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

Cynthia A. Challener

Active  pharmaceutical ingredients (APIs) are most conveniently developed and delivered  as solid dosage forms that contain a single crystalline form of an API. Cocrystals alter the physical properties of APIs without  chemical modification of the API while retaining the benefits of a crystalline  solid form.  For compounds that do not form useful salts and still require  improved physical properties, such as solubility or stability, cocrystals may  provide a viable solution. A cocrystal may thus represent a crystal form with  better pharmacokinetic properties, and therefore a better market opportunity.
  

In April 2013, however, FDA issued its  final regulatory guidance on cocrystals, including the classification of a pharmaceutical cocrystal as a “drug  product intermediate.” This classification may help some product development  strategies while, at least in the short term, it may limit the ability to use  cocrystals efficiently in other cases.  

Improved  pharmacokinetic properties and more

“The  selection of a specific crystal form for a given API is a profoundly important  step  from clinical, legal and regulatory perspectives: the dissolution  rate and intrinsic solubility of different crystal forms is variable and can  strongly influence bioavailability; stability to temperature and humidity are  critically dependent upon crystal packing; and the unpredictability (i.e. lack  of obviousness) of crystal structures and physical properties means that there  are legal issues and challenges in terms of obtaining and maintaining patent  protection for an API,” says Ning Shan, director  of preclinical development at Thar Pharmaceuticals. 
  

Highly lipophilic APIs have dissolution problems that may be overcome using a cocrystal because the new crystalline lattice being formed often has very different dissolution properties compared to those of the crystals of the API alone, according to Chris Frampton, chief scientific officer of SAFC Pharmorphix. The formation of a new crystal lattice with a pharmaceutically acceptable coformer often enables other issues, such as compaction problems and difficulties with hygroscopicity, to be overcome. In addition, for nonionizable APIs, for which it is not possible to make a salt, the option to form a cocrystal can be advantageous over finding alternative formulation methods or changing the API molecular structure to include the acid or base functionality required for salt formation, which can have an impact on the activity of the API. In addition, increasingly, adds Frampton, cocrystals are also being seen as a lifecycle management tool, because their use can delay or divert generic competition. 

If a  cocrystal is to be used, it clearly must have  physical properties that offer key improvements over the properties of the API  itself. “Cocrystals have been used to create crystalline forms of APIs that  were previously known in only amorphous forms, improve melting points, enable improved  chemical purity, reduce chemical degradation when exposed to light, remove the risk  of hydrate formation in a formulation, and improve the solubility to enable  higher bioavailability,” says Scott L. Childs, president and CSO of Renovo  Research.  For Frampton, improved stability  is one of the most desirable  attributes of a cocrystal API. “The physical stability, chemical stability, and  melting point can all be tuned to achieve a new form that is more desirable  than the crystal form of the API alone,” he explains. 
  

In  addition, the preferred cocrystal will not only meet the physical property  improvement requirements, but also contain a "pharmaceutically  acceptable" coformer that is nontoxic, has a high weight percent of API  compared to coformer, be chemically and physically stable under all relevant  conditions, be manufacturable using low-cost approaches in existing production  equipment, and remain stable over the shelf life of the product.

Inexperience and  need for screening pose challenges

While  cocrystals offer many potential benefits, it is important  to recognize that they have not yet been fully embraced or adopted by the  industry, according to Childs. There are both skeptics and advocates. It is his  opinion that cocrystals are viewed as a “last resort” in some laboratories, and  this perception will persist in many minds until FDA approves a product  containing a cocrystal that delivers improved performance. “I am convinced,  however, that the first FDA-approved cocrystal product is an issue of 'when'  and not 'if'’,” he asserts.
  

Shan  adds that the requirement in the FDA guidance that the developer of  a cocrystal drug product must provide “assurance that complete dissociation of  the API from its excipient occurs prior to reaching the site of action for  pharmacological activity” could be a real challenge. He also notes that the  prediction of cocrystal structures remains semiempirical, but he anticipates  that with more powerful computational equipment, better calculation approaches  and estimations will be possible.
  

The difficulties that are faced with  respect to the selection of appropriate coformers for a given API is a real  challenge, agrees Frampton. “The one thing we cannot do is predict the outcome  of the properties of cocrystals until they are generated. Although we can  recognize that there might be an interaction between the API and a conformer, there  is no guarantee that this interaction will lead to cocrystal formation, or if  the cocrystals that do form will have desirable properties. A lot of screening  is required, and sometimes what we get does not reflect the crystal lattice we  intended.” He does note, though, that with respect to melting points, he has  found that a lower melting point coformer will tend to reduce the melting point  of the cocrystal while a higher melting point conformer will increase it. 
  

Of course, Frampton also points out that  this lack of predictability is necessary to some degree. “As soon as it is  possible to predict cocrystal structures, we have a problem. Patent  applications for salt forms are already being questioned on the grounds that  they are predictable and obvious due to the pKa of the API and the limited list  of pharmaceutically acceptable salt formers. The lure of the cocrystal remains  the questionable and unexpected successful outcome.”

New formulating,  analytical and modeling techniques

There have  been some advances in various techniques and technologies that are beginning to  address some of these issued. Shan points to the solvent-drop grinding process  as a way to manage mechanochemistry issues and advances in computational  chemistry that are enabling improved crystal structure prediction. 
  

At SAFC Pharmorphix, the ability to perform crystal  structure analyses on really small crystals has been very advantageous. “Knowing  the crystal structure makes it possible to see how the two materials come  together to form a supramolecular complex. Then we can go back to our list of  potential coformers, tune them slightly, and follow an iterative process to  develop the optimum cocrystal,” observes Frampton.
  

Renovo, meanwhile, has developed a new approach to formulating cocrystals that uses the  improved cocrystal solubility more effectively. Traditionally, as is commonly  done with salts, cocrystals are used in 

 pre-clinical studies as  "neat" material (pure crystalline powder), according to Childs.  This approach is not optimal because cocrystals are  highly susceptible to solvent-mediated transformation into the poorly  soluble form of the API when dosed as neat material.  
  

“We  have developed our new approach, demonstrated with a danazol cocrystal, that  utilizes solubilizing excipients and precipitation inhibitors to create an  optimal supersaturation level and controlled dissolution event,” Childs  explains. The model system achieved a 10-X increase in the area  under the concentration time curve (AUC) in  animal studies using the formulation, compared to only a 1.7-X increase in the AUC  using the neat cocrystal solid.  “The  results of this study suggest that the appropriate formulation of cocrystals  can be an important part of the cocrystal development process,” he concludes.

While the  European Medicines Agency and the Ministry  of Health, Labor, and Welfare in Japan have not published any  official guides with respect to cocrystal development, FDA’s final guidance was  issued in April 2013 (1). 

 The  most important aspects of the guidance, according to Frampton, are that FDA  determined that a cocrystal is a drug product intermediate, and it defined two  types of interactions. 
 

 First, the  coformer used to make the cocrystal is defined as an excipient, which can form  a simple physical mixture with the API or interact with the API on the  molecular level within the crystal lattice. Frampton compares the latter  interaction to that of an API incorporated into a cyclodextrin excipient to  enhance bioavailability and solubility, in which the API sits within the barrel  of the beta-cyclodextrin. For salt forms, on the other hand, where it is  important to know where the proton sits in a complex and if it has been  transferred to the API because if that occurs, the salt form is no longer  considered to be the ‘same’ API from a regulatory perspective. 
 

 “Many in the  industry find it quite surprising that FDA has not regarded cocrystals as new  APIs because the properties can vary widely from one coformer to another. It  also makes it easier for generics companies to compete with branded products by  utilizing these materials,” Frampton comments. “Indeed, the guidance leaves a number of open questions and perhaps some concerns  within the pharmaceutical industry about how the use of a cocrystal could  impact development timelines, the drug product manufacturing process, and the  intellectual property position of a product containing a cocrystal,” adds Childs.
 

 First, cocrysals are treated like the polymorphs of a drug,  rather than as salts. As polymorphs they are considered to be the ‘same’ API, whereas  different salts are treated as different APIs from a regulatory perspective.  Secondly, drug product intermediates have different regulatory reporting  requirements than salts or polymorphs. Finally, there are IP issues. Because  cocrystals are considered to be, in effect, the same drug substance, generic-drug  companies can introduce them as competitive products that are still considered to  be the same API, whereas the use of a different salt would not be substitutable  as the same active ingredient.

Shan does point out, though, that a cocrystal still has the same active ingredient as the original drug. “Even so, under the patent protection of the original drug, a cocrystal version of the same drug cannot circumvent the original patent,” he notes. “However, a cocrystal with desired properties can be patented, intellectual-property protected, and marketed, as mentioned previously in the context of lifecycle management.”


“The current perception of cocrystals is analogous to that of amorphous materials during the period when that technology was emerging. While the basic understanding of cocrystals as materials has advanced at a steady pace, there are important questions remaining concerning the regulatory issues, impact of the coformer molecule on toxicology studies, the ability to produce some cocrystals at scale, the cost of production if advanced formulation is needed, and the ability of crystal form IP protection to enable product exclusivity,” says Childs

“All of these areas have been addressed to some degree in the available literature, but I would caution anyone who thinks that cocrystals will remain an inferior solid form, and that the real impact of cocrystals has yet to be demonstrated. With the appropriate time, effort, and investment, our ability to effectively use cocrystals to create improved pharmaceutical products will reach the point where cocrystals will be a desirable and useful solid form rather than a ‘last resort’”, he continues.

“I envision the ability of cocrystals, in specific situations, to be able to compete directly with amorphous dispersions in terms of efficacy but create a superior product because of the lower risk and improved stability of the crystalline state compared to the amorphous state,” Childs concludes.

1. FDA, Guidance for Industry: Regulatory Classification of Pharmaceutical Co-Crystals (Rockville, MD, April 2013), http://www.fda.gov/downloads/Drugs/Guidances/UCM281764.pdf, accessed June 20, 2013.

Articles

Cocrystals can enable the formulation of solid dosage drugs, but the FDA's final guidelines have left concerns about how their use could impact development timelines, the drug product manufacturing process, and the intellectual property position of products containing cocrystals.

Cocrystals Can Improve API Properties, But Will the Recent FDA Guidance Slow Adoption of This Technology?

In the fight  against counterfeit pharmaceuticals, governments are aiming to harmonize  legislation across borders. As a result, several new regulations will be coming  into effect in the coming years, including the European Union’s Directive  2011/62/EU and California’s e-pedigree. This  legislation will impact all areas of the pharmaceutical supply chain as a drug product must  be tracked as it travels from the point of manufacture all the way to the  pharmacy. Track-and-trace systems will require the participation of stakeholders  from all areas of the business, such as packaging, distribution, marketing,  regulatory, IT, and executive management. Although the challenges are  significant, and track-and-trace programs will impact all processes from  packaging through distribution, the benefits can be as equally rewarding.


  A number of major track-and-trace regulations are currently  in the process of being implemented globally. EU Directive 2011/62/EU requires the  implementation of uniquely coded, serialized packs for almost all prescription  drugs, as well as tamper-proof closures, according to Franz Ludwig, product manager track  & trace, Bosch Packaging Services. The new product serialization is  scheduled for application across Europe in 2016. Turkey has had regulations in  place since 2009, and China’s Good Supply (Distribution) Practices (GSP) rules  went into effect June 1 of this year.


In the US, there are several different  state regulations; California’s e-pedigree is the most stringent and requires item level serialization at the unit level  along with full aggregation from the item to the case, and depending on the  distribution model, the pallet, according to John DiPalo, chief operating officer  for Acsis, a provider of enterprise serialization solutions. Furthermore, each  individual saleable unit must be tracked from the manufacturer through to the  point at which it enters the dispensary using an electronic, interoperable  system. By 2015, 50% of all drug products sold into  California must be serialized, with all products in compliance by 2016.  Wholesalers and pharmacies, meanwhile, must meet the requirements by 2016 and  2017, respectively.   

In addition, there are two bills pending at the federal level in the US that are intended to eliminate the patchwork of state requirements, according to Margot  H. Drees, executive director of global  strategy with GHX. On June 3, 2013, the US House passed the Safeguarding  Americas Pharmaceuticals Act (H.R. 1919), and on May 28, 2013, the US Senate Committee on Health, Education, Labor, & Pensions marked up and combined the Drug  Supply Chain Security Act (S. 957) and the Pharmaceutical Compounding Quality  and Accountability Act (S. 959). The Senate is set to vote on the new act sometime  in July. “The intent is to reconcile the two bills and have something on the president’s  desk before the August recess,” notes Drees. She adds that both federal  proposals extend the time horizon in which industry must comply and propose a  lot-level tracking system with individual unit serialization.

The  driving force behind the regulations is to enhance patient safety by reducing  the number of counterfeit, adulterated, diverted, or stolen products in the  marketplace,” asserts Drees. Counterfeiters are increasingly targeting the  pharmaceutical supply chain, which is a dangerous trend that can have fatal  consequences, according to Ludwig. “Counterfeit medicines are thought to  account for up to 10%  of the global  medicines market, and are responsible for many instances of serious illness and  death. All supply-chain players can contribute to increasing the safety of drug  supply,” he says.
  

Enhanced patient safety isn’t the only  positive outcome for companies that implement effective track-and-trace  programs. Pharmaceutical manufacturers and distributors can also gain a host of  operational and supply-chain efficiencies, according to DiPalo. “An integrated serialization system that can combine serialization  data with business data can serve as a targeted visibility solution that  benefits other business activities, such as returns management, recall  management, and chargeback management, as well as helping to ensure 100% order  accuracy. Expired  product and new product introduction management  and forecasting replenishment are other business processes that can also benefit  from track-and-trace/serialization programs once the units are identifiable and  there is visibility across the supply chain, according to Drees. DiPalo also notes that serialization can provide the  ability to spot inefficiencies in the supply chain that are impossible to detect  when items are managed at the lot or batch level.

The  key properties of a track-and-trace system include data security, and for a  cloud-based model, a very strict data governance policy both of which protect  the integrity of the data and the product, as well as the proprietary  information of supply chain trading partners, according to Drees. 
  

The technology alone, however, will not  result in a successful track-and-trace program. “Stakeholders  from all areas of the business must be involved when designing an effective track-and-trace  system, including packaging, distribution, marketing, regulatory, IT and  executive management, and there needs to be cross-functional participation and  buy in,” DiPalo observes. 
  

Furthermore,  Drees believes, data  standards are needed to ensure interoperability across the supply chain. “These  standards include GS1 serialized Global Trade Item Numbers (sGTINs) to identify  individual products and cases of products, GS1 Global Location Numbers (GLNs)  to identify participating facility locations, and the electronic product code  information standard (EPCIS) for event capture.”
 

Another  major key is to fully understand the multiple paths that a product can go  through in both internal and external supply chains.  “Mapping this ‘day in the life’ is very  important for understanding all of the possible exceptions that a system will  need to be able to handle,” notes DiPalo. Finally, according to Drees, the system  must be scalable and highly available to meet the vast data access/storage and  retrieval/reporting that will be required as the industry matures. 

Many of the challenges in  implementing a serialization system are related to the changes required in the  flow of product in order to capture the serial number data and the new systems  required to capture and store the data. In addition to technology gaps and  business process changes, ambiguity about implementation requirements and  timelines, cost, evolving data standards, and the considerable variation in the  level of sophistication of the parties involved are some of the primary  challenges facing the industry today. Incorporation of third-party processing,  such as contract packaging and distribution  operations, into an overall serialization solution is also an area of risk that  companies need to be aware of, because many third-party service providers are not  serialization ready.

Early  adopters are serializing product lines, conducting pilot programs with their  trading partners, and participating in GS1 workgroups to define implementation  best practices and guidelines, according to Drees.
 

In Germany, the association “securPharm  e.V.” is developing technical and organizational solutions in compliance with  the EU directive within a pilot project that was launched earlier in 2013. “For  this purpose, the data matrix code was determined as the data carrier, which  must contain a unique product number and serial number for end-to-end  verification. In addition, because the serial numbers must be randomized for  tamper-proof allocation, secure and reliable software is essential,” explains  Ludwig.
 

In the US, some major healthcare  industry players have been working with GHX to develop a network-centric,  traceability information-sharing system (leveraging a variety of industry data  standards) in an effort to standardize the process for all parties involved and  provide access to actionable supply chain data, according to Drees. Using  secure connections, the various parties along the supply chain can use the GHX  cloud-based repository to capture key drug product events (i.e. commission,  aggregation, shipping, receiving and disaggregation.) The repository processes  the event data using a business rules engine and makes it available to the  authorized trading partners for tracing and chain of custody reporting. The GHX  system also retains a copy of all supply chain data submitted and can provide  redundancy in the event a trading partner experiences a system failure.
  

In a recent traceability pilot referred  to as “Track-and-trace for Serialized Product,” products that were manufactured  and serialized at AbbVie were tracked as  they were delivered to a McKesson Corporation distribution center and then  distributed to the Veterans Health Administration (VHA) Consolidated Mail  Outpatient Pharmacy. The GHX pilot was awarded the 2013 Distribution Management  Award from the Healthcare Distribution Management Association, recognizing GHX,  these three companies, and platform development partners AmerisourceBergen  Corporation, Cardinal Health and Johnson & Johnson Health Care Systems for their efforts in the pilot study.

Serialization regulations in California and the EU come into effect in 2015 and 2016, and two US federal bills are moving through Congress. What are the implications for the pharmaceutical industry?

Track-and-Trace Compliance Looming, but Once Implemented Will It Bring Benefits?

Aseptic fill–finish activities for  parenteral drugs include numerous operations, such as final formulation,  filtration, product transfer, and dose determination. With the success of  single-use systems in biopharmaceutical API manufacturing and the consequent  increased confidence in the technology, it is not surprising that  pharmaceutical companies have growing interest in extending the benefits of  disposable equipment to downstream operations, such as aseptic filling.  Advances in pump designs and other technologies have made it possible for  equipment manufacturers to respond, and single-use components for mixing,  filtration, fluid transfer, dosing, and sampling are now available.
 

 Magnetic, disk,  lightning, and levitating impeller mix systems can be used with disposable bioprocess  containers, and technology is available for the addition of both powders and  liquids in closed or open systems. Single-use filtration systems have been used  in aseptic fill–finish operations for many years and are based on  well-established technologies. For filling, the choice of pump technology is  important. Advances in peristaltic pump designs have made these systems  attractive for single-use aseptic dosing as an alternative to reusable  positive-displacement pumps or time/pressure filling systems. Newer diaphragm  technology is receiving growing interest, however, as a way to reduce particulate  formation, according to Justin  Hutchinson, associate director, product management for cell culture and  bioprocessing at Thermo Fisher Scientific. Some manufacturers offer pre-validated,  pre-assembled, and pre-sterilized single-use diaphragm pumps that fit into  existing stainless-steel pump housings. For sampling, systems that have been  used in biopharmaceutical API manufacturing are suited to aseptic applications. 

There are many benefits to  adopting single-use technology for fill–finish operations, many of which are  similar to those associated with manufacturing operations. “At the top of the  list are the reduction in validation requirements and the elimination of clean-in-place/steam-in-place  processes,” notes Hutchinson. “Indirect benefits are realized as well, such as  a dramatically reduced fill and finish process turnaround times,” he adds.  “Overall, compared to permanent setups, single-use aseptic filling systems  require much less space and labor to operate, generate less waste, have a  shorter turnaround time, require less documentation, and have a reduced risk  for cross-contamination. Together, these benefits add up to increased efficiency  and productivity, and reduced cost,” notes Brandon Pence, Thermo Fisher  Scientific’s director of global marketing for cell culture and bioprocessing.

Many of the same challenges that  stainless-steel parenteral filling operations face also exist for single-use  aseptic filling, namely, particulates and extractables/leachables. Advances in  peristaltic pumps in terms of rate of filling have led to their widespread  adoption in single-use aseptic-filling systems. These single or multiple head pumps operate by pushing liquid through a flexible tube with a hardened roller. Natural  variations (diameter, wall thickness, and flexibility), stretching,  compression, fatigue, and wear of the tubing can lead to inaccurate filling and  the generation of particulates, however. 
  

“One solution is the use of  disposable diaphragm pumps that rely on pressure to move the liquid, thus  eliminating the particulates issue while maintaining filling accuracy,” says  Hutchinson. For the filling of larger bags or containers, a system based on a gravimetric volumetric fill principle that does not rely on any type of pump or  pressurization can be implemented. In this case, the level of the sterile  parenteral drug in a reservoir (fed by a sterile holding tank) and in several  measuring tubes is carefully controlled using sensors to ensure accurate and  reproducible filling volumes.
  

For all of these filling methods, recent developments  in single-use filling needle technology have also led to improvements in speed  and accuracy.

The components of single-use  aseptic fill–finish systems are provided by many different vendors. An  integrator such as Thermo Fisher Scientific, which is familiar with the range  of components from these numerous suppliers, is situated to assemble customized  systems for the final formulator. “Integrators help customers design single-use  aseptic-filling systems that meet their needs in terms of volume, filling rate,  and level of automation. We then assemble the systems, sterilize them using  gamma irradiation, and then package them for use in the customer’s cleanroom,”  Hutchinson explains. He also notes that, with a packaged single-use system, a  manufacturer can begin aseptic-filling operations in about 8-10 hours, a much  shorter time frame than is necessary when using permanent equipment.

Once a customized aseptic-filling system has been assembled,  it is the job of the integrator to sterilize the system using validated gamma  irradiation processes. For some single-use systems, and after gamma  irradiation of an assembled system, Thermo Fisher Scientific conducts verification  testing that involves filling samples with tryptic soy broth (TSB) and exposure  of the system to  incubation for a two-week  period. The sample system is determined to be sterile if no organism growth is  detected. All sterile single-use systems that the company provides come with gamma  sterilization certificates.

One challenge for both integrators  and their customers is the fragmented nature of the supplier base for  single-use aseptic filling components. “As demand for single-use systems  increases, growing numbers of vendors are entering into this market. Each  single-use manufacturer has its own proprietary technology, and as a result  there is a lack of standardization, which can be an issue on many levels,”  Pence observes. “For end-users, it is critical that the industry strive toward a  minimum level of standardization in order to mitigate risks associated with the  use of single-use components from different suppliers while still providing  systems that can be uniquely configured to a particular operation or  application,” he continues. Standardization would also increase the efficiency  of system development, and ultimately lower the cost of disposable aseptic  filling systems.
 

“We believe that the key to  increasing standardization will be the continued move toward greater automation  in single-use aseptic filling systems. In the end, the choice to use, and thus  the acceptance of, single-use systems comes down to performance, quality, and  process economics,” remarks Hutchinson. “In parenteral manufacturing, there is  a natural evolution toward more automated systems in order to ensure reduced  risk of contamination and exposure of workers to highly potent drugs.  Single-use systems are a natural fit for both automation and risk reduction.  Therefore, these two complementary drivers will lead to implementation.” 

Pence predicts strong growth for  single-use aseptic filling systems over the next 5-10 years. “There is growing  confidence in the use of disposable technology for upstream processing. As  increasing numbers of people become comfortable with single-use systems and the  awareness of the numerous benefits of this approach expands, more single-use  aseptic-filling systems will be validated. In turn, confidence in these  downstream systems will increase, and their adoption will spread.” He adds that  the growing percentage of biopharmaceutical candidates (and thus parenteral  drugs) will also drive interest in single-use systems throughout the parental  production cycle, including aseptic fill–finish, as will continued pressure to  further reduce contamination risks while increasing efficiencies and lowering  costs.

The benefits of single-use systems are being realized for downstream unit operations, including aseptic filling.

Aseptic Filling is the Next-Generation Application for Single-Use Systems

Glycosylation of  biologic drugs affects their  mechanism of action, pharmacokinetics, and efficacy. Glycan analysis, as a  result, is critical for determining the quality of these biotherapeutics. Until  recently, analytical methods have been slow and tedious and largely limited to  use in the later stages of product development. The introduction of  high-throughput techniques is, however, creating opportunities for the  application of glycan analysis early in bioprocess development. 


Scott A.  Siegel, chief operating officer, and Hidehisa Asada, vice president of research  and development, both with Ezose Sciences Inc., spoke with Cynthia Challener,  editor of the 

Pharmaceutical Sciences,  Manufacturing & Marketplace Report

, about the importance of glycan  analysis for biotherapeutic development, the impact of recent advances in  high-throughput analysis capabilities, and what further advances such glycan  analysis technology might enable in the future. 

Pharmaceutical  Sciences, Manufacturing and Marketplace Report

:  Why is  the characterization of the glycosylation patterns on glycoproteins important? 
  

:   More than half the proteins expressed in  humans are post-translationally modified by glycosylation. Differences in  glycosylation can affect protein structure, stability, immunogenicity, and  function. Thus, glycomics plays a significant part in health and disease. In  fact, glycans play a key role in normal cellular function. They mediate  interactions among cells and between cells and pathogens. They enable immune  responses. They also have a role in tumor progression. 
  

In addition,  biotherapeutics, including monoclonal antibodies (mAbs), are often glycosylated,  and the extent, type, and location of the glycan structures present on the  proteins impact the mechanism of action, pharmacokinetics, and efficacy of these  drugs. In some cases, when non-human glycan structures are present, they can  trigger an undesirable immune response. Glycosylation is, therefore, often a  critical quality attribute in bioprocessing, and these biotherapeutic molecules  have become a mainstay of the biotech and pharmaceutical industries. 

:  How is  glycan analysis related to the development of novel biomarkers? 
  

:  Glycomics is a natural extension of genomics and proteomics. Glycan patterns  distinguish otherwise similar proteins, so we believe these patterns may serve  to distinguish between healthy and diseased tissue samples, between a disease  that is indolent and a disease that is aggressive, and between a patient in  whom a drug is effective and a patient in whom it isn’t. There are already  examples of marketed, glycan-related products, such as tests to manage cases of  pancreatic and liver cancer. And yet the lack, until recently, of  high-throughput techniques for the analysis of glycans has meant that the  glycome today remains mostly uncharted territory.

Limited by slow and  laborious techniques

:  What are  the challenges in completing effective glycan analyses? 
 

:  Traditionally, glycan analysis has been a slow, labor-intensive process, and  has been more routinely applied late in bioprocess development. Glycan analysis  early in bioprocess development has been limited to the analysis of small  numbers of samples. With the ever-growing focus on biotherapeutics, including  the increased investment in “biosimilars” and “biobetters,” there is, however,  a need to improve the process by monitoring glycosylation patterns across large  sets of samples generated during cell-line selection and early bioprocess  development. The challenge has always been to achieve speed and scope—a  high-throughput technology platform. Glycan analysis and glycomics in general  lacked laboratory techniques comparable to those that accelerated progress in  genomics. But now we’re getting there. 

New technologies  enable high-throughput solution

:  What  recent advances in technology (in general) have been introduced that help  address these issues? 
  

:  Mass spectrometry, automation, sample preparation, and separation methods  continue to improve, permitting more rapid and sensitive glycan analysis. Our  Glycan

 platform leverages several of  these advantages. We can now analyze more than 150 samples a day, and that  number is scalable as the demand grows. That kind of throughput would once have  taken weeks or even months to complete.

Automated enrichment,  mass spec detection, and bioinformatics

 technology, and why did you combine automated chemoselective, bead-based,  glycan enrichment with quantitative matrix-assisted  laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry coupled  to custom bioinformatics in your system? 
  

 technology was  originally developed by researchers at Hokkaido University and has been further  refined for commercial use by Ezose. The platform combines three basic  components: automated glycan enrichment, detection by mass spectrometry, and  custom bioinformatics that convert the mass spectrometric data into glycan  identities and concentrations. 
  

The chemoselective  bead-based enrichment provides rapid clean-up and capture of glycans from  complex samples, including cell culture media and biological fluids, such as  serum, urine, and cell and tissue lysates. Automating this once time-consuming  and tedious step accelerates the analysis and improves reproducibility,  resulting in a more industry-friendly solution. 
  

Mass spectrometry is  used for glycan detection because its mass accuracy provides accurate  identification of the oligosaccharide composition of each glycan, and it  provides a throughput that cannot be attained today with high-performance  liquid chromatography (HPLC). The mass spec data is then analyzed using our  proprietary bioinformatics system, which identifies glycan compositions and  converts peak intensities to absolute concentrations (in µM).

Facilitating  biopharmaceutical production

:  What  further advances might be expected in the near future? Longer term? 
  

:  For the immediate future, we are focused on a new option that makes our Glycan

 platform services routinely  available to researchers, particularly in bioprocessing, who need to rapidly  screen major glycans for cell-line selection and early bioprocess development.  At the same time, we have also driven down our cost for running the service,  and we are passing this savings along to our clients in the form of lower  pricing. We call our new CRO service Glycan

 We continue  to also see value in applying our technology services to discovering novel  biomarkers, particularly those for use in developing new diagnostics for  disease or companion diagnostics to stratify patient cohorts and monitor  response to medicines. Whatever the application, our technology can be applied  broadly to many different types of biomolecules and across therapeutic areas. 
  

So, we have an ambitious research program that  keeps us busy. We can, however, imagine still other avenues of investigation.  Some of our partners are interested in applying our technology to vaccine  development, for example, or using glycomics to develop a deeper understanding  of disease biology and drug mechanisms, and potentially to discover new drug  targets.

Until recently, glycan analysis has been a slow, labor-intensive process more widely used late in bioprocess development. New high-throughput methods are changing that.

New High-Throughput Glycan Analysis Improving Cell-Line Selection and Early Bioprocess Development

    In biological systems, complex molecules are constructed by forming multiple carbon-heteroatom bonds with the aid of enzymes and using modular building blocks (amino acids). First introduced by K. Barry Sharpless of The Scripps Research Institute in 2001, click chemistry encompasses reactions that mimic this modular approach. The most famous example is the Cu(I)-catalyzed version of the Huisgen 1,3-dipolar cycloaddition reaction between azides and terminal alkynes to form 1,2,3-triazoles.

These reactions have several features in common that qualify them as click chemistry reactions. First, they are modular, wide in scope, give very high yields, and generate minimal byproducts that are nonhazardous and readily removed without the need for complex separations (i.e., chromatography), according to Dr. Prakasam Thirumurugan, who previously worked in the Laboratory of Medicinal Chemistry and Neuroengineering at the Medical University of Lublin in Poland and who currently is a postdoctoral associate at the New York University Center of Science and Engineering in the United Arab Emirates.

In addition, click reactions are stereoselective (but not always enantioselective), can be run using simple reaction conditions (ideally in air at room temperature) and readily available and inexpensive starting materials, require no or a benign solvent such as water, and generate products that are easy to isolate using only simple purification processes if necessary, such as crystallization and distillation. Furthermore, the products of click chemistry reactions are stable under physiological conditions.

Significant impact on synthetic efficiency


In recent years, drug development has become more focused on biology-oriented synthesis with the goal of producing molecules and systems that exhibit well-understood biological activity. In addition, the sequencing of the human genome and the genomes of numerous types of harmful microbes has uncovered large numbers of potentially novel biological targets with no obvious and efficient synthetic routes.  

“Conventional organic synthetic approaches are often slow, costly, and labor-intensive, and thus very inefficient for the practical preparation of these complex compounds,” Thirumurugan notes. “Click chemistry, because it mimics the synthetic approach used in biological systems, is a particularly promising strategy for the synthesis of carbon−heteroatom−carbon bonds in an aqueous environment, especially when used in conjunction with combinatorial and high throughput techniques,” he explains. He adds that because click reactions are facile, selective, and reliable, they enable faster discovery and optimization of drug candidates, and should be readily scalable for commercial manufacture. 


    Probably the most widely recognized click reaction is the Cu(I)-catalyzed [2+3] cycloaddition of azides with terminal acetylenes to form 1,2,3-triazoles. “This reaction is very attractive because it proceeds at room temperature, is highly dependable, provides complete specificity to the 1,2,3-triazole scaffold, and uses reactants that are biocompatible,” explains Thirumurugan. As importantly, the triazole structure is found in many different natural products that exhibit biological activity.


    An interesting development in click chemistry has been its used in target-guided synthesis (TGS). In this approach, different building blocks are brought together in the presence of the biological target (an enzyme, DNA, a protein) so that it can act as a template and lead to formation, ideally, of the best ligand. The target is incubated with a mixture of reagents. In thermodynamic TGS (also known as dynamic combinatorial chemistry), different pairs of building block reversibly join, and those that bind most tightly to the target are retained, thus driving the reaction. With kinetic TGS, the building blocks combine irreversibly at different reaction rates that are influenced by the target, and the combinations that react the fastest are produced in higher concentrations. 

It has been suggested that, in the future, it might be possible to use such 

 click reactions to have a drug assembled inside the patient in response to the specific conditions of the disease within that particular patient. This approach has been investigated particularly for the development of enzyme inhibitors for the treatment of a number of medical problems, such as AIDS, cancer, anthrax poisoning, and Huntington’s disease, according to Thirumurugan. 

) click chemistry has also been used for the fragment-based assembly of a number of compounds with specific enzyme inhibition activity, including ultra-potent inhibitors of serine hydrolase. Other compounds, including agonists, antagonists, and selective ligands for receptor−ligand binding studies, have also been readily prepared using click reactions. “What is attractive about these bioorthogonal click chemistry reactions is that the reagents can form covalent bonds within functionalized biological systems. As a result, they have even been used for the site-specific bioconjugation of proteins, glycans, lipids, and cell surfaces inside living animals under physiological conditions,” observes Thirumurugan.

Of course, the click reactions used in biological systems do not involve copper, which is a limitation of the [3+2] cycloaddition reaction described above. For 

 applications in particular, the development of copper-free, and preferably metal-free-ligation chemistries, is important. Copper-free reactions involving azides and strained alkynes, and strain-promoted cycloadditions of cyclic alkenes and alkynes have been shown to be powerful tools in bioconjugations, according to Thirumurugan. 


    Because click reactions are highly selective and modular and involve the use of reactants that do not interfere with biological systems, they are also ideal for the labeling of biomolecules, both inside and outside of living systems. Azides are useful functional groups that can be incorporated into biological molecules and then tracked based on their click chemistry. The reaction of the azides with strained alkynes, such as cyclooctynes, leads to the formation of triazoles without the use of a toxic metal and enables the determination of their abundance, location, and dynamics. More recently, according to Thirumurugan, other metal-free click reactions have been introduced that are useful for this application, including thiol−ene reactions, alkene−azamethine cycloadditions, and the cycloaddition of tetrazine and trans-cyclooctene. 

Additional information on click chemistry in pharmaceutical synthesis may be found in this recent review: “Click Chemistry for Drug Development and Diverse Chemical−Biology Applications”. P. Thirumurugan, D. Matosiuk, and K. Jozwiak, 

10.1021/cr200409f, Publication Date (Web): March 27, 2013. 

Click chemistry has become a powerful tool in drug discovery, chemical biology, and proteomic applications.

Mimicking Nature with the Click of a Reaction

  Spectroscopy—the analysis of the properties of substances based on their interactions (absorption, transmission, reflectance) with electromagnetic radiation—is a fundamental analytical tool in the pharmaceutical industry. Commonplace methods, including infrared (IR) and ultraviolet-visible (UV-vis), and newer methods, such as near-infrared (NIR) and Raman, spectroscopy, use radiation of different wavelengths (i.e., energies) to provide unique information about the structure and electronic configuration of different compounds. Terahertz spectroscopy is the newest entrant on the block, and this low-energy technique is attracting a lot of attention due to its ease-of-use, rapid analysis time, depth of penetration into solid materials, and its ability to provide a wide range of physical and material property information.

Terahertz waves fall on the electromagnetic spectrum between the infrared and millimeter wave regions (300 gigahertz to 10 terahertz, see 

). They are transmitted through many types of materials, such as tablets, capsules, gels, and even liquids and slurries, which enables the analysis of their internal structure. Notably, the intermolecular vibrations between small molecules and the intramolecular vibrations of larger molecules (peptides and proteins) as they fold around themselves occur at this wavelength. Terahertz spectroscopy can thus be used to determine the chemical composition of substances, the presence of impurities, and other properties. In addition, by comparison with a known spectrum, differences between pharmaceutical products such as tablets can be analyzed nondestructively. 

Recently, the cost and size of terahertz instrumentation has decreased significantly to the point where it is now a practical tool for industrial applications, including for pharmaceutical development and manufacturing, according to Ed King, an R&D fellow at Advantest Corporation. He also points to the increasing number of presentations given at conferences and papers appearing in the published literature as evidence that the utility of terahertz spectroscopy is being recognized in the academic community.


  Like many other spectroscopes, terahertz instruments generally have an emitter and a detector (see 

). In this case, femtosecond laser pulses are shined on the detector, causing the emission of energy in the terahertz region. These sub-picosecond pulse duration emissions are directed at the sample and are transmitted, reflected or absorbed. A second laser is then used to digitally sample the waves that are generated after interaction with the substance being analyzed. A single scan can be processed in various ways (e.g., Fourier transforms, time-of-flight analysisto generate a useful spectrum in as little as 8 milliseconds.

“The spectrum of a sample is a unique fingerprint of the interactions (hydrogen bonding, dipole-dipole, and Van der Waals) between small molecules and within larger molecules, providing, for example, crystal lattice information,” observes David Heaps, a principal scientist with Advantest.

Most importantly, rapid, nondestructive imaging and analysis of pharmaceutical samples in the liquid or solid state is possible. “Crystalline qualities, the characteristics of constituents, and even the thickness, density, and internal structure of tablet coatings—previously impossible to analyze nondestructively—may be evaluated and visually rendered in two or three dimensions,” adds King. 

The Advantest system uses a proprietary, in-house-developed broadband emitter using an optical rectification-type, terahertz-wave generation scheme to generate spectral coverage from 0.5 to 7 THz. In addition, spectrometry tailored to various dosage forms—from liquids to solids—and the analysis of physicochemical properties are made possible by simply exchanging the measurement module from transmission to reflectance or to attenuated total reflection, according to senior R&D scientist Mark Sullivan at Advantest (see 


  Tablet coatings can be highly complex systems that consist of multiple layers, contain APIs, and serve a functional purpose (i.e., sustained-release, taste masking) beyond providing the appearance of the dosage form. As the complexity of such coatings increases, the uniformity of the thickness and distribution of active ingredients, interactions between multiple layers and the tablet core, and coating stability all become critical quality parameters, according to King.

 By analyzing the time-delay and amplitude of terahertz wave pulses reflected by different coating layers with different refractive indices, the thickness and density of the layers can be evaluated as can the interfacial adhesion between the layers and the tablet. Changes in the results obtained over a period of time can indicate migration of ingredients within the coating or from the coating to the tablet. 

Furthermore, because terahertz waves can penetrate up to a few millimeters into a solid, the chemical composition of the tablet itself can be evaluated(see 

). In addition to the distribution of ingredients in a tablet, the density and porosity can be determined. This analysis gives results similar to those that are obtained using the hardness test, which requires destruction of the tablet. Terahertz spectroscopy has also been proposed for the evaluation of the uniformity of the density of ribbons produced by roller compaction.

Analysis of polymorphs is also possible. Terahertz spectroscopy gives results similar to those obtained using X-ray powder diffraction, but there are no safety issues associated with low-energy terahertz radiation, and the results are obtained much more rapidly, according to Sullivan. 

Absorption in the THz frequency range is sensitive to crystal-lattice vibrations. These absorptions result in a characteristic spectrum for each crystal form, thus enabling the analysis of polymorphs. “Rapid evaluation is becoming increasingly important as poorly soluble drugs begin to be formulated as cocrystals. In solid-form discovery, maybe 10 different pure polymorphs, hydrates, or salts will be screened to select the best candidates for development. The number of possible forms for cocrystals is significantly higher, perhaps even comprising a combinatorial library of possible forms,” he notes.

Terahertz spectroscopy is also useful for the evaluation of the physical stability of amorphous dispersions of poorly soluble drugs. In all of these cases, being able to extensively characterize the solid form of APIs is critical for establishing a strong intellectual property position.


  The ability to rapidly acquire spectra makes terahertz spectroscopy suited for use in process analytical technology (PAT) applications, according to Heaps. “In many of the aspects of pharmaceutical manufacturing where continuous processing is being actively explored, there is a great need for vary rapid analytical techniques that can be used to provide real-time information.  Terahertz spectroscopy is very attractive for this application because it can be used to monitor multiple different properties very rapidly, and can be used equally effectively for R&D, pilot plants, and commercial-scale production,” he adds.


  A key advantage of terahertz spectroscopy is that it enables the analysis of many material properties that traditionally have required the destruction of the sample. The greater penetration of terahertz waves probes sample depths that are not obtainable using most spectroscopic techniques, and its high sensitivity allows for limits of detection of < 1% for minor components in a sample. In addition, it does not present any safety hazards, unlike X-ray analysis, and extensive operator training is not required with the instrumentation available today. Finally, terahertz spectroscopy can be used to complement FTIR, NIR, Raman, and X-ray powder diffraction, and its speed of measurement makes it attractive for online measurement.

Now at a practical size and price, terahertz spectroscopy is advancing as a nondestructive method of analysis for solid forms and tablet coatings.