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

Cynthia A. Challener

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. 

Articles

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

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. FDA is in the process of reviewing the applications received to determine who will be accepted 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.

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.

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, 

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.

The Role of NMR and Mass Spectroscopy in Glycan Analysis

The International Society for Pharmaceutical Engineering (ISPE) Facility of the Year Awards (FOYA) program recognizes state-of-the-art pharmaceutical manufacturing projects that use new and innovative technologies to enhance the delivery of quality products and reduce the cost of producing high-quality medicines. It also spotlights the accomplishments, shared commitment, and dedication of individuals in companies worldwide to innovate and advance pharmaceutical manufacturing technology for the benefit of all global consumers.

Novartis was recognized with the overall FOYA award in 2013 because its influenza cell- culture technology is a significant departure from the traditional approach to flu vaccine production, which relies on the growth of the virus in eggs. Cell-culture technology enables the use of raw materials that are readily available and not threatened by pandemic events, as well as closed-system bioreactors that reduce the required biosafety level for the manufacturing space. In addition, it is possible to provide rapid response to potential pandemic influenza threats while fulfilling the need for seasonal influenza vaccines.

First step: facility construction in partnership with HHS


  Following the 2009 outbreak of the H1N1 pandemic flu and the numerous delays in producing vaccines against the virus, the US Department of Health and Human Services (HHS) recognized the need to invest in new vaccine technologies that can ensure national preparedness for a pandemic influenza or other diseases. 

The dedication of the Novartis Holly Springs flu cell-culture plant in November 2011 was a first step in increasing the level of preparedness. Development of the cell-culture technology was funded in part by the HHS Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Contract No. HHSO100200600012C. Construction of the Holly Springs facility was also funded in part by the same HHS agency under Contract No. HHSO100200900101C. The total public/private investment in the technology development and facility was more than $1 billion, according to Novartis. HHS and Novartis have committed to maintaining the facility under a public–private partnership for at least 25 years.

The Holly Springs facility has the capability to produce seasonal flu cell-culture vaccine, prepandemic vaccine, and 150 million doses of pandemic vaccine within six months of an influenza pandemic declaration, according to Novartis.  A fill–finish set up for the production of both flu and non-flu products has also been installed in the facility.

Second step: FDA approval of cell-culture vaccine

Novartis received FDA approval for Flucelvax, the first cell-culture vaccine in the US designed to protect against seasonal influenza in individuals 18 years of age and older, in November, 2012. Clinical trial results indicated that Flucelvax was well tolerated with an acceptable efficacy against antigenically-matched strains and WHO-identified strains of the influenza virus compared to placebo for people aged 18 and older. Adverse reactions were found to be similar to those observed with the administration of other seasonal influenza vaccines (1). 

Third step: first shipments of seasonal flu cell-culture vaccine


  The first shipments of the cell-culture flu vaccine Flucelvax were made in August, 2013, along with shipments of the company’s conventional Fluvirin vaccine. The majority of shipments were completed in October in advance of the peak of influenza season. “We are pleased to offer a portfolio of flu vaccines that includes Flucelvax, which reflects how we are rethinking flu vaccination with a new approach to vaccine production and which offers a choice to consumers,” says Brent MacGregor, president of US Vaccines and Head of Region North America, in a press release.


  Traditional vaccine manufacture begins with a ‘seed” virus identified and provided by the Centers for Disease Control and Prevention. This virus is introduced into fertilized chicken eggs. It then reproduces and builds up in the white (the allantoic fluid) of the egg, which is collected and purified. It takes two eggs to generate enough vaccine for one dose, and thus large numbers of eggs must be produced in advance of vaccine, which is always a challenge. Another difficulty with this approach is the fact the seed virus is not always accurately replicated, and as a result the virus in the vaccine may not be the same as the infective strain, and thus provides reduced immunity. Extensive DNA and protein analysis is required to help avoid this problem, and further testing is conducted to ensure that no pathogens from the eggs are transferred to the vaccine. All of this testing extends the production time and increases manufacturing cost.


  The limited availability of eggs prevents the rapid production of vaccine, which is a major concern in a pandemic situation. It takes, in fact, many months to organize egg supplies, incubate the virus, obtain produced vaccine that can be delivered, according to Novartis.

That is where cell-based flu culture has the advantage. Typically immortalized canine kidney cells are employed, and these cells can be stored in advance until needed and then rapidly amplified, enabling the production of large quantities of vaccine in a much shorter period of time than is possible using fertilized eggs. Cell-culture technology also enables more robust virus production, and therefore the virus in the vaccine is more consistent and more closely resembles the seed strain, which can be increased efficacy. In addition, the size of the bioreactors required to produce large numbers of doses is much smaller than the space required to produce a similar number of doses using eggs. Furthermore, the use of bioreactors ensures a closed, sterile, controlled environment, and thus the risk of potential impurities is reduced. As a result, Novartis’ Flucelvax vaccine does not contain any preservatives, such as thimerosal, or any antibiotics. Finally, unlike vaccines produced in fertilized eggs, cell-culture-derived vaccines can be administered to patients who are allergic to eggs.

For its use of innovative technology for the production of seasonal and pandemic influenza vaccines, the Novartis Holly Springs, North Carolina facility was the Overall Winner of the 2013 Facility of the Year Award issued by the International Society for Pharmaceutical Engineering (ISPE).

Novartis' US Influenza Cell-Culture Facility Recognized

The Suzuki−Miyaura coupling reaction has become a widely used method for creating carbon–carbon bonds. The palladium-catalyzed cross coupling between organoboron compounds (boronic acids, potassium trifluoroborates, organoboranes, or boronate esters) and halides (aryl, alkyl, alkenyl, and alkynyl) or pseudohalides (i.e., triflates). This reaction is attractive because it proceeds under mild conditions, the boron compounds are generally easy to prepare or readily available, and separation of the desired product from the inorganic byproducts is readily achieved. In addition, boron compounds are less toxic than other metallic species use in carbon–carbon coupling reactions, such as organostannanes, and the reaction is fairly tolerant of functionality. In fact, the Suzuki−Miyaura coupling reaction can also be effective for the coupling of functionalized methyl boronates with functionalized methylene groups bearing dialkylamine, ether, or thioether substituents.

Researchers at GlaxoSmithKline (GSK) recently developed a synthetic route to a drug candidate that involved this type of Suzuki−Miyaura coupling reaction, and as a result needed multikilogram quantities of potassium bromomethyltrifluoroborate, which is the key intermediate for preparation of functionalized methyl boronates (1). “The Suzuki−Miyaura coupling reaction was an early step in the overall synthesis, and we, therefore, needed to develop a rapid, economical, and practical method for preparing potassium bromomethyltrifluoroborate on a large scale,” notes Toby Broom, a chemist with GlaxoSmithKline Research and Development. 

The conventional method for the synthesis of this compound, which involves treatment of dibromomethane (CH

BuLi) in tetrahydrofuran (THF) to form a lithium salt intermediate followed by treatment with potassium bifluoride (KHF

), was not suitable for large-volume manufacturing. Cryogenic temperatures are necessary to obtain high purity product, but the reaction is very exothermic and heat removal is challenging on a large scale, according to Broom. In addition, he notes that the quantity of solvent required upon scale-up is not practical.


The highly exothermic nature of the reaction led the GSK researchers to adopt a continuous-flow strategy for the synthesis of bromomethyltrifluoroborate (1). Continuous manufacturing is attractive for such reactions because it generally involves small reactor volumes combined with enhanced control of reaction conditions. Specifically, because materials are introduced continuously and react on contact with continuous removal, there is better control of process variables, and the risk of side reactions is lower. For this particular reaction, a flow process was also attractive because maintaining a cryogenic temperature (-78 °C) in a microreactor would be more cost-efficient, and continuous consumption of the unstable lithium intermediate would avoid its decomposition, according to Broom.

Initial laboratory studies in a plate reactor demonstrated the feasibility of preparing the lithium intermediate using a flow approach (1). The biggest challenge with this reaction was the side reaction of 

B, which led ultimately to the formation of butyltrifluoroborate, a product that was very difficult to separate from the desired bromomethyltrifluoroborate. “This problem was overcome by using an excess of dibromomethane and just one equivalent of triisopropyl borate,” notes Broom.

 also proved to be a challenge (1). This reactant has limited solubility in water, and thus a very dilute solution was required that led to unacceptable workup volumes, according to Broom. “At this stage, we needed to find an alternate method for converting the intermediate lithium triisopropylborate salt to the desired bromomethyltrifluoroborate. We found that 20 molar aqueous hydrofluoric acid (HF) was effective for obtaining the lithium rather than the potassium salt.”

    In this reaction, however, the formation of small amounts of insoluble lithium fluoride clogged the reactor. After extensive evaluation, therefore, the researchers elected to carry out this step in a stirred reactor (1) rather than maintain an entire continuous process. Even though the quenching reaction wasn’t fully continuous, Broom emphasizes that the addition of HF in the plant would be automated, thus reducing operator exposure. An added benefit of the use of HF was its lower cost compared to that of KHF

Addressing reactor plugging on a larger scale

   With a successful laboratory-scale process in hand, the GSK researchers moved to the pilot plant, where a less efficient, but more economical, tube-and-shell reactor was employed. With this reactor, however, the temperature could not be maintained sufficiently low enough, and precipitates formed that blocked the reactor (1). “Fortunately, once we went back into the lab and ran the reaction using a similar type of reactor, we were able to address this problem. Use of a more dilute triisopropylborate solution, a faster flow rate, and the addition of half of the 

BuLi to each of two reactors in series helped prevent the temperature form rising above acceptable levels,” Broom explains.

  In the pilot plant with these modifications, the GSK researchers completed a 240 hour continuous run with 10 kilograms (kg) of lithium bromomethyltrifluoroborate obtained per day (total 100 kg). This material was then subjected to a cation exchange reaction with potassium fluoride to convert it to the potassium salt (1). Broom notes that this last conversion step was time-consuming and could potentially be achieved with a continuous flow separation method as well, but the GSK researchers did not have time to investigate this approach. “Direct use of the lithium salt in the Suzuki−Miyaura coupling reaction is also worth investigating,” he notes.

One conclusion the researchers reached was that a hybrid approach involving the use of each type of process—continuous and batch—where most appropriate was very successful in achieving the goals for the project. “Combined use of continuous flow and batch-type processes was very effective at meeting our needs for the rapid, practical, and efficient large-scale manufacture of this key intermediate,” Broom concludes.

 online, DOI10.1021/op400090a, June 10, 2013. 

Use of a continuous-flow reaction made it possible to scale up a highly exothermic reaction for the production of a key Suzuki−Miyaura coupling reagent.

GlaxoSmithKline Adopts Continuous Manufacturing for Preparation of Key Intermediate

In sterile fill–finish operations, the monitoring of critical quality parameters, such as oxygen content, moisture content, and container-closure integrity, has been accomplished off-line using multiple destructive technologies, including electrochemical analyzers and gas chromatography for oxygen analysis and Karl Fischer titration for moisture analysis, or in-line methods, such as spark testing and vacuum decay that suffer from limited dynamic range and/or high false reject rates. Laser headspace analysis, which involves measurement of the gas phase using light, provides several advantages over these conventional techniques, according to James Veale, president of Lighthouse Instruments, a provider of headspace inspection systems.


    In laser headspace analysis, the light from a tunable diode laser is shined through the headspace of a sterile product container (above the product and below the stopper). The wavelength of light is tuned to match the unique absorption wavelength of a headspace gas molecule (e.g., oxygen or moisture). Measuring both the amount of laser light absorbed at a specific wavelength and the range of wavelengths absorbed provides information about the gas concentration and gas pressure. Specifically, the amount of light absorbed at a specific wavelength provides quantitative information about the gas density while the range of wavelengths absorbed provides quantitative information about the gas pressure. 

   “Analytical instruments incorporating laser headspace technology are available commercially and are proven, fully validated, and approved by regulatory agencies for oxygen, moisture, and leak detection applications,” Veale states.  He adds that these commercial systems are configured either as laboratory instruments useful for development and quality-control laboratory implementation or as fully automated inspection systems useful in manufacturing for 100% inspection of batches.


    The ability to measure headspace gas composition and pressure rapidly and nondestructively allows sterile product manufacturers to simultaneously monitor a number of important quality parameters, including oxygen content, moisture content, and container-closure integrity.

 “For example,” says Veale, “oxygen-sensitive, sterile liquid pharmaceutical products are compounded and filled into vials and ampuls at rates of 10,000 containers per hour. This high speed presents a challenge to personnel responsible for assuring product quality.”  In fact, off-line process monitoring of headspace oxygen occurs only at periodic intervals, typically 3–10 containers per hour, which can lead to significant production losses and potential customer complaints if process upsets occur between tests. The rationale for testing only 0.03–0.1% of manufactured product, according to Veale, is that a validated process that runs continuously should have consistent performance. “Workflow is, however, periodically interrupted due to jams, line-speed variations, and operator error.  Such process upsets increase the probability for manufacturing significant amounts of out-of-specification product.” 

Moisture monitoring of freeze-dried products using Karl Fisher analysis also suffers from limitations. Typically less than 1% of manufactured product (20–200 samples from batches of 20,000) are destructively analyzed for residual moisture content, again under the assumption that a validated process should produce uniform product within specifications, according to Veale.  “We have found, however, that [when] using nondestructive analysis of entire batches, even after extensive lyophilization cycle development, a significant number of vials in a batch can be out of specification for moisture content due to process defects and variability from, for example, stopper seating and shelf positioning.”  

    Finally, Veale points out that the vast majority of lyophilized product manufactured today are not tested in-process for container-closure integrity. “The reasons are an historic lack of available in-process analytical methods and a generally held belief that once a package is developed and has shown container closure integrity (CCI) in laboratory tests, in-process testing is no longer necessary.” The increasing number of product recalls in recent years that have been related to package integrity defects, however, indicates that package defects often occur in-process and there is a need for new test and measurement technologies for the inspection of 100% of a batch in order to understand where and why defects occur and enable the removal of defective packages from fill and finish lines.

“In general, laser headspace analysis can provide more detailed knowledge of sterile fill and finish processes and will help to locate where quality defects are occurring,” Veale asserts. “This ability will in turn allow improvements to be made that minimize the amount of rework, reduce customer complaints, and reduce the risk of recall.”   

Case study one: deep cold storage of sterile products


    Many products are candidates for transport on dry ice, which keeps product vials at -80 °C.  A recent article described customer complaints coming from the field concerning vials of liquid product that had not only lost vacuum, but also developed an overpressure (1).  When a syringe was inserted into the vials to remove product for injection, the overpressure caused product to spray out of the needle holes.

“The vials were specified to have a certain headspace gas composition and air pressure of 1 atmosphere after filling. Laser headspace analysis of the vials revealed that they were filled with a carbon dioxide (CO

)/air mixture and the pressure was 1.5 atmospheres,” notes Veale.  Further studies showed that when the stoppered vials were cooled to below the glass transition temperature of the stoppers, the stoppers lost elasticity and leaks developed.  In a dry ice (CO

) environment at -80 °C, the vials then filled with dense cold (CO

) gas to a total pressure of 1 atmosphere.  Subsequently, when the vials were warmed the stoppers regained elasticity and the seals reformed.  As the temperature continued to rise, the headspace gases expanded and increased the headspace pressure to 1.5 atmospheres.

 “Careful attention must be paid during package and process development regarding stopper materials and capping forces in order to avoid container-closure failures at low storage temperatures. The use of headspace analysis can help ensure that the types of issues described above are avoided,” Veale concludes.

Case study two: in-process leak detection


    Specifications for an oxygen-sensitive sterile product were established at < 1% headspace oxygen and < 0.2 atmospheres of pressure in the vial headspace because exposure to oxygen resulted in degradation and discoloration of the product. Despite these specifications, customer complaints regarding the quality of the product led to an investigation and subsequent recall and quarantine of several batches. After headspace analysis of these batches, the source of the problem was identified, the problem was addressed, and production was allowed to resume. “From this experience, the manufacturer came to recognize the value of in-line headspace analysis and as a result installed an automated laser headspace analysis system capable of simultaneously measuring for container-closure integrity and oxygen content at production speeds,” Veale notes. 


    1. B. Zuleger et al., PDA J. Pharm. Sci. Technol. 66 (5) 453-465 (2012).

Measuring headspace gas composition and pressure allows sterile product manufacturers to simultaneously monitor important quality parameters rapidly and nondestructively.