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

Cynthia A. Challener

Mass spectrometry (MS) is a powerful and sensitive technique used to detect, identify, and quantify molecules separated by their mass to charge (m/z) ratio in the gas phase. In a recent publication by FDA reviewing MS usage trends over the past 10 years of biologic license application (BLA) filings, the agency determined that MS is now ubiquitous in filings and that the number of ways it is being used is increasing (1).

	The demands of life-science applications have led to the improvement of MS technologies and rapid growth of new types of instruments that feature powerful analytical capabilities-- sensitivity, selectivity, resolution, throughput, mass range, and mass accuracy, according to Gang Huang, vice-president of analytical development and regulatory affairs with WuXi Biologics. In the biopharmaceutical industry today, mass spectrometry finds applications at the earliest discovery stages (e.g., imaging of biomarker/protein binding in cells), during process development, and for product characterization throughout the development cycle, including for release testing.

	Challenges with sample preparation and data processing remain, however. Biologics manufacturers and analytical laboratories are working closely with instrument suppliers to address these issues. Improvements are continually introduced, and as a result the potential for additional use of this analytical technology for biologic drug development and manufacturing is significant.

	Mass spectrometry is widely used for the characterization of protein therapeutics in the development stage. More specifically, MS is a effective tool for intact protein analysis (measurement of the molecular weight of the protein), fragment analysis, peptide mapping, and identification of post-translational modifications (PTMs), according to Tiffani Manolis, segment manager for pharma at Agilent Technologies.

	As a characterization tool, MS is used to assign the primary structure of biologic drugs, including amino acid sequencing for peptides/proteins, disulfide bond mapping, N-glycan profiling of monoclonal antibodies (mAbs), and nucleotide sequencing for oligonucleotides, adds Jie Ding, associate director of mass spectrometry services at PPD Laboratories’ GMP laboratory.

	Peptide mapping can also be applied for determination of other critical quality attributes (CQAs), such as physicochemical properties of oxidation, glycosylation, deamidation, and isomerization and the presence of N-terminal cyclization as well as confirmation N- and C-terminal groups, according to Hillary Schuessler, an investigator with GlaxoSmithKline (GSK) R&D Platform Technology & Science. She adds that characterization of product variants and higher-order structure (HOS) analysis via hydrogen-deuterium exchange (HDX) are also important applications of MS.

	In addition, MS can be used for media and feed characterization, such as quantitative targeted metabolomics for amino acids, vitamins, lipids, central carbon metabolites, sugars and sugar bases, etc. In this case, the absolute quantities of these compounds are measured as they are depleted/excreted from cells. “A challenge is to differentiate extracellular from intracellular metabolites. It is possible, albeit challenging, to measure the flux of metabolites for use in predictive modeling of protein product quality attributes such as glycoform variants and amino acid misincorporation,” says Greg Kilby, manager of biopharm analytical sciences with GSK R&D Platform Technology & Science.

	Profiling of process-related impurities such as host-cell proteins (HCPs) is another application for MS. “The identification of the most abundant HCP species provides valuable information to process scientists for developing a tailored process for removal of these critical impurities,” notes Huang. Sanofi uses MS to support process development and manufacturing, including impurity identification and tracking and the collection of detailed, high-level, advanced structural information to confirm that the product is as intended, according to Jianmei Kochling, director of analytical science and technology for the company.

	Sequence variant analysis of production cell lines used for biologic production is an important part of process development. The potential presence of sequence variants, which can result from DNA mutations and amino acid misincorporations, is analyzed at the protein level using high resolution MS and data analysis software. From multiple candidate clones, the one without mutations or low level mutations will be chosen as the final clone for further development, according to Huang.

	At early development states, MS is used in cell imaging applications, as well as for characterization of biomarkers and determining drug metabolism and pharmacokinetic (DMPK) profiles (clearance/lifetimes).

	These applications are just the beginning, however. “The roles for MS are rapidly expanding to more hybrid qualitative/quantitative applications and the examination of higher-order biotherapeutic structure,” notes Scott J. Berger, senior manager of biopharmaceuticals in the Waters Corporation’s pharmaceutical business group.

	Other newer applications include biosimilar development, where MS is an enabling technology to show that an innovator and biosimilar have identical sequence and comparable variant profiles. Similarly, Berger says, the rise of antibody-drug conjugates (ADCs) has required more advanced liquid chromatography (LC)-MS laboratory workflows for challenging separations, mass detection, and data processing to determine the average number of drug conjugates (DAR) on a molecule, their distribution across the many possible sites of reaction, and individual occupancy levels for each of these sites on these hyper-complex molecules.

	Mass spectrometry is a preferred analytical technique in many of these applications because it is a more targeted method that provides detailed information about protein structure/conformation, whether for the desired product or impurities like HCPs that are present at low concentrations.

	The sensitivity and specificity, high mass accuracy at low part per million levels, and ability to return precise chemical information on the molecule of interest are main drivers for using mass spectrometry, according to Kilby. Additional attractive characteristics include compatibility with most chromatographic methodologies, the ability to gain both qualitative and quantitative [relative and absolute] information, and the lack of any theoretical limitations on the size of proteins that can be analyzed.

	“MS is an ideal tool for supporting process development and a quality-by-design approach. For process impurity identification, it is much more specific than ELISA (enzyme-linked immunosorbent assay) testing, which is the conventional method (and is still used for rapid product release),” Kochling says.

	Mass spectrometry is also preferred in these applications because it offers superior sensitivity and specificity without the need for a large volume of samples, according to Ding. “Furthermore,” she adds, “there is a plethora of knowledge and well-established procedures for LC-MS/MS analysis of biologic drugs, from sample preparations to software-assisted data interpretation.”

	Berger also notes that MS provides more confident qualitative mass analyses and makes it possible to identify a single peptide or modified peptide in the presence of the many other peptides that are generated in the digest of a biotherapeutic protein, even if that peptide is fully or partially co-eluting with other components. “Monitoring of multiple mass channels simultaneously allows for monitoring of several components at the same time, and in many cases without the need for optimizing the separation conditions to get valuable quantitative information,” he says.

	In addition, the ability to detect components at levels lower than optical detection-based assays expands the dynamic range of detection of an assay for the peptide mappeak of a peptide or its variant, according to Berger.

	Overall, observes Manolis, MS is an indispensable tool for peptide and protein analysis due to its speed, sensitivity, and versatility. “MS is particularly useful for gaining knowledge about the location of disulfide bonds and amino acid sequences,” asserts Kochling. Adds Mario DiPaola, senior scientific director for Charles River Laboratories: “No other analytical technique can provide the extent of information obtained by mass spectrometry, nor the selectivity or sensitivity. Previously it would have taken months to determine or confirm the entire sequence of a protein by first collecting enzymatic digests and then performing Edman degradation, but now the same analysis can be performed in a matter of days with mass spectrometry while using micrograms of product rather than milligrams.”

	Several advances in technology have been enabling the wider use of MS. Examples include developments in high-resolution mass spectrometry, such as quadrupole time-of-flight (QTOF) and orbitrap technology, and workflow-driven software development, according to Ding. Ion mobility methods have made MS useful for HOS analysis, cysteine variant determination, and N-Glycan profiling, while top- and middle-down analyses, which have been made possible through the introduction of electron transfer dissociation (ETD) fragmentation, are useful for determination of CQAs and identification of product variants, says Schuessler.

	While not very recent, DiPaola points to the introduction of tandem mass spectrometry and the electrospray ionization (ESI) interface as key advancements in the field of mass spectrometry. ESI has allowed for easy coupling of a high-performance LC (HPLC) system to a mass spectrometer, as a key advancement because it permits separation of species followed by direct in-line mass analysis. Hybrid mass spectrometry has made it possible to obtain very detailed information on PTMs and protein primary structures.

	In addition to ETD, the introduction of a variety of other alternate methods to collisional induced dissociation (CID), including electron capture dissociation (ECD), higher-energy collisional dissociation (HCD), electron transfer in the higher-energy collisional dissociation (EThcD), ultraviolet photodissociation (UVPD), and surface induced dissociation (SID), among others, have led to significant improvements in fragmentation technology, according to DiPaola. Some of these fragmentation methods can be used independently or in-series to garner as much information about protein analyte as possible.

	For Huang, one of the most powerful developments in the evolution of MS technology is the commercialization of hybrid instruments. “Hybrid MS instruments are made by combining two different types of mass analyzers together in tandem; one can choose almost any combination of quadrupole, time-of-flight, or ion-trap hybrids. These hybrid instruments promise the ability of combining the best features from the different components and allow tandem mass spectrometry experiments and unique scanning modes that are not possible on a single instrument,” he explains.

	Huang points to Thermo Scientific’s Orbitrap Fusion mass spectrometer as one example. It is designed to include quantification using isobaric tags, low-level PTM analysis, and data-independent acquisition (DIA). The instrument, according to Huang, features enhanced sensitivity resulting in improved analyte detection, characterization, and quantitation in advanced biopharma, proteomics, and metabolomics applications.

	Berger adds that the increasing focus on hybrid quantitative/qualitative workflows favors TOF-based platforms that do not suffer the lower-end dynamic-range limitations of automatic gain control (AGC)/orbitrap-type instruments. “Time-of-flight and quadrupole time-of-flight MS technology has been the primary high-resolution MS tool used for biopharmaceutical characterization and monitoring due to its ability to maintain high resolution and sensitivity independent of the mass of a species, and the ability to do so with increasingly rapid LC and CE [capillary electrophoresis] separations on the front end of these analyses,” he explains.

	Various bioinformatics tools associated with MS analyses, such as pathway mapping/analysis, network association, and ADC calculators, have had an impact on the use of MS as well, notes Kilby. “Each major instrument vendor has software that works specifically with its instrument and uses complex algorithms to process its proprietary data files. Vendors constantly seek feedback from users to improve software features, such as chemical intelligence, batch data process, automation, report generation, Title 21 

 Part 11 compliance, and so on,” agrees Ding.

	More specifically, Manolis notes that dedicated data analysis software has been developed for biopharma applications, and the workflow-specific design this software has streamlined the process. “In addition,” she says, “walkup software has been developed to enable MS novice users, such as biologists, to have access to high-end MS instruments. Special consideration has also been given to providing total workflow solutions to address sample preparation all the way to reporting.”

	Overall, Berger believes that the expanded use of MS in more targeted biotherapeutic CQA monitoring experiments, even in later (regulated) development and quality control  (QC) environments, is related to the increased robustness of the instrumentation, growing usability of these systems, and deployment on informatics platforms designed for regulated environments. “The ability to follow specific product variants in a peptide map, intact mass profile, or released glycan profile enables targeted quantification of the amount of that modification as an organization develops and matures its manufacturing processes or sets specific limits of a variant in a QC release test,” he explains.

	The other area of great expansion has been in HOS analysis, according to Berger. The development of ion mobility MS has introduced the ability to measure collisional cross-section (CCS) data for molecules, bringing an added level of separation to MS analysis and generating data based upon gas-phase cross-sectional area and shape, in addition to the traditional mass and charge characteristics measured by a mass spectrometer. Biologic folding interactions and stability can be screened and assayed using this type of ion mobility information. In addition, Berger notes that more resolving HOS information can now routinely be provided by hydrogen deuterium exchange MS (HDX-MS), which measures the accessibility of backbone amide hydrogens to exchange with deuterated water in solution. HDX-MS is being used  to define the interaction of a mAb and its target (epitope mapping) for intellectual property (IP) filings and to demonstrate structural comparability between innovator and biosimilar biotherapeutics.

	With current MS technology, there is a disconnect between the practical and actual time it takes to complete complex analyses, according to Kochling. “The analysis of proteins is very complicated, and even with current mass spec instrumentation and software, a significant amount of manual labor is required, and in some cases it can take up to one month to complete an analysis, which is not practical in an industrial setting,” she states. “Although mass spec technology has been ever improved in sensitivity and dynamic range, the hardware capability is ultimately limited by the complexity of samples. Sample preparation technology and procedure improvement can compensate for the instrument capability in sensitivity and dynamic range,” she continues.

	Current mass spectrometers were designed and optimized for the analysis of smaller molecules, and as a result large protein molecules often suffer from poor data quality resulting from limited resolution, sensitivity, and mass range, adds Manolis. She notes that additional alternative and optimized fragmentation methods for large molecules for top-down and middle-down analysis are desired. On the other hand, Berger believes that MS systems are designed to be general-purpose platforms for both large- and small-molecule studies, with compromises on some specific performance attributes for large molecules to accommodate a wider range of applications in the lab.

	At GSK R&D Platform Technology & Science, poor parallelization is an issue; currently it is not possible to highly multiplex MS analyses without buying extra mass spectrometers (compared with genomic and microarray technology, for example). In addition, as resolution and scan speed increase, files sizes are getting very large such that current informatics suites struggle with data analysis, especially for large experiments, according to Schuessler. “With the enormous volume of data being generated, data processing and analysis become increasingly important and remain a bottleneck,” agrees Huang.

	Furthermore, according to DiPaola, the analysis of these files requires sophisticated and expensive software, as well as highly skilled and knowledgeable users. “Both of these scenarios present recruiting and financial challenges for laboratories and companies,” he asserts.

	Kilby also points to the incompatibility of MS with commonly used biologic matrices/buffers/detergents, issues with samples that have extended dynamic ranges (HCPs, serum proteomics, etc.), integration in process analytical technology and continuous flow manufacturing, and the fact that response factors are not universal (unlike for UV, charged-aerosol, and evaporative-light-scattering detectors).

	The need to label or spike analytes with an appropriate isotopically labeled species to obtain highly quantitative results due to the ionization variability of species is one issue for DiPaola. Another is the difficulty of detecting low-level impurities (<1-2% in abundance) in biological samples without some prior knowledge of their type. A third issue for DiPaola is the need to confirm isobaric amino acids when conducting sequencing by MS/MS using Edman degradation, which becomes a bottleneck because the peptides containing such isobaric amino acids must be collected and individually sequenced.

	Finally, DiPaola notes that the high cost of mass spectrometers presents another obstacle, especially when dealing with high-resolution, tandem systems. “Affordability is a sizable issue for small laboratories and companies with limited financial capacity. Additionally, these systems are rather complex and delicate, so that any repairs can only be performed by skilled engineers, adding additional costs,” he explains.

	Berger asserts that many of these limitations with mass spectrometry today do not involve the instrument itself, but other elements of the analytical workflow that interact with the system. “Faster and more robust sample preparation is needed to match the improvement in back-end data processing throughput for many applications of MS in the biopharmaceutical industry,” he notes.

	For instance, Berger points to clone screening and other early development activities, where reproducible microscale and nanoscale separations remain a challenge to many MS users. “Waters has developed a chip-based microfluidic platform, the ionKey/MS System, that simplifies the process of microscale LC-MS of proteins and peptides, but the industry is looking for further innovations that raise usability of systems at the microscale to that of analytical scale LC-MS,” he observes.

	Berger also notes that innovative software products such as those from Waters for automating biotherapeutic peptide mapping and intact mass analysis, intact mass, peptide mapping, and released glycan analysis characterization and monitoring have been key to advancing the productivity of labs using LC–MS instrumentation. He agrees, though, that “analysts are looking for smarter software, automated processing, custom calculations for critical results, and efficient reporting tools that simplify communication of the results, and they now want to be able to use this software in regulated environments.”

	To address throughput issues, there is, according to DiPaola, an effort to transition from serial data acquisition to parallel acquisition. “In combination with novel data interpretive software tools, this move should make possible the multiplexing of data acquisition and analysis for peptides, intact proteins, and whole protein complexes,” he says. He also expects new data formats, compression methods, and storage architectures to be introduced to address file size, storage space, retrieval, and speed of data analysis issues.

	Researchers at GSK R&D Platform Technology & Science are excited about a number of new developments in MS technology, including platform-neutral, multi-vendor solutions such as the software suite for biopharma applications from Protein Metrics; integrated analyzers for process monitoring, such as the Waters ACQUITY QDa Mass Detector; commercial solutions for ion mobility, HDX, and ETD fragmentation; and capillary electrophoresis devices that can be attached directly to the MS source such as 908 Devices’ ZipChip, which are fast and sensitive and offer the potential for rapid CE-MS analysis of peptide maps, intact proteins, charge variants, and metabolites, according Kilby.

	DiPaola also expects high-resolution, top-down or bottom-up systems combined with novel fragmentation techniques and improved analysis software to be available in a year or two that will overcome the isobaric amino acid issue for de-novo protein sequencing by mass spectrometry. He also notes that new dissociation methods such as CID, HCD, ETD, and infrared multi-photon dissociation (IRMPD) are generating new acquisition paradigms that require more systematic data collection. “New software tools and algorithms with compliance to 21 

 Part 11 requirements are in development to address these new data acquisition paradigms,” he asserts.

	In essence, major developments efforts can be divided into two disciplines, according to Manolis. “From a physics perspective, significant advances in instrument design are very close to release that will greatly improve not just the capability to analyze large biomolecules, but also the reproducibility of such analyses. On the biology front, advancements in data analysis software that can make clear biological sense of the rich data collected by mass spectrometry is critical for multiple applications. Combining these efforts with the latest capabilities in automated sample preparation and separation techniques allows for a big step forward in the total confidence in using mass spectrometry for biopharmaceuticals,” she states.

	Despite the advances in MS technology achieved to date, those in the industry expect further notable improvements in the long term. “Research in electron transfer dissociation (ETD), top-down and bottom-up proteomics, high-resolution mass spectrometry (HRMS), and software-assisted structural elucidation will have lasting impact on biologics analysis,” Ding observes.

	Huang also believes that multi-attribute methods that utilize LC/MS for separation, identification, and quantification of post-translational modifications will have potential to replace multiple purity methods currently used for protein product release testing. “With this approach, there will be fewer methods for product release testing but richer product critical quality attribute information obtained,” he comments.

	While current MS technology has been primarily limited to the analysis of in-vitro proteins, proteoforms, and multi-protein complexes, the future drive in mass spectrometry is to study the same entities in vivo to determine native biological structures and their dynamics within biological processes, according to DiPaola. “New MALDI [matrix-assisted laser desorption ionization] imaging spectrometers are available and more advanced models will be soon introduced for spatial mapping of entities of interest, including proteins, signaling molecules, etc., in tissues and eventually in single cells,” he says. Data files for imaging experiments are starting to approach “Big data” definitions, though, and thus challenge most existing software tools, according to Kilby.

	In a similar vein, Kilby notes that the application of MS to quantitative whole body imaging, which is currently very expensive and requires the use of radiolabeled compounds, would allow analysis of all compounds present in a given location, not just the radiolabeled ones, without the need for radiation. “There is a long way to go, however, to extend dynamic ranges and improve compatibility with salts and other materials present in untreated biologic samples, and it is not yet known whether it will be possible to return quantitative results,” he says.

	One of the biggest challenges Waters is taking on, according to Berger, is to enable more people in biopharmaceutical organizations to leverage the resolution, sensitivity, and dynamic range offered by mass spectrometry. “We also want to raise the dependability of higher-performance instruments to match that of optical detectors that are routinely used in biopharmaceutical labs today. Photodiode array UV/Vis detectors were once viewed as too complicated for regulated environments, but today we rarely give them a second thought about their applicability for the analysis of pharmaceuticals. My hope is that high-performance MS analysis will soon be seen the same way,” he says.

	The goal on the horizon, according to Manolis, is to extract the most information for the least amount of effort. “Can we get to a point where we can directly analyze a biopharmaceutical sample without any time-consuming sample preparation and get all the information about the sample we need?” she asks. “To do so will require improved fragmentation techniques, greater dynamic range, and software tools that can pull more information out from a single sample, as well as aggregate data across the development cycle,” Manolis asserts.

	Kochling certainly expects greater applicability for mass spectrometry in the biopharmaceutical industry. “MS is already providing great benefits to biologics development and manufacturing. Although improvements in data processing software for throughout enhancement are still needed, mass spec technology has advanced dramatically in the past 25 years. It is exciting to think about how its capabilities will be expanded over the next 25.”

	1. S. Rogstad, A. Faustino, A. Ruth, and J. Park, 

	When referring to this article, please cite it as C. Challener, "Mass Spec Sheds New Light on Biologics Drug Development," 

Articles

Although limitations must be overcome, mass spectrometry is having a great impact on biologic development and manufacturing.

Mass Spec Sheds New Light on Biologics Drug Development

Following two years of near-record numbers of new drug approvals by FDA, with 41 in 2014 (1) and 45 in 2015 (2), 2016 appears to be seriously bucking the trend, with only 20 approvals issued as of Dec. 14, 2016 (3). The sharp decline was not expected. What might be the reasons? John Jenkins, director of FDA’s Office of New Drugs, attributed the difference to a decline in the number of submitted applications, more complete response letters in 2016, and the fact that five approvals originally scheduled for 2016 were finalized earlier in 2015 (4). On a positive note, Jenkins reported that the number of applications received by FDA through Dec. 9, 2016 was 36, surpassing the average number (35) of new molecular entity filings for the past decade.

	Notably, the decline in approvals does not seem to be having an impact on growth expectations for the global API market. Mordor Intelligence projects the global API market to expand at a compound annual growth rate of 6.5% from $154 billion in 2015 to $225 billion in 2021 (5).

	While chemical compounds continue to account for the greatest percentage of APIs, biologic drug substances are growing at the fastest rate. APIs were intended slightly more often for branded drugs compared to generic drugs (43.5%) in 2015, and APIs for cancer treatments are experiencing the fastest growth vs. APIs for other therapies. North America remains the largest market for APIs, but demand is growing most rapidly in Asia-Pacific (5).

	The 20 approvals in 2016 included treatments for asthma, type II diabetes, different cancers, hepatitis C, plaque psoriasis, multiple sclerosis, muscular dystrophy, and several rare diseases. Eight of the approvals were for biologics, including seven monoclonal antibodies (mAbs) and one hormone. Two were radioactive diagnostics agents and two were for the treatment of hepatitis C. Merck, Eli Lilly, and Sanofi each received two approvals. Half of the new drugs approved by FDA in 2016 received one or more special designations. Three are considered new classes of drugs, and three are the first-ever treatments for the diseases they target.

	With passage of the FDA Safety and Innovation Act (FDASIA) in 2012, FDA was granted authority to implement expedited review and approval programs to accelerate the introduction of novel medications to the market. Four programs are currently in use. Manufacturers can qualify for fast track and breakthrough therapy designations, and accelerated approval and priority review processes depending on the characteristics of their drug candidates. Breakthrough therapies provide substantial improvement over currently available treatments. Fast Track drugs meet unmet medical needs and treat serious conditions. Accelerated approval is available for similar reasons. Priority review is granted to drugs that have improved safety and effectiveness compared to current medicines.

	Drug companies are utilizing these pathways. In 2014 and 2015, respectively, 66% and 60% of the 41 and 45 new drugs approved by FDA received one or more special designation or was granted an expedited review of some kind (6,7).

	In 2016, five of the newly approved drugs were awarded the breakthrough therapy designation, three received fast track status, five received accelerated approval, and eight underwent priority review. In addition, six of the newly approved treatments were classified as orphan drugs. One product-Exondys 51 (eteplirsen, Sarepta Therapeutics) for the treatment of patients with Duchenne muscular dystrophy (DMD)-received priority review for a rare pediatric disease. Notably, two of the approved drugs in 2016 had a total of five special designations (8).

	Several of the newly approved drugs are the first new therapies approved by FDA for the treatment of different diseases. Exondys 51 is the first drug approved to treat patients with DMD that have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13% of the population with DMD (8).

	In addition to receiving the seventh rare pediatric disease priority review voucher issued by FDA since the program began, Exondys 51 was granted fast track and orphan drug designations and was approved under the accelerated approval pathway based on the surrogate endpoint of dystrophin increase in skeletal muscle observed in some Exondys 51-treated patients. FDA is requiring Sarepta Therapeutics to conduct a clinical trial to confirm the drug’s clinical benefit.

	Nuplazid (pimavanserin) from Acadia Pharmaceuticals is the first drug approved to treat hallucinations and delusions associated with psychosis experienced by as many as 50% of people with Parkinson’s disease. An estimated 50,000 Americans are diagnosed with Parkinson’s disease each year, according to the National Institutes of Health, and about 1 million Americans have the condition. Nuplazid received priorty review and was designated a breakthrough therapy (9).

	Defitelio (defibrotide sodium) marketed by Jazz Pharmaceuticals is the first FDA-approved therapy for the treatment of severe hepatic veno-occlusive disease (VOD), a rare and life-threatening liver condition that can occur in adults and children that receive a stem cell transplant from blood or bone marrow (hematopoietic stem cell transplantation or HSCT). Fewer than 2% of patients develop severe hepatic VOD after HSCT, but as many as 80% of patients who develop severe hepatic VOD do not survive. Defitelio was granted priority review and designated an orphan drug (10).

	Epclusa (fixed dose combination of sofosbuvir and velpatasvir) from Gilead Sciences is the first regimen to treat all six major hepatitis C virus (HCV) genotypes and was approved for adult patients with chronic HCV. Velpatasvir is the new drug; sofosbuvir was approved in 2013. Epclusa was granted priority review (11).

	Lartruvo from Eli Lilly is the first new therapy approved by the agency for the initial treatment of soft-tissue sarcomas (STS), cancers that develop in muscles, fat, tendons or other soft tissues and cannot be cured with radiation or surgery, since doxorubicin’s approval more than 40 years ago. This platelet-derived growth factor (PDGF) receptor-alpha blocking antibody blocks PDGF receptors that cause tumor growth. The National Cancer Institute estimates that 12,310 new cases of STS and nearly 5000 deaths are likely to occur from the disease in 2016. Lartruvo (olaratumab) with chemotherapy drug doxorubicin was granted accelerated approval and priority review, as well as fast track, breakthrough therapy, and orphan designations (12).

	Other drugs approved in 2016 fall into new classes of compounds that act by new pathways. Genentech’s Tecentriq (atezolizumab) is the first FDA-approved PD-L1 inhibitor and the latest in the broader class of PD-1/PD-L1 targeted biologics approved by FDA in the past two years. This mAb was approved for the treatment of patients with locally advanced or metastatic urothelial carcinoma, the most common type of bladder cancer.  It blocks PD-1/PD-L1 interactions between the body’s immune cells and some cancer cells, potentially helping the body’s immune system to fight cancer cells. Tecentriq received priority review and accelerated approval and was designated a breakthrough therapy (13).

	Venclexta (venetoclax) from AbbVie is the first FDA-approved treatment that targets the B-cell lymphoma 2 (BCL-2) protein, which supports cancer cell growth. The drug is for the treatment of patients with chronic lymphocytic leukemia (CLL) who have a chromosomal abnormality called 17p deletion and who have been treated with at least one prior therapy. According to the National Cancer Institute, CLL is one of the most common types of leukemia in adults, with approximately 15,000 new cases diagnosed each year. The relevant chromosomal abnormality occurs in approximately 10% of patients with untreated CLL and in approximately 20% of patients with relapsed CLL. Venclexta was granted priority review and accelerated approval and designated a breakthrough therapy and orphan drug (14).

	Netspot from Advanced Accelerator Applications USA is the first kit for the preparation of gallium Ga 68 dotatate injection, a radioactive diagnostic agent for positron emission tomography imaging. The radioactive probe helps locate tumors in adult and pediatric patients with the rare condition somatostatin receptor positive neuroendocrine tumors by binding to receptors on the tumors, which develop in the hormone-producing cells of the body’s neuroendocrine system. Netspot received priority review and was designated an orphan drug (15).

	Several of the approved drugs in 2016 are intended for the treatment of diseases with existing therapies, but in some cases the new drug is of particular interest. For instance, Ocaliva (obeticholic acid) from Intercept Pharmaceuticals is only the second drug approved to treat patients with primary biliary cholangitis. Ursodeoxycholic acid, the only other approved drug, is effective in just over 50% of patients on its own and is not tolerated by many. An oral drug, Ocaliva binds to the farnesoid X receptor, a receptor found in the nucleus of cells in the liver and intestine, reducing bile flow from the liver and suppressing bile acid production in the liver. It received accelerated approval and fast track and orphan drug designations (16).

	Merck’s ZINPLAVA (bezlotoxumab) is approved for the reduction of the recurrence of Clos

 infection (CDI) in patients 18 years of age or older who are receiving antibacterial drug treatment for CDI and are at high risk for CDI recurrence. CDI is caused by bacteria that produce toxins, including toxin B. Developed by researchers at the University of Massachusetts Medical School’s MassBiologics Laboratory in conjunction with Medarex (now part of Bristol-Myers Squibb) and licensed to Merck in 2009, Zinplava, a human monoclonal antibody, binds to 

 toxin B and neutralizes its effects (17).

	Developed by Elusys Therapeutics in cooperation with the US Dept. of Health and Human Services’ Biomedical Advanced Research and Development Authority, Anthim (obiltoxaximab) is a mAb that neutralizes toxins produced by 

 and was approved for the treatment of inhalational anthrax in combination with appropriate antibacterial drugs. Inhalational anthrax is a rare disease that can occur after exposure to infected animals or contaminated animal products, or as a result of an intentional release of anthrax spores. Notably, the drug was approved under  FDA’s Animal Rule, which allows efficacy findings from adequate and well-controlled animal studies to support FDA approval when it is not feasible or ethical to conduct efficacy trials in humans (18).

	It is also worth noting that FDA’s Center for Drug Evaluation and Research (CDER) approved 72 “first generic drugs” in 2016 as of Nov. 16, 2016 (19). “First generics” are the first approval by FDA that permits a manufacturer to market a generic drug product in the United States and receive prioritized review.

New Molecular Entity and New Therapeutic Biological Product Approvals for 2014

New Molecular Entity and New Therapeutic Biological Product Approvals for 2015

New Molecular Entity and New Therapeutic Biological Product Approvals for 2016

,  accessed Dec. 14, 2016.
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,” presentation at FDA/CMS Summit, Dec. 14, 2016, accessed Dec. 14, 2016.
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Global Active Pharmaceutical Ingredients Market-Growth, Trends & Forecasts (2016-2021),

” September 2016, accessed Dec. 14, 2016.
	6. FDA,  

. January 2015, accessed Dec. 14, 2016.
	7. FDA, 

. January 2016, accessed Dec. 14, 2016.
	8. FDA, “FDA Grants Accelerated Approval to First Drug for Duchenne Muscular Dystrophy,” Press Release (Silver Spring, MD, Sept. 19, 2016).
	9. FDA, “FDA Approves First Drug to Treat Hallucinations and Delusions Associated with Parkinson’s disease,” Press Release (Silver Spring, MD, April 29, 2016).
	10. FDA, “FDA Approves First Treatment for Rare Disease in Patients Who Receive Stem Cell Transplant from Blood or Bone Marrow,” Press Release (Silver Spring, MD, March 30, 2016).
	11. FDA, “FDA Approves Epclusa for Treatment of Chronic Hepatitis C Virus Infection,” Press Release (Silver Spring, MD, June 28, 2016).
	12. FDA, “FDA Grants Accelerated Approval to New Treatment for Advanced Soft Tissue Sarcoma,” Press Release (Silver Spring, MD, Oct. 19, 2016).
	13. FDA, “FDA Approves New, Targeted Treatment for Bladder Cancer,” Press Release (Silver Spring, MD, May 18, 2016).
	14. FDA, “FDA Approves New Drug for Chronic Lymphocytic Leukemia in Patients with a Specific Chromosomal Abnormality,” Press Release, April 11, 2016.
	15. FDA, “FDA Approves New Diagnostic Imaging Agent to Detect Rare Neuroendocrine Tumors,” Press Release, (Silver Spring, MD, June 1, 2016).
	16. FDA, “FDA Approves Ocaliva for Rare, Chronic Liver Disease,” Press Release (Silver Spring, MD, May 31, 2016).
	17. Merck & Co., “FDA Approves Merck’s ZINPLAVA (bezlotoxumab) to Reduce Recurrence of Clost

Infection (CDI) in Adult Patients Receiving Antibacterial Drug Treatment for CDI Who Are at High Risk of CDI Recurrence,” Press Release (Silver Spring, MD, Oct. 21, 2016).
	18. FDA, “FDA Approves New Treatment for Inhalation Anthrax,” Press Release (Silver Spring, MD, March 21, 2016).
	19. FDA, 

FDA issued approvals for less than half the number of new drugs in 2016 compared with 2015.

New Drug Approvals Slump in 2016

Cross-coupling reactions enable the synthesis of complex building blocks and intermediates with structural motifs common in many classes of APIs. Numerous cross-coupling methodologies that allow the reaction of unsaturated substrates (acetylenes, alkenes, aryls) with a wide range of functionalized organohalides have thus been employed in the commercial manufacture of APIs. These reactions, however, are typically mediated by expensive and often toxic palladium catalysts with specially designed ligands. Consequently, there is significant interest in developing alternative catalyst systems that avoid these issues.

	Advances in cross-coupling chemistry have had a significant impact on API synthesis at commercial scale, according to Chris Senanayake, vice-president of chemical development in the United States at Boehringer Ingelheim Pharmaceuticals. “The expanded substrate scope, the availability of a variety of novel ligands, and the in-depth mechanistic understanding of cross-coupling reactions have led to shorter development times and enhanced process robustness,” he notes.

	The most commonly used cross-coupling reactions, most of which are catalyzed by palladium-based, transition-metal complexes, include Suzuki and Sonogashira coupling, according to Jeff Song, director of development, also with Boehringer Ingelheim Pharmaceuticals.

	When first developed, the Suzuki reaction involved the cross coupling of aryl organoboronic acids with halides. Today, the substrate scope is much broader and includes alkyl, alkenyl, and alkynyl compounds. In addition, potassium trifluoroborates and organoboranes/boronate esters can be used instead of boronic acids, while in some cases triflates or other pseudohalides can be used instead of actual organohalides.

	The Sonogashira reaction involves the coupling of terminal alkynes with aryl or vinyl halides. In addition to the palladium catalyst, a copper(I) cocatalyst and an amine base are required. When first introduced, maintaining anhydrous and oxygen-free conditions was essential for this coupling method, but newer versions do not require such strict control of the reaction environment.

	Suzuki reactions at Boehringer Ingelheim
	In the past few years, scientists at Boehringer Ingelheim have employed Suzuki coupling chemistry for the preparation of FLAP (1), HCV Polymerase (2), and 11-β-HSD 1 inhibitors (3). In the first case, a Suzuki reaction was used to form the key biaryl bond between an aryl bromide and aminopyrimidine boronic acid. In the second example, a one-pot sequential borylation−Suzuki coupling process was devised to efficiently couple 2-iodo-5-bromopyrimidine and a 2-bromoindole substrate. The third reaction involved a Suzuki coupling using a pyridone boronic acid on scale.

	“Non-precious-metal catalysis is a new horizon for the field of cross coupling,” observes Senanayake. “Iron, copper, cobalt, and nickel catalysts for cross-coupling reactions have begun to be applied on large scales,” he adds.

	In fact, Boehringer Ingelheim, Pfizer, and AbbVie have formed a cross-company consortium with the aim of jointly driving research in this area. The goal of the Non-Precious-Metal Catalysis Alliance is to advance the development of abundant-metal catalysts and replace precious-metal catalysts in the synthesis of APIs.

	“The use of non-precious-metal catalysis on large scales will greatly improve the economics and sustainability of chemical processes,” asserts Song. He points to a recent example from Boehringer Ingelheim that involves an iron-catalyzed Kumada coupling reaction under flow conditions (4). In the reaction, 2-chloropyrazine can be coupled with various aryl Grignard reagents with catalyst loadings as low as 0.5 mole percent. Switching from batch to continuous processing avoided large exotherms, providing a longer catalyst lifetime and higher yields. The scale-dependent issues often observed with Kumada coupling reactions were also eliminated, making scale up easier.

	A key area for additional development in coupling chemistry, according to Song, is their use in the synthesis of chiral compounds. “In the next few years, we expect to see further advancement in methodology development for asymmetric cross couplings, including for biaryl and sp

 coupling reactions. In fact, significant progress has recently been made, as exemplified by the asymmetric Suzuki reactions using ligands developed at Boehringer Ingelheim,” he notes.

	For instance, novel monophosphorous ligands that are chiral at the phosphorus atom were employed for the palladium-catalyzed asymmetric Suzuki-Miyaura coupling of aryl boronic acids with aryl halides in the presence of tripotassium phosphate to afford functionalized chiral biaryls compounds under mild conditions and up to 96% enantiomeric excess (5). These reactions were applied to the asymmetric synthesis of an atropisomeric HIV integrase inhibitor on large scales (6). Similar asymmetric coupling reactions were used to achieve the first total syntheses of biaryl natural products korupensamine A and B (7).

	Silicon-based cross-coupling reactions have recently been shown to have potential advantages over existing cross-coupling chemistry, particularly with respect to alkyl-aryl and alkyl-alkenyl (sp

) cross-couplings, according to Gerald L. Larson, a senior research fellow at Gelest.

	Because the energy barrier for these couplings is high, reactions with transition-metal catalysts must be run under extreme conditions or using highly reactive substrates, which prevents the incorporation of many functional groups.

	The newer silicon-based reactions involve both photoredox and cross-coupling catalysts to facilitate cross-coupling reactions of a variety of substrates, such as trifluoroborates and carboxylic acids. Unlike traditional cross-coupling reactions mediated by palladium, these dual-catalyst reactions proceed via radical mechanisms. As a result, they are not plagued by the high-energy barriers that exist for conventional cross-coupling reactions.

	Gary A. Molander and his research group at the University of Pennsylvania have been very active in this area (8). Organosilicates such as alkylbis(catecholato)silicates serve as excellent radical precursors for nickel/photoredox dual catalysis. The organosilicates undergo single-electron oxidative fragmentation upon exposure to visible light in the presence of a ruthenium catalyst. The generated radicals then participate in single-electron transmetallation mediated by more environmentally friendly and less expensive nickel complexes.

	One benefit of this approach, according to Larson, is the greater stability of the nucleophilic partners used in the cross-coupling reactions. In addition, the organosilicates are easily prepared from readily available precursors. “The ability to cross couple alkyl and, particularly primary alkyl groups in sp

 cross-coupling protocols, as well as the ability to cross couple nucleophilic substrates containing unprotected amino groups are key advantages,” he adds. The use of nickel rather than palladium also reduces the overall cost and eliminates the need to remove the metal from the final reaction mixture, according to Larson. “As importantly,” he observes, “there is no need to synthesize boronic acids, silanols, trifluoroborates, or zinc reagents, and both preparation of the silicate reagents and the cross-coupling reactions are fully scalable.”

. 80 (3) 1651-1660 (2015).
	2. Y. Zhang et al. 

. 16 (17) 4558-4561 (2014).
	3. Y. Zhang et al. 

. 81 (6) 2665-2669 (2016).
	4. F. G. Buono et al., 

. 2016 (15) 2599-2602 (2016).
	5. W. Tang et al., 

. 14 (9) 2258 (2010).
	6. K. R. Fandrick et al., 

. 54 (24) 7144 (2015).
	7. G. Xu et al., 

. 136 (2) 570-573 (2014).
	8. J.C. Tellis et al., 

Alternatives to expensive palladium catalysts are gaining acceptance for commercial API synthesis.

New Catalysts Enable Cross-Coupling Reactions

	When referring to this article, please cite it as C. Challener, “FDA New Drug Approvals Down Significantly in 2016," 

As of mid-December, less than half the number of new drug approvals were issued by FDA in 2016 compared with 2015.

FDA New Drug Approvals Down Significantly in 2016

Although pharmaceutical manufacturers prefer to formulate APIs in solid-dosage form, many drug candidates do not readily form stable crystalline solids. In addition, a growing percentage of APIs in development suffer from poor bioavailability and/or other undesirable properties. There are many approaches for addressing these issues, such as the formation of amorphous dispersions, salts, and cocrystals.

	Cocrystals are attractive because, like solvates and hydrates, they allow the modification of an API’s properties without altering its chemical structure. They also provide an option for APIs that lack ionic functionality and thus cannot form salts. The synthesis of cocrystals may also be advantageous from a cost standpoint (1).

	There has been, however, significant hesitation in the pharmaceutical industry regarding the use of cocrystals since the issuance of FDA’s initial guidance on cocrystals in April 2013 (2).

	In that guidance, FDA defined cocrystals as “drug product intermediates (DPIs)” rather than APIs. This decision raised questions about expiration dates, intellectual property protections, and the consistency of the US approach with those adopted by other regulatory bodies (1).

	A new draft guidance issued by FDA in August 2016 (3) redefines cocrystals as APIs. Classified as a special case of solvates and hydrates in which the coformer is nonvolatile, cocrystals are now to have a regulatory classification similar to that of polymorphs of APIs.

	Cocrystals have crystalline lattice structures that are different from the pure API and consequently often have different physical and chemical properties. The formation of cocrystals can reduce hygroscopicity and change the particle size and shape and melting point to help overcome compaction problems for improved manufacturability. The stability of an API can also be enhanced. As importantly, dissolution properties in the body can be significantly modified. In addition, because cocrystals are neutral combinations of APIs with conformers, they can be prepared for compounds without acid or base functionality, which is required for salt formation.

	The most common property targeted for improvement via cocrystal formation is an improved aqueous dissolution rate compared to an API free acid or base, according to G. Patrick Stahly, chief operating officer at Triclinic Labs. “Cocrystals are like amorphous materials in that they provide a supersaturated concentration of an API in the body for a time sufficient for absorption to occur (a supersaturating drug delivery system),” he explains. He adds that it needs to be realized, however, that a cocrystal typically does not affect the equilibrium solubility of an API; cocrystals usually dissociate in solution because they are held together by relatively weak chemical bonds.

	Cocrystals also may offer benefits from an intellectual property perspective, according to Stahly. “At the current level of the science, it is not possible to predict cocrystal properties based on the molecular structures of the components. Solid-form screening, including cocrystal screening, is an empirical process. Since the formation of cocrystals is currently unpredictable, cocrystals that are discovered and found to have advantageous properties can be patented,” he notes.

	It should be recognized, however, that cocrystals are still not regarded as new APIs. “Drug products that are designed to contain a new cocrystal are considered analogous to a new polymorph of the API,” according to the 2016 FDA draft guidance.

	The redefinition of cocrystals as APIs is important for several reasons, according to Stahly. First, it allows the use of existing FDA guidance to provide information necessary for development. Second, existing manufacturing facilities and supply chains that make and distribute APIs can be used, and there is no need to add cocrystal manufacturing capabilities to existing drug-product manufacturing facilities. Third, API expiry dating begins at the time of cocrystal manufacture. Fourth, there is now consistency among FDA guidance, international guidance, and scientific reality.

	“After the first FDA guidance was issued, there was confusion within the pharmaceutical industry about the viability of cocrystals. My personal experiences led me to believe that scientists (myself included) were unhappy because they felt the guidance was wrong from a scientific standpoint. I believe the guidance presented unnecessary obstacles to development of cocrystals, which would discourage some companies from utilizing them. Those dealing with the facilities and regulatory issues seemed to be more pessimistic. There was talk that cocrystals were ‘dead’ in the pharmaceutical industry,” comments Stahly.

	Triclinic Labs did cocrystal screening and other development activities for clients in the period between issuance of the old and new guidance, indicating that not everyone felt that the original guidance eliminated the use of cocrystals.

	With the new guidance, however, cocrystals were, if ‘dead’ before, now ‘reborn’, according to Stahly. “The development pathway is clear and cocrystals should now be considered another tool in the box of tools used to improve solid-state properties,” he says.

	An issue addressed by the new FDA guidance is the nature of the interaction of components in a cocrystal where one component is acidic and the second component is basic. The drug manufacturer must demonstrate that there are no ionic interactions in such a cocrystal, which can be done either using the acid dissociation constants of the API and the coformer or via spectroscopic analysis.

	It is important, according to Stahly, to realize that there are various analytical techniques that can be used to probe the extent of proton transfer. “A common method is single-crystal x-ray structure determination, which is an excellent technique only if used properly. It is easy for a novice to believe protons have been actually located when often they are simply left to ride on the atom to which they are bonded; the protons are in assumed instead of refined positions,” he notes. A single-crystal study is not always necessary, however, and other techniques such as infrared (IR) spectroscopy can often be used.

	What drove the agency to make this significant change to its guidance on cocrystals? Stahly believes the strong response from industry was the major driving force. In 2012 at an Indo-US Bilateral Meeting held from Feb. 2-4, 2012, 46 attendees co-authored a publication presenting their consensus “… on the need to define cocrystals more broadly and to classify them like salts” and their recommendation “… that the classification of cocrystals in pharmaceutical science should be consistent with current scientific thought” (4).

	Also in 2012, a working group of the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ Consortium) submitted a comment paper to FDA recommending that “FDA considers classification of co-crystals within existing guidance[s], which provides discrete, industry understood points where quality attributes are measured and controlled to ensure product quality and patient safety (i.e., the drug substance and drug product)” (5).

	“The primary messages from those groups were that the original guidance was inconsistent with both current scientific understanding of the nature of cocrystals and existing FDA guidance relating to drug development. In addition, the guidance would lead to negative regulatory and manufacturing consequences and be inconsistent with the European Medicines Agency provisional guidance outlining submission requirements for cocrystals. It appears that FDA seriously considered industry’s comments and revised the guidance,” Stahly observes.

Guidance for Industry: Regulatory Classification of Pharmaceutical Co-crystals

 (Silver Spring, MD, April 2013).
	3. FDA, 

Draft Guidance for Industry: Regulatory Classification of Pharmaceutical Co-crystals

 (Silver Spring, MD, Aug. 2016).
	4. S. Aitipamula, et al., 

.12, 2147-2152 (2012).
	5. IQ Consortium, “IQ Comments on FDA Draft Guidance Regulatory Classification of Pharmaceutical Co-Crystals,” March 1, 2012.

Previous hesitation by pharma industry to use cocrystals may change with FDA’s new guidance that classifies cocrystals APIs.

New Opportunities for Cocrystals

	When referring to this article, please cite it as C. Challener, “New Horizons for Cross-Coupling Reactions," 

Non-precious-metal catalysts are increasingly employed for commercial API synthesis.