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

Cynthia A. Challener

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

	1. Catalent Applied Drug Delivery Institute,  

Non-invasive Macromolecule Drug Delivery Guide 

Articles

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

Seeking Solutions for Oral Delivery of Biologic APIs

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

PAT for Continuous API Manufacturing Progresses


	Vol. 41, No. 3
	March 2017
	Pages: 24–28

	When referring to this article, please cite it as C. Challener, “Oral Delivery of Biologic APIs: The Challenge Continues," 

Effective solutions for overcoming the high molecular weight, hydrophilicity, and instability of large biomolecules have yet to be identified.

Oral Delivery of Biologic APIs: The Challenge Continues

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,” 

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.