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

Cynthia A. Challener

Cannabinoids have great potential to treat a wide variety of diseases, leading to tremendous interest among pharmaceutical companies in accessing pharmaceutically pure cannabinoids for the development of novel therapies. Several companies are developing cannabinoid-based treatments, including Vitality Biopharma, GW Pharmaceuticals, Zynerba Pharmaceuticals, INSYS Therapeutics, Teewinot Life Sciences Corporation, and Nemus Bioscience.

	Some cannabinoids bind to the same receptors that are bound by endocannabinoids, which are naturally produced in human cells. The human endocannabinoid system (ECS) is a group of endogenous cannabinoid receptors located in the mammalian brain and throughout the central and peripheral nervous systems that are controlled by neuromodulatory lipids, according to Richard Peet, executive vice-president and research director at Teewinot Life Sciences Corporation. “The ECS is involved in a variety of physiological processes, including pain sensation, appetite, memory, and mood. It is for this reason that cannabinoids frequently influence various physiological functions when administered to humans,” he notes.

	Cannabinoids such as delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), tetrahydrocannabivarin (THCV), and cannabidivarin (CBDV) have many potential uses, such as the treatment of nausea associated with chemotherapy, epilepsy, appetite control, diabetes, and inflammation, among many others.

	Why biocatalysis for cannabinoid synthesis?

 plant via biosynthetic pathways consisting of several enzyme-catalyzed steps. The 

plant produces only small quantities of many of these cannabinoids including the varin series of cannabinoids (THCV, CBDV, and cannabichromevarin [CBCV]), and cost-effective chemical synthesis of these molecules is not available, according to Peet.

	“Traditional chemical manufacture of these complex, chiral molecules typically requires several steps and elaborate purification from impurities,” observes James Lalonde, senior vice-president of research and development for Codexis. Chemical synthesis of cannabinoids such as THC also generates copious amounts of organic waste, takes many weeks to complete at the kilogram scale, and is very expensive, adds Peet. “Practical methods for chemical synthesis for many of the 111 known cannabinoids have not been developed,” he states. Enzymes that have been evolved in nature, in contrast, can synthesize these molecules cleanly with the exclusion of impurity-generating side reactions, according to Lalonde.

 plant is also not practical for production of large quantities of pharmaceutically pure cannabinoids. Extraction of targeted cannabinoids from the more than 100 related compounds in the plant is laborious and time consuming, according to Lalonde. “The 

 plant must be grown for three to four months and requires large quantities of water and energy inputs. Then obtaining pharmaceutically pure cannabinoids from plant tissue is very expensive, because many cannabinoids are produced in very small quantities in the 

 allows the synthesis of targeted single distinct compounds, rather than a complex mixture. “Production of these compounds can be controlled for purity and regulatory controls in the same manner as any active pharmaceutical. In addition, the biocatalytic process is independent from the plant growth cycle and the constraints of marijuana cultivation. Scale up of the synthesis of rarer, but desirable compounds would also be made possible in an enzymatic, 

 process in which enzymes that catalyze formation only of the desired compounds could be cloned and purified in high yield,” Lalonde explains.

	Teewinot has developed biosynthetic processes for the production of large quantities of cannabinoids including the varin series of cannabinoids. “Compared to chemical syntheses, these processes are less expensive, more efficient, and produce minimal amounts of chemical waste. As a result, biosynthetic processes make possible the production of pharmaceutically pure cannabinoids in much larger quantities than is practical through plant cultivation and cannabinoid extraction,” Peet asserts.

	The company uses bioinformatics and metabolomics to facilitate the development of synthetic biology and biocatalytic routes to cannabinoids. Genes in the 

 plant, as well as chemical compounds in plant tissues, are identified, characterized, and quantified if relevant. “By cloning specific gene sequences from the plant and incorporating them into microorganisms, we are able to develop new expression systems and processes for the production of naturally-occurring and specific cannabinoids,” Peet explains. Specifically, Teewinot has cloned genes from the Cannabis plant that code for cannabinoid biosynthetic enzymes and incorporated them into yeast and other microorganisms.

	Teewinot refers to its cannabinoid biosynthetic processes as CannSynthesis. Using its synthetic biology and biocatalytic processes, the company can now biosynthetically produce 18 different cannabinoids that have the identical chemical structures to those produced in the plant, including tetrahydrocannabinolic acid (THCA), THC, tetrahydrocannabivarinic acid (THCVA), THCV, cannabidiolic acid (CBDA), CBD, cannabidivarinic acid (CBDVA), CBDV, cannabichromenic acid (CBCA), CBC, cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabinol (CBN), and cannabicyclol (CBL), according to Peet.

 enzymes THCA synthase and CBDA synthase in a microorganism. THCA synthase converts the substrate CBGA into THCA and/or CBCA, while CBDA synthase converts the substrate CBGA into CBDA and/or CBCA. During biocatalysis, both reactions proceed in a pH-dependent manner in a bioreactor. The CBGA used in this biocatalytic process can be produced in any number of ways, including synthetic biology or chemical synthesis. Alternatively, the entire process can be done in a microorganism by means of synthetic biology.

	Teewinot can also produce next-generation, proprietary cannabinoid pro-drugs and cannabinoid analogs, many of which are novel and have never been produced in large quantities in pharmaceutically pure form. “Some of these compounds have improved efficacy and bioavailability and other beneficial characteristics that improve therapeutic outcomes,” Peet notes. He adds that it is now possible to test the efficacy of these compounds in the treatment of a wide variety of diseases, including inflammatory bowel disease, Crohn’s, epilepsy, cancer, migraine, fibromyalgia, and many others, according to Peet. For instance, in 2016, Teewinot licensed its patent-protected biosynthetic processes to Nemus Biosciences for use in the manufacture of its THC pro-drug for treatment of glaucoma.

	The company expects to undertake GMP production of cannabinoids using biocatalysis within the next three to six months, and is in conversations with a large number of pharmaceutical companies seeking to purchase cannabinoids produced by its biosynthetic processes for use in pre-clinical and clinical trials.

	In March 2017, Teewinot signed a letter of intent with Noramco, a producer of controlled-substance APIs, to commercially produce cannabinoids using its CannSynthesis technology. The agreement provides Noramco access to Teewinot’s patent-protected processes for the production of pure cannabinoids using biocatalysis and the company’s assistance with rapid implementation of the technology.

	Noramco will create 10-15 cannabinoid reference standards and evaluate the feasibility of using the technology to produce Dronabinol on a commercial scale. Dronabinol is known for its effectiveness in pain management, as an antiemetic, in the control of vomiting, as an appetite stimulant in the treatment of AIDS and for reducing the side effects of chemotherapy.

	Although biosynthetic processes like those developed by Teewinot are new and improved methods of production that reduce cost and production time, increases purity, and allow for the synthesis of a much greater diversity of molecules that can be incorporated into drug formulations, the approach is different from traditional extraction or chemical synthesis. As a result, Teewinot has found that bringing this new technology to market has created the need to educate researchers and others in the pharmaceutical cannabinoid field. “Educating researchers includes describing the broad array of cannabinoids that can now reliably be manufactured, tested, and put into clinical trials for development of potential new therapies,” Peet comments.

	He does note, however, that because synthetic biology and biocatalysis are common pharmaceutical manufacturing processes, pharmaceutical companies and regulators are generally familiar with the technical aspects of these methods.


	Vol. 41, No. 6
	June 2017
	Pages: 20–22

	When referring to this article, please cite it as C. Challener, “Accessing Cannabinoids Using Biocatalysis," 

Articles

Efficient synthesis of complex cannabinoids is possible while avoiding marijuana cultivation. 

Accessing Cannabinoids Using Biocatalysis

Dramatic increases in protein titers from cell culture processes have enabled downsizing of upstream bioprocesses and facilitated the adoption of single-use bioreactors for biopharmaceutical manufacturing.

	Manufacturers of disposable bioreactors have continually worked to improve their systems to meet evolving needs for biologic drug substance production.

	This article explores recent advances in disposable bioreactor technology, the limitations that remain, and efforts that are underway to address them.

The decision to use disposable bioreactors is now driven by commercial rather than technological considerations.

Single-Use Bioreactors Have Reached the Big Time

	Continuous API manufacturing has the potential to provide tremendous benefits to the pharmaceutical industry-smaller plant and environmental footprints, reduced costs, and improved processes with more consistent quality. Extensive process and product knowledge and the ability to control process conditions in real time are necessary for achieving these goals. Effective process analytical technology (PAT) for online monitoring during research, development, and commercial manufacturing is therefore essential. While notable advances have been made, there are numerous challenges yet to be overcome. Manufacturers, instrument suppliers, and data analysis and process control system vendors are working collaboratively to design effective solutions.

	PAT can be used for many different objectives, including reaction/process characterization, process development, and long-term, robust installations. At Eli Lilly, PAT implementation is used to pursue all three, according to Todd Maloney, principal research scientist. During process development, adds Nuno Matos, head of continuous manufacturing within R&D with Hovione, the main driving force for using PAT is to enhance knowledge gathering. Therefore, more versatile, complex technologies with high dynamic ranges are used.

	Spectroscopic methods are most widely used because they are non-invasive and non-destructive. Chromatographic separation combined with various detection techniques is increasingly used due to the additional specificity, higher sensitivity, and resolution it offers and with the advent of faster technologies such as ultra-performance liquid chromatography (UPLC) or ultra-high pressure LC (UHPLC), according to Ernie Hillier, principal systems manager, Waters Corporation.

	Early efforts typically emphasize elucidation of the chemical mechanism along with gaining kinetic information relevant to the reaction. Eli Lilly heavily leverages flow-nuclear magnetic resonance (NMR) along with other 

spectroscopic measurements including Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. “These data-rich experiments drive process development by providing critical information at an early stage and also serve as inputs to reaction modeling efforts,” says Gordon Lambertus, senior research scientist with Eli Lilly. At Hovione, the most widely use technologies also include mid-IR and Raman spectroscopy, in particular for reaction and crystallization monitoring, according to Matos. Chemometrics are applied to collected spectra to maximize the information that can be extracted.

	As chemical mechanisms become understood, more emphasis is placed on process development, with efforts focused on monitoring reaction conversion and tracking process-related impurities to optimize reactor platforms and reaction conditions. “Process monitoring attributes of interest at this stage are typically focused on supporting the overarching control strategy for the process, commonly including removal of many low level process impurities,” Maloney observes. He adds that online chromatography is the single most important platform technology used to support process development because no other analytical tool offers the specificity and sensitivity required for analysis of these low-level species. Eli Lilly largely employs online high performance liquid chromatography-ultraviolet (HPLC-UV) detection, but also uses HPLC-mass spectrometry (MS), gas chromatography-thermal conductivity detection (GC-TCD), and GC-flame ionization detection (GC-FID) when appropriate.

	Spectroscopic tools such as FTIR, Raman, near infrared (NIR), UV, refractive index (RI), and fluorescence are strategically implemented for specific applications, as are simple sensors that cover additional analytical space and often offer more robustness over the course of a long-term installation, according to Lambertus. These sensors include some spectroscopic measurements (UV, RI, fluorescence), monitoring of specific chemical entities (e.g., pH, dissolved oxygen), and physical property measurements (e.g., turbidity, viscosity, conductivity). “A key point is that there is no single solution, so we have built quite an extensive toolbox that includes a wide range of analytical technologies,” asserts Lambertus.

	Process dynamics in continuous manufacturing should be well understood, namely how process parameter variability will affect the quality attributes of interest or how predetermined changes in process parameters and material attributes will influence the outcome of the continuous step, according to Matos. “Capturing this information requires analytical technologies that can output in real-time the data of interest (either directly or via prediction). As a result, experiments for determining residence time distribution, building kinetic models, or executing design of experiment approaches will be data- and information-richer,” he explains.

	The more data that can be collected from a single experiment, the more efficient the process development model becomes, agrees Lambertus. The two most widely used PAT tools in his department are flow-NMR and online HPLC because of the amount of information they provide.

	“Flow-NMR provides information relevant to understanding chemical reaction mechanisms through elucidation of transient species or monitoring reactive intermediate species, readily generating kinetic information and providing valuable inputs to chemical reaction models that help in optimization,” Lambertus notes. It also allows for access to reactions that could not be run in static NMR tubes (e.g., heterogeneous reactions, high pressure reactions).

	Online HPLC is used in R&D and manufacturing at Eli Lilly to support various control strategy elements. Online measurements are required because there are few isolated intermediates in most continuous synthetic routes that offer traditional control points where measurements can be made, according to Lambertus. He adds that HPLC is the preferred measurement technique because many of the control elements of a process are related to species present in the solution matrix at low levels. “No other analytical technique is capable of detecting and quantifying at the low levels necessary to show that the process is performing as expected with respect to those impurities (e.g., residual starting material, genotoxic impurities, identified impurities with poor rejection efficiency),” Lambertus says.

	 “During the past decade, the PAT user community has been seeing important technological developments; equipment manufacturers are not only improving established technologies but also adapting complex technologies for real-time monitoring,” Matos asserts. There are still unmet analytical needs in the continuous API manufacturing space, however.

	Matos notes that NMR spectroscopy is a good example of an analytical technology that is seeing important developments for going from bench to process applications, with manufacturers racing to make these units smaller while not losing too much resolution. Online UHPLC is a reality, but even runs of a few minutes can hinder real-time monitoring, and interfacing with process equipment requires complex automated sampling devices, so this technology is currently more often used in an at-line fashion.

	The separations piece is still needed, though, according to Hillier, and UPLC provides the high sensitivity and resolution required to gain greater process knowledge. Introducing new technologies is always challenging, he adds. “It is difficult to generate interest when there are existing technologies that are well understood, respected, and do an adequate job. From time to time, we’ve had to contend with perceptions that liquid chromatography is a slow process that requires extensive technical skill to perform,” Hillier comments.

	For Eli Lilly, online metals analysis remains a significant need. “Integrated continuous processes, by nature, eliminate many of the intermediate isolation points of traditional pharmaceutical manufacturing processes. Without these isolation points, inorganic species (salts and metals) can potentially be carried through many subsequent unit operations. Continuous extractive workups are incorporated as rejection points for these types of materials, and currently we use at-line instrumentation based on x-ray fluorescence (XRF) technology to do analysis of metals around these workup unit operations. There is a strong desire to have instrumentation for online, quantitative metals analysis available to support continuous process development,” Lambertus explains. He would also like to see more traditional oxygen and pH sensors that have the chemical compatibility and robustness required for use in aqueous/organic mixtures or neat organic solvents.

	Nanoparticles--with a median particle size typically in the range of 100-600 nm--also present online analytical challenges, according to Matos. “Applications involving nanoparticles are growing in number and the manufacturing technology is often continuous in nature (e.g., co-precipitation, wet-milling, etc.). While there are established technologies for determining concentration in solution of the compound of interest, the real-time measurement of particle-size distribution for the nano range is still not trivial,” he states.

	The biggest challenge is interfacing PAT to a process, regardless of whether it is batch or continuous. “For the latter, the challenges are of different type, in particular when dealing with small setups; in-line probes may disrupt the process by creating a constraint and/or an opportunity for deposition/precipitation, flow-cell dead volumes may promote back-mixing, etc.,” Matos observes. He notes that the opportunity to use higher concentrations (in some cases even running solvent-free reactions) forces the development of strategies to cope with high analyte signals such as automatic dilution strategies or signal attenuation via special probe or flow-cells configurations.

	Automated sampling systems that are capable of sampling, diluting, and delivering a representative sample are essential for sensitive PAT techniques such as online HPLC, HPLC-MS, or online GC, agrees Maloney. Due to the variety of different processes that require sampling, a combination of different sampling techniques are often utilized. For example, suspended solids or precipitates in a process may lead to plugging of small-diameter tubing associated with automated sampling devices. Sampling of supersaturated solutions can be challenging and often requires quenching or dilution at process temperatures to prevent solids fouling. For optical technologies such as FTIR, Raman, or focused beam reflectance measurement (FBRM), location and orientation of the optical probe and bench relative to the process can be difficult.

	Part of the challenge is also determining what tools are needed when. “In order to achieve the optimum process performance, a ‘kitchen-sink’ approach is generally used, with as many tools as possible used to investigate every conceivable thing that might happen in a reaction,” says Hillier. The other piece is coordination, collection, and dissemination of the disparate data so that it can be analyzed and applied for process control. It is important to assure that the sampling frequency will be able to capture the expected process dynamics and thus process knowledge, and allow proper and timely feedback control, Matos agrees.

	Translating PAT data into meaningful information that enables process decisions is another significant challenge. Simple sensors that provide two-dimensional data (pressure, temperature, flow rate) can easily be incorporated into a distributed control system (DCS), according to Bradley Campbell, a research scientist with Eli Lilly. Multi-dimensional data (HPLC, HPLC-MS, FTIR, Raman, etc.) are much more challenging to integrate into a DCS.

	“Many analytical technologies that were designed to be bench-top instruments are being adapted for online applications in support of continuous process monitoring. While the analytical instrument hardware was designed with flexibility in mind, the same is not always true for the software. Analytical instrument control, data processing, and reporting software were not designed to communicate with and export data in a format that can be consumed by a DCS,” Campbell says. There are currently several vendors looking into this issue and providing solutions for testing, but there is still not a single platform to integrate all data types into a single data package to use on the manufacturing floor.

	All of the pharmaceutical industry is working on continuous processing. Waters, according to Hillier, is collaborating closely with not only its pharma customers, but providers of other sampling and software and control systems to develop comprehensive PAT solutions. “Much of our time is spent working with other companies. We are taking an agnostic approach because our customers often have individual preferences for which sampling devices, data analysis software, and control systems they select, and our chromatography solutions need to work with them all,” he states.

	It is worth noting, according to Matos, that manufacturers of PAT instrumentation are diligently working to improve both the technology and the sampling interface, in some cases via successful partnerships with continuous processing equipment manufacturers. “PAT is not optional for continuous processing. It is necessary for gathering information and establishing an effective control strategy for commercial manufacturing. The increasing interest in continuous processing is creating a favorable environment for PAT equipment manufacturers to push technological advancements in three directions: improving the spec sheets to increase the analytical power of existing and established technologies, adjusting bench equipment for real-time measurements, and achieving further simplification to drive costs down,” Matos asserts.

	Eli Lilly has focused its efforts on engineering better ways to develop robust process interfaces for integrating analytical instruments to continuous processes and investigating ways to link complex analytical data to DCS systems for the purpose of trending, process monitoring, and control. “In a regulated environment where ideally the technology designed in development can be readily transferred to a cGMP manufacturing facility, care must be taken to ensure that computer systems, PAT, and sample delivery systems are designed to operate in a fully validated state,” Campbell notes. Communication between these systems is also essential to enable real-time decision making when more than one data source is providing information on the state of control.

	As challenges in process engineering and analytical technologies are overcome, it is vital that PAT data are available to the DCS, process historian, and process control models to enable the development of an integrated process control strategy. PAT data must be visualized within process control charts with the appropriate automation in place to minimize operator intervention and enable real-time decision making when appropriate, according to Campbell.

	In addition to the development of control strategies based on PAT, the data-rich environment of continuous processes with online monitoring should eventually allow for real-time release testing (RTRT), according to Matos. “Using physical, chemical, or mathematical analyses, companies will benefit from RTRT from increased assurance of quality (decisions based on more data) and improved manufacturing efficiency (reduced cycle-times, inventory, etc.),” he says.

	While the science, technology, and regulatory landscape for PAT use are the same for contract development and manufacturing organizations (CDMOs) and drug manufacturers, the CDMO business model does imply other considerations. “When investigating the implementation of PAT, CDMOs must be assured of a return on their investment beforehand, and thus the technology should be versatile and not product- or application-specific. The concept of modularity and the need for considering the operating costs are certainly part of the decision checklist so that CDMOs can maximize the use of these technologies,” Matos observes.  On the flip side, the exposure to different products in a high-paced environment creates the opportunity for intensive learning by CDMOs on the use of a given real-time analytical technology for certain applications.

Advances in equipment, instrument, and control systems are enabling online monitoring of continuous API manufacturing.


PAT Developments Move Continuous API Manufacturing Ahead

While most basic research occurs in academia, contributions from industry are also undeniably significant and impactful, according to Jeff Song, director of development at Boehringer Ingelheim (BI) Pharmaceuticals. Several pharmaceutical companies have established strong R&D organizations that routinely discover innovative and practical chemistries, and technology companies such as Johnson Matthey and Codexis also engage in original research to provide new chemistries for API synthesis.

	In addition, both academia and industry are increasingly seeking opportunities for collaboration in precompetitive spaces, either through individual research agreements or multi-party consortia, according to Song. He notes that BI was one of the founders of the BI-Pfizer-AbbVie Consortium for Non-Precious Metal Catalysis, Flow Hydrogenation, and Computer Aided Synthetic Design. BI is also an industrial sponsor of the National Science Foundation’s Research Center, Center for Rational Catalyst Synthesis, and is intimately involved in its projects.

	Two areas of technology have emerged in recent years as particularly important for API manufacturing: flow chemistry and catalysis. “Both can be applied to a broad scope of APIs. Catalysis provides shorter/more atom-economical, more cost-effective, and greener processes, while also providing novel reactivities that were not accessible before. Flow chemistry is another green technology that expands the horizon of the types of chemistry that can be used for making bulk APIs,” states Chris Senanayake, US vice-president of chemical development for BI.

	Chemistries routinely practiced in the pharmaceutical setting include amide bond formations, common acid/base promoted hydrolytic reactions to access amide and carboxyl moieties, carbon-carbon bond formations (Grignard, Suzuki-Miyaura, Negishi, Sonagishira, Heck), oxidations, reductions (classical and asymmetric), halogenations, esterifications, alkylations, aminations, nitrations, and classical and enzymatic resolutions, according to Marlon Lutz, a process development and flow chemist with Regis Technologies.

	 “Advances in catalysis, including both chemical and biocatalysis, have been the most impactful for API synthesis,” asserts Senanayake. Specifically, he points to the design and discovery of new (chiral) phosphine ligands that have allowed many efficient transformations such as cross couplings and the installation of chiral centers to be performed on large scales (1-3).

	Carbon-carbon bond forming organometallic chemistry is another chemistry worth highlighting, according to Stephen A. Munk, president of Ash Stevens, a division of Piramal Pharma Solutions. “Scalable palladium-catalyzed coupling reactions, such as Sonogashira and Suzuki reactions, allow for efficient carbon-carbon bond formation at the larger volumes required for API manufacturing,” he observes. (See an example in reference 4.)

	In biocatalysis, the use of directed evolution technology has resulted in the engineering of highly efficient enzymes for a wider range of chemical reactions than ever before, according to Song. Multiple examples of biocatalysis in API synthesis on the commercial scale have been reported (5-6).

	 “With lower processing times, solvent volumes, and reduced waste streams, flow chemistry (continuous processing) has enabled processes to be operated more efficiently, safer, cleaner, cheaper, and faster,” Lutz states. In addition, process analytical technologies (PATs) have enabled flow chemistry processes to be executed with higher confidence and reproducibility and generate products that are of high purity and yield. Furthermore, FDA supports the use of a continuous flow approach for producing APIs.

	 “Many pharmaceutical chemical processes are still performed in batch vessels, which are strictly limited by the process conditions (e.g., boiling points of solvents at atmospheric pressure) and often require long processing times for heating/cooling of large batch reactors. There has, however, been an increasing expectation for safer, more efficient, cost effective and greener synthetic routes. Flow chemistry has attracted attention as realization of its numerous benefits for achieving this goal has spread,” Lutz adds.

	In particular, flow chemistry is highly applicable for processes that require high mixing and heat/mass transfer, control of processes operating in the thermal runaway regime, and safely conducting hazardous chemistry that is difficult to handle in conventional batch reactors.

	At BI, flow chemistry has been employed for reactions that would not be possible using conventional batch protocols, according to Song. “Several of our programs involving high-energy reagents (azides), unstable intermediates (organolithiums), and extreme reaction temperatures (low or high) have benefited from flow technology,” he comments. In one example, approximately one metric ton of a propargyl borolane reagent was manufactured in good yield and high quality using flow chemistry (7).

	In addition, the ability to automate commercial flow reactors has impacted how process development is performed and translated into manufacturing, according to Lutz. “The use of flow chemistry enables process development and reaction optimization to be performed significantly more quickly than traditional approaches using batch vessels,” he explains. Employing PAT with flow reactor systems extensively reduces development times with elimination of the bottleneck of offline analysis (8).

	Furthermore, translating flow processes from research-scale models to commercial scale is relatively simple; if the geometries and design of the flow reactor are maintained, the number of flow reactors can be increased to achieve the desired product output (‘numbering up’), according to Lutz. He also notes that many of the commercial-scale flow reactors are designed to offer enhanced mixing and heat/mass transfer phenomena when increasing throughput.

	It is also worth noting, according to Lutz, that implementing flow chemistry can be beneficial for contract manufacturing organizations (CMOs) because they can easily use flow chemistry for problem steps that are low yielding, require harsh conditions, are medium- to high-risk due to the reaction conditions, or use excesses of expensive reagents.

	It isn’t only advances in chemistry and processing technology that have improved API manufacturing. The evolution of a contemporary data-rich laboratory environment with new equipment, software, process modeling techniques, quality-by-design methodology, multidisciplinary collaborations, more sensitive analytical instruments, enhanced safety analyses using reaction calorimetery, accelerating rate calorimetry (ARC), and thermal screening units (TSus) is providing a deeper understanding of chemical processes and their predictability at scale, according to Munk.

	“Today it is not only possible to improve yields and limit the formation of impurities; in-depth understanding of processes leads to better process control, safety, reliability, and robustness, along with improved product quality,” he explains. “It also allows for continuous improvement of the process and understanding where things fail and the consequences of the failure using statistical data without having to do hundreds of experiments,” Munk adds. New safety equipment/instrumentation and software allow for safer scale up of processes, while new laboratory reactor systems better model plant equipment and stirring conditions. A more multidisciplinary approach to process development looks at processes from more perspectives (modeling, statistically, engineering, safety, and environmental impact).

	Enhanced chromatography equipment and more

	While catalysis and flow chemistry are having the greatest impact on API manufacturing, there are many other advances providing measurable improvements. More efficient, environmentally compatible, and cost-effective production-scale chromatography equipment, including simulated moving bed (SMB) and supercritical fluid chromatography (SFC), is one example, according to Munk.

	Greater understanding of solid-state forms and genotoxic impurities are also important. Access to greater knowledge through rapid and effective solid form screening leads to the selection of more developable APIs (9). Identification of impurities and their sources makes it possible to develop more efficient, optimized processes with higher yields and purity profiles (10).

	Highly potent and complicated drugs such as antibody-drug conjugates and proteolysis targeting chimera are more prominent in the industry and are requiring significant containment infrastructure, purification equipment, and experienced chemists, according to Munk. The existence of different potency categorization schemes and the need to ensure operator and environmental safety pose significant challenges (11).

	Returning to specific chemistry examples, Song points to Knochel’s Turbo Grignard reagent as an important chemistry enjoying wide application in industry and academia. “This chemistry revolutionized the way in which Grignard reagents are generated. Due to its unique reactivity, Turbo Grignard offers benefits such as wider functional group compatibility and improved safety and cost-effectiveness for Grignard formation (12-13). Organozinc reagents have shown wider functional group compatibility as well, according to Marlon. (See reference 14 for an example.)

	While the advances discussed above are indeed significant, challenges in API synthesis and manufacturing remain. Fortunately, further developments are ongoing and provide significant potential for the future. For example, useful methods for fluorination and trifluoromethylation (15-16), which are needed given that approximately 20% of drugs contain at least one fluorine atom, are allowing medicinal chemists to access novel structures for structure-activity relationship studies and to secure intellectual property for new drug candidates, according to Senanayake. These methods are not ready for large-scale application, however. More work is required in both academia and industry to address substrate scope, catalyst loading, and general practicality issues. Lutz also points to greener metal-catalyzed fluorinations as showing great promise for the replacement of common and harsh fluorinating reagents (17-18).

	Direct functionalization of unactivated C-H bonds is another field showing promise for simplification of synthetic routes through reduction of the number of required steps, according to Lutz. He also points to palladium-catalyzed cyanations with potassium ferrocyanide (19) as providing convenient access to amides, amidines, esters, and carboxylic acids while eliminating the concerns associated with current cyanide-based reactions.

	Much work is also being directed at the development of non-precious metal catalysis, which can reduce costs and toxicity and enable greener processes, according to Senanayake. He notes that significant strides have been made in this area in the past five years, with the development of nickel (Ni)- and iron-catalyzed sp2-sp3 couplings of particular interest to the pharmaceutical industry (20-21).

	Microwave reactors, according to Munk, offer enhanced reaction times and enable transformations that will only work under microwave conditions. Photo-redox reactions, meanwhile, allow the use of the cheapest and cleanest energy source (i.e., solar power) for chemistry, according to Song. In addition, this technology can be combined with chemistry to deliver greener processes (22-23).

	 “API manufacturing requires not only effective chemical technologies, but seasoned, experienced process chemists who understand the necessary requirements for the development of GMP processes, including route selection, cost efficiencies, robustness, reaction mechanisms, environmental impact, and product quality, among other attributes,” asserts James M. Hamby, vice-president of business development for Ash Stevens. These process chemists many times develop and report interesting modifications, efficiency enhancements, and uses of established chemistries to effect large-scale transformations.

	 “Each process presents a unique set of challenges. No one chemistry or technology really stands out as above all the rest. That is why experienced chemists working in a multidisciplinary team deliver the best results,” Hamby concludes.

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	When referring to this article, please cite it as C. Challener, “Catalysis, Flow Chemistry Impact API Manufacturing," 

Advances in chemical synthesis are enabling greener, more cost-efficient processes for API manufacturing.

Catalysis, Flow Chemistry Impact API Manufacturing

Oral delivery of drugs is the preferred route of administration by both patients and manufacturers. Ease and convenience are the greatest requirements for patients, which leads to higher medicine adherence. Production costs for oral dosage forms also tend to be lower than for other options and offer the greatest versatility with respect to optimization of drug delivery.

	For biopharmaceuticals, however, oral administration has been limited to a few relatively smaller molecules, including peptide products such as desmopressin and cyclosporine for systemic treatment, and a number of other products for local treatment. Many attempts have been made to develop oral insulin and other treatments for diabetes, but none have yet been successfully commercialized. Novo Nordisk, for example, halted development of its oral insulin product in October 2016.

	The difficulty lies in the physical and chemical properties of large biomolecules and the physiology of the gastrointestinal (GI) tract. Overcoming these fundamental characteristics is challenging. “Numerous approaches have been explored to enable the oral delivery of biologic APIs. These range from using permeation enhancers to support permeation through the tight junctions of the epithelium, to nanocarriers to force active transport while protecting the biologic API from the gut, to making pro-drugs of the biologic APIs to enhance passive permeation,” observes David Lyon, director of research at Capsugel’s Bend, OR facility. He notes that each of these approaches has had modest success in animal models, but has yet to develop into a commercial product.

	The greatest challenge to the oral delivery of biologic APIs is absorption across the GI tract, according to Ronak Savla, scientific affairs manager, Catalent Pharma Solutions. Most oral drugs are absorbed in the small intestine, which has the highest permeability compared to the large intestine and stomach. Characteristics of the GI tract that influence absorption include the surface area and mucosa permeability of the particular segment; the pH; the presence of food, bile, enzymes, and bacteria; and the number of structures such as Peyer’s patches and lacteal ducts.

	For biomolecules, however, their large size and polar surface make it difficult for them to diffuse across the epithelial layer. “Molecules with molecular weights of less than 700 Daltons are relatively easily absorbed, which does include peptidomimetics; while those above 1000 Daltons, regardless of size, are not typically absorbed to any extent,” Savla says. In addition, most biologic molecules, including peptides, violate Lipinski’s Rule of Five, which is used to determine if a drug molecule has physicochemical properties that will allow it to be therapeutically active when orally administered. “Drug lipophilicity has the strongest correlation with oral bioavailability; there appears to be a minimum degree of lipophilicity required for oral absorption,” Savla adds.

	A second important issue for biologic APIs is their susceptibility to enzymatic and chemical degradation in the GI tract. “Specifically, the physicochemical delivery challenges include hydrolytic sensitivities of the molecules to the acidic media in the gut, as well as the enzymatic activity of the intestine,” explains Lyon. Once the active is absorbed, there are additional opportunities for degradation inside both intestinal epithelial and liver cells, which in the latter is referred to as the hepatic first-pass effect.

	The main consequence of the poor absorbance and degradation of biologic APIs administered orally is low bioavailability. In some cases, this issue can be overcome by using higher doses, but doing so raises concerns about both potential side effects and excessive costs.

	Drug formulation efforts focus on overcoming the various physicochemical challenges to increase bioavailability. Permeation enhancers and enzyme inhibitors are commonly used. Encapsulation and/or the use of protective (enteric) coatings can help prevent chemical and enzymatic degradation. Modification of the biologic API structure through either covalent or non-covalent bonding with lipophilic substances is another approach.

	The stability of biologic drug substances must also be considered when developing formulations. For instance, some techniques used for the production of small molecules--such as those that introduce heat or organic solvents--may not be suitable for biologic products. In addition, according to Savla, the prevention of aggregation upon storage can be a challenge, particularly for biologic products in solution. “Temperature, pH, and--in some cases--light can have an important impact on protein structure during manufacturing, which can lead to a delicate balance between the formulation, process conditions, and stability of the biologic API,” Lyon comments.

	A common theme across current approaches is the use of functional excipients such as permeation enhancers. “The larger size and hydrophilicity of biologic molecules hinders their passage across cells. Permeation enhancers are typically used to increase the space between epithelial cells in the GI tract lining,” Savla explains. Common enhancers include bile salts, fatty acids, surfactants, salicylates, chelators, chitosans, and zonula occludens toxin (1). Use of these excipients does, however, have the potential to cause damage to the mucosa and to allow absorption of other substances, such as other prescribed APIs in patients taking multiple drugs or even gut bacteria and their toxins.

	Enzyme inhibitors are also used to prevent degradation of biologic APIs by enzymes in various parts of the GI tract, according to Savla. Examples include sodium glycocholate, bacitracin, and soybean trypsin inhibitor (1).

	In addition to the use of functional excipients as formulation solutions, common approaches to increasing the oral bioavailability of biologic APIs include modifying the drug molecule and/or the biological system.

	A range of technologies have been explored for modifying the structure of biomolecules, including the use of polymers, the formation of nanoparticles, and their inclusion in lipid systems. In many cases, all three approaches are employed. “Each technology has its pros and cons and may be more suitable for certain molecules or applications over others. Lipid systems are interesting because there have been a lot of studies completed with small molecules and they can both increase the permeability of biologic molecules and protect them against hydrolysis and enzymatic degradation,” says Savla. Examples of lipid systems include liposomes, archaesomes, and emulsions.

	Encapsulated biologics often exist as nanoparticles. Encapsulation protects the active against degradation, while the size of nanoparticles allows for better penetration through the stomach lining, according to Savla. In addition, tailored receptors that target the GI tract lining for enhanced absorption can also be attached to encapsulated proteins. Polymers used for the preparation of such nanoparticulate formulations include chitosan and chitosan derivatives and other polymers commonly used for the preparation of small-molecule drugs (1).

	More exotic solutions include “robotic” pills, such as those being developed by Rani Therapeutics. Tiny needles comprised of a permeation enhancer, which are pushed by self-inflating balloons that function under specific conditions in the intestine, deliver the biologic active through the intestinal wall.

	Actual modification of the protein structure through conjugation with lipophilic compounds (fatty acids) or oligomers such as polyethylene glycol (PEG) has also been investigated. Crystallization of proteins can improve oral delivery, but typically is quite difficult for most biologics (1).

	“Manipulation of the structure of biologic drug substances using any of these approaches must be pursued with caution, however, given the fact these structures are not only complex but crucial to the efficacy and safety of the drug product,” Savla asserts.

	In addition to modifying the structure of the biologic API, some approaches focus on manipulation of bacteria that are commonly found in the intestine. Many of these bacteria are designed to tightly bind to the intestinal mucosa. Through genetic engineering it is possible to create versions of these bacteria that can produce and secrete protein APIs and deliver a high concentration to the absorption mucosa, avoiding the possibility of degradation, according to Savla. In addition, these bacteria can deliver the biomolecules throughout their lifetimes in the intestine. This technology can be combined with modification of protein structures to increase lipophilicity and resistance to degradation.

	There are also promising oral technologies being developed by companies such as Applied Molecular Transport (AMT) that use poorly immunogenic transporter proteins covalently linked to biologic APIs to actively transport them across the epithelium, according to Lyon. AMT’s Transint platform provides targeted delivery of peptides and proteins to cells in the GI tract or liver using the non-toxic portion of the protein cholix toxin, which delivers a toxic compound produced by 

across the gut epithelium. The carrier protein is covalently bound to the biologic API for delivery to the GI tract, while a linker inserted between the two allows delivery to the liver following enzyme cleavage.

	“These types of approaches are elegant at delivering peptides and proteins, but still require protection of the construct from the stomach and intestinal environments,” Lyon says. He notes that the combination of such a transporter technology and Capsugel’s intrinsically enteric capsule technology, or enTRinsic drug delivery technology, may prove effective for providing both physicochemical protection and permeation. “Our enTRinsic drug delivery technology, which uses an enteric polymer in the capsule shell thereby avoiding the need for enteric coating and the high temperatures associated with coating processes, provides acid-, water-, and enzyme-impermeable capsule characteristics that allow the cargo to be delivered intact to the site of absorption while protecting the active molecules,” Lyon explains.

	Despite the high level of interest in formulating biologic drugs for oral delivery, it remains uncertain when commercial products will eventually be available. “Several programs have reached and failed in Phase III. While there are a few current programs in Phase II and III clinical trials, it is difficult to predict whether these trials will be successful. Most of these programs face competition from injectable formulations--a high bar to match or surpass,” Savla observes.

	Lyon is more hopeful when it comes to biologic APIs that treat local gut disease states, such as Crohn’s diseases and irritable bowel syndrome, and expects that oral delivery for these drug substances will be fairly common within the next five years. Although he notes that great progress continues to be made translating “IV drugs to oral,” he says that significant breakthroughs have yet to be made if currently marketed biologic APIs are to be converted without covalent modification from IV delivery to drug products delivered orally.

	To tackle this challenge, Catalent is taking a parallel screening approach. “We understand that there is no one-size-fits-all solution and it is difficult to predict oral bioavailability a priori. We first assess a molecule’s permeation through the oral and sublingual/buccal routes using the permeation enhancer sodium caprate. If the molecule demonstrates potential for one of these routes, we work on developing formulation candidates,” Savla explains.

	The Catalent Applied Drug Delivery Institute’s Non-invasive Macromolecule Delivery Consortium also brings together academic and industry experts from a variety of disciplines to discuss and develop possible ways to address challenges faced by everyone in the area, according to Savla. The Institute organized a conference focused on routes of non-invasive macromolecule delivery in February 2017 in San Diego. The conference was designed to bring together leading experts from academia and industry to discuss challenges, share results, and pave the path for the next steps in the field.

	“As a specialized contract service provider with a focus on product design,” says Lyon. “Capsugel has invested in technologies that solve some of the challenges associated with the non-invasive delivery of biologic APIs, including oral solutions.” As mentioned above, the company’s enTRinsic drug delivery technology overcomes the need to protect biologic APIs from the GI tract environment. “The enTRinsic technology can be used in conjunction with standard biologic APIs and pro-drugs for either systemic or local gut delivery, and we have ongoing development and clinical programs with a number of pharmaceutical and biopharmaceutical partners,” Lyon notes.

	The oral delivery of biologic APIs--mainly peptides and proteins--has been a “holy grail” of pharmaceutical drug delivery for the past several decades, according to Lyon. “Oral delivery remains the preferred route for delivery of medicines. Biologic APIs are growing faster as a class of molecules than traditional small molecules and have enjoyed shorter development times and approval times over the past decade. While the driving factor is improved therapies, biotherapeutics also provide innovative companies with increased differentiation in their product lifecycle strategies. It is reasonable to expect that the interface of these two areas will remain an exciting and active area of research and investment in the coming years,” he states.

	One potentially exciting direction of research for Savla is the intentional design of orally administered biologic APIs. “Given that most biologic drugs are injected, there is no guidance or rules of thumb for oral delivery. To date the focus has been converting existing biologic APIs initially developed specifically for parenteral administration to alternative oral delivery formulations. It is certainly possible that better success with oral delivery can be achieved if the biologic APIs are designed from the start to function effectively within the intestinal tract. Of course, there are significant risks in taking this approach given the numerous issues with the absorption and degradation of large biomolecules in the GI tract. It may in fact take initial successes with modified existing APIs before someone is willing to develop and orally administered biologic drug from scratch,” he concludes.

	1. Catalent Applied Drug Delivery Institute,  

Non-invasive Macromolecule Drug Delivery Guide 

Researchers continue efforts to overcome challenges of effective oral delivery of biologic drugs.

Seeking Solutions for Oral Delivery of Biologic APIs

	When referring to this article, please cite it as C. Challener, “PAT for Continuous API Manufacturing Progresses," 

Advances in process analytical technology have been achieved, but significant challenges remain.