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

Cynthia A. Challener

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). 

Articles

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 

Pharmaceutical Sciences, Manufacturing & Marketplace Report

,  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.

Triple Quadrupole Mass Spectrometry Advances Pharmaceutical Analysis

Chiral  heteroatom-containing compounds have become highly useful reagents for the  enantioselective synthesis of important building blocks and intermediates used  for the preparation of pharmaceutical compounds. Stephen Han, Chris Senanayake,  and other coworkers from the Department of Chemical Research and Development at  Boehringer Ingelheim Pharmaceuticals in Ridgefield, Connecticut, recently  reported on the development of new and more efficient routes to two types of  important sulfur- (1) and phosphorous-based reagents (2).

Need for a new chiral sulfinyl transfer agent

Chiral  sulfinamides are widely used for the synthesis of chiral amine-containing  compounds, and they also serve as useful chiral ligands for catalytic  asymmetric transformations. The number of routes to these useful compounds is  limited, and few methods offer the accessibility for structurally diverse sulfonamides  (1). The researchers reported that their goal was to develop a route for the  synthesis of hindered sulfinamides on a large scale in a practical manner (1).  Previously, the researchers at Boehringer Ingelheim had developed a versatile  method for the preparation of sulfinamides by means of cyclic-oxathiozolidinone-based  chiral sulfinyl transfer agents. Even though this method was used to prepare  many hindered substrates, it suffered from the need to use excess NH

 using a large excess of solid Li metal and anhydrous NH

  and presents both safety and waste generation issues (2-4) Therefore, the group  set out to develop an efficient, cost-effective, and practical method for the  synthesis of hindered sulfinamides (1). 

They  reported that the first step was to replace NH

  with a safer and greener reagent, such as lithium bis(trimethylsilyl)amide  (LHMDS), for the final S-O bond cleavage. In order to do so, they needed to  increase the reactivity of the S-O bond, which meant developing a new template  (1). To investigate the reactivity of the S-O bond, the researchers reported  that various 

-butanesulfinates  were reacted with LHMDS. Not surprisingly, the reactivity correlated well with  the electronegativity of the oxygen, which was related to the basicity of the  leaving group (RO-). Thus, sulfinates derived from phenol, particularly those  bearing electron-withdrawing substituents, reacted very quickly and  quantitatively. Importantly, it was also noted that the reaction proceeded in  an enantioselective manner (1).

Based  on these preliminary studies, the researchers reported that optically pure 

-chlorobenzothiazine 2-oxide was  prepared as a new chiral sulfinyl transfer agent on a large scale (2 kg) in 87%  yield and 99.6:0.4 diastereomeric ratio (d.r.) 

 configuration at sulfur) from the corresponding commercially  available chiral aminophenol, SOCI

Preparation of hindered, enantiopure sulfinamides

The researchers  reported that the reactivity of the transfer agent was first tested by treating  it with 

-butylmagnesium chloride  in tetrahydrofuran (THF) to cleave the S–N bond to generate a  diastereomerically pure, stable, crystalline sulfinate ester, to which was  subsequently added LHMDS to cleave the S-O bond to provide enantiopure 

BSA) in high yield and high enantiomeric excess, even  when the two steps were run at 10 °C and 0 °C, respectively (1). This reaction was also run on a 100-g scale (15 °C and 5 °C). After the  two steps, the reaction mixture was quenched with phosphoric acid to  provide a mixture of the starting chiral aminophenol  and (

BSA. The template was crystallized from water and recovered in 99%  yield while the product was extracted using methylene chloride and obtained in  85% yield and 99.5:0.5 enantiomeric ratio (e.r.) (1).

The researchers reported that the  reaction is applicable to the synthesis of a wide variety of structurally  diverse and sterically hindered alkyl and aryl sulfonamides. In some cases, a  lower temperature was required to maintain the very high enantioselectivty. In  addition, they found that the ester intermediate does not need to be isolated,  and a one-pot process is effective (1). 

Extension to hindered, chiral sufinyl ketimines

Furthermore, in  the same study, the researchers applied the methodology to the preparation of related  sterically hindered chiral sufinyl ketimines, which are key intermediates for  chiral amines and typically prepared from ketones and sulfinamides in the presence  of a Lewis acid. Yields for sterically hindered ketones with this reaction are,  not surprisingly, low. With the new chiral sulfinyl transfer agent, however, the  researchers reported that imine nucleophiles were added to the sulfinate ester  intermediate, making it possible to access hindered products (both alkyl and  aryl) in good yields and high enantiomeric purities (1).

The researchers then investigated  the value of the chiral sufinyl ketimines as substrates for the preparation of  chiral amines. They reported that reduction of the ketimines with NaBH

 in THF proceeded in very high yields with high selectivity (diastereomeric  ratios > 90:10). The sulfinyl group was also found to clearly influence the  selectivity, and the researchers are investigating further the preparation of  different chiral amines under different reaction conditions (1).

Wanted: structurally diverse P-chiral phosphines 

Chiral phosphines are widely recognized as important ligands for asymmetric transition-metal  catalysts and as effective organocatalysts for enantioselective transformations.  The researchers, however, noted that there are few efficient methods for preparing  optically active 

-chiral phosphorous  compounds, and those that are used are generally limited to nonsterically  hindered substrates (5).

Recently, Han and his colleagues at  Boehringer-Ingelheim reported on the successful development and use of MeO–BIBOP,  MeO–BOP, and P-chiral biaryl ligands in a number of important asymmetric  reactions, such as propargylations (6), hydrogenations (7), and Suzuki-Miyaura  couplings (8). The ligands were all prepared from a bulky P-chiral phosphine  oxide that, at the time of their research could only be obtained using a  resolution-based synthetic route. Han, Senanayake, and coworkers, therefore, set  out to develop a general method for the efficient and stereoselective synthesis  of 

-chiral phosphine oxides with diverse  structures and functionalities (5).

Taking a page  from their research notebook on the chiral sulfinyl transfer agent, the researchers focused their efforts on developing a chiral phosphinyl transfer agent based on  the 1,3,2-benzoxazaphosphinine-2-oxide structure (5).

The researchers reported that the challenge was to overcome the low reactivity of P–N  and P–O bonds. This goal was achieved by activating the P–N bond (increasing  its eletrophilicity) with an arylsulfonyl group (specifically a tosyl group) on  the N atom, and activating the P–O bond with a phenol derivative that could act  as an effective leaving group (as was the case with the sulfinyl transfer  reagent). The researchers noted that the design also ensured that the P−N and  P−O bonds have differentiated reactivity toward nucleophilic substitutions, thereby  enabling their sequential cleavage with two different organometallic reagents. They  then described how the desired (

)-1,3,2-benzoxazaphosphinine-2-oxide  derivative was prepared from an intermediate aminophenol-based auxiliary that  was easily synthesized on a large scale from the readily available compound (

)-2-(1-aminoethyl)-4-chlorophenol.  The transfer agent was obtained in high yield 

 addition of 1-methylimidazole  (1-MeIm) to the chlorophenol in dichloromethane with a diastereomeric ratio of  98:2, and was then recrystallized to give the pure isomer. The yield on a 100-g  scale was 85% (5).

Han et al.  reported that the chemoselective cleavage of the P–N bond proceeded upon  treatment with Grignard reagents or 

BuLi  in THF at low temperature (-20 °C) (5). This reaction occurred with inversion  of configuration at phosphorous and generated diasteromerically pure (

-butyl and mesityl derivatives, in moderate  to good yields. When 4 equivalents of the Grignard reagent 2-MeO-PhMgBr were  used, the reaction was complete in 15 minutes at -10 °C (5). The researchers  reported that cleavage of the P–O bond proceeded using a variety of reagents to  generate a wide variety of (

)-chiral  phosphine oxides. While bulky Grignard reagents (

-BuMgCl) reacted  slowly, addition of the corresponding lithium compounds proceeded in good  yields with good enantioselectivity. Primary alkyl and vinyl Grignards were also  effective, as were alkynyl and ferrocenyl lithium reagents. Chiral versions of several Buchwald-type ligands were also effectively prepared. It should also be noted that the free phosphine ligands could be obtained in excellent yield without  erosion of the enantiopurity via reduction with polymethylhydrosiloxane (PMHS)  in the presence of Ti(OiPr)

The researchers  asserted that numerous types of functionality can be incorporated into the  chiral phosphine oxides prepared using this new method, making it possible to  synthesize a wide variety of interesting chiral phosphines for use as ligands  and/or organocatalsyts (5). As an example, the researchers reacted a  vinyl-substituted chiral phosphine oxide with dialkyl- or diarylphosphine−borane  adducts to generate enantiomerically pure bisphosphine compounds in high yield  (5).The new approach was also used  to prepare the bulky P-chiral phosphine oxide from which Boehringer-Ingelheim’s  new P-chiral ligands were derived as earlier described. In this case, a different  (

)-1,3,2-benzoxazaphosphinine-2-oxide  derivative was first prepared by reacting the chiral template with  dichloro(2,6-dimethoxyphenyl)phosphine followed by H

-BuLi to this  compound followed by reaction with MeLi/MgBr

-OEt provided the  desired chiral phosphine oxide with a 98.3:1.7 er. The researchers asserted that this new method is a practical,  stereoselective, and high-yield route to P-chiral phosphine oxides, which are  of interest as intermediates for preparing P-chiral phosphine ligands as well  as applicability for novel chiral ligand structures for asymmetric synthesis (5).

New routes enable the efficient synthesis of enantiopure sulfinamides and structurally and sterically diverse P-chiral phosphine oxides.

New Chiral Sulfur and Phosphorous Chemistry from Boehringer Ingelheim

Risk  management is always a priority in pharmaceutical manufacturing. With highly  potent drugs, the level of risk management required is even higher. For oral  solid dosage forms based on highly potent APIs, rigorous care must be taken  throughout the production process (granulation, mixing, tableting, etc.) to  ensure the highest level of quality. Shielding operators from exposure to these  highly potent drugs is paramount. Initially, for operator protection, the emphasis  was placed on the use of extensive personal protective equipment (PPE), but  more recently, it has shifted to the use of contained equipment and automation.

Most large  pharmaceutical companies in general are well prepared in terms of providing developmental  and commercial manufacturing capabilities to support their pipelines of highly  potent drugs, according to Clemens Stief,  team leader/PPD development for Pfizer's Freiburg, Germany facility, which is  part of Pfizer  Global Supply (PGS). The requirements for the handling of highly potent drugs  are quite demanding with respect to environmental, health, and safety  considerations, and the necessary investment for appropriate facilities is significant.  Some CMOs, however, have identified the capabilities required for handling  highly potent drugs, and there are a few companies out there offering  containment capabilities, he adds.

With  exposure of operators a real concern, separating them from the drug product is  critical. “The real equipment breakthrough for the manufacture of highly potent  solid dosage drugs in terms of both safety and efficiency has come in the form  of automation,” says Stief. “With automation, it is possible to achieve a 100%  remotely controlled manufacturing facility with no human interaction during the  manufacturing process,” he notes. Stief points to the fully automated solid-dosage  form manufacturing suites used for large-volume products such as smoking  cessation drugs as prime examples.

These  benefits can be harder to realize with smaller-volume products, which are often  oncology drugs. Automated equipment is often not designed for smaller-scale  operation. Thus, it may only be a solution if platform technologies are applied  to a variety of compounds, such as dry granulation followed by capsule-filling  for more than just one product in the portfolio, according to Stief.  Here, of course, the issue of  cross-contamination must then be addressed.

Although  single-use and  disposable systems  have not yet penetrated the solid dosage manufacturing sector to the degree of  their usage in liquid  and aseptic manufacturing, the idea of using disposable parts is promising.  “The use of such systems could allow for a tremendous reduction in cleaning  verification and validation efforts as well as related analytical method  development work. In addition, it could minimize the potential for contamination  of operators and/or the environment,” he observes. 

Although  disposable systems have advantages in certain applications, the CDMO Metrics  elected to install purpose-built, hard-wall isolation technologies in the  dedicated potent and cytotoxic facility it constructed in 2008, according to director of pharmaceutical  development Joe Cobb. “Local ventilation and laminar/down-flow technologies  are versatile and comfortable, but they are quite dependent on work practice.  Similarly, flexible barrier solutions are practical and achieve good  containment, but pose challenges from a cleaning standpoint, or, if disposable,  a cost-of-goods standpoint,” he explains.

With a  limited physical  footprint at its site, Metrics could not house each piece of manufacturing  equipment (granulators, mills, blenders, etc.) in its own customized isolator.  The company thus engineered a series of multi-use, hard-wall isolators that can  accommodate up to three individual pieces of processing equipment, for a total  of eight individual equipment bays. Standard processing equipment was modified  by installing it through a metal baffle, which fits into any bay on any  isolator.  “As a result, we have general-purpose  isolators that can be customized to meet any client request or operation. That  flexibility is very appealing to pharmaceutical companies,” Cobb comments.   

The isolation barriers for its purpose-built  potent facility have performed so well that Metrics has worked closely with its  vendors to design similar isolation barriers for use elsewhere in its  operations in order to provide an extra measure of safety for its employees  even when projects don’t require that level of protection. “We are taking the concepts  of containment that we successfully engineered for our potent facility and  pushing them out elsewhere in development and manufacturing to make those  operations as safe as possible for our operators,” says Cobb.  

Little  has changed over the last couple of years regarding the design principles for containment  facilities, according to Steif, with the leading paradigm being ‘technical  solutions first’ for commercial facilities. “Containment and isolator  technology are the technologies of choice, followed by PPE, laminar-flow booths,  or restricted access barrier systems (RABS), with only some exemptions in  development facilities. This approach means that there is nothing like well-designed  containment for individual pieces of equipment and/or manufacturing suites,” he  asserts. 

Meanwhile, negative-pressure and  respective-pressure cascades continue to be considered the state-of-the-art. Separately,  the shift to smaller batch size is product-related and applies mostly to  oncology drugs with highly selective working principles.

With that key emphasis on  well-designed containment equipment comes the challenge, according to Cobb. “While  the pharmaceutical industry has a good handle on the technology of isolation (Metrics’  hard-wall containment systems are rated down to occupational exposure levels  (OEL) of 30 nanograms per cubic meter of room air), the size and scale of the  equipment needed for commercial production can be an issue. In addition, there  is a real need for containment-related vendors with first-hand knowledge of the  needs and opportunities of the pharmaceutical industry,” asserts Cobb.

When constructing its high-potent  facility, the company had to adapt the technologies of vendors that provided  equipment for the production of dangerous substances, such as biologicals and hazardous  dusty materials. “The onus was on us to seek out vendors who specialized in  containment systems for dangerous substances and encourage them to apply their  knowledge to our needs within pharmaceutical development and formulation. It  would be helpful to have vendors who better understand the pharmaceutical  industry and can present ideas for containment advances to us, instead of the  other way around,” Cobb says.

Manufacturers of highly potent solid dosage drugs are also dealing with the issue of segregation and the need  to comply with stricter regulations introduced by several regulatory  authorities, according to Stief. “Such requirements not only influence the facility  layout and HVAC design, they also trigger activities related to industrial  hygiene and monitoring and have an impact on overall equipment efficiency,  thus, making the containment business even more challenging and expensive.” 

Process  analytical technology (PAT) can definitely make a difference, according to  Stief, because it provides many opportunities for preventing operator exposure  to highly potent drugs and drug products. These techniques are also attractive  because they are mostly noninvasive and support process monitoring without  direct product contact or sampling procedures. He points to the use of Raman analysis  for compound identification, which can be achieved through a polyethylene bag. “Near-infrared  (NIR) methods are also available for online blend uniformity measurement in  blending operations, and NIR assays may eventually even support a real-time release  strategy,” he notes.

Both  Cobb and Stief believe that the flexibility of their highly potent solid dosage  manufacturing facilities provides a real benefit to the industry. “With our custom-engineered,  hard-wall isolators, clients have the flexibility to select only those  capabilities needed for their particular projects,” explains Cobb. He notes  that such flexibility is proving increasingly important as greater numbers of APIs  (active pharmaceutical ingredients) are becoming increasingly more potent. “In  addition, many of the new APIs are more difficult, challenging, and hard to  handle, which means those of us who work with them need to be nimble and  flexible in order to respond appropriately.”

The DevCon Facility (Development  Containment) for Product and Process Development at Pfizer´s Launch Site in  Freiburg, Germany can cover batch sizes from 10 to 150 kg and provides full GMP  compliant services.  This flexibility of  scale is important in the development arena, according to Stief. He adds that  the facility can also be used for the production of launch and commercial  quantities of small volume products. PGS is also currently evaluating investment  in additional laboratory-scale equipment in response to the urgent need to have  GMP-compliant lab-scale equipment in a containment environment to cover the initial  phases of the development cycle, according to Stief.

Risk management guides decisions in facility design and operation for highly potent drugs.

Technology Choices in Highly Potent Solid Dosage Manufacturing

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.