less extreme temperature conditions and control of very short reaction times, even at production scale, allowing the production of unstable intermediates that cannot be obtained under batch conditions-and with a reduced energy cost. These technologies are rapidly expending and Novasep has built a strong expertise in this domain,” says Guillermin.

	Late in 2017, Novasep also initiated a €4-million ($4.7-million) investment to expand its low temperature capability at its Chasse-sur-Rhône facility and address the increasing market demand for cryogenic processes. The company now has a total low-temperature capacity of 35 m3. The investment includes the installation of a new cGMP cryogenic production line capable of operating at temperatures as low as -80 °C and is equipped with a 4000L Hastelloy reactor, filter drier, and cleanroom. The cGMP pilot-plant capabilities were also expanded with the addition of a new stream comprising a 400L Hastelloy reactor, filter drier, and cleanroom.

	PCI Synthesis, meanwhile, added a 1000L jacketed reactor specifically designed for cryogenic chemistry to meet the needs of two recent projects including a new chemical entity moving from Phase I to Phase II and a generic API for which its client wished to de-risk the supply chain by adding PCI as an approved alternate supplier with in-house cryogenic capacity.

	In addition to the 1000-gallon Hastelloy C reaction vessel, the system comprises three separate heat exchangers for liquid nitrogen, steam, and glycol; a specially designed pump; and control valves and control logic. The reactor is housed in a special suite and is paired with a 50-gallon glass-lined reactor for workups and additional processing and a 3-m2 Hastelloy pressure drier.

Vol. 42, No. 8
	August 2018
	Pages: 22–25

	When referring to this article, please cite it as C. Challener, “Increasing API Complexity Drives Demand for Cryogenic Capabilities" 

Low-temperature chemistry enables performance of more challenging and selective chemistry.

Increasing API Complexity Drives Demand for Cryogenic Capabilities

Regulatory agencies have significant interest in the implementation of more advanced methods for the detection of adventitious agents. This interest, according to Horst Ruppach, director of viral clearance and virology at Charles River Laboratories, comes from recent experiences in which adventitious viruses were found in a marketed rotavirus vaccine in 2010 and in an insect production cell line (SF9) in 2014. In both cases, the standard testing methods failed to detect these contaminants. Concerns about more advanced methods still remain to be addressed.

	Overall, the methods used in adventitious agent testing have not changed dramatically in recent years and are performed as recommended and described in guidances and regulations. Antibody detection assays and nucleic acid amplification techniques are used to screen source materials for specific viral contaminants in the plasma product industry, according to Ruppach. A panel of cell-based 

 assays combined with antibody detection, and quantitative polymerase chain reaction (qPCR) assays for specific viruses is used in the vaccine and recombinant protein industry.

	Protocols have been, in some cases, partly modified or complemented when necessary, such as prolonging incubation times for some in-vitro assays to detect slow-growing viruses like Vesivirus, according to Ruppach. In the plasma-product industry, he notes that screening for emerging viruses such as Zika and Hepatitis E has also partly been added to meet country-specific recommendations. A shift from culturing and

 assays to qPCR-based assays for mycoplasma testing of recombinant products has also occurred to some extent.

	In general, however, release testing is still performed using slow, but long-accepted culture-based tests, according to Michael Brewer, head of pharma analytics with Thermo Fisher Scientific. The exception, he adds, is the much broader use of qPCR for in-process testing during cell-culture processes (e.g., mycoplasma, mouse minute virus [MMV], and Vesivirus for Chinese hamster ovary [CHO] culture) and even for conditional release to downstream processing while waiting for results of the culture-based tests.

	There are real issues with existing cell-based methods that are driving interest in the development of alternative techniques. “Cell based 

 assays can detect a broad range of viruses, and even unknown viruses, but the methods are not standardized and can be run in different ways, which significantly impacts the assay specificity and sensitivity,” Ruppach says. In addition, some potential contaminating viruses don’t grow in the detector cells, while others do grow but cannot be detected. “On top of these issues is the fact that positive results cannot be assigned to a virus type or even virus family; subsequent identification assays are necessary,” he observes.

 assays encounter the same challenges and limitations, and there is a high failure rate due to the route for test sample administration. Studies have also shown a low detection rate for these tests compared to 

 assays, according to Ruppach. Ethical reasons are an additional concern, with growing emphasis placed on minimizing animal testing. One of the biggest issues with both 

 testing, however, is the time it takes to obtain results, which can be as long as five to six or even more weeks. “An additional complicating factor is the fact these assays cannot be validated following International Council for Harmonization (ICH) Q2 due to the undefined specificity and sensitivity,” he states.

	While these limitations are overcome with qPCR, Brewer points out that, at the moment, available assays exist only for a small set of viruses that represent the biggest threat to CHO cell culture. “Rapid testing for viruses and mycoplasma remains a critical need for the industry to achieve the goal of rapid, real-time or near-real-time lot release. For continuous processing, time to result is even more important,” he asserts.

	There have been some advances in instrumentation for adventitious agent testing that have the potential to impact testing in the near future. Ultra-high multiplex PCR is enabling rapid workflows when combined with high-recovery nucleic acid extraction, automated sample prep, and high-throughput DNA sequencing technologies, according to Brewer.

	There is much discussion in the industry and among regulators about the potential advantages of new nucleic acid-based technologies and other high-throughput sequencing (HTS) methods, also known as massive parallel sequencing or next-generation sequencing (NGS). “These technologies have the power to detect any virus with a level of sensitivity comparable to virus-specific qPCR and, unlike 

 assays, deliver sequence data that can identify the contaminant,” says Ruppach.

	There are other benefits as well. “Replacing traditional adventitious agent tests with NGS-based tests would provide the highest level of confidence in negative and positive results,” states Brewer. “In addition, time to result for such tests is aimed to be less than two days, and the ability to automate much of the workflow could reduce hands-on time to less than three-and-a-half hours. Furthermore, while traditional culture testing is limited to sampling points in cell banks and harvest media only, rapid molecular testing enables sampling, testing, and decision-making throughout cell-culture processes,” he continues.

	There are major concerns about this technology, however, that must be addressed. The presence of virus-specific nucleic acid does not necessarily indicate a replication-competent contamination, and thus most current nucleic acid assays cannot differentiate viable from non-viable contaminations, which is the case for both in-vitro and in-vivo assays, according to Ruppach. “Some HTS-based methods have been recently developed that can differentiate replication-competent and non-replication-competent viruses, which will help eliminate this important drawback,” he notes.

	For instance, Brewer notes that qPCR and NGS techniques that offer quantitative values or target enumeration capability can be applied to assess viability by measuring changes in target nucleic acid levels at multiple time points in the case of a positive initial test.

	Managing the large amounts of data produced when using HTS technologies presents another challenge. “Data processing becomes an important factor in results evaluation. The algorithm used can lead to both false-negative or false-positive results and significantly impact the outcome of the analysis,” Ruppach explains. No regulations or standards have yet been established regarding the data-processing process.

	Brewer notes that qPCR has been improved by the inclusion of controls that help reduce the risk of false positives due to inadvertent cross contamination of test samples with positive controls.  He does agree that a full solution will require designed-for-purpose software to enable data analysis and results reporting, including enabling compliance with 21 

	The concept of nucleic-acid testing has been well received by industry and regulators, and solutions are in development. “The goal is to supplement or replace the traditional adventitious agent tests with rapid NGS-based detection and identification of a predefined panel of viruses and broad range of bacteria, including mycoplasma,” says Brewer.

	Initiatives are underway to grow regulatory understanding of the capabilities of these new technologies that are supported by various regulatory agencies. For instance, Ruppach points to the Advanced Virus Detection Technologies Interest Group, which was established by the industry and FDA to address specific challenges and provide solutions to make these technologies ready for use under GMP conditions.

	The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB), established by the Massachusetts Institute of Technology Center for Biomedical Innovation, has also had a positive impact. “The CAACB allows for open communication on contamination events that have impacted the industry and provides a forum for experts to discuss how viral testing can be improved and the overall risk of viral contamination reduced,” according to Brewer. The consortium has also helped to develop a more comprehensive understanding of the adventitious agents that are encountered.

	“More advanced NGS-based techniques are in development and offer the potential to supplement or replace the 28- or 14-day culture-based test following validation and regulatory review and acceptance,” asserts Brewer. “Given that a stated goal of the pharmaceutical industry is to move to real-time or near-real-time lot release in the future, replacement of the 28- or 14-day culture-based adventitious agent test with a rapid test will be required,” he adds. 

	It is worth noting that following validation, regulatory filing, and review, regulators have accepted qPCR-based mycoplasma testing for lot-release testing applications across multiple therapeutic modalities (e.g., manufacturing cell culture, cellular therapy, tissue therapy), according to Brewer.

For more advanced NGS-based methods, regulatory acceptance and validation are still needed.