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

Cynthia A. Challener

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.

Articles

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.

Collaboration Formed to Develop Large-Scale Bioreactor Production of Stem Cells

Tetracyclines  comprise a group of antibiotics that are recognized as safe and effective and  are thus commonly used to treat serious bacterial infections and other less  severe conditions such as acne. Unfortunately, because tetracyclines are  commonly used, many bacteria have developed resistance to the older versions of  these drugs. Recent efforts have thus been directed at developing new tetracycline  derivatives. 

Scientists at Tetraphase Pharmaceuticals  are overcoming this barrier by implementing a new synthetic route first  reported by Myers in 2005 (1). This approach involves the coupling of a cyclohexenone  intermediate that contains the key tetracycline functionalities with a second  functionalized aromatic intermediate via a Michael-Dieckmann reaction, thus enabling the incorporation of a variety of different  substituents at various positions in the tetracycline skeleton. Using this  methodology, Magnus Ronn, vice-president of CMC at  Tetraphase Pharmaceuticals and his colleagues at the company recently reported  the successful preparation of eravacycline, a fully  synthetic broad spectrum 7-fluorotetracycline in clinical development, in multihundred  gram quantities (2). A summary of their work is presented below.

The  advantage of this approach to the synthesis of tetracycline analogues is that a  single key intermediate can be used to access a wide range of substituted  tetracycline active pharmaceutical ingredients (APIs),” says Ronn. This key  intermediate is a tricyclic cyclohexenone with three chiral centers  (the synthesis of this compound was reported  previously (3)).  The enone is reacted  with a suitably functionalized phenol bearing an 

-methyl  substituent. Other functionalities are included as needed to produce the  desired tetracycline analogue. 

This aromatic compound, referred to by  the researchers as the lefthand piece (LHP), is deprotonated with a strong base to  form a benzylic anion, which then undergoes diastereoselective 1,4-conjugate (Michael) addition to the enone moiety when added to the cyclohexenone. The  ketone enolate that forms from this step undergoes a Dieckmann-type  condensation with the phenyl ester to produce the protected tetracycline  compound. To obtain the desired tetracycline analogue, this intermediate is  subjected to subsequent silyl-ether cleavage and hydrogenolysis of the benzyl  protecting groups with concomitant reductive ring opening of the isoxazole (2).

The  LHP selected for the preparation of eravacycline is a benzyl-protected phenol with a fluorine atom and a dibenzylamine substituent. It was prepared from a commercially available starting material in seven steps, the synthesis of which  will be published in the future (2).

One  of the hurdles that the researchers had to overcome in developing the  large-scale synthesis of eravacycline was the sensitivity of the Michael−Dieckmann  transformation to the reaction conditions, according to Ronn. Not only the  order of addition, but the strength of the base was important for the two  different deprotonation steps (2). Thus, the researchers reported that it was  necessary to first deprotonate the LHP (1.04 equivalents of LHP is used) with  lithium diisopropylamide (LDA, 1.13 equivalents) and then add the generated  anion to a solution of the cyclohexenone and the weaker base lithium  bistrimethylsilylamide (LiHMDS) at -70 °C. The desired adduct was isolated  after workup and trituration with methanol in > 90% yield a 98% purity (using  high-performance liquid chromatography), even on the 200-g scale (2).

Because both the  deprotonation and the Michael−Dieckmann reaction should be performed at -70°C, two cryogenic reactors are required. The  researchers reported that attempts to eliminate one of those reactions by  raising the temperature of the cyclohexenone solution to -20 °C led to increased production of impurities (2).

To obtain eravacycline, the first step after the Michael-Dieckmann reaction involved  cleavage of the 

-butyl silyl (TBS)  protecting group. Despite the issues associated with using hydrofluoric acid in  commercial manufacturing, the researchers reported that this reagent gave  better results than other investigated alternatives and it was thus selected  for scale-up (2). 

Reductive ring opening of the  isoxazoline group and removal of the four benzyl groups using palladium on carbon(Pd/C)/hydrogen to give the 9-amino-7-fluoro-sancycline required extensive  investigation by the researchers (2). A mixed solvent system of tetrahydrofuran  (THF) in methanol (1:3) was required because of solubility issues. An acid additive was also needed to improve the rate of the hydrogenation reaction, but  epimerization at the C-4 position and reduction of undesired groups led to the  formation of impurities, including one that was very difficult to separate from  the desired product. The reaction was optimized using concentrated aqueous  hydrochloric acid (HCl) because it is a stable reagent with a reliable  concentration. The palladium on carbon was removed using Celite, and residual palladium  was eliminated with the metal scavenger (SiliaBond DMT, Silicycle). The desired  hydrochloride salt was precipitated from water/ethanol in approximately 80%  yield and high purity (< 2% of the undesired impurities), even on a large  scale (2). 

Next, the hydrochloride salt of the fully  deprotected penultimate intermediate was coupled with the desired side  chain to prepare eravacycline. The reaction was carried out in acetonitrile and water. To achieve complete  conversion, several charges of the acid chloride were necessary, and it was  also found that adjustment of the pH from approximately 3 to approximately 7 after  the second charge aided the complete dissolution of the starting material, allowing  the reaction to go to completion. After the completion of the coupling, the pH  of the reaction solution was brought to pH 6.8 to ensure hydrolysis of any over-acylated  compounds to the desired tetracycline product.

Eravacycline was extracted using  dichloromethane at pH 7.4. As an added benefit, the researchers found that the  undesired C-4 epimer was partly removed in the aqueous layer and when the dichloromethane  solution was dried with sodium sulfate prior to evaporation, thus increasing  the purity of the tetracycline product (2). Finally, the bis-hydrochloride salt  of eravacycline was prepared using an ethanol−methanol mixture  containing an excess of hydrogen chloride and precipitated with addition of  ethyl acetate. 

“While some of the steps presented  challenges, this overall route to eravacycline has enabled the production of sufficient  quantities of the API for clinical testing. This tetracycline derivative has  completed Phase II clinical studies and has been shown to be active against  multidrug resistant bacteria and is therefore a candidate as a broad spectrum  antibiotic for serious hospital infections. We are continuing to improve the  process for future larger-scale manufacturing and are also developing an  isolation procedure that will be suitable for commercial production of  eravacycline,” Ronn notes.

M. Ronn et al., Org. Process Res.  Dev. 17 (5) 838-845 (2013). 

Synthesis of tetracycline derivatives with novel substituents has been challenging. Using an approach based on a Michael-Dieckmann reaction, new compounds with enhanced antibiotic properties are now being prepared on a large scale.

Tetraphase Pharmaceuticals Achieves Process-Scale Synthesis of New Tetracycline Derivative

The  pharmaceutical industry is experiencing a period of major changes. The age of  the blockbuster drug seems to be over, and many major products have lost patent  exclusivity in recent years. The focus now is on niche therapies designed for  smaller-volume, targeted patient populations. Drug manufacturers are also under  increasing pressure to reduce costs. As a result of the shift to smaller-volume  products and as a consequence of large acquisitions over the last decade or so,  there is significant equipment/facility redundancy, and companies are looking  to achieve cost recovery through the proper management of these assets. Matt  Hicks, chief operating officer of Federal Equipment Company, a supplier of used processing  equipment and asset management services to the pharmaceutical and other  industries, spoke with Cynthia Challener, editor of the 

,  about different strategies for surplus asset management and provided real-world  examples of how they were implemented.

Pharmaceutical Sciences, Manufacturing & Marketplace Report: 

What changes in  the pharmaceutical industry are impacting asset management for manufacturers?

There  are identifiable market forces impacting manufacturing-asset management within  the pharmaceutical industry. First, pharmaceutical manufacturers are under intense public and political pressures to provide lower-cost products. Second, the  market has seen major blockbuster drugs lose patent exclusivity, and third, mergers  and acquisitions within the industry have created redundant manufacturing  capabilities within several large organizations.   

In terms of pressures to lower costs,  pharmaceutical manufacturers that have redundant manufacturing capacity for  blockbuster drugs can no longer afford to sit on idled equipment for market-demand  or manufacturing risks that may never materialize. Then, losing that  blockbuster to generic competition creates a hole in the manufacturers’  production schedule.  Ideally, they fill that hole with the next branded  product.  However, new products and processes generally require new  equipment. Finally, as the dust settles from many of the industry’s  mergers and acquisitions, many of the larger manufacturing companies are  finding redundant manufacturing capabilities within their networks. They  realize that they may not need 10 different sites making tablets.  So they  transfer products and reduce the number of manufacturing sites. 

Pharmaceutical  Sciences, Manufacturing & Marketplace Report: 

As the industry restructures, what are the  different asset management strategies available to pharmaceutical manufacturers? 

There  are three primary strategies for dealing with manufacturing equipment surplus—often  referred to as “investment recovery services.”  We see some clients  managing their surplus assets with an in-house team dedicated to investment  recovery. Others outsource investment recovery services via their purchasing  departments. Finally, some use a combination of an in-house, global investment  recovery program manager with external service providers. 

What are the challenges that pharmaceutical  manufacturers face in pursuing these different strategies? 

An  in-house program requires personnel, physical storage locations, an advertising  budget, and systems for handling and managing the surplus inventory and  transactions.  Investment recovery as an outsourced service will put  almost all of the burdens of an “in-house” program on the service  provider.  The challenge then becomes identifying the service provider  that is the best fit for the program and determining who owns and manages the  program site-by-site – will it be purchasing, engineering, manufacturing,  etc.?  If a combined strategy is employed with an in-house, global program  manager and external service providers, some resources are required internally  to manage and champion the program while placing the burdens for managing  transactions, advertising, and the movement and storage of inventory on the  service provider. 

Where/how does selling/buying of used equipment  fit into these strategies? 

The  actual selling/buying of equipment is best handled on a case-by-case  basis.  There are several key factors to consider with each new project,  including location, project timeframe, removal costs, and the return that the  owner expects from the equipment.  The investment recovery provider (whether in-house or outsourced) should be able to customize a plan for each  project and propose the best solution that will maximize the goals of the  program – from demolition and scrap through complete plant and single machine  sales. 

 Do you have any specific  examples you can share that demonstrate these different strategies and issues?

We  can apply these general principals to specific case studies for solid-dose  pharmaceutical manufacturing.  In 2012, a major pharmaceutical  manufacturer lost patent exclusivity on one of the top branded drugs in the  market.  The company correctly anticipated the loss of revenue from this  product and planned for the surplus manufacturing equipment, which included an  entire production facility with multiple production lines dedicated to that  product.  Several months before the loss, the company commissioned a study  and created and launched a marketing campaign to sell the entire primary  production facility as a going concern.  They were unsuccessful, and the  decision was made to liquidate the equipment.  

Once the facility was closed, a team  of investment-recovery providers was able to bring several buyers to the site  and sell over 70% of the equipment with value.  Most of the laboratory and  facility equipment was sold via online auction while production equipment was  sold by private sales. The remainder of the equipment was removed and shipped  to a warehouse managed by the investment recovery service providers, where it  is currently being marketed and sold piece-by-piece on a consignment  basis.  The real estate and building were then sold to one of the site’s  physical neighbors.  The project returned  millions to the owner. 

In a different scenario, an over-the-counter products  manufacturer restructured its manufacturing operations to reduce redundant  manufacturing capacity.  In all, three manufacturing sites were designated  to be closed and liquidated.  Each site has managed its own closure and  liquidation with its own request for proposal, service provider evaluation,  contract, and marketing process.  Two of the three sites have had  successful liquidations to date with good returns to the owner and quick  wind-downs of activities at each site. 

Finally, after many acquisitions, a major pharmaceutical  manufacturer determined that there was a great deal of redundancy within its  global manufacturing network.  A plan was created to close certain sites  and reduce manufacturing capacity at other sites.  A global investment  recovery manager was appointed to oversee this multiyear investment recovery project.  

This manager developed a set of rules and processes for the  sites to follow, including internal redeployment and external sales.   Service providers were selected matching the needs for different classes of  equipment (lab equipment and production equipment).  The team developed  the disposal program for each site slated for reduction or closure.   Additionally, the team presented the investment recovery platform to other sites  within the network in order to encourage redeployment of surplus equipment,  which has saved the company millions of capital expenditure dollars by avoiding  the purchase of new equipment. The program has returned millions to the  owner annually. 

Pharmaceutical industry restructuring has created different strategies for drug manufacturers to consider in managing surplus laboratory and manufacturing assets.

Strategies for Managing Surplus Assets in Pharmaceutical Manufacturing

Mass spectrometry (MS) is an  important analytical tool for the evaluation of both small- and large-molecule  pharmaceuticals. In small-molecule drug manufacture, very high sensitivity is  required for the detection of impurities and other contaminants, such as  extractables and leachables in parenteral formulations. Speed of the analytical  technique and the uptime of the instrument are also critical. High resolution  and mass accuracy are also important for the analysis of biopharmaceuticals,  and MS systems are used throughout the development and optimization process. As  the complexity of drug therapies increases, regulatory requirements become more  stringent and many active pharmaceutical ingredients (APIs) become  increasingly potent, there is a continual need for improvement of both the  sensitivity and speed of analytical techniques. New mass spectrometers from  Thermo Fisher Scientific address these issues through a combination of  technology advances and software developments.

Mass spectrometers are typically quite expensive and thus have generally been used where alternative  analytical techniques cannot provide the level of performance required. In small-molecule manufacturing, therefore, MS  is used for the analysis of impurities in drug products. Detection of extremely  low levels of extractables and leachables in large-volume parenteral  formulations, for example, is testing the limits of conventional analytical  techniques, and there is growing interest in MS for this application. It is  also becoming a valuable technique for the analysis of highly potent drugs due  to the very low API concentrations found in these types of products. “In these  cases, triple quadrupole mass spec instruments provide the resolution,  specificity, and speed that is needed,” says Patrick Bennett, marketing  director for pharmaceuticals and biopharmaceuticals with Thermo Fisher  Scientific.

In  biopharmaceutical manufacturing, there is a need to analyze with high  resolution and accurate mass very complex molecules. As a result, MS is widely  used through the development, optimization, and manufacturing of large-molecule  drugs. “Whether identifying which clone to use, evaluating the impact of  different nutrients on production, or characterizing the protein or peptide  product, high resolution, accurate mass spectrometry is used in order to  correctly determine the structures and quantities of the compounds of interest  in very heterogeneous and complex systems,” Bennett observes.

  Regardless of the application, increased  resolution, mass accuracy, and speed are always desired.   “The pharmaceutical industry is evolving, and  analytical techniques must develop to meet the changing environment. While a small-molecule  pharmaceutical manufacturer using a triple quadrupole instrument might obtain  adequate chromatographic resolution and sensitivity by pushing an older  instrument to its limits, the consequences of doing so are often more  challenging method development and sample analysis, and more frequent  maintenance resulting from larger injection volumes. In biopharmaceutical  manufacturing, given the complex systems that need to be analyzed, there is  always demand for greater sensitivity and improvements in the quality and  quantity of the data, and getting all of that information in less time,”  explains Bennett. Outside of the performance of the instruments, ease-of-use is  another aspect that can impact the utility of mass spec as an analytical technique. 

Two new triple quadrupole instruments  from Thermo Fisher Scientific (TFS) were recently introduced to address the  need for increased sensitivity for small-molecule analysis. The TSQ Endura  triple-stage quadrupole mass spectrometer has a new ion source design and  interface. In addition, an ion-beam blocker that prevents neutral ions from  contaminating components improves the robustness and dramatically increases  uptime, according to Bennett. First, the ion source is self-contained, with all  gas, electrical, and other connections made automatically as it is plugged in.  Secondly, the system was designed to be easy to clean. No tools are required to  maintain the system up to the point of the first quadrupole. “This system is  ideal for workhorse applications requiring trace level quantitation, including  pharmaceutical quality analysis and quality control, where long maintenance intervals are important,” Bennett notes. 

The TSQ Quantiva  triple-stage quadrupole mass spectrometer has the same new source and  simplified maintenance design as the TSQ Endura, plus several other significant  engineering changes, according to Bennett. First, active ion management (AIM)  is used to optimize ion creation and transmission from the source to the  detector, resulting in extreme sensitivity to the attogram level (see 

). “The opening for the ions  was changed to more of an oval shape, and as a result provides much more  efficient solvent evaporation, and thus provides much lower chemical noise. The  ion path, optical transfer line, and the collision cell were also modified,” he  explains (see 

). The increased scanning speed of the  new design also makes it possible to screen several hundreds of compounds in a  single analysis. 

Thermo Fisher Scientific has also introduced a novel tribrid system that is the first instrument to combine three  mass analyzers—quadrupole, Orbitrap, and linear ion trap – in a new architecture  (see 

). The Thermo Scientific Orbitrap Fusion Tribrid offers improved biomolecular  characterizations and can collect twice as many scans per unit time at  significantly greater sensitivity than was previously possible, according to  Bennett (see 

). “We have found that we get much  greater proteome-wide coverage with much greater quantitative accuracy than  ever before using the Orbitrap Fusion Tribrid system,” says Steven Gygi,  professor of cell biology at Harvard Medical School.   

In addition,  Bennett notes that there are three possible fragmentation methods available  that can be run in tandem or in combination in ways that were never possible  before, thus making it possible to dramatically reduce the number of individual  experiments that must be completed. “A typical mass spec run might last 1-2  hours. If a company needs to do 100 runs for a given product, with the Orbitrap  Fusion Tribrid, that number could potentially be cut in half, saving 50-100  hours of analysis time.”

The new technologies were  introduced in early June at the American Society for Mass Spectroscopy Conference on Mass Spectrometry and Allied Topics  and have since garnered significant interest, according to Bennett. As might be  expected, most of the requests for demonstrations are coming from existing  customers or new customers looking to use the new instruments in expected  applications. Bennett comments that the Orbitrap Fusion Tribrid, which would  typically find use in biopharmaceutical analysis applications, is also  attracting the attention of people who would normally use a triple quadrupole  system for quantitative analysis. “There seems to be growing recognition that  the ability to use three different fragmentation methods with three types of  analyzers to obtain very high resolution results very rapidly would be, despite  the higher price of the tribrid system, highly beneficial for metabolic studies  for large molecules as well as other quantitative analyses,” Bennett observes.

Making all of the new systems easy to use was a prime goal for Thermo Fisher Scientific. The user interface  is simple with click-and-drag operations. All of the systems also include new  software that provides templates for rapid method development and data acquisition. There are also special software programs designed to assist with protein and  small-molecule identification  as well as  mass spectral databases for the identification and structural elucidation of unknowns.   

“Our goal was to develop highly sophisticated systems that provide  the resolution, speed, and mass accuracy needed for various pharmaceutical  industry analyses, but make them easy to use by both those with extensive training  and those that are new to mass spectrometry. Regardless of their backgrounds,  researchers, and operators do not have time to spend preparing to run  experiments. They need to be spending that time reviewing the results and  making decisions,” states Bennett.

Mass spectrometry is making  advances in adoption with the pharmaceutical industry. “We are right at the  initial stages of market adoption in pharmaceutical manufacturing. With  continued technology evolution, we will see growing use of MS analysis  throughout the development, optimization, production, and downstream  applications in pharmaceutical manufacturing.”

Bennett even  sees mass spectrometry having a role in process analytical technology (PAT) applications in the future. “We are working with partners to develop fully  automated systems that can take advantage of the rapid analysis times and high  sensitivity that mass spec offers.” He notes that PAT systems for small  molecules do not present as much of a challenge as those for large molecules  because biopharmaceuticals require a digestion step that must also be automated  in some way.

For both small- and large-molecule applications, advances in mass spectrometry are leading to interest in this technique as an alternative to liquid chromatography.