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

Cynthia A. Challener

Recently adopted regulations in the European Union, United States, and Japan require pharmaceutical manufacturers to ensure that the excipients they use are produced in compliance with current good manufacturing practice (cGMP) regulations. Doing so can be quite challenging; however, drug makers can use hundreds of excipients from as many different suppliers. In addition, the pharmaceutical market accounts for only a fraction of the sales for many excipient producers. Several different voluntary standards and guidance materials have been developed by industry to help both drug companies and excipient suppliers meet GMP requirements, and regulatory agencies encourage the use of third-party auditing of excipient suppliers against these standards to ensure that they are using excipients that meet cGMPs. Adding to this challenge is the continual development of excipient regulations in emerging markets, such as China and Brazil.

	Meeting these more stringent and evolving global requirements for excipients requires close collaboration between pharmaceutical companies and their excipient suppliers. Two-way exchange of information is necessary to ensure transparency and security of the excipient supply chain.

	There has been an overall increase in regulation of the pharmaceutical market, some of which impact excipient use, according to Christian Becker, pharmacist, regulatory and external affairs for the global segment management pharma solutions at BASF. “There are new regulations that directly apply to the manufacturers of pharmaceutical excipients (e.g., the bundling procedure in China) but more often, excipient manufacturers are indirectly affected by guidelines that apply to drug manufacturers. To comply with such guidelines, drug manufacturers depend on forming close collaborations with reliable excipient manufacturers,” he says.

	The US FDA Safety and Innovation Act (FDASIA) and the European Falsified Medicines Directive (FMD) are probably the most far-reaching regulations impacting pharmaceutical excipients in recent years, according to James Morris, director, pharma biotech with NSF International. “The need under FMD to complete formalized excipient risk assessments by March 2016 set an aggressive timeline for the pharmaceutical industry. And while risk assessment guidance is available, it is up to pharmaceutical excipient customers to ensure that excipients purchased meet the appropriate GMP requirements for the products they manufacture,” he observes.

	The International Council for Harmonization Q3D 

(ICH QD3) (1) has also presented issues for excipient suppliers and their customers. It applies a risk-based approach that creates challenges with respect to determining how to assess and quantify risks with non-API materials, including excipients, according to Patricia Rafidison, global regulatory compliance manager with Dow Corning.

	Excipient manufacturers are expected to provide a risk-management plan or analytical data on intentionally added elements to enable drug manufacturers to comply with the guideline; however, the declaration of metal catalysts may become an issue if the identity of the metal constitutes crucial production know-how, according to Becker. “Many discussions are ongoing to determine the best risk management/risk assessment approaches and the most effective strategies to be adopted by the pharmacopoeias around the world,” Rafidison says.

	Changes in excipient regulations are also occurring more rapidly than expected in developing regions, often with adoption of regulations modeled after those in the US and Europe, according to Rafidison. For instance, the new Chinese regulation came into force in August 2016 and replaces the existing Import Drug License (IDL) system for excipients imported into China. Going forward, excipients will be reviewed only in conjunction with a drug approval application (bundling), similar to the process in the US and Europe.

	In addition, Rafidison notes that pharmacopeias have now been established in many different countries, and despite some successful harmonization efforts most have differing specifications, which presents difficulties for companies looking to sell their products in multiple markets.

	Overall, there has been a greater focus on excipient quality and the security of the total excipient supply chain. “Drug manufacturers are actively working to comply with newer regulations, which has translated into increased requirements for excipient suppliers ranging from the completion of numerous questionnaires to greater numbers of customer audits,” states Rafidison. She adds that voluntary industry standards, such as 

NSF/IPEC/ANSI 363: Good Manufacturing Practices (GMPs) for Pharmaceutical Excipients 

(2) and the EXCiPACT GMP and GDP standards are helping companies overcome compliance challenges and providing opportunities for both drug manufacturers and excipient suppliers that proactively implement them.

	“It is important to keep in mind that new regulations generally are introduced to enhance patient safety or otherwise improve things for patients, and the entire supply chain is responsible for meeting the new requirements,” asserts Gary Lord, global strategic marketing director, medical solutions, with Dow Corning. He adds that the best relationships involve two-way dialogues, with excipient suppliers providing more than products at the lowest cost. “Everyone needs to be vested in helping develop the best medicines for treating unmet patient needs,” he says.

	Indeed, in the highly regulated pharmaceutical market, both parties depend on a close and forward-looking collaboration to be successful, according to Becker. “Reliability is key for long-term partnerships, which are beneficial in a business that involves protracted processes such as drug development and approval,” he notes.

	Another ongoing difficulty, according to Morris, is the fact that the pharmaceutical sector represents a small portion of the total excipient market, since excipients are also used in foods, cosmetics, and other products. “Even so, regulators expect pharmaceutical companies to justify their selection of excipients and ensure that excipients are manufactured in compliance with appropriate GMPs. In practice it is often not easy since the pharmaceutical client may represent a small fraction of the excipient manufacturer’s total sales,” he observes.

	To be able to justify excipient selection during development, therefore, pharmaceutical companies must form strong partnerships with their excipient manufacturers. “It is incumbent on both suppliers and their buyers to establish mutually beneficial supply agreements and leverage third-party audit and certification programs to meet regulatory expectations in a cost-effective manner,” Morris concludes.

	The regulatory requirement to perform supplier risk assessments, in fact, warrants the use of excipient GMP certification schemes to demonstrate ongoing assurance that the most current GMP requirements are being met by the excipient manufacturer, according to Morris. “GMP certification by an accredited certification body provides a level of quality assurance which will, in many cases, satisfy and preclude an on-site company audit,” he asserts.

	Leaders, including large and small companies committed to the pharmaceutical industry, actively seek open communication and transparency with their excipient suppliers. They also clearly define the intended applications for the excipients they purchase, according to Rafidison. “It is essential for the excipient supplier to understand the intended use so the appropriate grade/product can be recommended. There must be trust in the supplier-customer relationship; limited information sharing leads to limited understanding, which only creates further issues,” she notes.

	These companies leverage industry resources through IPEC, NSF International, and Rx-360 to understand and comply with the new regulations, according to Morris. Drug manufacturers that are proactively working to comply with the newer excipient regulations are also organized and typically have systems in place to conduct supplier risk assessments, either themselves or through third-party certification programs. “These companies are working closely with suppliers like Dow Corning to find mechanisms for minimizing the work load and optimizing the output,” asserts Rafidison.

	Lord would like to see harmonization/standardization of questionnaires and quality agreements, so that resources could be freed up for more important work. Becker, meanwhile, would like to see regulatory-affairs groups on both sides becoming involved at earlier stages of new development projects not only to help identify solutions for potential regulatory hurdles in advance, but also to allay ungrounded concerns.

	Selecting the right excipient supplier is crucial given the heightened emphasis on ensuring security of the entire excipient supply chain. Suppliers should be knowledgeable about the unique needs of the pharmaceutical industry and its complex regulatory landscape and share that knowledge through active participation in industry conferences, working groups that influence the development of new regulations, and trade associations that prepare guidance documents and tools, according to Rafidison. The best excipient suppliers also willingly provide information needed for customers to complete risk assessments and take the initiative to ensure compliance through the establishment of comprehensive quality programs. Morris adds that pharmaceutical manufacturers should look for excipient suppliers that have proactively evaluated their level of GMP compliance against one or more of the voluntary industry standards and ideally sought certification from an accredited certification program. “These excipient suppliers will ultimately reduce their audit burden, increase GMP awareness internally, and increase the value they offer,” states Morris.

	Many excipient suppliers are constantly working hard to improve the quality of their materials, according to Keith Horspool, vice-president of pharmaceutical development in the US for Boehringer Ingelheim Pharmaceuticals. “They are also making robust efforts in conjunction with FDA to improve the content of the Inactive Ingredient Database (IID),” he adds.

	Suppliers with long-term experience in the development and manufacture of excipients, a broad product portfolio that provides solutions for nearly any formulation problem, and a good reputation with regards to high quality standards and regulatory compliance will be the best choice for drug manufacturers to be successful in a highly regulated and competitive market, says Becker. “Instead of pure product supply, we support our customers with expertise in R&D, drug formulation, and regulatory affairs,” he adds. Asserts Rafidison: “Working with excipient suppliers who invest in innovation and provide targeted expertise can help pharmaceutical companies speed up new drug development and shorten time to market all around the world.”

	In fact, novel excipients are taking on increasing importance as enablers of product development, according to Horspool. “The need for such materials has never been greater based on the ability of new excipients to improve product profiles (more consistent performance), to facilitate new product processing concepts such as continuous manufacturing, and to enable new therapeutic entities to be formulated into efficacious products,” he states.

	To realize the potential and value of novel excipients, however, the regulatory process for their acceptance needs to be modernized. At present, novel excipients are approved as part of a new drug approval that creates a huge challenge and risk to excipient developers and pharma companies in adopting a new excipient in conjunction with an important new therapeutic. “This situation is becoming increasingly difficult and is leading innovators in new excipients to question whether investments in this area are commercially viable. Both pharma/biotech and excipient developers regard this as a critical topic to address and a joint IQ/IPEC-Americas Initiative is ongoing to solicit support from FDA to revise current regulatory processes that are an impediment to future development and adoption of important novel excipients,” observes Horspool. In addition, a request has been made for a Critical Path Initiative Meeting in 2017 to discuss a potential qualification process in early clinical development for novel excipients outside of the drug approval process.

, Step 4 version (Dec. 16, 2014).
	2. NSF International, 

NSF/IPEC/ANSI 363: Good Manufacturing Practices (GMPs) for Pharmaceutical Excipients

	When referring to this article, please cite it as C. Challener, "Collaboration Key to Meeting Excipient GMP Requirements,” 

Articles

Meeting evolving global requirements for excipients requires close collaboration between pharmaceutical companies and excipient suppliers.

Standards Set Excipient GMPs, But Collaboration is Needed

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, 

Mass spectrometry plays and important role in advancing on biologic drug development and manufacturing, but limitations still exist.

Mass Spectrometry Measures Up to Biologics Drug Analytical Challenges

Drug manufacturers need to work closely with excipient suppliers to ensure supply chain safety.

Collaboration Key to Meeting Excipient GMP Requirements

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

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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Global Active Pharmaceutical Ingredients Market-Growth, Trends & Forecasts (2016-2021),

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	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.”

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	3. Y. Zhang et al. 

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. 2016 (15) 2599-2602 (2016).
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. 54 (24) 7144 (2015).
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. 136 (2) 570-573 (2014).
	8. J.C. Tellis et al., 

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