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

Cynthia A. Challener

The complexity of both small-molecule and large-molecule drugs is increasing as pharmaceutical companies seek therapeutics with new mechanisms of action. In small-molecule therapeutics, increasing complexity often translates to larger numbers of stereocenters. Because only the isomer of a chiral drug that exhibits activity must be produced in high purity, much effort has been invested over the past 20 to 30 years in the development of tools for the asymmetric synthesis of pharmaceutical intermediates. Classical resolution and other methods for the separation of isomers, including large-scale chiral chromatography, and advanced chemocatalysts that mediate a wide variety of enantioselective transformations and enable more rapid and efficient synthesis of complex pharmaceutical intermediates and APIs are widely used today. More recently, technology for the development and commercialization of a diverse array of enzymes that can catalyze stereoselective reactions has led to their increasing use for pharmaceutical synthesis. While advances continue to be made in chemocatalysis, the area of chiral chemistry generating the most excitement is synthetic biology, or the use of multiple enzymes in one-pot reactions (and ultimately in a single organism) to carry out sequential reactions that result in the production of an advanced intermediate or even an API.

While there haven’t been any major new breakthrough chemical or biological transformations reported in recent years, there continue to be incremental improvements in existing technologies. For the pharmaceutical industry, these advancements include improvements in processing capabilities, such as flow chemistry, that make large-scale implementation of these technologies possible, according to John Caldwell, editor-in-chief of

and professor emeritus at the University of Liverpool. He also notes that the development of effective chiral transformations that are “greener” and proceed at large scale in water, use low levels of catalysts that can be recycled, require lower temperatures and pressures, reduce the number of steps through carefully designed cascade sequences, are more atom economical, and use less hazardous materials has become important in the pharmaceutical industry. “We now have a lot of choices of methods for obtaining pharmaceutical intermediates with extremely high enantioselectivities in the range of 99.9%, and part of the challenge now is choosing the best method for a given API,” he notes.

Route development based on good process chemistry is in fact crucial and should be addressed prior to choosing a chiral technology, according to Garrett Hoge, chiral technology manager at the ChiroTech Technology Center, part of Dr. Reddy’s Custom Pharmaceutical Services business. “It is important not to use chiral technology just for the sake of it. Chiral chemistry must be implemented in an effective manner. It is as important to have an efficient route to the prochiral substrate as it is to have a highly effective chiral catalyst,”  Hoge says.


The “greening” of chemical manufacturing is one trend that has led to the growing use of biocatalysts for asymmetric synthesis, because these reactions often proceed in water under mild conditions, produce less waste, and are highly selective. Biocatalysis has also passed the proof-of-concept stage, according to Hoge. In addition, Tom Moody, head of biocatalysis and isotope chemistry with Almac and a professor of chemistry at the University of Belfast, notes that a diverse array of enzymes are now commercially available in quantities suitable for pharmaceutical manufacturing. There are a large number of transaminases and dehydrogenases, for example, available for commercial-scale use that enable the enantioselective production of key building blocks and intermediates needed for API synthesis, according to Stefan Mix, Almac’s biocatalysis team leader.


The increased availability of enzymes can be traced back to significant advances that have been achieved in molecular biology, metabolic engineering, and bioinformatics, according to Moody. “The design and engineering of enzymes for highly selective chiral transformations is now possible under tight timelines,” Mix notes. “In fact, a customized enzyme for a specific application can be developed in a few months rather than with extended periods of time and cost,” Mix explains. In many cases today, the cost of chiral ligands is greater than the cost of the enzyme that can provide the same product,” he adds. Hoge also points out that the identification of the gene of interest within a specific organism is also much easier today, and genes can be synthesized for just s few $100.


The availability of large quantities of enzymes at reasonable cost, combined with the greater sustainability of processes based on biocatalysts, has led to their use at earlier stages of the drug development process. “There is definitely a growing acceptance of the use of enzymes for the synthesis of APIs according to GMP,” Moody says. However, unlike for chemocatalysis, for which there are established procedures and guidelines for GMP manufacturing, a scientific approach has been undertaken for biocatalysis. Selected industrial specialists are now getting together to prepare a detailed document for the application of enzymes under GMP that will be published in 2014, he notes. Mix adds that, even though medicinal chemists are slow to adopt novel technologies, where once it was common to use enzyme catalysis only in second-generation synthetic routes, today biocatalysts are being considered from the earliest phases of new product development because of the benefits they provide in terms of cost, productivity, and efficiency.

One leading example of the implementation of a second-generation biocatalytic route was achieved by Merck & Co. A highly active and stable immobilized transaminase catalyst for the reduction of ketones to chiral amines created using directed evolution techniques served as a replacement for a rhodium-catalyst used for asymmetric enamine hydrogenation in the production of the antidiabetic drug Januvia (sitagliptin) (1). Development of the enzyme was accomplished in 10 months through cooperation with Codexis. The process demonstrated the potential to improve productivity by 56%, increase yield by 10-13%, and reduce overall waste generation by 19%, according to Merck. A pilot plant process was completed in 2009, and FDA granted approval for the new route in April 2012 (2). 

The work that Frances Arnold, the Dick and Barbara Dickinson professor of chemical engineering, bioengineering, and biochemistry at the California Institute of Technology, demonstrates the versatility and potential of enzyme catalysis. Arnold has engineered a bacterial cytochrome P450 enzyme to catalyze highly diastereo and enantioselective carbene transfers from diazoester reagents to aryl-substituted olefins, enabling cyclopropanation through a route not observed in nature (3).


  More recently, the idea of synthetic biology has taken hold, according to all three scientists. “Pharmaceutical companies, contract manufacturers, and academic researchers are all investigating the possibility of engineering systems that consist of multiple enzymes that work in tandem and/or in sequence to generate complex chiral molecules with high selectivity,” Hoge explains. Further down the road, Moody says the ultimate goal is to engineer whole cells that switch on and off genes for different enzymes and can take basic raw materials and convert them into isomerically pure, complex, highly value added pharmaceutical intermediates, and ideally APIs themselves. “Investigations are still at the very early stages, but the interest in biomimetic synthesis is very high, and there is a wealth of transformations possible given the biological complexity of nature,” Mix observes.

ChiroTech, for example, has developed a two-enzyme system for the dynamic kinetic resolution of nonnatural α-amino acids. The company engineered N-acetyl amino acid racemase (NAAAR) to catalyze the racemization of many N-Acetyl amino acids, which when used in combination with an L- or D- acylase enzyme, enables their dynamic kinetic resolution. “Because the NAAAR enzyme is general in its ability to racemize many N-Acyl amino acids, we have found many applications for this system and typically achieve an enantiomeric purity of 99%,” Hoge comments.


  Chemocatalysts will always have a place in API manufacturing, even though much of the excitement today is centered around biocatalysis. “Asymmetric hydrogenation in particular is a very important reaction in the pharmaceutical industry and will continue to be so,” Hoge asserts. He adds that there are still many new applications yet to be discovered. The fact that many chiral ligands are coming off patent will also promote the use of chemocatalysis because the cost of catalyst systems will decline significantly. In addition, chemocatalysts are very attractive for generics manufacturers, who can capitalize on asymmetric hydrogenation technology by improving on innovator routes that might not access a chiral technology. “These new and improved routes are enabled with technology allowing for more efficient implementation and a lower cost of goods than the originals,” Hoge notes.


  Organocatalysts also show potential for use in chiral transformations, particularly when used in cascade reactions, such as those being developed by David MacMillan, the James S. McDonnell distinguished university professor of chemistry at Princeton University. “We have a much greater understanding of the molecular environment required for catalysts, and therefore there is greater potential to design effective organocatalysts,” Caldwell says. In fact, ChiroTech has found that industrial application of this technology is possible and plans to bring an organocatalytic route to the plant within the next several months, according to Hoge.

Fitting the theme of synthetic biology, MacMillan’s approach to organocascade catalysis is based on the biochemical blueprints of biosynthesis. According to MacMillan, it includes the successful merger of different reactivity platforms, allowing access to intricate structural motifs directly applicable to natural product synthesis and enabling the straightforward generation of multiple stereocenters in a single operation. His group is using this strategy to construct the chiral frameworks of several complex natural products. Most recently, they achieved the enantioselective total synthesis of (−)-minovincine in nine chemical steps (4).

Caldwell believes that the design of cascade reactions through the assembly of known chemistry--whether involving organo-, chemo-, or biocatalysis--in unique sequences that leverage the best properties of each provides tremendous opportunities for advancing asymmetric synthesis in general and developing practical, efficient, and more sustainable commercial-scale routes to enantiomerically pure pharmaceutical intermediates and APIs.


  1.	C. K. Savile et al., Science, 329 (5989), 305-309, (2010).
  2.	Merck, Case Study: A Journey To Revolutionize Drug Manufacturing. In an industry charged with helping the world’s populations be well, green chemistry is a critical tool. http://www.merckresponsibility.com/wp-content/uploads/2013/07/green_chemistry.pdf . Accessed 1/30/2014.
  3.	P. S. Coelho et al., Science 339 (6117), 307-310, (2013).
  4.	B.N. Laforteza et al., Angew. Chem. Int. Ed. 52 (43), 11269 -112, (2013).


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

Articles

The commercial availability of an increasing diversity of enzymes has led to the growing use of biocatalysts for API synthesis.

Synthetic Biology: The Next Frontier in Chiral Chemistry for API Synthesis

With the growth in manufacturing of APIs and formulated drugs around the world, there has been increasing pressure on government agencies to ensure that all products entering the drug supply chain meet necessary safety and quality requirements. In fact, several incidents with dire consequences have brought attention to the need for increased security within the global drug supply chain. Governments and regulatory agencies have responded with the adoption and implementation of both legislated and voluntary programs designed to address many of the issues that have caused problems in the past. While some of these initiatives are focused particularly on formulated drug products or other raw materials (e.g., excipients), others target APIs and will thus have a direct impact on API manufacturers.

The US: increasing resources and streamlining operations  


  In the United States, the Drug Quality and Security Act (DQSA) was signed into law by President Obama on November 27, 2013. This law will largely impact drug manufacturers and most notably contains requirements in Title II for the identification and tracing of certain prescription drugs that are distributed in the US (1). FDA is responsible for administering this and other programs established by the DQSA. While the DQSA does not directly affect API manufacturers, John DiLoreto, executive director of the Bulk Pharmaceuticals Task Force (BPTF) of the Society of Chemical Manufacturers and Affiliates (SOCMA) is glad to see that GMPs are being expanded to compounded drugs. “We recognize that any safety problems with a drug product reflect on the entire drug industry and thus have a negative impact on all members of the supply chain,” DiLoreto notes. 

FDA has also been actively developing guidance and other materials associated with the Food and Drug Administration Safety and Innovation Act (FDASIA), which was signed into law on July 9, 2012. Most relevant to the global drug supply chain is Title VII of the statute, which includes provisions that give FDA new authorities to detain drugs of questionable origin, penalize companies for delaying, denying, limiting or refusing inspection, and increase the fines for adulterated and counterfeit drugs (2). 

The FDASIA also grants FDA the authority to establish mechanisms for more rapid approval of drugs for patients with serious or life-threatening diseases and collect user fees from various sectors of the pharmaceutical industry (e.g., prescriptions drugs, generic drugs, biosimilars, and medical devices) that will be used to fund reviews of innovator drugs, medical devices, generic drugs, and biosimilar biological products and inspections of international manufacturing facilities. 

The BPTF is particularly encouraged by this development, because it has been working closely with members of Congress, FDA staff, and industry leaders to address FDA’s lack of resources for conducting GMP inspections of foreign drug manufacturers. “Industry is eager to see FDA increase its inspections of foreign manufacturing sites, not only to increase the safety of the API supply chain, but also to level the playing field for companies that already invest in programs and processes that ensure the safety and quality of their products,” DiLoreto observes. DiLoreto is gratified to see that FDA has also been able to work with the Chinese government, which seems to be establishing an inspection protocol for Chinese sites that is similar to the one the agency uses for domestic firms. Section 705 of the FDASIA, in fact, requires FDA within its two-year drug inspectional frequency requirement to use a risk-based scheduling approach for domestic and foreign drug facilities (2). Inspection criteria include the establishment’s compliance history and the inherent risk of the drug being manufactured. The agency is currently developing risk-based methodologies for inspections to reflect the statutory criteria and is working to establish common risk principles across the various centers. 

FDA is also revising existing cGMP regulations to update and harmonize requirements and improve detection of and response to emerging product safety and quality signals (2). Finally, Section 712 of FDASIA allows FDA to enter into written agreements with foreign governments to recognize the inspection of foreign drug establishments, and the agency is currently determining how this authority can be used to advance existing efforts in this area and further FDA’s goal of relying on foreign government inspections.

Most recently, FDA began accepting applications for its voluntary Secure Supply Chain Pilot Program (SSCPP), which is intended to enable 100 qualified firms to expedite the importation of up to five APIs and/or finished drug products into the US while allowing the agency to focus its import surveillance resources on preventing the entry of high-risk drugs that are the most likely to compromise the quality and safety of the US drug supply, according to the agency. The program is related to section 713(4)(B)(i) of the FDASIA, which covers the required submission of compliance information. 

Participating firms in the SSCPP program must be the new drug application/abbreviated new drug application sponsor or the foreign manufacturer of the product, comply with cGMP regulations and be in compliance with registration and listing requirements outlined in sections 510(i) and 510(j) of the Federal Food, Drug and Cosmetic Act, and commit to securing their drug-supply chains as participants in the Customs-Trade Partnership Against Terrorism (C-TPAT). The SSCPP is jointly administered by FDA’s Center for Drug Evaluation and Research (CDER) and Office of Regulatory Affairs (ORA) and will run from February 2014 through February 2016. On Feb. 18, 

FDA announced the first 13 companies prequalified for acceptance into the pilot program

The agency will use the information gathered, such as the timeframe for passage of products through the imports entry process, the level of adherence to program requirements, and the overall impact of the program, to develop its approach to implementing the FDASIA. Janeen Skutnik-Wilkinson, vice-president of Health Sciences at NSF International explains, “FDASIA, DQSA, and SSCPP codify into law the practices and expectations that FDA has been emphasizing since 2008.  It is important to recognise that the FDASIA has far-reaching implications for the pharmaceutical industry. To date, most pharmaceutical manufacturers and the media have focused on FDASIA’s high-profile provisions related to user fees, but several provisions of the law focus on ensuring safety and quality in the increasingly global pharmaceutical supply chain. After the 2008 Heparin contamination incident and the questions it raised about pharmaceutical safety, it’s no surprise that global supply chain safety provisions found their way into the pages of the FDASIA.

Europe: refining and expanding legislation


In the European Union, the European Medicines Agency (EMA) has implemented the API import provisions of the Falsified Medicines Directive (FMD) and has revised its GMP Annex 16, which outlines the responsibilities for Qualified Persons (QPs) (3). The main new requirement of the FMD is that pharmaceutical companies can only purchase APIs from suppliers that have received GMP certificates. “The original hope was that the amount of work might be reduced, since API suppliers would all be inspected by the authorities,” says Christine Morris, an associate with ISIS Pharmaconsulting. “However, that is not the case; pharma manufacturers still must conduct their own audits. Therefore, we don’t expect any real changes in product quality, other than perhaps for material coming from manufacturing sites outside of the EU and the US.” It appears, however, that an unintended consequence of the FMD is an increase in API prices as suppliers respond to the increase in audit and compliance requirements.

Fortunately, according to Pete Gough, executive director of Health Sciences at NSF International, the impact of implementing the FMD has not been as problematic as some had feared with respect to reported shortages of medicinal products due to issues with importing APIs from outside of the EU.  However, it is possible that many companies imported and stockpiled APIs ahead of the July 2013 deadline for implementing the new import requirements, and therefore, some problems may still emerge once these stockpiles are exhausted. Many companies in the US were concerned about meeting the requirements of the FMD, according to DiLoreto. “These concerns have been addressed with an agreement between the US authorities and the EMA,” DiLoreto explains. 

The proposed changes to Annex 16 are largely welcomed, according to Gough, because there was a real need to harmonize QP responsibilities across the EU. However, there are areas that still must be addressed, such as harmonization of terms within the Annex with other parts of the GMP Guide.

China: will regulatory changes have an impact? 


In October 2010, the new version of the 

 was adopted that included new and revised drug monographs as well as newer production technologies and analytical techniques that are used internationally for drug quality control. This action was followed by the introduction of revised GMP regulations by the Chinese State Food and Drug Administration (CFDA) in March 2011 (4). These regulations are modeled after those in the US and Europe and include more specific requirements for drug manufacturing than the previous version. However, it remains to be seen if these changes will have any impact. “We still have yet to determine if these new GMP expectations can be effectively applied and if the CFDA can enforce them,” Gough comments. 


Moves to harmonize international GMP requirements continue to progress, but very slowly, and Gough believes recent moves to share inspection outcomes across various countries and regions, and particularly between the EU and US, might be of more practical interest. “Such sharing will enable authorities to focus GMP inspections on the regions that pose the highest potential risks for both GMP non-compliance and of counterfeiting, e.g., Asia,” Gough says.  SOCMA’s BPTF strongly supports the sharing of information across countries to the greatest extent feasible as a way to eliminate duplicate work and free up resources for increased inspections where they are most needed, according to DiLoreto. Morris, however, believes that while harmonization of regulatory requirements is possible, the actual acceptance of other agency inspection results won’t take place for some time, because the different authorities have very different approaches.


  Even so, Morris does believe that the global drug and API supply chains are more secure than in the past. “There is much more awareness within pharmaceutical companies of the importance of supply-chain management and the need to have greater knowledge of the supply chain,” she states. DiLoreto adds that the industry is headed in the right direction, with better regulations and more inspections across the globe. Gough points to India’s plan to increase inspections and China’s closure of alleged producers of counterfeit drugs as further good signs. Both Gough and DiLoreto, however, note that it is too soon to judge which initiatives will be effective, because many of the legislative changes are in the early stages of implementation. 

“It is imperative though,” asserts Gough, “that pharmaceutical companies comprehensively map their supply chains and then conduct a risk assessment to identify potential vulnerabilities.  It is surprising just how many companies still do not have a clear picture of their supply chains.” API manufacturers must ensure that they can demonstrate compliance with cGMP regulations for APIs and other requirements for APIs as outlined in the new and proposed legislation discussed above. 

FDA, Food and Drug Administration Safety and Innovation Act 

(FDASIA). Accessed Jan. 17, 2014.
  3.	EC, 

. Accessed 1/17/2014.
4.	Chinese Food and Drug Adminstration, 

In order to ensure a safe drug supply chain, governments have implemented both legislated and voluntary programs designed to address problem areas.

Regulatory Initiatives Spur API Supply Chain Security

The global market for pharmaceutical excipients, or inert ingredients such as fillers, binders, disintegrants, glidants, colorants, flavoring agents, and coatings, is estimated by various market research firms to be approximately $6 billion in 2013 and growing at 5-7% annually through 2018 (1,2). There are several trends driving this growth, including the increasing size of the branded pharmaceutical market, the expansion of the generics and biosimilars market as patents expire, the reformulation of other drugs to achieve patent extensions, the use of excipients to overcome the poor solubility and bioavailability of many new drugs, and the rise of coprocessing technologies to increase productivity and reduce costs. Recognition of the important role of excipients and the development by excipient manufacturers of substances with specific functionalities designed for pharmaceutical formulations is also leading to the development of more complex products. 

At the same time, however, issues regarding the safety of the supply chain have brought attention to the need for increased regulatory controls and quality specification—for example, quality by design (QbD)—while the overall drive to reduce healthcare costs is placing significant downward pressure on excipient prices. Combined with an increasingly competitive environment in this sector, these factors are all contributing to a decline in the profit margins of many excipient manufacturers.


"Today, excipients are coming to be regarded for their true importance by the pharmaceutical industry," says Paul Evers, a market research analyst with BCC Research (Wellesley, Mass.), a publisher of technology market research reports.  In the past, excipients were typically substances produced initially for other markets, such as the food or personal care industries, and then adopted for use in pharmaceuticals. With the realization that excipients can play a major role in the performance of formulated drugs, there is now growing interest in developing substances specifically for pharmaceutical applications. These products typically have the advantage of being manufactured using more robust, carefully controlled processes that provide products of consistently high quality.

The need for the use of production processes that meet high standards is in fact another consequence of the fact that regulatory agencies have also taken notice, following a number of high profile safety/quality issues, of the potential impact that excipients can have on drug quality.  "As a result, quality and safety regulations extend to deeper levels of the industry today, starting with the design of excipients and including their manufacture, distribution, and storage. Smaller businesses in the industry have been most affected, because non-compliance has resulted in severe economic losses for these players," observes an analyst from MarketsandMarkets.

Some actions that have been taken to address these issues include the publication of the European Medicines Agency Risk Assessment Guide for Excipient GMP (Good Manufacturing Practice) as part of the Falsified Medicines Directive (3) and Revision of the EU GMP Guidance of Chapter 5 (Production)– New Supplier Qualification Requirements, which includes excipient manufacturers (4). The China Food and Drug Administration also has developed a preliminary list of high-risk excipients (5) and is developing new excipient regulations. 

Industry has taken steps on its own as well. The EXCiPACT Excipient GMP program, which was developed by industry experts from the European Fine Chemical Group and several other trade groups, was launched in January 2012 and is a voluntary, international scheme for the high quality certification of excipient manufacturers and suppliers to current GMP (cGMP) and current Good Distribution Practice (cGDP) standards (6). ANSI NSF-IPEC 363: Good Manufacturing Practices for Pharmaceutical Excipients is also being developed by the International Pharmaceutical Excipients Council (IPEC) and standards company NSF International in conjunction with the US FDA and will provide a stand-alone, quality system-based GMP standard that industry and regulatory agencies can use in evaluating, auditing, and certifying excipient manufacturing and quality (7). 

Excipient suppliers desired such guidelines because currently, novel excipients must receive approval as part of the final drug formulation; therefore, excipient manufacturers must share production information, including possible proprietary data, with the drug formulator. With the ability to demonstrate compliance with GMPs, such data provision won’t be necessary.  

Even so, there will be additional compliance requirements, particularly in Europe, for both drug formulators and excipient manufacturers, according to an analyst from MarketsandMarkets. Pharmaceutical companies will need to conduct risk assessments for pharmaceutical excipients and ensure that they meet quality standards and conduct audits of their excipient suppliers. Excipient manufacturers, meanwhile, will need to implement quality management systems that meet the more detailed requirements of the pharmaceutical industry and prepare for a large number of audits. Most importantly, increased interaction between drug and excipient manufacturers is required to achieve effective quality risk assessments and the development of common control and risk mitigation strategies, the analyst notes. 

Despite the challenging regulatory environment, there is a significant desire for the development of innovative excipients, according to Evers. "It is quite possible that the level of excipient innovation will double over the next 5 years, depending on how soon the economy recovers," he  says. BCC Research points in particular to growing interest in coprocessing for efficiency and the increasing use of oleochemicals (materials derived from biological oils or fats that are lipophilic and often have a high molecular weight) for improving the delivery of poorly soluble drugs. Other technologies, such as low permeability solubilizers, absorption enhancers, stabilizers for nanoscale carriers and orally disintegrating tablets, and replacements for animal-derived substances, are also under development. Continued innovations based on microcrystalline cellulose, which BCC believes is the most important excipient introduced in the last 50 years, will also be important in the future. Coprocessed excipients consisting of more than one substance and can be prepared using a number of different processes, such as granulation, spray drying, melt extrusion, milling, and others. The method depends on the physical characteristics of the individual excipients and the desired properties of the coprocessed excipient. Both excipient manufacturers and drug formulators must keep in mind, however, that the properties and safety profiles of coprocessed excipients may not be the simple sum of the characteristics of the individual excipients alone. 


The excipient market can be broken down by product type and by functionality. There are three main categories of products: organic, inorganic, and other (largely USP water). Organic excipients, including carbohydrates, proteins, oleochemicals, petrochemicals, and other organic chemicals, account for the largest percentage of the market (85-95% depending on the market analysis). Oleochemicals are not surprisingly expected to experience the largest growth rate of all of the organic excipients. Inorganic chemicals, including calcium compounds (phosphate, calcium sulfate, and calcium carbonate), metal oxides, and halides, account for 5-10% of the excipients market, according to MarketsandMarkets. By functionality, fillers and diluents, binders, suspension and viscosity agents, and coating agents are the leading categories of pharmaceutical excipients, and all are expected to show healthy growth through 2018.

It should also be noted that the pharmaceutical excipients market is highly fragmented, in large part due to the fact that most excipients, as mentioned above, were not originally developed for pharmaceutical applications. MarketsandMarkets reports that in 2012, none of the leading excipient manufacturers, such as BASF, Ashland, The Dow Chemical Company, FMC, Roquette, and JRS Pharma held more than a 10% share, and the top 10 companies accounted for just 60% of the market. The remainder was comprised of numerous smaller companies, with many located in emerging markets such as China, India, and Brazil.

, (Wellesley, Mass., October 2013)
  2. Markets and Markets, 

Pharmaceutical Excipients Market [By Products (Carbohydrates, Oleochemicals, Petrochemicals, Polymers, Microcrystalline Cellulose, Sugar, Calcium carbonate), By Functionality (Fillers, Binders, Lubricants, Preservatives)] – Global Forecast to 201

, (November 2013).
3. European Commission Health and Consumers Directorate,

Draft Guidelines on the Formalized Risk Assessment for Ascertaining the Appropriate Good Manufacturing practice for Excipients of Medicinal Products for Human Use

, Feb. 2, 2013. Accessed Feb. 12, 2014.
4. European Commission Health and Consumers Directorate-General, 

Proposed Draft of Chapter 5: Production in The Rules Governing Medicinal Products in the European Union (EudraLex) Volume 4: Good Manufacturing Practice: Medicinal Products for Human and Veterinary Us

, Accessed Feb. 12, 2014.
5. China Food and Drug Administration, 

Preliminary list of high-risk excipients (in Chinese)

NSF/ANSI 363 – 20xx, Issue 1, Draft 1 (April 2011) 

Draft Standard for Pharmaceutical Excipients Good Manufacturing Practices for Pharmaceutical Excipients

Drug formulators are looking for new excipients to address solubility and bioavailability issues and extend patent protection, but there are hurdles to using novel, specialized ingredients.

Demand Increases for Novel Excipients, but Manufacturers Still Face Challenges

Although proteins are known to be linear chains of amino acids that can form three-dimensional (3D) structures and enable binding interactions with specific biologic molecules, only recently have biopharmaceutical companies begun to pay more attention to their higher-order structure (HOS) of proteins. The previous lack of attention to this area is surprising given the crucial role this specific attribute plays with respect to the stability, efficacy, and safety (e.g., immunogenicity) of protein-based drugs. While there are numerous effective analytical methods for determining the chemical composition of proteins, such as the order of amino acids in linear chains, the weakest link in the development of protein therapeutics, according to Steven Berkowitz, a senior principle scientist at Biogen Idec, is HOS characterization. Much of this problem is a result of the limited capability of biophysical methods to obtain this information for routine purposes.

While there are several types of analyses that can give low-resolution structural information (e.g., fluorescence, ultraviolet-visible [UV-VIS] analysis, circular dichroism [CD], etc.) and even more detailed data (e.g., differential scanning calorimetry [DSC], vibrational circular dichroism [VCD], and analytical ultracentrifugation [AUC]), only two methods to date—hydrogen deuterium exchange-mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy—are capable of providing detailed high-resolution fingerprint data on the HOS of proteins. Of the two, HDX-MS (which provides this high-resolution fingerprint indirectly) is becoming increasingly adopted as advances in instrumentation, methods, and software for data analysis are making this technique more accessible. NMR, while capable of determining the 3D structure of a protein directly, is slowly finding more frequent use due to the need to overcome several hurdles.

A breakdown on higher-order protein structure


  Proteins have multiple layers of structure. The primary structure consists of the linear order of amino acids. When this linear structure folds on itself, it can take the form of an α-helix, β-sheet, or other shape, which comprises the secondary structure. The secondary structure can also fold to different degrees, and this level of folding is considered to be the tertiary structure of the protein. Although this folding can to some extent be stabilized via the formation of permanent interactions, (e.g., covalent disulfide bonds) folded protein structures are predominantly stabilized through weak interactions, such as hydrogen bonding and van de Waals interactions, which can be easily disrupted with changes in temperature, pH, protein concentration, and other conditions. HOS includes everything but primary structure, with some emphasis on tertiary structure.


  Higher-order protein structure is crucial for the performance of protein-based drugs. Because some aspects of the HOS of proteins can change in response to changes in the protein environment, it is important that the behavior of the protein structure under different conditions and the actual structure of the protein in the formulated product be as well-defined as possible. This sensitivity of the HOS of proteins to the environment can cause problems with some biophysical characterization techniques. These technologies require the use of a different solvent, high dilution, high-salt concentrations, and/or the removal of interfering excipients, or require other conditions that are different from the environment of the formulated product, says Berkowitz. As a result, the structure being analyzed may not be the structure of the protein in the drug product. Therefore, biophysical characterization techniques that do not significantly alter the sample environment are highly valued. The results from analytical methods that do require changes to the formulation matrix of a therapeutic protein must be carefully designed so that meaningful comparisons can be made and the actual presence of real differences detected.


  Although common and simple routine biophysical characterization techniques such as calorimetry, absorbance, and fluorescence provide low-resolution information on protein structure, collectively this information provides a coarse fingerprint picture of the HOS of protein drugs. 

Because different types of protein structural elements are known to melt at different temperatures, observing the melting behavior of a protein using DSC can provide some basic information about these structural elements in the protein, which generally correspond to higher-order structural elements, such as domains or possibly super-secondary structures, explains Berkowitz. Analysis of the UV–VIS absorption and fluorescence spectra of a protein provides general information about the local environments of the aromatic amino acids. Information about the amides in the peptide bond present along the polypeptide backbone of the protein can be obtained using fourier transform infrared spectroscopy.

 “The overriding problem here, unfortunately, is the limited resolution. For example, the signals from all of the aromatic acids in the UV–VIS or fluorescence spectra just overlap too much to see the individual peaks. In fact, it is this low-resolution that initially lulled researchers into not paying attention to the HOS of these drugs,” Berkowitz observes.



  Determination of secondary structure with VCD


Another technique based on the vibrational motion of atoms in the protein is VCD spectroscopy, which is useful for determining the secondary structure of proteins, notes Wasfi Al-Azzam, a scientist in the Bioanalytical Sciences department at GlaxoSmithKline. VCD is sensitive to the interactions of dipoles, and the information in VCD spectra (e.g., peak frequencies, signs, patterns, and band shapes) makes it possible to distinguish between different secondary structures.

“This technique has the advantages of being effective at high concentrations and in various liquids, including water and buffer solutions,” Al-Azzam notes. “It is, in fact, specific for high concentrations and is being increasingly used as a result, because growing numbers of protein therapeutics are being formulated at high concentration for subcutaneous delivery. The minimal level of sample manipulation is a real benefit,” he continues. 

VCD is also being used more frequently for comparability studies (e.g., under varying temperature and pH conditions). The spectrum obtained provides a fingerprint of the secondary structure of the protein, and can indicate the formation of fibrils and other folded forms. Conformational changes can also be seen to some extent, according to Al-Azzam.

The only real limitation for this method is the concentration, which has a lower limit of < 100 mg/mL, although taking longer scans can extend that level somewhat. As protein-based drugs have increased in size, the fact that there is no limit on the size of the proteins that can be analyzed has become important. Even insoluble proteins and aggregates can be evaluated. Al-Azzam also points out that advances in instrumentation have contributed to the increased use of this analytical method, including improvements in the laser and photomultiplier, as well as the detectors, which have led to improvements in the quality of the spectra and the sensitivity.


  The use of AUC by the biopharmaceutical industry has been growing since the 1980s and early 1990s when its use as a complement to size-exclusion chromatography (SEC) was shown to be invaluable for the detection of aggregates that often are not observed using the former method, comments Tom Laue, a professor at the University of New Hampshire and cofounder of Spin Analytical, a developer of recently commercialized next-generation AUC instrumentation

AUC can provide two types of data—the sedimentation velocity (most common) and the sedimentation equilibrium. In both experiments, the material is placed in the centrifuge and its movement from the top to the bottom of the centrifuge tube is monitored with various optics systems/detectors, including UV–VIS and fluorescence detectors and refractometers. A chromatogram-like graph is produced with a baseline and peaks. The x-axis, rather than time, is the sedimentation coefficient, which is directly related to the ratio of the material’s molar mass and its size, and thus its state of aggregation. Sedimentation coefficients for monomeric monoclonal antibodies (mAbs) typically range from 6.1-6.9, while the value is around 9 for mAb dimers and 12 for mAb trimers. Thus, the data can be used to quantify the amount of dimer and trimer present, although it is not accurate at very low levels (<1%).

With sedimentation equilibrium, the data are directly proportional to the material’s molar mass. For either velocity or equilibrium sedimentation, samples with different protein concentrations are analyzed to determine if the fractional amount of aggregate increases or remains constant with protein concentration. A lack of change in the amount of aggregate formation indicates the presence of irreversible bonding, while an increase in aggregate formation with concentration proves the presence of reversible interactions. 

The advantage of AUC over SEC is that aggregates can be unknowingly retained on the column, and even if present at just 1–2%, these protein structures can be a real problem in the formulated drug, Laue points out. In addition, the high salt concentrations used with SEC have the potential to influence the protein HOS, as does the significant dilution of the sample that passes through the column. “AUC can be carried out in any liquid and does not suffer from these issues. As a result, it has become an excellent orthogonal method for confirmation of SEC results,” Laue states.

There is also growing interest in using AUC to evaluate the behavior of protein therapeutics in different media, such as serum. “In some cases, proteins are designed to interact with components like albumen to influence the pharmokinetics, while in other cases interactions are not desired. AUC can be used to determine if these types of interactions are occurring,” Laue explains.

The biggest limitations are the complexity of the data workup and the length of time for each analysis. Laue, however, notes that there are numerous workshops offered on a regular basis that provide training on data analysis, and he also expects that the software will continue to improve. “Throughput is the biggest issue. Only 7-8 samples can be done per day,” he says. John R. Engen, professor of bioanalytical chemistry at Northeastern University, notes that multi-angle light scattering is also being increasingly used along with SEC to obtain information similar to that available from AUC, but more rapidly.


Unfortunately, methods that provide high-resolution structural information suffer from lengthy analysis times and require training in data interpretation. According to Berkowitz, this situation, however, is changing for proteins that crystallize, x-ray crystallography can provide the most information, and depending on the protein, the analysis can be achieved rapidly. For proteins that do not crystallize, advances are being made particularly for HDX-MS, and to some extent, NMR. 

“NMR can provide massive amounts of information,” says Berkowitz. For some large and complex proteins, however, too much overlapping information is obtained such that it is difficult to deconvolute the spectral data. “Advances in analytical tools that make NMR more amenable for larger molecules and more complex structures are needed,” he adds. “Despite the high cost of NMR instruments, the high level of training required, and the complex data analysis, NMR plays a useful role in characterizing the HOS of protein biopharmaceuticals in certain cases. In the future, however, NMR should eventually be a dominant and valuable tool in the biopharmaceutical industry for the characterization of the HOS of proteins. We just need more innovation.”

HDX–MS has seen wider adoption than NMR due to significant advances in both the instrumentation and software tools, observed Engen. “The introduction by Waters Corporation of an instrument available for HDX-MS that includes the instrumentation, additional specialized equipment, and software tools for data analysis has greatly increased HDX-MS use in the marketplace.  Better processing power and software for data analysis have also made the experiment more accessible to people,” he notes. 

In addition, as the number of biotherapeutic proteins has increased, so has the use of HDX-MS because it provides useful information that is hard to obtain using other methods in a relatively short period of time for a wide variety of proteins, says Engen. “For example, proteins, and even specific conformational states of proteins that won’t crystallize can be studied under conditions that mimic physiological conditions. Furthermore, the location of effects in proteins that are hard to see with other techniques, such as the allosteric effects of binding, can be obtained because HDX-MS has higher spatial resolution than most classical biophysical tools (except NMR and x-ray crystallography),” he explains.  

There are some limitations, though. In HDX-MS, backbone amide hydrogens are detected through exchange of hydrogen and deuterium, and some sites involved in complex formation are in side-chain hydrogen’s, which are generally not detected by MS. Not all interactions (such as binding) alter the environment of backbone amide hydrogens, particularly in folded proteins, and a good understanding of the theory is essential to make sense of the data. In addition, analysis of large proteins (>500 kDa) is challenging because large numbers of peptides are produced during the analysis, and the data processing can be difficult or the spectrum can be compromised due to suppression of some signals as a result of complex mixes of peptides.  

Obtaining single-amino acid resolution is also a challenge. “Methods like electron-capture dissociation and electron-transfer dissociation fragmentation inside the mass spectrometer try to address this issue but they are not without problems. Most importantly, there is yet very little software available to evaluate the data obtained from these analyses,” Engen says. He also notes that creating many overlapping peptides, a technique that has been known for many years, can be used to increase resolution. By altering the digestion conditions, the mix of peptides produced can be slightly altered, and then subtractive analysis can be used to extract the data. This method, however, has some downsides and does not totally solve the problem.

Despite these issues, both Engen and Berkowitz see a bright future for HDX–MS. “This method is already leaps and bounds ahead of where it was 10 years ago, and great strides have been made in just the last 5 years as well.  We used to be afraid of proteins larger than 30 kDa, and now we are routinely working with analytes that are 200 kDa, and HDX-MS is now a common analytical technique,” Engen asserts.

In fact, HDX-MS data are starting to be used more extensively in the biopharmaceutical industry. “HDX–MS data appear in filings with FDA and are used to check formulation and comparability, to look at small molecule binding, and to map epitopes,” Engen observes. He goes on to add that academic research groups continue to further advance the limits of the method by expanding into the membrane protein space, enabling the analysis of ever larger complexes and improving the analytical aspects at every step. 

Automated sample handling, advanced glycan analysis, and specially designed columns are help speed up confirmation of the biosimilarity.

Advances in UPLC Techniques and Column Chemistry Aid the Confirmation of Biosimilarity

Participating firms in the SSCPP program must be the new drug application/abbreviated new drug application sponsor or the foreign manufacturer of the product, comply with cGMP regulations and be in compliance with registration and listing requirements outlined in sections 510(i) and 510(j) of the Federal Food, Drug and Cosmetic Act, and commit to securing their drug-supply chains as participants in the Customs-Trade Partnership Against Terrorism (C-TPAT). The SSCPP is jointly administered by FDA’s Center for Drug Evaluation and Research (CDER) and Office of Regulatory Affairs (ORA) and will run from February 2014 through February 2016. FDA is in the process of reviewing the applications received to determine who will be accepted into the pilot program.

As a leading industry event for the fine chemical and pharmaceutical industries, DCAT Week 2014 (March 10-13 in New York) is anticipated by fine-chemical suppliers, contract manufacturers, and pharmaceutical/biopharmaceutical firms. This year, the Drug, Chemical & Associated Technologies Association (DCAT), which organizes the event, is celebrating the 125th anniversary of its founding. In addition to the annual dinner, which is scheduled for Thursday, March 13, DCAT Week is widely recognized for the valuable educational sessions offered at the event.        

The first educational session on Monday, March 10 is DCAT’s 2014 PharmaChem Outlook, sponsored by PharmSource, which will cover how pharmaceutical companies are adapting their organizations, pipelines, marketing, and innovation strategies to meet the varying challenges in today’s marketplace, including the increased healthcare needs of an aging and expanding global population, new, complex managed-care programs, patent expirations, global economic risks, evolving manufacturing technologies, and difficult and potentially more costly new product launches.          

On Tuesday, March 11, sessions will cover value creation with excipients (e.g., product differentiation, lifecycle extension) and through the formation of strategic alignments with suppliers. The presentation on excipients will discuss the opportunities for innovation, product enhancement, and cost savings through excipient development and the impact that regulatory uncertainty can have on realizing them. Those interested in procurement strategies will learn about a new approach designed to transform how procurement organizations manage supplier relationships and deliver value to their companies. Finally, Dr. Ilisa Bernstein, deputy director, FDA’s Office of Compliance, Center for Drug Evaluation and Research (CDER), and other representatives from FDA will discuss the agency’s priorities for meeting the challenge of evaluating increasing numbers of complex and innovative products and suppliers  and discuss ways the pharmaceutical industry can collaborate to assure the delivery of high-quality medicines in an ever-increasing complex and global marketplace.          

Presentations on Wednesday, March 12 will tackle issues associated with quality by design and regulatory and business trends in China.


  In the European Union, the European Medicines Agency (EMA) has implemented the API import provisions of the Falsified Medicines Directive (FMD) and has revised its GMP Annex 16, which outlines the responsibilities for Qualified Persons (QPs) (3). The main new requirement of the FMD is that pharmaceutical companies can only purchase APIs from suppliers that have received GMP certificates. “The original hope was that the amount of work might be reduced, since API suppliers would all be inspected by the authorities,” says Christine Morris, an associate with ISIS Pharmaconsulting. “However, that is not the case; pharma manufacturers still must conduct their own audits. Therefore, we don’t expect any real changes in product quality, other than perhaps for material coming from manufacturing sites outside of the EU and the US.” It appears, however, that an unintended consequence of the FMD is an increase in API prices as suppliers respond to the increase in audit and compliance requirements.

Recent regulatory initiatives designed to secure the global pharmaceutical supply chain will directly impact the global supply chain and API manufacturers.

Securing the Global API Supply Chain

In biological systems, most peptides and proteins are glycosylated. Differences in glycosylation can affect protein structure, stability, immunogenicity,

 half-life, and function. Glycans, in fact, are important to normal cellular function, mediating interactions within cells and between cells and pathogens, enabling immune responses, and influencing tumor progression. “In mammals, sugars are most commonly found as glycoconjugates, the most abundant being the glycoproteins, proteoglycans, and glycolipids. These important biological substances are predominantly located on cell membranes, but are also found in secreted fluids where they modulate or mediate a host of events in cell-cell and cell-matrix interactions,” notes Brady Clark, founder and CEO of Sussex Research Laboratories, a Canadian CRO focused on the synthesis of glycans and glycoconjugates, including glycans on functionalized linker/spacers, glycoamino acids, glycopeptides, glycolipids, and glycans on carrier proteins. In addition, Sussex Research is also active in the determination of free (or non-conjugated) reducing sugars in biological samples.The glycosylation of biologic drugs affects their mechanism of action, pharmacokinetics, and efficacy. Glycoconjugation of a biotherapeutic (peptide, protein, or antibody) may, therefore, confer increased therapeutic efficacy by increasing the stability, aqueous solubility, and/or bioavailability; enhancing the target resolution; and/or extending the 

 half-life. “Glycans are involved in nearly all aspects of the biochemistry of life, and proteins and peptides in the body can’t be evaluated without looking at glycans,” states Garnet McRae, director of bioanalytical services with Sussex Research Laboratories in an interview with 

 “Recently, people have begun to realize the importance of glycosylation, and thus there has been more interest in the synthesis and analysis of glycans,” he adds.

Clark, McRae, and Dennis Whitfield, who sits on the scientific advisory board at Sussex Research, spoke with 

 about the analytical methods used at Sussex Research for both the structural determination of glycans during complex synthetic processes and the quantitative evaluation of free sugars involved in biological processes.

Why is structural determination so important during glycan synthesis?

 It is as important to be able to characterize the actual structures that we create as it is to know how to synthesize them. Unlike peptides, which are linear structures composed of amino acids with just two reactive sites, glycans are comprised of monosaccharides with up to five hydroxyl groups per molecule that can be reactive. They can, therefore, be linear or highly branched, contain multiple sites of glycosylation, and be linked in either an alpha or beta fashion. It is imperative to know exactly how each saccharide has been linked, which means confirming that the reaction occurred at the correct hydroxyl group and with the right stereochemistry.

We use highly effective and, in general, well-understood chemical, enzymatic, and chemoenzymatic methods for the synthesis of complex glycans that are often drug candidates. These methods may also be used as standards at biotechnology and biopharmaceutical companies as well as research laboratories. While we have extensive experience in synthesizing glycans and generally obtain the desired products, no process is 100% fool proof. Therefore, we comprehensively confirm the structure of each intermediate throughout a synthesis.

What are the major analytical instruments that you use for structural glycan determinations and why?

We use methods such as high-resolution proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, including both electrospray ionization and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF).

NMR analysis is the key to analyzing the structure of complex glycans. By evaluating the chemical shifts and coupling details, it is possible to confirm the site of attachment and the stereochemistry of the saccharide linkage, which are the two crucial factors in glycan synthesis. With the advanced two-dimensional (2D) NMR techniques available today, it is possible to decipher the structures of very complex glycans. 

Because mass spectrometry does not provide real structural information, it is mainly used to confirm that we have synthesized the expected compound since we use well-established methods to prepare the glycans. 

Other methods that were previously commonly used for structure determination, such as optical rotation and elemental analysis, are now only used on very rare occasions and only when requested by a client. Much more useful and specific information can be obtained via NMR analysis in combination with mass-spec evaluation.

 Which advanced 2D NMR analyses do you use and what do you learn from each of them?

 For some of the most complex glycan structures we synthesize, it wouldn’t be possible to determine the structures without 2D NMR techniques. The methods we use for nearly every sample are 2D 

H-gCOSY (gradient correlation spectroscopy), which takes approximately 15-30 minutes, and 

H-gHSQC (gradient heteronuclear single quantum coherence) spectroscopy, typically the edited version, which distinguishes CH and CH

There are several additional methods that are used frequently, but not for every sample. These techniques include 

H-gHSQC with proton coupling for measurement of 

H-gHMBC (gradient heteronuclear multiple-bond correlation) spectroscopy and 

H-NOESY (nuclear overhauser effect spectroscopy) (or ROESY [rotating frame effect spectroscopy]) for determination of the connectivities across linkages and positions of protecting groups (3-4 hours each), and 

H-TOCSY (total correlation spectroscopy) for the determination of inter-ring connectivities if the COSY analysis is incomplete (3-4 hours).

We have found, however, that the higher digitization of selective 1D (1Dsel) experiments, such as 1Dsel-

H-NOESY (or ROESY) allow for the determination of coupling constants and work better then 2D for polymer-bound and other complicated samples. They are thus used to determine inter-ring connectivities if the COSY analysis is incomplete (2 hours) and are particularly applicable if the anomericity (alpha versus beta) cannot be determined from the coupling constants, and most commonly for derivatives of L-iduronic acid and D-mannose. In addition, for phosphates or other similar phosphorous-containing species, 

It is important to note that there are many variants of these spectra, and each is applicable to certain circumstances. In addition, there are many so-called fast NMR techniques that greatly reduce the time to acquire the data and complete these types of experiments, which will have a significant impact on turnaround times.

 What are some of the challenges that you face when carrying out structural determinations of complex glycans?

Challenges are mostly presented by the compounds themselves. Larger, more complex glycans, such as those that contain numerous glucosamine groups, can have poor solubility. Glycans with charged structures and a large number of heteroatoms, such as iduronic acids, glucosamines, O-sulfates, and amine-sulfates, can also be difficult to analyze. Often, in these cases, various solvents must be investigated to find a suitable one for NMR analysis. In some cases, the NMR spectrum is so complex that individual peaks cannot be discerned. In many of these situations, smaller sections of the molecule are analyzed independently, and the NMR spectra of the smaller segments are then used to assist in obtaining the complete spectrum of the glycan.

Quantitative analysis of sugars
PharmTech: 

What information can be gained from the quantitative analysis of sugars in biological systems?

While determining the structure of glycans is crucial for confirming the synthesis of appropriately glycosylated compounds, the quantitative determination of free sugars in biological systems, as well as the total sugars (bound plus free, which is obtained after cleaving the bound sugars), can also provide valuable information about biological processes. In cellular and fermentation systems, various sugars are used as feeds, and other sugars are produced as by-products. By monitoring the consumption and production of these various sugars over time during reactions run under various conditions, it is possible to optimize a biological process in order to obtain the maximum amount of desired product. 

What instrumentation do you use for the quantitative determination of sugars and why?

Liquid chromatography-mass spectrometry (LC–MS); in particular LC-MS/MS, is ideally suited for this type of analysis. Mass spectrometry is crucial in this application because it is highly specific and sensitive in the detection of sugars at very low levels. Because the compounds are already known and standards are used for quantitation, LC–MS/MS is a very powerful targeted technique for quantitation.

 What recent advances in technology have been of importance for carbohydrate analysis?

 The increasing resolution and sensitivity of mass spectrometers and the growing choice of instrument types has had a real impact on glycan and sugar analysis. The development of some new derivatizing agents has also been important.

Why is derivatization required for the quantitative determination of sugars?

 Derivatization is a key step in the quantitation of free reducing sugars in solution when using reversed-phase LC–MS/MS. There are many different isomeric sugars with the same molecular weight and only slight differences in structure. There are, for example, three common six-carbon sugars that occur naturally in biological systems—galactose, mannose, and glucose. All have five hydroxyl groups, and it is only the stereochemistry at the hydroxyl groups (axial or equatorial) that differentiates these compounds. While this slight difference can lead to very different reactivity for each molecule, it is not easy to separate them from one another on a reversed-phase column unless they are derivatized.

A common derivatizing reagent for carbohydrates is 1-phenyl-3-methyl-5-pyrazolone (PMP). Not only are the derivatives of isomers prepared using this reagent separable by reversed-phase HPLC, but also up to 100 times more sensitivity during mass-spec analysis can be achieved. The presence of the nitrogen groups in the derivatizing reagent are important for increased ionization and fragmentation efficiency while the added phenyl and aliphatic character provides increased reversed-phase HPLC retention. 

Two additional compounds, 2-aminobenzoic acid (2-AA) and 2-aminobenzamide (2-AB), are also classically used as derivatizing agents for free reducing sugars, particularly for quantitative glycoprofiling of biopharmaceuticals such as therapeutic proteins and monoclonal antibodies. 

NMR analysis provides crucial structural information of synthesized glycans while LC-MS/MS is ideal for quantitation of free sugars in biological matrices.