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

Cynthia A. Challener

Plants, like human and animal cells,  are capable of producing complex and folded proteins, and plant-based protein  expression technology may provide advantages over cell-culture systems. Bio-Manguinhos/Fiocruz,  the  technical scientific unit of the Oswaldo Cruz Foundation (Fiocruz), which produces  and develops immunobiologicals for public health needs, primarily in support of  programs run by the Brazilian Ministry of Health, is taking plant-based  expression technology to the next level. It recently signed an agreement with  GE Healthcare for the design of a new plant-based multipurpose  biopharmaceutical and vaccine manufacturing facility using a plant-based  expression and production platform from iBio, a developer of such systems.

Plant-based  protein expression is not new technology. The first human test of a therapeutic protein made in plants was conducted by NeoRx  Corp. in 1997, according to Robert Erwin, president  of iBio. The experimental cancer  therapeutic Avicidin incorporated a monoclonal antibody produced in transgenic  plants by a unit of Monsanto. Although there were no problems with the quality  of the plant-made antibody itself, the Avicidin product was not ultimately  commercialized. About the same time, Planet Biotechnology tested a transgenic  plant-derived antibody for the prevention of tooth decay in collaboration with  Guy’s Hospital in London. 

Transgenic  plants have since been used to produce several products that have entered human clinical trials, including gastric lipase, interferon, and insulin.  Plant-produced human glucocerebrosidase, produced in transgenic carrot cells by  the biopharmaceutical company Protalix BioTherapeutics, received FDA approval. In  May 2012, Protalix and its partner received FDA approval for Elelyso (taliglucerase alfa), a plant  cell-produced glucocerebrosidase enzyme  replacement therapy (ERT) for the long-term treatment of adult patients with  Type 1 Gaucer disease. Protalix is also developing an oral form of glucocerebrosidase; Elelyso is parenterally delivered. Viral  vectors were used to produce proteins for individualized treatment of  non-Hodgkin’s lymphoma in a Phase I clinical trial conducted in 2002 (1), and Icon  Genetics is currently conducting a Phase I clinical trial of individualized  cancer therapy based on its transient expression technology for producing  proteins in plants. 

Compared to the traditional use of animal cells grown in bioreactors, plant-based protein expression systems have  several advantages, according to Erwin. Because they do not require the time-consuming  identification and isolation of high-producing cell clones that need to be  scaled up for manufacture, plant-based systems can be operational in much less  time. In addition, the plants are grown in a soil-free, hydroponic medium under  strict climatic and lighting controls for the highest degrees of  reproducibility (see  Figure 

). As a positive consequence, no animal-derived  products or growth factors are used, thereby eliminating the risk of the  introduction of human pathogens into the manufacturing process. Thus, the  expensive aseptic liquid-handling process steps for cell culture are not  required.  

The iBioLaunch technology from iBio  is a third-generation plant-based protein expression platform that uses unmodified  green plants. The first-generation system, which some companies still use, relies on transgenic (i.e., genetically modified organisms)  plants, a process that is slow and relatively  low in yield. Second-generation systems, also still used by some companies, are based on the transient virus-delivery of genes into nontransgenic plants. Although this  technology works rapidly, there are yield constraints and target product-size limitations that restrict its applicability, according to Erwin.

“Harnessing  the combined biological features of bacterial, viral, and plant biology to  achieve the speed of transient production without the yield and size  limitations of older technology, iBioLaunch technology has the advantages of  speed, simplicity, flexibility, and lower cost,” Erwin asserts. He says that unlike  more conventional systems that fundamentally alter a host cell’s DNA, there is  no need to isolate and expand rare, high-producing cell clones to production  level. A gene of interest can be cloned  and inserted into the iBioLaunch vectors and be ready for production at scale  within a month’s time. In addition, the transient nature of the expression system  allows the accumulation of massive amounts of desired protein over a four- to seven-day period, after which time purification can begin.

“The  simplicity of the iBioLaunch system comes from the use of whole plants for the  manufacture of proteins,” explains Erwin. “These plants can be grown in  chemically defined media in the open air or climatically controlled greenhouses  and do not require the expensive aseptic-culture systems required to prevent  contamination of cell-culture production methods. Furthermore, the system is  highly flexible, thereby allowing the expression of vaccine antigens,  monoclonal antibodies, replacement enzymes, blood-cell growth factors, and many  other products that cannot be produced by more traditional expression systems,”  he adds. Other important benefits include the ability to produce proteins  that are difficult to obtain using other methods and the ability to scale-up capacity  readily, for example, in developing treatments for pandemic disease. Also, using whole plants to provide the biomass for manufacturing  obviates the need for bioreactors and media and also reduces development times  associated with traditional cell-culture manufacturing.

Erwin notes that various proteins  have been successfully produced with iBio’s platform, including antibodies,  interferons and cytokines, growth factors, protease inhibitors, clotting  factors, and enzymes for replacement therapy (specifically alpha-galactosidase  A). In addition, a variety of antigens for vaccines against diseases, such as  influenza, anthrax, malaria, yellow fever, sleeping sickness, plague, and human  papilloma virus have been produced, as well as several therapeutic antibodies  for the treatment of acute influenza and anthrax infections. “Virtually any  protein that can be produced in prokaryotic or other eukaryotic expression  systems can be made in plants with the iBioLaunch platform with significant  cost advantages,” Erwin states.

The iBioLaunch technology was validated through the construction of a cGMP pilot manufacturing  facility in Newark,  Delaware that has provided clinical-trial  material for multiple FDA-approved studies, according to Erwin. Human testing  of products made using the iBioLaunch platform began in 2010 with the  initiation of Phase I clinical trials of an H5 avian influenza vaccine  sponsored by the Bill & Melinda Gates Foundation and a vaccine for H1N1  influenza sponsored by the US 

Defense  Advanced Research Projects Agency

,  the results of which were published in 2012 (2). 

Bio-Manguinhos/Fiocruz is interested  in and investing in the iBio plant-based technology." “ "In order to  accomplish our mission and to meet the Brazilian Ministry of Health’s demands,  we are constantly investing in innovative technologies,” says Artur Couto,  director of Bio-Manguinhos/Fiocruz. “The agreement with GE Healthcare is  important because it will allow us to build a new facility based on a very  innovative platform and address an important need of the Brazilian Ministry of  Health,” he says. 

Bio-Manguinhos/Fiocruz  also  has an additional co-development agreement with iBio and its research and  development collaborator Fraunhofer USA Center for Molecular Biotechnology  (FCMB), which will continue to play a key role in advancing iBioLaunch,  according to Erwin. The contract with Bio-Manguinhos/Fiocruz  in Brazil for the design of a new plant-based biomanufacturing facility is the  first for the global alliance between GE Healthcare and iBio, which was  announced in 2012. The agreement with Bio-Manguinhos/Fiocruz and GE Healthcare  for the new plant-based production facility was announced in April 2013.

Although  the details of the process and facility design are confidential, a typical  plant would be based on the following approach. The front-end of the process  would involve technology proprietary to iBio, including the production of green-plant  biomass using hydroponic systems, the transfer of appropriate recombinant  protein expression vectors into the plants, and the extraction of protein from  the biomass (See Figure 

). Downstream operations,  including separation and purification of the target protein and final product  formulation, could be based on the proprietary products and expertise of GE  Healthcare.

, 105 (29), 10131–10136 (2008). 
  2. J. A. Chichester et al., 

Articles

GE Healthcare's partnerships with iBio and Brazil's Bio-Manguinhos/Fiocruz for a new plant-based multipurpose biopharmaceutical and vaccine manufacturing facility move plant-based protein production to the next level.

GE Healthcare, iBio, and Bio-Manguinhos/Fiocruz Advance Large-Scale Plant-Based Protein Production

For pharmaceutical manufacturers, finding the cause of product-quality failures and ensuring product quality are  crucial, and various methods can be used. Industrial X-ray imaging is used in  the pharmaceutical industry in a variety of ways, from product-integrity and  materials-distribution analysis to improving product-delivery methods and the  integrity of packaging.  It is also used to address manufacturing  challenges that may occur in the hardware and machinery of a pharmaceutical  manufacturing plant. In short, industrial X-ray imaging can be used to provide  an inside perspective of a product or the mechanisms used to produce that  product.  

  There are two primary modes of  industrial X-ray imaging.  The first is two-dimensional (2D) digital  radiography, which involves capturing static or in-motion "real-time"  images of the inside of an object (or living organism) by passing X-rays  through that product and capturing them on a digital detector.  “This technique  is often used for the simple detection of foreign materials and contaminants in  a product. It is also effective for investigating the distribution of materials  within a delivery device, such as looking at a drug inside of a syringe, or the  active ingredients and excipients in a solid dosage form,” says Jeffrey Diehm, comanaging director of Avonix Imaging, a provider of industrial  X-ray and computed tomography (CT) imaging services and systems. 

The  second, more advanced X-ray imaging technology used in the pharmaceutical  industry is three-dimensional (3D) computed tomography imaging.  With this  method, the same X-ray imaging technology that is used for 2D imaging is also used,  except that instead of capturing a single static image, the sample is rotated 360  degrees while capturing a series of images. These images are then  "reconstructed" into a true three-dimensional representation of the  internal and external features of the sample.  “This imaging method is  quite valuable because it provides the only means for capturing the internal  geometric data of a sample as well as the density distribution within,” asserts  Brian Ruether, Diehm’s business partner and resident technical expert. 

The most common uses for X-ray  imaging in the pharmaceutical industry include process and design verification,  product-quality analysis, foreign-material determination (metal, glass,  organics), fill-level confirmation, and missing-components detection. These  analyses are applied to the various types of solid and semisolid dosage forms,  including tablets, capsules, and gel caps. Packaging materials–from medicine  bottles to syringes–can also be evaluated using digital X-ray and computed  tomography.

One  of the main advantages of X-ray imaging is that it is a nondestructive  analytical technique. Its depth of penetration and reasonably high resolution are  also critical aspects of the technology that make it very useful for  pharmaceutical analysis. There are no other methods that can define the internal  geometry of an object or provide a true three-dimensional view of a finished  product in a nondestructive manner, according to Ruether. In  addition, he points out that X-ray imaging offers high internal and external dimensional accuracy and the ability  to distinguish between materials of varying densities within a sample while  also being fast and cost-effective. 

X-ray imaging can be used to  determine the thickness of layers in multilayer tablets and the shape and size  of the interface between layers, investigate the microstructure of  fast-dissolving tablets and the features of embossed markings on tablet  surfaces, evaluate the morphology and pore-size distribution of granules, and  identify defects in compacted tablets.

In  addition, as long as the materials that comprise individual pills and capsules generate  sufficient X-ray contrast, high-resolution  computed tomography can be used to determine the distribution of the ingredients  (APIs and/or excipients) throughout the pill or capsule by segmenting the components  by size and/or density (see 

 1). The higher energy of the X-rays enables them to readily penetrate most  pharmaceutically relevant excipients and APIs. In addition, with the short-wavelength  radiation, it is possible to get high-resolution images with minimal  diffraction.

The  information available from such an analysis can be used for several purposes.  Comparative evaluation of the internal structures of two supposedly branded  tablets using X-ray imaging can, for example, be used to identify counterfeit  products without damaging the questionable tablets. Changes in the structure of  solid and semisolid drug products over time (i.e., during storage) or under  different stresses can also be identified using X-ray imaging.

Even  the performance of manufacturing processes can be evaluated, and potential  sources of product-quality inconsistencies identified. In one case, a  pharmaceutical company observed that a drug product produced at different  manufacturing sites exhibited inconsistent efficacy depending on the production  location, according to Diehm. To determine the cause of the variation, X-ray  imaging was used to evaluate the level of crystallinity of the interior of tablets  manufactured at each site because the API was crystalline while the binder was  not. “Using high-resolution CT scans, we were able to  determine that there was a significant quantifiable difference in the makeup of  the pills coming from the different plants.  This information was used by  the manufacturer to modify the manufacturing process and achieve a higher level  of consistency between plants,” he explains. 

Improving  delivery and packaging systems

X-ray inspection in pharmaceutical  manufacturing can also be used to simultaneously inspect products for multiple  quality control issues, which safeguards the integrity of the drug product and  brand while ensuring regulatory compliance, according to Diehm. In addition to  formulated dose analysis, X-ray imaging can be used to detect product defects ranging  from insufficient fill levels to damaged seals to misshapen containers to  missing components (See 

 2). “We  have worked with pharmaceutical clients to identify the sources of  inconsistencies in their manufacturing processes and help them validate process  improvements in order to ensure that the products being produced meet the  stringent requirements of the industry,” he comments.   

For  one pharmaceutical company, Avonix Imaging used X-ray  analysis to identify the cause of inconsistencies in the delivery performance  of their syringes. Variation in the syringe- manufacturing process was  resulting in patients not receiving  the prescribed dosage of liquid in the right amount of time. “By analyzing CT  scans of the syringes, it was determined that there was not only a difference  in the inside diameter of the needle but, more importantly, there was also an  inconsistency in the shape of the syringe opening, which sometimes resulted in the  formation of a small burr or ridge that led to the inconsistency in the flow  rate,” Diehm says. With this information, the company was able to modify the  manufacturing process and eliminate these issues.

Even minor flaws in basic product packaging can cause  real problems for drug manufactures and their customers. In many cases, X-ray  imaging can help identify these defects as well. Diehm offers a case in point from a liquid antacid manufacturer that was having a problem with the sealing  of one of its bottles, which benefited from the technique. In this example, the  defective seals were leading to leakage once the  bottles were opened for the first time.  Avonix Imaging scanned numerous  bottles made using different plastic molds and found variation in the wall  thickness of the bottle at the top surface.  “This thickness variation made  it difficult to properly seal the bottle unless enough force was applied, which  was often difficult for elderly patients in particular. Here again, using the  dimensional information provided by the CT scans, it was possible to identify  the source of the problem. As a result, the manufacturer changed the problem  molds and gained more control over its process.”

Recent advances in X-ray imaging  technology have included new, higher-resolution X-ray tube and imaging detector  combinations, along with stronger computing platforms, which enable the imaging  of smaller internal features.  In addition, advanced 3D  analysis software can now provide automated void and particle-density  distribution calculations from the CT data, according to Ruether.

Currently,  the one major limitation of X-ray imaging is that it is not ideally suited yet  for in-line analysis, particularly for certain features of interest. “The  technology is constantly evolving, though,” stresses Ruether. “Higher-resolution and  higher-speed detectors, in combination with advancements, in software analysis  will help overcome the issues that are limiting the use of X-ray  analysis in production environments.”

Additionally,  education will encourage further adoption of X-ray imaging technology. “Our  main goal is to help educate the pharmaceutical industry about how Xray and CT  imaging can be used as a tool for overcoming the challenges that drug  manufacturers face every day,” Diehm says.  “As they learn more and see  first-hand how they can improve the quality and consistency of their products  through the use of X-ray imaging technology, they begin to think of other  manufacturing challenges that might be better addressed through the use of this  nondestructive method," he observes. 

Greater sophistication in 3D X-ray imaging technology raises its utility for QA/QC in manufacturing.

Addressing Manufacturing Challenges with Nondestructive X-Ray Imaging

In the second half of the 2000s, Fujifilm was extending its  business interests outside its traditional camera and digital products. The  company already had a strong presence in medical diagnostics, and felt that many  of the proprietary technologies cultivated in these businesses could be used in  pharmaceuticals, according to Steve Bagshaw, managing director of Fujifilm  Diosynth Biotechnologies, UK.  Thus, in 2008, the company created a  Pharmaceutical Products Division that includes a growing portfolio of companies,  such as Toyama Chemical Company and Fujifilm Kyowa Kirin.

Fujifilm Diosynth Biotechnologies is another  part of the Pharmaceutical Products Division. It was formed following the  acquisition by Fujifilm of the Merck Biomanufacturing Network.  Merck had created the Biomanufacturing  Network following its acquisition of the former Avecia Biologics in February  2010, which it partnered with the former Diosynth Biotechnologies (previously owned  by Schering Plough).

The company consists of sites in both the UK  and the US, which according to Bagshaw, have complementary technologies and facilities and work closely  together to share ideas, services, and expertise in order to benefit their customers.  “While separate business units with individual leaders, the business is  marketed as a single entity with a shared sales team, and the key R&D and quality  functions are also unified under single leaders,” he notes.  The decision on where a customer’s product is  developed and manufactured depends on a number of variables, including  geography, technical fit, and capacity. 

When Fujifilm acquired Fujifilm Diosynth  Biotechnologies, the stated intent of the company was to become a leading global  biologics CMO. “Our two former companies were recognized as world leaders in the  development and manufacture of microbial-based biopharmaceuticals, but had somewhat  limited capabilities and capacity in mammalian-derived products. To meet  growing market demand, over the last two years we therefore upgraded our sites  in both the US and UK,” Bagshaw explains.
  

Specifically,  cGMP mammalian facilities at its Research Triangle Park, North Carolina site were  expanded with the addition of a 1000-L single-use bioreactor (see 

) that complemented the firm’s existing  2000-L stainless steel bioreactor and process-development capabilities at that  site. At its Billingham, UK  site, Fujifilm  Diosynth expanded its  mammalian capabilities with a new cell line and process/analytical development  services and established a dedicated team of 30 people in this area.  The latest high-throughput technologies are used  to enable the rapid transition of biologics from discovery to clinic, according  to Bagshaw.

In addition, a dedicated mammalian cell-culture  manufacturing facility at the company’s Billingham site is currently under  construction and due for completion in the fourth quarter of this year.  This facility will primarily use single-use  technologies, including a 1000-L single-use bioreactor; a second 2000-L  bioreactor is also already planned for 2014. Furthermore, Fujifilm  Diosynth’s Cell Banking Facility  opened in May 2013 and is working on its first customer programs.  
  

The company has not neglected its long-established  microbial-based business either. Over the last two years, it has upgraded a  number of development and pilot-scale laboratories at its Billingham site, with  the installation of new equipment, including eight 

 sterilizable 20 L Sartorius Stedim Biotech fermenters and a  number of fully instrumentable DASGIP parallel bench-top high throughput units.  The R&D teams at both the UK and US sites have also been significantly  increased over the last 12 months.
  

Finally, both sites have seen upgrades in  their analytical development laboratories, with the installation of new  equipment (see 

), including ultra  performance liquid chromatography (UPLC), capillary  electrophoresis-sodium dodecyl sulfate (CESDS), and imaged isoelectric focusing (IEF) instruments that  deliver rapid results in support of the cGMP manufacture of customer drug  products or as a stand-alone service, according to Bagshaw.

Taking advantage of single-use technologies

Much of the new capabilities that Fujifilm Diosynth  Biotechnologies has installed incorporate single-use technologies. “Our  decision to invest in single-use technologies and equipment at the two facilities  was based on three factors: compliance, flexibility and speed,” Bagshaw  observes. He points to the fact that single-use technologies are much more  compatible in a multi-product facility because the risk of cross-contamination  is much reduced, no matter how potent the product. Much reduced cleaning times  also allow the company to offer a much faster turnaround between customer  campaigns, and simplified materials and waste management are further benefits.  
 

 “In addition, a new facility using single-use  technologies can be on the ground in a much shorter timeframe than a  traditional stainless steel facility. The new facility at the Billingham site is  taking less than 18 months from ground-breaking to validation, which can be compared  to the 2-5 years that is required for a stainless steel facility,” says Bagshaw.  The same is true for capacity expansion to  meet customer demands.

To reach its goal of becoming a leader in the biologics CMO  industry, Fujifilm Diosynth Biotechnologies has extended its enhancement activities  beyond its own facilities. Most notably, it recently formed a key alliance with  Piramal Healthcare UK Ltd. “This partnership focuses specifically on the  contract development and manufacture of antibody drug conjugates (ADCs), a  growing niche in biopharmaceuticals. It brings together the expertise of  Fujifilm Diosynth’s biologics development and manufacturing with Piramal’s  world-leading experience in ADC, offering customers a ‘one-stop-shop’ approach,  thereby reducing complexities in the supply chain and vendor management  relationships and leading to shorter time to clinic,” Bagshaw says.  
  

Fujifilm  Diosynth has already received substantial interest in this alliance and is in  discussions with a number of customers for the development and manufacture of ADCs.  The company is also exploring other opportunities. “We are always looking for  ways to enhance our offering to meet customer needs, either by adding  facilities ourselves, or working with third parties,” Bagshaw comments.  In fact, the firm is currently in discussion  with two companies regarding potential alliances, but he is not in a position  to provide details at this time.

Since its inception in 2011, Fujifilm Diosynth Biotechnologies has added capabilities and single-use production capacity for mammalian-derived product manufacturing, upgraded development and pilot-scale microbial laboratories, and installed advanced analytical instrumentation.

Fujifilm Diosynth Biotechnologies in an Expansion Mode

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.

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.