Patricia Van Arnum was executive editor of Pharmaceutical Technology.

Patricia Van Arnum

Antibody drug conjugates (ADCs) represent a niche but important area in biotherapeutic drug development. ADCs, which consist of a monoclonal antibody (mAb) chemically linked to a small-molecule therapeutic, are a promising modality for certain therapeutic areas such as oncology. ADCs are attracting the interest of both small and large pharmaceutical companies in their drug-development efforts as well as contract manufacturers specializing in high-potency API manufacturing and related conjugation services.  

In August 2011, FDA approved Adcetris (brentuximab vedotin), codeveloped by Seattle Genetics and Millennium Pharmaceuticals (now part of Takeda Pharmaceutical), making it only the second ADC approved by FDA. Adcetris was approved for treating Hodgkin's lymphoma and systemic anaplastic large-cell lymphoma. Adcetris consists of three parts: the chimeric IgG1 antibody cAC10, specific for human CD30, the microtubule-disrupting agent monomethyl auristatin E (MMAE), and a protease-cleavable linker that covalently attaches MMAE to cAC10 (1, 2). Before the approval of Adcetris, the only other ADC approved by FDA was Mylotarg (gemtuzumab ozogamicin), approved more than 10 years ago in 2000. The drug, an anti-CD33 mAb conjugated to the cytotoxin calicheamicin, was developed by Wyeth (now part of Pfizer) and was granted accelerated approval in 2000 but was voluntarily withdrawn by Pfizer in 2010 because a required Phase III trial failed to demonstrate a survival advantage for Mylotarg plus chemotherapy compared with chemotherapy alone (1). 

Other ADCs are advancing. Roche's Trastuzumab emtansine (T-DM1) is an ADC in Phase III development. The drug combines trastuzumab, (Herceptin), which targets human epidermal growth factor receptor 2 (HER2) receptors in breast and stomach cancer, with a maytansine derivative DM1, a small-molecule cytotoxin that binds to tubulin to prevent microtubule formation, through a nonreducible bis-maleimido-trixyethylene glycol linker (1, 2). Trastuzumab was developed by Genentech (now part of Roche) and was approved by FDA in 1998 for use in women with metastatic breast cancer who have tumors that overexpress the HER2 protein. The maytansine derivative DM1 and linking technology were developed by ImmunoGen (1).  Genentech submitted a biologics license application for trastuzumab emtansine to FDA for use in people with HER2-positive, unresectable locally advanced or metastatic breast cancer, and Roche submitted a marketing authorization application to EMA for the same indication. Roche presented data showing encouraging efficacy, safety and quality of life results for T–DM1 at the American Society of Clinical Oncology annual conference in June 2012. Roche has a total of nine ADCs in its development pipeline. It includes RG7593, a humanized IgG1 anti-CD22 monoclonal antibody (anti-CD22) conjugated to an antimitotic agent in Phase I development.  

Pfizer also is advancing ADCs. Its lead ADC product is inotuzumab ozogamicin, consisting of a mAb targeting CD22, a cell-surface antigen expressed on approximately 90% of B-cell malignancies, linked to a cytotoxic agent. As of March 2011, Pfizer had 10 ADC programs in preclinical development (3).  

In October 2012, the biotechnology company Seattle Genetics expanded its ADC collaboration with Abbott. Under the expanded deal, Abbott will pay an upfront fee of $25 million for rights to use Seattle Genetics' auristatin-based ADC technology with antibodies to additional oncology targets. Seattle Genetics's proprietary technology uses synthetic cytotoxic agents, such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), and stable linker systems that attach these cytotoxic agents to the antibody. In addition in its deal with Abbott, Seattle Genetics may receive up to $220 million in potential milestone payments per additional target upon achieving predetermined development and commercial objectives as well as mid-to-high single-digit royalties on worldwide net sales of any resulting products under the multitarget collaboration. Seattle also is partnered with Genentech for ADC development and with Takeda for Adcetris.  

Other companies specializing in ADC technology are partnering with pharmaceutical companies. Earlier this year, Spirogen announced a multiyear collaboration and license agreement with Genentech for the discovery and development of ADCs using Spirogen's pyrrolobenzodiazepine-based drugs and associated linker technology. Spirogen will be primarily responsible for synthesizing and manufacturing drug reagents while Genentech will use Spirogen's drug reagents to generate ADCs and evaluate their potential therapeutic utility. Genentech will have the exclusive license to fully develop and commercialize licensed products that contain these ADCs. 

Mersana Therapeutics, which is developing ADCs using its Fleximer polymer and customizable linker chemistries, formed a $270-million collaboration with Endo Pharmaceuticals in March 2012. The Fleximer polymer is customized to improve the pharmacokinetics of targeting moieties, such as antibody fragments, and also to optimize the quantity of cytotoxic payload carried per targeted conjugate and the stability of the drug–polymer linkage as well as the targeting moiety–polymer linkage. In July 2012, Mersana received $27 million in venture-capital funding, which included investment by Pfizer Venture Investments.  

Biotecnol and PolyTherics formed a collaboration last month for the development of multispecific tribody drug conjugates (TDCs). The companies will combine Biotecnol's tribody technology with PolyTherics' proprietary site-specific linker technologies to attach cytotoxic payloads to various tribodies for targeted cancer therapy. Tribody molecules are multispecific antibody products that, from a single molecule, enable the targeting of two or three tumor antigens in the same disease or two or three epitopes on the same tumor target. 

 studies conducted by Biotecnol have demonstrated tribodies to have targeting abilities and high tumor-to-blood ratios, which make them suitable for delivering toxic payloads in certain disease settings. Biotecnol has a portfolio of multivalent antibodies and capabilities in antibody development and antibody engineering, which will be directed toward this collaboration. 

PolyTherics' ThioBridge linker technology is a conjugation technology for creating more stable, less heterogeneous ADCs through site-specific linkage and rebridging of disulfide bonds. It has been developed to support the conjugation of a range of therapeutic payloads to antibodies, antibody fragments, or scaffolds through specific sites without impacting functional domains. The companies will test the potency of the ThioBridge tribody-drug conjugates in preclinical cancer models. 

Contract API manufacturers offering high-potency manufacturing and related conjugation services are expanding their offerings in response to increase demand for ADCs and other highly potent compounds. For example, Carbogen Amcis and ADC Biotechnology recently announced a partnership to provide customers with development and manufacturing services for ADCs. ADC Bio will provide access to proprietary solid-phase immobilization technologies for conjugation and long-term storage of ADCs, and Carbogen Amcis will focus on small- to large-scale GMP supply and on the formulation of ADCs. 

In October 2012, Fujifilm Diosynth Biotechnologies formed a strategic alliance with Piramal for ADC production whereby the two parties will offer contract development and manufacture of ADCs. Piramal offers ADC production at its site in Grangemouth, Scotland. Fujifilm recently announced an expansion of its cGMP manufacturing facilities at its sites in Research Triangle Park, North Carolina, and  Billingham, United Kingdom.  

Novasep recently announced an EUR 3 million ($3.9 million) invesment to expand its highly potent API (HPAPI) manufacturing capabilities at its Le Mans, France, facility. The plant expansion is expected to be fully operational by the beginning of 2013. The new Le Mans facility adds to Novasep's strategy for total synthesis of ADCs, including toxin and mAb production, mAb-toxin coupling, downstream processing, and formulation. 

Lonza recently partnered with the biopharmaceutical company Intellect Neurosciences to provide the future development and manufacture of Intellect's ADC Conjumab-A. Lonza will supply the preclinical study material for the drug optimization and drug selection for Intellect's Conjumab-A. The new program includes 

 studies to evaluate both therapeutic and prophylactic potential in age-related macular degeneration testing different chemical variants manufactured by Lonza.  

 485 Route One South, Bldg F, First Floor, Iselin, NJ 08830 tel. 732.346.3072,  

 2. FDA, "Label for Adcetris, BLA 125338," FDA Approved Drug Products: Drugs@FDA, accessed Dec. 20, 2011. 

 3. M. Dolsten, presentation at the Barclay  2011 Global Healthcare Conference, Mar. 17, 2011. 

Articles

The promise of antibody drug conjugates is creating a network of partners among large pharma companies and specialized players.

Partnering in Antibody Drug Conjugates

Pharmaceutical companies continually grapple with ways to make drug development more cost- and time-effective. The shared goal among companies is to identify and allocate resources to the most promising drug candidates, a process that to be effective, has to begin in early-stage development. Increasingly, the large pharmaceutical companies are seeking to create more flexible R&D organizations tied to more stringent requirements for return on investment (ROI) as a way to foster greater R&D productivity. As the strategies of pharmaceutical companies evolve, including a greater reliance of external partnerships, so do the strategies of contract service providers and other partners to meet this changing paradigm. 

The necessity for pharmaceutical companies to evolve their R&D models and improve R&D productivity is evident by the cost and time of drug development. It takes on average 10–15 years to bring a new drug to market at an estimated cost of $1.2 billion, according to data from the Pharmaceutical Research and Manufacturers of America (PhRMA). Only two of 10 marketed drugs return revenues that match or exceed R&D costs, notes the PhRMA data. Although the number of compounds in development has increased significantly during the past decade, the number of approvals of new molecular entities (NMEs) has not increased commensurately. In 2011, there were 3240 compounds in development, a 58.8% increase compared with 2040 compounds in development in 2001, according to PhRMA. In four of the past seven years (2005–2011), fewer than 30 NMEs have launched, according to the IMS Institute for Healthcare Informatics. According to the KMR Group, based on industry success rates from 2006–2010, approximately 95% of the compounds that entered Phase I development failed to achieve regulatory approval. The failure rate for compounds that entered Phase II development was approximately 90%, and for compounds that enter Phase III development, it was approximately 54%. In 2011, research-based pharmaceutical companies (defined as members of PhRMA) collectively spent $49.5 billion on R&D, down slightly from the $50.7 billion spent in 2010. 

 Facing a need to improve success rates, Pfizer, like other large pharmaceutical companies, is redirecting its R&D strategy. "...We have sharpened the focus on certain key areas, emphasized strategic externalization, which includes select CRO partners as alliances for the future, additional biotech acquisitions and collaborations, and [are] also building a real strong network with academia," said Mikael Dolsten, president of Worldwide Research & Development at Pfizer at a UBS Global Life Sciences Conference on Sept. 20, 2012.   

Part of that focus includes earlier integration of science and finance into R&D decisions, which for Pfizer has resulted in the termination of approximately 90 projects in the pre-proof-of-concept phase, according to Dolsten in his recent presentation. Overall, Pfizer spent $9.1 billion on R&D in 2011, slightly down from $9.4 billion in 2010, according to company information. As of year-end 2011, Pfizer had 262 projects in R&D ranging from discovery through registration, of which 95 programs were in Phase I through registration (including 22 programs in Phase III), with the remainder of the projects in preclinical development. Its research primarily focuses on five high priority areas with a mix of small and large molecules: immunology and inflammation; oncology; cardiovascular, metabolic and endocrine diseases; neuroscience and pain; and vaccines. In addition to reducing the number of disease areas of focus, Pfizer is realigning and reducing its R&D footprint and outsourcing certain functions. On the CRO side, Pfizer has transitioned from 17 functional service providers to two strategic partners for clinical trials, ICON and Parexel.  

Pfizer also is stressing partnerships with academia as part of its R&D model. Beginning in 2010, Pfizer established several Centers for Therapeutic Innovation (CTI) as a new entrepreneurial research unit for fostering global partnerships with academic medical centers (AMCs). The CTI is an open-innovation partnering model to facilitate early science and translation into clinical applications for biotherapeutic modalities (antibodies, peptides, and proteins) across all therapeutic areas. CTI laboratory staff include Pfizer employees working with basic and translational science investigators and post-docs from the AMCs. CTI has established partnerships with 21 AMCs in the US and supports collaborative projects from four dedicated laboratories in Boston, New York City, San Francisco, and San Diego. Pfizer also has established disease-focused research units in Cambridge, Massachusetts, and Cambridge, United Kingdom as part of  a restructuring of its R&D sites following its acquisition of Wyeth in 2009.  

 In 2010, GlaxoSmithKline (GSK) said it became the first major pharmaceutical company to publish an internal R&D ROI to measure the investment choices within its R&D organization. In early 2010, the company estimated the ROI it made on its recently launched products and late-stage pipeline at 11%. This ROI increased to 12% in 2011, and the company has a long-term goal to improve returns to approximately 14%. 

GSK changed its R&D business model in 2008, moving away from industrial-sized therapy groups to smaller, more agile and focused discovery performance  units (DPUs) consisting of 5–70 scientists. Each DPU works on a particular disease or pathway and is responsible for potential new medicines through early-stage clinical trials (up to the completion of Phase IIa). As part of this new model, DPUs were given their own budgets and a three-year window to complete specific tasks, which included opportunities for collaboration with external organizations. The three-year mark for most DPUs was reached in 2011, and progress of all 38 DPUs was reviewed by GSK's discovery investment board (DIB), a panel consisting of senior management from R&D and commercial operations and external experts. Based on this review, in 2011, four new DPUs were created, and three were closed. Of the remaining DPUs, six received increased investment, and five had investment decreased. Based on this review, GSK believes that it can progress up to 30 medicines into late-stage development during the next three years.  

External partnerships are an important component of GSK's R&D model In 2011, GSK had more than 50 external discovery alliances, compared to 17 in 2007, and it has been active again in 2012. In August 2012, GSK completed its $3.6-billion acquisition of Human Genome Sciences and in September 2012, increased its share to 15.2% in Response Genetics, which performs companion diagnostic tests and other related activities for GSK's immunotherapies and oncology pipeline candidates. Earlier this year, GSK increased its ownership in the biopharmaceutical company Theravance from 18.3% to 26.8%; the companies are developing compounds for respiratory disease. 

In academia, in May 2012, GSK and Yale University established a research collaboration that combines GSK's expertise in medicinal chemistry with Yale's work on proteolysis targeting chimeric molecules or PROTACs. Under the agreement, a joint research team will work to show that PROTACs can be turned into future medicines. GSK will have the right to use this technology for multiple disease-causing proteins, and for each protein-degrading drug that is discovered and developed, Yale will be eligible for milestone and royalty payments. GSK also has several collaborations with UK-based universities that also entail such joint working toward common milestones combined with an element of risk sharing by the parties.  

Sanofi also is restructuring its R&D activities, and in September 2012, it provided further detail about its plans for its R&D sites in France during the next three years. The development activities in Vitry/Alfortville, Chilly-Mazarin/Longjumeau, and Lyon will continue in their current configuration. The Montpellier site will  evolve toward a strategic center focused on development, and the research activities at Vitry/Alfortville and Chilly-Mazarin/Longjumeau will  be increased. The Strasbourg site will maintain its collaborative platform open to academic research and biotechnology companies. Sanofi had 64 NMEs and vaccines in its portfolio in development as of July 2012, which included 29 products (four vaccines and 25 NMEs) in Phase I and 17 products (3 vaccines and 14 NMEs) in Phase II.  

Sanofi has emphasized several goals in its R&D model: improve R&D cost structure, execute on late-stage projects, use medical value and translational feasibility to guide early-stage portfolio prioritization, establish new models of external innovation, enhance the value of external partnerships, and create open and creative models of pharma/biotechnology partnerships.  

One example of such a partnership is Sanofi's collaboration with Warp Drive Bio, which specializes in microbial genomics for natural products research. Warp Drive Bio was founded by venture capitalists and scientists from Harvard Medical School and the University of California, San Francisco. In a deal announced in January 2012, Sanofi and private funders are providing $125 million in initial funding, including a $75-million equity investment in although Warp Drive  Bio retains strategic direction, operational management, and full rights to select assets. This approach enables Warp Drive Bio to advance its programs in collaboration with Sanofi while maintaining the ability to secure future partnerships.  

Also, earlier this year, Sanofi formed a two-year agreement with Massachusetts General Hospital aimed at furthering translational medicine research to develop new treatments for hematological malignancies and solid tumors. The academic and industry cross-organizational expertise will include preclinical and clinical translational research to elucidate questions on proof-of-concept, tolerability, efficacy, and effectiveness.  

 Novartis, while essentially using the same model as the traditional drug-development process, has tailored it to consist of two parts: exploratory and confirmatory development. Exploratory development consists of clinical proof of concept and involves small clinical trials (typically 5–15 patients) that combine elements of traditional Phase I/II testing. These customized trials are designed to give early insight into safety, efficacy, and toxicity. Once a positive proof of concept has been established, the drug moves to the confirmatory development stage, which has elements of traditional Phase II/III testing aimed at confirming the safety and efficacy of the drug in the given indication.   

Novartis is investing $600 million for new laboratory and office space in Cambridge, Massachusetts, close to its research facilities and announced restructuring of some of its R&D sites and staffing. With regard to partnerships, in August 2012, Novartis and the University of Pennsylvania (Penn) formed an exclusive global collaboration to research, develop, and commercialize targeted chimeric antigen-receptor immunotherapies for treating cancers. In addition, the parties will jointly establish a new R&D facility on the Penn campus.   

Other companies are pursuing a path of R&D restructuring and external partnerships. In June 2012, Roche announced a streamlining of its R&D activities within the pharmaceuticals division, which involves closing its site in Nutley, New Jersey, by the end of 2013, with a reduction in the workforce of approximately 1000 people. The R&D activities at Nutley will be consolidated at existing sites in Switzerland and Germany and at the planned Translational Clinical Research Center in the US. Merck& Co. increased the level of its partnerships with several recent pacts with Ablynx, AiCuris, Chimerix, and Yamasa. Bristol-Myers Squibb formed several academic partnerships this year in drug development with separate pacts with Vanderbilt University and Emory University and formed the Immuno-Oncology Network, a global collaboration between industry and academia with 10 leading cancer-research institutions participating.  

AstraZeneca announced further restructuring in its R&D operations earlier this year, including the creation of a new "virtual" neuroscience innovative medicines unit (iMed). The neuroscience iMed is one example of an iMed. iMeds focus on particular disease areas and work across discovery and early development. Eli Lilly established the Center for Cognitive Neuroscience, an industry–academic consortium and post-doctoral fellowship program focused on increasing the probability of clinical success for drugs to treat cognitive impairment. It is also participating in the Innovative Medicines Initiative, a public–private collaboration to speed drug development. Finally, Johnson & Johnson plans to open four innovation centers in California, Boston, London, and China, with the aim of accelerating early innovation.  

As pharmaceutical companies adjust their development strategies, contract service providers are responding in kind to provide targeted services for early-stage development. For example, in October 2012, Catalent Pharma Solutions formed a global alliance with the CRO Parexel to help streamline the clinical-trial supply process. The formation of this alliance follows Catalent's February 2012 acquisition of the clinical-trial supplies business of Aptuit. In August 2011, Bend Research and Xcelience formed a collaboration to provide oral solid-solubilization formulation solutions for early-phase development. In February 2012, Covance signed a three-year, exclusive integrated drug development agreement with BioPontis Alliance to manage early development of scientific discoveries from academia.  

Collaborative models for drug development 			

Almac's solid-state services group recently created a series of preclinical screening packages for clients to identify their candidate's chances of success much quicker than using traditional mechanisms. Experimental efforts concentrate on establishing the physical, chemical, solubility, stability, and polymorphic characteristics of each drug candidate. Separately, Almac launched a qualified person facilitation program that is designed for clients importing investigational medicinal products into the European Union for clinical trials.  

EMD Millipore announced the opening of its advanced GMP bioproduction facility in Martillac, France, which will serve users of EMD Millipore's Provantage biodevelopment and clinical supply solutions, an open-source manufacturing option for upstream and downstream processes. The open-source approach is designed to provide customers with greater control over production. The facility offers GMP production of mammalian proteins for preclinical to Phase II production at 50 L to 1250 L scales. For Phase III and commercial production, customers can transfer manufacturing at any scale. 

Companies are augmenting their testing services to support early-drug development. In June 2012, Metanomics Health, part of BASF, launched MetaMap Tox, a service evaluating specific metabolomic patterns 

 enabling customers to better and faster identify potential safety risks of test compounds in 

 studies of rats. SAFC a contract API manufacturer and providers of biosciences services and products, acquired BioReliance, a provider of biopharmaceutical testing services for $350 million. BioReliance provides biologic, specialized toxicology, and animal-health testing.  

Pharmaceutical companies and contract service providers adapt strategies and capabilities to reduce costs and accelerate drug-development timelines.

Optimizing Early-Stage Drug Development

Sharanya Reddy, PhD, application scientist with PerkinElmer, describes the use of time-of-flight–mass spectrometry for impurity analysis of an over-the-counter drug product. Robert Mattes, application scientist with Foss NIRSystems, and Stephen Hoag, professor,  and Ravikanth Kona, PhD candidate, both in the Department of Pharmaceutical Sciences, University of Maryland, explain the use of in-line moisture analysis in a laboratory-scale fluid-bed dryer using diode array near-infrared spectroscopy. Jeffrey P. Kiplinger, president, Paul M. Lefebvre, director of laboratory operations, Michael J. Rego, staff scientist, and John H. Tipping, staff scientist, all with Averica Discovery Services, explain a chiral biotransformation analysis using supercritical fluid chromatography–mass spectrometry. 

Impurity analysis with time-of-flight mass spectrometry

Sharanya Reddy, PhD, application scientist, PerkinElmer

Time-of-flight (TOF) mass spectrometry (MS) provides high resolution and exact mass information over a wide mass range to identify unknown compounds. This case study describes a workflow to identify impurities in over-the-counter (OTC) drugs using proprietary technology (TrapPulse, PerkinElmer) of the AxION 2 TOF mass spectrometer (PerkinElmer). In conventional orthogonal TOF instruments, the ions are lost between pulses. In the TrapPulse mode operation of the AxION 2 TOF mass spectrometer, the ions are collected as packets before they are pulsed, which results in signal enhancement. Analysis in the TrapPulse mode prevents potential "wraparound" of higher mass ions into subsequent spectra, thereby eliminating spurious peaks in the spectrum and potentially incorrect mass assignments. Using this technology, the author improved detection limits and identified 10 impurities in OTC melatonin, including previously uncharacterized ones. 

The AxION 2 TOF mass spectrometer also provides mass accuracy and isotope ratio-profile accuracy, both of which are required for elemental composition identification of unknowns. The author predicted the elemental composition of several unknown compounds in melatonin using information provided by the TOF in conjunction with elemental composition matching software (AxION EC ID, PerkinElmer). Accurate monoisotopic mass and isotope ratio information is used by the AxION EC ID software to search against databases, such as the PubChem database (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) or other databases for potential molecular formula matches. The software provides a ranked summary of the potential matches and suggestions for possible compound structures for a given elemental composition. 

The following materials and conditions were applied for liquid chromatographic (LC) analysis:  

LC system: Flexar FX-10 high-performance LC

LC column: PerkinElmer Brownlee Supra column C18 (1.9 μm, 2.1 × 50 mm) 

Mobile Phase A: water with 0.1% formic acid

Mobile Phase B: acetonitrile with 0.1% formic acid

Gradient: 20% B to 40% B over 5 min, to 70% B over 2 min.

The following were applied for mass spectrometric analysis:  

Mass spectrometer: PerkinElmer AxION 2 TOF 

Ionization source: PerkinElmer Ultraspray 2 (Dual electrospray ionization [ESI]source)

Pulse mode: mass-to-charge ratio (m/z) 100–700

An OTC melatonin tablet containing 1 mg of melatonin was crushed by a mortar and pestle and dissolved in 10 mL of water. The mixture was vortexed and centrifuged at 6000 rpm for 10 min. The supernatant was collected and injected on the column.  

Figure 1 (TOF MS): (a) Analysis of melatonin in conventional pulse mode; (b) Analysis of melatonin in TrapPulse (PerkinElmer) mode. (FIGURES 1â3 (TOFâMS) ARE COURTESY OF THE AUTHOR (REDDY)) 

Initial analysis in traditional pulse-mode operation showed few impurities in the melatonin tablet present at a low signal-to-noise ratio (S/N) (see 

). When the analysis was performed in TrapPulse mode, the increase in S/N was almost fivefold to sevenfold higher, which resulted in the identification of 10 different impurities in melatonin (see Peaks A through J, see 

). The comparison highlights the advantage of analyzing the sample in TrapPulse mode versus pulse mode.  

Figure 2 (TOF MS): (a) Base peak ion chromatogram (BIC) (mass-to-charge ratio (m/z) of 265.132 Â± 0.050 Da) shows the elution position of the Impurity Isomers A and D in a melatonin tablet. (b) BIC (m/z of 265.132 Â± 0.050 Da) shows the co-elution of N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) with Peak D when spiked into a melatonin tablet extract. (c) Spectrum of Peak A Impurity. The accurate mass of [M + H]+ and the fragments of Peak A are within 2.0 ppm of theoretical value. (d) The mass spectrum of Peak D. The accurate mass of [M + H]+ and the fragments are within 2.5 ppm of theoretical value. (FIGURES 1â3 (TOFâMS) ARE COURTESY OF THE AUTHOR (REDDY)) 

Using accurate mass, isotope-profile information, and EC ID software, the author was able to identify all unknown drug impurities. The workflow used for identification of unknowns with the example of the drug impurities labeled Peak A and D are highlighted (see 

) have identical accurate masses, but have different spectra and elution times, which suggest that they are most likely isomers (see 

). The elemental composition predicted by the EC ID software is C

 studies with melatonin have shown a minor metabolite, 

2-formyl-5-methoxykynuramine (AFMK), to be produced, which has an elemental composition and exact mass identical to Peak D. For these reasons, the author suggests that the structure of Peak D to be AFMK. The structure of Peak D was confirmed by analyzing a synthesized standard of AFMK, which matched both the retention time and accurate mass spectrum of Peak D (see F

). Using a similar approach, the author identified the remaining impurities Peaks B to J as summarized in 

Figure 3 (TOF MS): Software (AxION EC ID, PerkinElmer) lists the elemental composition of C13H16N2O4 with the top score for the accurate mass 265.1188 and the observed relative isotope abundances. (FIGURES 1â3 (TOFâMS) ARE COURTESY OF THE AUTHOR (REDDY))  

. Several impurities in OTC tablets of melatonin that were not visible in the conventional pulse mode were identified using the AxION 2 TOF in TrapPulse mode. High mass accuracy and accurate isotope-profile ratio information provided by the TOF and the elemental composition matching software (AxION EC ID) facilitated the identification of several unknown drug impurities. 

Table I (TOF MS): Structures of impurities identified by time-of-flight mass spectrometer (AxION, PerkinElmer). 

Robert Mattes, application scientist, FOSS NIRSystems. Stephen Hoag, professor, and Ravikanth Kona, PhD candidate, both in the Department of Pharmaceutical Sciences, University of Maryland 

Top-spray granulation in a fluid-bed dryer is a common method of increasing particle size to increase flow characteristics and API content homogeneity. After spraying the liquid into the formulation and forming the granule, the product must be dried to the proper moisture level. If the granules are overdried or underdried, damage to the formulation may occur, thereby causing problems with subsequent processing and problems with product stability during storage (1). Samples are typically withdrawn from the fluid bed with a thief during processing and analyzed off line in a laboratory for moisture content. Commonly, there is a delay before analysis results are available to the operator, which results in processing decisions being made without optimal product-moisture information. Top-spray granulation end point is often based on time or product temperature and not actual moisture content. 

Near-infrared spectroscopy (NIRS) is a rapid, nondestructive technique for in-process analysis of moisture in a manufacturing environment (2). Real-time measurements are made with no sample preparation, and data can be analyzed and stored automatically. NIRS fits in well with FDA's process analytical technology   initiative (3). Using NIRS, the process can be monitored for low levels of residual moisture and other process constituents to yield better process control and end-point determination (4). Laboratory-scale fluid-bed dryers are often used in research at the university level and in process development to better understand formulation processing. This study shows the use of NIRS for monitoring residual moisture in laboratory-scale equipment. 

 The NIR instrument used to collect spectra was the ProFoss Diode Array (FOSS NIRSystems). Spectra were collected in the reflectance mode from 1100 nm to 1650 nm with 0.5-nm data intervals, and 32 scans were co-added to produce a single spectrum. A fluid-bed probe, specifically for fluid-bed dryer applications, was inserted into a fluid-air granulator at a 45 ° angle to the central axis of the product container as seen in 

. The collection "spoon" and purge vents are located on the probe tip (see 

). After each NIR spectrum was collected, the software sent a "data complete" signal that energized an air purge exiting through the ports in the probe, thereby clearing the "spoon" for a new sample. The insert in 

 shows the probe with the sample collected. 

Figure 1 (NIRS): The fluid-bed dryer with the near infrared  probe inserted at a 45Â° angle. Also shown is the black purge line on the left and the sample thief on the right. (FIGURES 1â5 (NIRS) ARE COURTESY OF THE AUTHORS (MATTES ET AL.)) 

A charge of lactose monohydrate (Pharmatose 110M, DMV-Fonterra Excipients) and microcrystalline cellulose (Avicel PH 102, FMC) was prepared and loaded into the product container. The product was fluidized for 5 min to blend and dry the mixture to homogeneity. An aqueous solution of 10% polyvinyl pyrrolidone (Kollidon K30, BASF) was added by top spray. NIR spectra were collected every 50 s during the blending operation. Samples for loss on drying (LOD) analysis were withdrawn with the sample thief at approximately 5-min intervals to be later correlated with spectra acquired at the same time. 

Figure 2 (NIRS): The specially designed spoon probe with purge ports. The insert shows sample collected on the probe. (FIGURES 1â5 (NIRS) ARE COURTESY OF THE AUTHORS (MATTES ET AL.)) 

 shows the second derivative of the sample spectra. The second-derivative mathematical treatment is commonly used in NIR spectroscopy to minimize baseline offset caused by scattering and to enhance absorbance peaks. Due to the second derivative treatment, the moisture increases downward in this region. Water absorbs strongly in the NIR between 1400 and 1450 nm as evidenced by the peaks in that region. 

Figure 3 (NIRS): Second derivative mathematically treated dryer spectra. (FIGURES 1â5 (NIRS) ARE COURTESY OF THE AUTHORS (MATTES ET AL.)) 

A two-factor partial-least-squares regression model was developed with spectra from a calibration run and LOD reference values. The second derivative intensity over the range 1100–1650 nm was used to develop a prediction model with an R

 value of 0.9519 and a standard error of calibration of 0.7358%. 

 shows a calibration plot of NIR predicted versus LOD % moisture. 

Figure 4 (NIRS): Scatter plot  of the near infrared (NIR) predicted values versus the loss-on-drying (LOD) values. (FIGURES 1â5 (NIRS) ARE COURTESY OF THE AUTHORS (MATTES ET AL.)) 

 is a typical analysis output trend chart showing the moisture decrease during the drying cycle. The operator is aided with real-time graphical output such as this in making the decision to end the drying operation before the product is damaged or degraded. The delay caused by waiting for laboratory results before the product can be released for subsequent processing can be minimized or eliminated. Output from the NIR computer is used by the fluid-bed dryer's programmable logic controller for closed-loop process-control decisions. The correct NIR probe must be placed in the product container in a manner that provides sufficient sample contact with the probe-tip window. Correct probe design and proper placement in process equipment is of high importance (4). 

Figure 5 (NIRS): Trend plot of moisture on subsequent run. (FIGURES 1â5 (NIRS) ARE COURTESY OF THE AUTHORS (MATTES ET AL.)) 

  1.  A.G. Rogers, "Granulation and Drying Principles," 

Hands-on  Postgraduate Course in Tablet Technology,

  2.  R.A. Mattes et al., "Process Analytical Technology" supplement to 

  4.  R.A. Mattes, D.E. Root, and A.P. Birkmire, "The Role of Spectroscopy in Process Analytical Technologies" special issue to 

Supercritical fluid chromatography–mass spectrometry

Jeffrey P. Kiplinger, president, Paul M. Lefebvre, director of laboratory operations, Michael J. Rego, staff scientist, and John H. Tipping, staff scientist, Averica Discovery Services

Supercritical fluid chromatography (SFC) is a well-characterized technology for analytical and preparative chiral resolution (1). It is useful for small- to mid-scale production of single enantiomers for pharmaceutical discovery, where competitive assays of enantiomers can help validate mechanisms and improve the precision of lead-compound assessment (2). Current FDA guidance speaks to the desirability of comparing enantiomers early in pharmaceutical R&D (3). SFC, however, has seen limited use in areas dominated by highly selective high-performance liquid chromatography assays due to perceptions of low sensitivity, interfacing difficulties with detectors such as mass spectrometers, and incompatibility with hydrophilic analytes and matrices. The authors present an example in which chiral SFC–mass spectrometry (MS) is shown to be an expedient analytical approach to solving a bioanalysis problem. 

 A compound developed in a pharmaceutical lead-optimization project as a racemic mixture was separated into constituent Enantiomers A and B using SFC with ultraviolet (UV) detection of 230 nm. The enantiomers were tested competitively in rats, and plasma was drawn from the animals for pharmacokinetic assays. In the course of the efficacy study, unexpected off-target effects were observed, and the team suspected 

 racemization. Unfortunately, insufficient compound remained for further 

 work. Only residual samples in storage vials of a mixture of the two enantiomers (not racemic, simply a mixture generated for testing) and of the active Enantiomer A were available. 

Developing analytical methods and performing related testing is crucial for ensuring the quality of a pharmaceutical product.

Analytical Applications

Ben Venue Laboratories has resumed production on a limited number of manufacturing lines in the company’s Bedford, Ohio, facilities. The company reported last week that it continues to make progress toward upgrading the company’s manufacturing facilities and anticipates that production will resume on additional lines as corrective measures to address facility and equipment issues are implemented. For most of the products it manufacturers, however, supplies will not be available for at least several months, according to the company.

Late last year, Ben Venue Laboratories announced that it was extending its voluntary suspension of manufacturing at its Bedford facility. The company had originally announced the suspension on Nov. 19, 2011, and announced the extension of the suspension on Dec. 23, 2011. Ben Venue had suspended manufacturing at the Bedford facility after an internal review indicated that routine preventive maintenance and requalification of some manufacturing equipment were overdue. These issues also were noted in inspection findings by FDA, EMA, and other global regulatory agencies. Ben Venue’s operations in Bedford include sterile injectable facilities with aseptic filling, lyophilization, and cytotoxic manufacturing in four factories.

, Ben Venue says it continues to work in close collaboration with FDA and other global regulatory agencies. Following FDA’s inspection of Ben Venue’s manufacturing operations in May 2011, the company began the implementation of a remediation effort and says it has invested more than $300 million to upgrade its facilities by making necessary improvements and changes to its processes to resolve the manufacturing-related issues that resulted in the voluntary shutdown last year.

EMA Says Ben Venue's Anticancer Drugs Should be Manufactured in Another Facility

Ben Venue Continues Suspension of Manufacturing Activity at Ohio Facility

Ben Venue Laboratories has resumed production on a limited number of manufacturing lines in the company's Bedford, Ohio, facilities.

Ben Venue Resumes Limited Production

The perception of buyers of pharmaceutical ingredients and contract services in how they see market conditions and their own individual business prospects is an important barometer in assessing the market for pharmaceutical outsourcing and ingredients. The results of a survey released this week at CPhI Worldwide in Madrid were largely positive as buyers assessed market and business conditions for the next 12 months.

		According to the survey, 97% of pharmaceutical buyers attending CPhI that were surveyed expect sales to increase or stay the same over the next 12 months (up from 90% over the last 12 months), and 90% rate their business opportunity in the global pharmaceutical market positively. The CPhI Worldwide survey, a pre-event survey conducted by UBM Live’s CPhI portfolio, polled nearly 1100 preregistered visitors representing pharmaceutical buyers. Attendees expect pockets of innovation to positively impact their overall business including GMP certification (64.8%), the rise in generics (58.6%), the move toward outsourcing (53.4%), and supply-chain security (50.4%).

		Other key results show that 61% of visitors said that the current economic climate will have no impact or a positive impact on their business, and 53.2% of visitors said that the move toward outsourcing will have a positive impact on their business. Also, 91% of visitors said they use Internet to source products and services. And sustainability is also important with 90% of visitors rating sustainability as important to the future development of their company and 71% having a sustainability policy in place or in progress.

		The results reflect improving pharmaceutical industry growth. Following several years of slowing growth, the global pharmaceutical market is expected to rebound from an expected low point of 3–4% growth in 2012 to 5–7 percent in 2016, according to the IMS Institute for Healthcare Informatics. Annual global spending on medicines will rise from $956 billion in 2011 to nearly $1.2 trillion in 2016, representing a compound annual growth rate of 3–6%, according to IMS. Growth in annual global spending is forecast to more than double by 2016 to as much as $70 billion, up from a $30 billion pace in 2012, driven by volume increases in emerging markets and an uptick in spending in developed nations.

		These signs are positive, but also have to be taken into consideration with how the pharmaceutical industry as a whole is changing. Greater growth in emerging markets and a more cost-constrained environment for healthcare spending in developed markets contribute to more competitive pricing conditions than historically have occurred in the pharmaceutical industry. How pharmaceutical companies deal with these challenges, and in turn, how suppliers respond in ways to deal with these new economic realities of their pharmaceutical customers will continue to have important implications for the market for pharmaceutical outsourcing and ingredients.

The perception of buyers of pharmaceutical ingredients and contract services in how they see market conditions and their own individual business prospects is an important barometer in assessing the market for pharmaceutical outsourcing and ingredients.

Gaining a Market Pulse at CPhI

As fine-chemical producers, contract manufacturers, and pharmaceutical/biopharmaceutical companies meet this week in Madrid at CPhI Worldwide, what are the key issues on the minds of industry players?

It is no secret that the pharmaceutical industry continues to be in a period of transition as it faces slower growth compared with historical levels. Increased generic-drug incursion, shifting growth patterns to emerging markets, and the erosion of the blockbuster drug model to more specialized drugs have all contributed to placing new demands on contract service providers and fine-chemical suppliers as their pharmaceutical customers deal with these new realities. In speaking with CMOs and fine-chemical producers here this week at CPhI, many point to the increasingly strategic and collaborative nature of outsourced relationships, the greater emphasis placed on project management in executing those partnerships, and the attendant need for suppliers to adapt their business models to the reality of their pharmaceutical customers in facing a more resource-constrained environment. 

The new reality, as one supplier explained, is that as pharmaceutical companies increase their level of external development and manufacturing, they are also obliged to deal with greater complexity in their supply chains. In general, suppliers cited an interest by pharmaceutical companies to manage their supply chains more efficiently by working with fewer suppliers, accessing contract service providers with broader capabilities to not only reduce the number of suppliers working on a given project but to also make technology transfer and project workflow more time- and cost-efficient. Suppliers also noted the increased importance of project management and the related expertise of contract service providers in not only managing their direct relationships with sponsor companies but also in facilitating the activities resulting from the external network of suppliers that a given project may entail. 

That emphasis on collaboration and project facilitation is also evident in the partnerships that contract service providers are forming with each other. For example, at CPhI this week, Almac and DSM announced a partnership in biocatalysis. The agreement grants both parties access to enzyme platform technologies, services and expertise, with Almac offering expertise in enzyme identification, scale-up, and implementation of early-phase projects and DSM providing capabilites for large-scale production. Earlier this year, Catalent Pharma Solutions and BASF formed a broad collaboration and open alliance to provide solutions to overcome bioavailability challenges of new molecular entities with solubility or permeability challenges. Last year, Hovione and Bend Research formed collaboration that married Hovione’s commercial-scale pharmaceutical manufacturing capabilities with Bend Research’s oral drug-delivery, formulation, and process engineering expertise.

These examples show that the collaborative and strategic partnerships are not only at play among sponsor companies and their suppliers but among the contract service providers themselves as they seek to meet the needs of their customers in an evolving pharmaceutical industry.  

The PTE team is currently in Madrid attending numerous meetings and presentations at CPhI and its collocated events. Read the thoughts of our editor on the highlights of the show so far.