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

Cynthia A. Challener

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

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

First step: facility construction in partnership with HHS


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

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

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

Second step: FDA approval of cell-culture vaccine

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

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


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


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


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

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

Articles

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

Novartis' US Influenza Cell-Culture Facility Recognized

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

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

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

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

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


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

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

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

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

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

Addressing reactor plugging on a larger scale

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

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

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

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

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

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

GlaxoSmithKline Adopts Continuous Manufacturing for Preparation of Key Intermediate

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


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

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


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

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

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

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

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

Case study one: deep cold storage of sterile products


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

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

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

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

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

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

Case study two: in-process leak detection


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


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

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

Laser Headspace Analysis Advantageous for Monitoring Sterile Fill-Finish Operations

Granulation is a key processing step in the production of many solid dosage drugs. It is often necessary to convert powdered drug formulations into a physical form that has improved flow and compaction characteristics to produce tablets with uniform content and consistent hardness and other properties. During granulation, powder particles adhere to one another to form agglomerates, and the size of the particles is increased. There are several methods that can be used for the granulation of active pharmaceutical ingredients (APIs) and partial or complete solid dosage formulations. The most widely used techniques include wet and dry granulation. There are numerous other approaches, however, that are used less frequently, such as melt granulation, fluid-bed granulation, spray-drying granulation, extrusion granulation, rotary granulation, slugging, foam granulation, and moisture-activated granulation. (Direct compression is not discussed here as it does not require a separate granulation step.) Selection of the most appropriate granulation process is important because the consequences can be significant with respect to the performance of the tablet.


    Wet granulation is the oldest and most common granulation technique and can be accomplished using different types of equipment, including high-shear, fluid-bed, and twin-screw granulators. It involves blending at high and/or low shear forces with the addition of a liquid. In fluid-bed granulation, for example, an atomized liquid is sprayed from the top or bottom directly onto the solids under a continuous air stream with little or no shear, 

“While wet granulation is used for a large number of pharmaceutical drugs, it does have the drawback of being a high-energy process, because drying is necessary once the wet granulation process is complete,” notes Dejan Djuric, manager of scientific operations at LB Bohle. The energy consumption can be slightly reduced using spray-dry granulation, in which a liquid containing dissolved or suspended solids is atomized and rapidly dried using a controlled air stream to produce a dry powder, which is agglomerated by forcing it again into the atomizing zone, according to Shaukat Ali, technical sales manager with BASF. Djuric adds that twin-screw granulation has the greatest potential of the wet-granulation methods because it is a robust, continuous process. 

   Dry granulation via roller compaction is, on the other hand, the granulation method of choice with respect to cost. It involves the direct physical compaction of the dry powder, resulting in densification or agglomeration. Dry granulation is also attractive because it can be applied in continuous granulation processes and is suitable for APIs that are moisture and/or temperature-sensitive.

Each of the more specialized granulation processes has special features that make them suitable for powdered APIs/formulations with specific physical characteristics or that need to be converted into granules with specific properties. For instance, in melt (or thermoplastic) granulation, a thermoplastic or low melting binder is melted and mixed with the dry powder at higher temperatures, and then the mixture is quenched by cooling, which allows the binder to hold the granules together via solid bridges, according to Ali. With extrusion/spheronization granulation, a pressure gradient force is used to wet or plasticize the mass through an orifice with a linear shear while in rotary granulation, a central rotating disk, rotating walls, or both create centrifugal or rotational forces that spheronize, agglomerate, and/or densify a wetted or non-wetted dry powder. Finally, in slugging, the formulation blend is slugged at a relatively lower compression force, and the resulting granules from the slugs are further compressed into the desired dosage while foam or moisture-activated granulation involves continuous exposure of the powder to a foam (moisture) generated by bubbling of air through a binder solution, thus, allowing the granulation to take place under nearly dry conditions. 

Consideration of API characteristics critical


    While often the granulation method is determined based on what techniques are readily available, the characteristics of the API, such as the melting point (for processing temperature), toxicity (containment issues), particle form and size (flowability), moisture/temperature sensitivity, compressibility/compactability (needed amount of shear), must be considered. In addition, production aspects, such as throughput, scale-up ability, and production time, must also be considered, according to Djuric. “Most importantly, an inappropriate choice of granulation method can lead to API destruction, an improper drug-release profile, compression problems, and more,” he adds. Thus, the characteristics of the final tablets depend on the granulation process, and it is important to identify the appropriate granulation technique for each drug formulation. 

APIs that are good to sparingly soluble in water and amorphous can be granulated with either wet or dry methods. For APIs with poor flowability and compressibility, fluid-bed granulation remains a viable choice, while APIs that are sensitive to moisture but stable at higher temperatures can be granulated using melt granulation. For insoluble and highly crystalline drugs with poor solubility, melt granulation and/or spray-drying granulation may be preferred. Slugging is an option for APIs with poor flowability that are also highly sensitive to hydrolysis, but Ali notes that fluctuations in the forces applied to the individual slugs can lead to variability in the particle size of the granules and, hence, result in reduced content uniformity. In such cases, roller compaction is a preferable choice.  Finally for APIs that will be formulated into tablets with taste masking and/or controlled release profiles, fluid-bed granulation is widely used because it enables the formation of coating layers.

 More than 70% of tablets are designed and developed using wet-granulation processes, according to Ali, in part due to the availability of a wide selection of desired fillers, binders, and disintegrants for these methods. He further points out that wet granulation is often used in the development of very low dose drugs that are challenging to consistently formulate as tablets with good content uniformity. 


    As with any type of formulation, the goal is to obtain the desired drug product with the fewest necessary processing steps and the lowest cost. For solid dosage drugs, that means using the granulation (if required) that provides a tablet with the desired properties (appearance, uniformity, drug-release profile) using the least number/amount of excipients possible and as rapidly as possible. Djuric adds that it is necessary to carry out small-scale experiments to confirm that the chosen method works for the specific formulation because computational tools, while helpful, are not sophisticated enough to be relied upon completely. “The recent introduction of small-scale equipment that accurately predicts granulation behavior for commercial-scale runs has been very advantageous for the more rapid development of granulation processes,” he says.

Identifying any special techniques or methods to ensure a robust granulation method for tableting is often challenging, according to Ali. “Successful granulation depends on the excipients and the API (or APIs), their compatibility in the formulation blend, and the granulation technique used,” he explains. In wet granulation, for instance, the physical characteristics of the powder mixture change with time as agglomeration and densification proceed. The characteristics of the formed granules also depend on the processing parameters, such as the amount and viscosity of granulating fluid, the rate of granulation, and the impeller speed, which must be determined for each granulation process in order to obtain a robust tablet with good quality and performance.  

Beyond these issues, it is important to determine the “end point” of a granulation process in order to obtain granules with the desired particle size and porosity, and with good flowability and compressibility, according to Ali. “Finding an adequate and precise method for determination of the right end point, which is critical for granulation in scale-up and production batches, is highly warranted due to the current lack of accurate and reproducible methods,” he observes.


    As granulation becomes more prevalent in the industry, the need for innovative technologies for monitoring the granulation process is also growing.  New on-line monitoring spectroscopic/microscopic methods, such as near infrared (NIR), acoustic emission, focused beam reflectance measurement (FBRM), and particle video measurement (PVM), for the determination of the end point of granulation are meeting some of these needs, according to Ali. With NIR, the frequencies of certain functional groups and their interactions in the granules during processing can be determined while acoustic emission detects sounds that are analyzed using high-frequency piezoelectric sensors. FBRM uses a focused laser beam to measure the particle size of granules, and PVM provides the microscopic images of the particles during the granulation process.

  Other instruments, such as the FT-4 powder rheometer (Freeman Technology) and the TC Probe (C-Therm Technologies), and general differential scanning calorimetry (DSC) have also been used to measure the energies and thermal properties of dry powders and granules, according to Ali. “These techniques are useful because they provide information on the flowability, compressibility, and permeability of the granules, and can be used to address process analytical technology and quality-by-design requirements.

The characteristics of tableted solid dosage drugs often depend on the granulation process, so selecting the most appropriate granulation technique and monitoring the process are crucial

Granulation Method and Process Monitoring Matter

Extractables  and leachables (E&Ls) associated with parenteral drugs present an ongoing challenge  to pharmaceutical manufacturers. Any time new materials are introduced into the  extensive system that comprises parenteral manufacturing, which includes any  and all filters, containers, and transfer tubing, and all components of the  final drug product packaging, there is the potential that unknown leachable  compounds will accumulate in the drug product. Even though leachables are  typically present in minute quantities, some can cause significant problems for  patients. Thus, E&Ls must be identified to the greatest extent necessary or  possible and, if found to be a problem, eliminated or reduced to acceptable  levels. The challenge, however, is clear: they can originate from any material  that comes in contact with a parenteral drug during filtration, purification,  formulation, transfer, filing, packaging, and storage, from a new additive in  tubing or a container seal or a new ingredient in a washing solution used  during syringe manufacture, and therefore can be continually changing.   

Recognition  of the issues posed by E&Ls throughout the supply chain has been one of the  most important advances with respect to managing this challenge, according to Carsten  Worsøe, a principal scientist with Novo Nordisk. “The  challenge is that every change in the composition and processing of a part of a  device or a container-closure system potentially can change the leachable  profile in the drug product. Such changes can be introduced by the supplier of the  device or container-closure system or any number of subsuppliers or converters.  The importance of bringing the changes forward to the pharmaceutical company can’t  be overstated. This information is critical to a drug manufacturer’s ability to  evaluate the risks associated with the change with respect to the leachable  profile of the drug product,” he explains.

Range of recent closure and device advances

The increased awareness in  the supply chain has led to several advances in container-closure system and  device technologies. “In each case, critical experiences with interactions of leachables  with drug products led to the development of alternative technologies that  minimize those interactions, and in turn, these examples have further underscored  the role that participants in the extended supply chain have to play in  managing extractables and leachables,” Worsøe observes.  

The introduction of coated stoppers for vial  products is one example of such a recent advance, according to Dennis Jenke, a  distinguished scientist in the Technology Resources Division of Baxter  Healthcare Corporation. These  coatings retard the leaching of substances from the stopper’s base rubber  material. In addition to the use of such barrier films and coatings,  elastomer manufacturers are more closely evaluating the raw materials they use,  including critical raw-material quality properties. “Enhanced controls on raw  materials and the use of quality-by-design principles when manufacturing  elastomers will result in cleaner, new elastomer formulations that should  exhibit both reduced levels of extractables and greater lot-to-lot  consistency,” notes Jennifer Riter, senior director of global analytical  services with West Pharmaceutical Services. “The use of new films and coatings  combined with these approaches will help further minimize extractables coming  from an elastomer used in primary packaging for injectables,” she adds. 

Advances have also been made in the  chemistry of UV-cured adhesives used in device manufacture and the process used  to form the needle channel of prefilled syringes, according to Worsøe. In the  former case, the incomplete curing of the adhesives used to glue device parts  together was leading to the interaction of unreacted acrylic acid with  proteins. The solution to this problem was the optimization of the adhesive  formulations and UV curing parameters to ensure that the amount of unreacted  acrylic acid monomer was minimized, which in turn reduced the leaching of acrylic acid-related leachables.       

For prefilled syringes  (PFS) made of glass, the formation of the needle channel is commonly performed  using a tungsten pin at high temperature (approximately 1200 °C). At this  temperature, according to Worsøe, tungsten can be deposited  on the surface of the glass as elemental/metallic tungsten or as tungstates/oxides. The presence of  these tungsten compounds has been shown to lead to  protein aggregation and oxidation in the PFS over time. Different approaches  have been taken to minimize the deposition of tungsten, including optimization  of the pin composition and design and more frequent replacement of the pins. 

The relatively recent commercialization of “low extractables”  plastics such as cyclic olefin copolymer is another example of the development  of new materials for use in the extended supply chain that are specifically  designed to address the E&L issue, according to Jenke.

To effectively screen for E&Ls, it is  essential to use a number of orthogonal analytical methods, or  those that will complement each other with respect to the detection of  different compound classes, according to Worsøe. Such  analytical methods include high-performance liquid chromatography–ultraviolet  spectroscopy (HPLC–UV), liquid chromatography–mass spectroscopy (LC–MS), gas  chromatography–mass spectroscopy (GC–MS), gas chromatography–headspace–mass  spectroscopy (GC–HS–MS), inductively-coupled plasma-atomic emission  spectroscopy (ICP-OES), inductively coupled plasma mass spectrometry ICP–MS, and ion chromatography (IC). Mass spectroscopy is often  coupled with chromatographic techniques for the identification and accurate  mass detection of organic E&Ls. He adds that recent improvements in gas chromatography time-of-flight  mass spectroscopy (GC–QToF–MS) have enabled this technique to be used as an  important method for the identification of unknown organic E&Ls. “More  recently, liquid chromatography-nuclear magnetic resonance (LC–NMR)  spectroscopy and liquid chromatography-solid-phase  extraction-nuclear magnetic resonance (LC–SPE–NMR) spectroscopy  have been shown to be valuable techniques for identifying unknown organic  E&Ls,” Worsøe  says.   

Many of these latest analytical  techniques used for extractables and leachables determinations are improvements  on current technologies; more modern equipment and electronics offer greater  sensitivity, according to Paul Cummings, laboratory operations manager for West  Pharmaceutical Services. West is implementing ultra-high pressure liquid chromatography (UHPLC), which is a  next-generation HPLC technique that requires less solvent and has higher  sensitivity, so also enables the use of smaller sample quantities. Accelerated  solvent extraction, which is an improvement over the reflux extractions used in  the past, is also providing West with more control over temperature, pressure,  and time, thus yielding more reproducible results.

For Jenke, it is the coupling of high-efficiency  chromatographic techniques with information-rich detectors that continues to be  an interesting area of development for E&L analysis.  He points to the  coupling of UHPLC with high-resolution MS detectors capable of providing accurate  mass information, which enables the determination of the empirical formulas for  otherwise unidentified extractables and leachables as being a noteworthy recent  advancement in E&L test methods.  “This development makes the LC  screening process more broad-scope and information-rich, and thus more  productive," Jenke comments.

Even with such advances in the  resolution and accuracy of analytical techniques, there are concerns that  remain. One of the most important issues, according to Jenke, is the fact that the screening  methods commonly employed for organic extractables (GC and LC) are not 100%  efficient in capturing all E&Ls. “Even if one were to identify all of the  peaks in GC and LC chromatograms, it is possible ̶ and in fact likely ̶ that  there would be extractables that the LC and GC methods did not detect.   The questions then are: ‘How does one know whether such uncaptured extractables  exist?’ and ‘How does one find these uncaptured extractables?’ Scientifically  sound and universally applicable answers to these questions are not currently  available.” 

Large-volume  parenterals (LVP) present additional issues when it comes to E&Ls in  the form of the so-called “'volume challenge.'” “The main  challenge for LVPs is the need to develop scientifically based toxicological  acceptance criteria for E&Ls that can be met using currently available  analytical techniques,” Worsøe  notes. With LVPs, such as a dialysis bag used by patients with kidney failure,  the daily volume of the parenteral drug that is consumed can be several liters  compared to injection volumes for a small-volume parenteral (SVP) down to 100  µL.   

If both an SVP and LVP each contain 0.1 mg/L (0.1 ppm) of a leachable  compound, and the two drugs are dosed at 0.05 L and 1 L per day, respectively, the  patient would be exposed to 0.1 mg/L x 0.05 L = 0.005 mg in the case of the SVP  and 0.1 mg/L x 1 L = 0.1 mg in the case of the LVP.  “Thus,” says Jenke,  “all things being equal, leachables would need to be measured in LVPs to much  lower levels than SVPs in order to establish the same degree of safety.”    

In fact, the ‘volume challenge’ can result in a detection limit for the same leachable that is 10,000 times  lower for an LVP compared to that for an SVP, according to Worsøe. “Because it is often the case that such low detection levels cannot be  achieved using existing analytical methods, the approach must be taken to  accept the level that is as low as practically possible,” he concludes.  Although such an approach is a practical  necessity, Jenke notes that adopting this approach increases the uncertainty of  a toxicological safety assessment based on the chemical data. It is appropriate  to address this uncertainty with alternate or additional testing. For example,  if a leachable in an LVP drug product cannot be measured down to a  toxicologically safe level, then it might be possible and appropriate to  measure the same compound as an extractable by extracting the test system with  an extraction medium that is more analytically viable than the drug product. 

Understanding the supply-chain challenge and coupling high-efficiency chromatographic techniques with information-rich detectors are leading to improvements in the management of extractables and leachables in parenteral drugs.

Experts from Novo Nordisk, Baxter Discuss Technological Advances in Addressing the Challenge of Extractables and Leachables

As stem-cell therapies progress from the R&D stage into clinical testing and  ultimately commercialization, the need for reliable and consistent bioreactor  technology for the production of these fragile cells has become more pressing.  Currently, most stem cells are produced using multilayer culture flasks, but  there are limitations with these systems when it comes to large-scale  production.

The BALANCE project, which is funded by  the Seventh Framework Program of the European Commission, the companies  Hep-Art, Pharmacell, and BioPredic, along with the Amsterdam Medical Center and  the University of Edinburgh, aims to develop bioartificial livers that will  provide patients with acute liver failure (ALF) a liver-support system while  they are waiting for a liver transplant or recovery. To do so, the companies  will need access to large numbers of hepatic cells known as HepaRG cells.

In April 2013, EMD Millipore and  Pharmacell entered into a collaboration to develop optimized large-scale  expansion and harvest of HepaRG cells using bioreactor technology. These hepatic progenitor cells will be used to support the BALANCE project. Robert Shaw, commercial  director of EMD Millipore's Stem Cell Initiative and Alexander Vos, CEO of  PharmaCell, spoke with Cynthia Challener, editor of the 

Pharmaceutical Sciences, Manufacturing & Marketplace Report

,  about the drivers for the development of HepaRG cells, the goals of the BALANCE  project, what each firm brings to the collaboration, and the technical hurdles  they face with respect to the large-scale production of stem cells in  single-use bioreactors.

Treatment needed for severe liver disease

Pharmaceutical  Sciences, Manufacturing and Marketplace Report

:  What is driving the interest in HepaRG cells?

  HepaRG  cells are a unique cell line that was isolated from the liver some time ago. Initially developed as a model for studying the hepatitis virus in  culture, the cells demonstrate many of the properties of human liver cells  (hepatocytes).  These cells are being  investigated by the BALANCE Consortium as a means to produce an artificial liver. That is,  to use large numbers of HepaRG cells to perform many of the functions of a  liver. Such an artificial liver would be used in a device designed for patients with failing livers or who need help in maintaining liver  function. 

: What are the expected deliverables for the BALANCE project?

 The project is expected to deliver scalable expansion for the production of  large-scale cell therapies, which is necessary for advancement into human  clinical trials and to deliver these quantities efficiently and effectively. To  be able to deliver a consistent and reproducible supply of cells, further  progress in the areas of cell culture and scale-up, characterization,  enrichment, and purification are required.

Combining microcarrier and human cell culture  technologies

: What expertise/capabilities does each company bring to the  collaboration? 

 EMD Millipore brings specific  knowledge in the area of microcarrier-based cell culture, bioreactor growth of  microcarrier cultures, and the scale-up and handling of large volumes of cells.  EMD Millipore is helping Pharmacell develop a method to produce the very large  numbers of cells (tens of billions) that are needed to generate such an artificial liver. Bioreactors have already been delivered to Pharmacell, and cell culture is underway to develop the process. Much communication by phone and email is needed to help work out all of  the technical details between the two companies.

PharmaCell provides GMP contract manufacturing services and process-development  solutions in the area of cell therapy and regenerative medicine. For this  project focused on HepaRG cells, PharmaCell’s advanced know-how in culturing human cells under GMP conditions is vitally important. 

: What are the critical challenges/hurdles that must be  overcome in order for the goals of the project to be met?

 As more cell-based therapeutics progress towards clinical testing, the  consistency, quality, and reproducibility of large-scale culture systems become  an imperative. The ability to ensure that a population of cells can be  reproducibly produced in a manner that can be validated is critical to the  success of clinical applications. The technologies developed as part of this  collaboration will help enable such consistent production and move the field  forward. 

The first challenge was the screening of microcarriers in order to select the right surface for the cells to grow on. The selection process involved the use of both static cultures and small-scale dynamic cultures in spinner flasks. Next, mixing studies with the bioreactors were initiated in order to determine the optimal mixing conditions for the microcarriers. The major challenge remains in translating the relevant parameters for microcarrier culture (shear stress and oxygenation) from small spinner flasks (approximately 125 milliliters) to the big media volumes used in the bioreactors (3-200 liters) without affecting the potency of the HepaRG cells. The optimal cell number per microcarrier for attachment and the best feeding and control strategies for the large vessels must be determined.  Finally, protocols to harvest (release the HepaRG cells from the microcarrier surface) and separate the detached cells from the carriers must also be developed.

: What other applications might the new technology that is  developed during the collaboration be used for? 

:  The advantage of using microcarriers  and bioreactors is that this technology can be applied to any adherent cell  line.  Although the specific cells from each line may require different  microcarriers, the attachment, growth conditions, etc, used in the general  approach remain the same. Therefore, the approach becomes a platform technology  that can be used with many other cell types.  

EMD Millipore and PharmaCell have entered into a collaboration to develop optimized large-scale expansion and harvest of HepaRG cells using bioreactor technology.