Selection of Analytical Technology and Development of Analytical Procedures Using the Analytical Target Profile.

A structured approach to method development can help to ensure an analytical procedure is robust across the lifecycle of its use. The analytical target profile (ATP), which describes the required quality of the reportable value to be produced by the analytical procedure, enables the analytical scientist to select the best analytical technology on which to develop their procedure(s). Once the technology has been identified, screening of potentially fit for purpose analytical procedures should take place. Analytical procedures that have been demonstrated to meet the ATP should be evaluated against business drivers (e.g., operational constraints) to determine the most suitable analytical procedure. Three case studies are covered from across small molecules, vaccines, and biotherapeutics. The case studies cover different aspects of the analytical procedure selection process, such as the use of platform method development processes and procedures, the development of multiattribute analytical procedures, and the use of analytical technologies to provide product characterization knowledge in order to define or redefine the ATP. Challenges associated with method selection are discussed such as where existing pharmacopoeial monographs link acceptance criteria to specific types of analytical technology.

Strategies for Setting Patient-Centric Commercial Specifications for Biotherapeutic Products.

Commercial specifications for a new biotherapeutic product are a critical component of the product's overall control strategy that ensures safety and efficacy. This paper describes strategies for setting commercial specifications as proposed by a consortium of industry development scientists. The specifications for some attributes are guided by compendia and regulatory guidance. For other product quality attributes (PQAs), product knowledge and the understanding of attribute criticality built throughout product development should drive specification setting. The foundation of PQA knowledge is an understanding of potential patient impact through an assessment of potency, PK, immunogenicity and safety. In addition to PQA knowledge, the ability of the manufacturing process to consistently meet specifications, typically assessed through statistical analyses, is an important consideration in the specification-setting process. Setting acceptance criteria that are unnecessarily narrow can impact the ability to supply product or prohibit consideration of future convenient dosage forms. Patient-centric specifications enable appropriate control over higher risk PQAs to ensure product quality for the patient, and flexibility for lower risk PQAs for a sustainable supply chain. This paper captures common strategic approaches for setting specifications for standard biotherapeutic products such as monoclonal antibodies and includes considerations for ensuring specifications are patient centric.

Expectations for Phase-Appropriate Drug Substance and Drug Product Specifications for Early-Stage Protein Therapeutics.

Early-phase specifications are established to ensure that materials used in clinical studies have appropriate product quality, reducing the risk of harm to patients. Currently, guidance is available for specification setting practices at commercial phase. With very limited data and manufacturing experience available, it is not possible to fully align to these expectations at the start of clinical trials. A survey was performed among 19 biopharmaceutical companies to gather information about the current practices for setting specifications in early-phase development. The results indicate that most companies develop platform approaches to support setting specifications at the first-in-human clinical trial stage of development. Based on shared learning across multiple companies, example specification approaches for monoclonal antibodies and antibody-drug conjugates are included. General principles of the example specifications can also be applied to other protein therapeutics and vaccines. Strategies for justification of acceptance criteria are described, along with discussion of considerations for some specific tests. Options for use of non-numerical acceptance criteria are also discussed. While specifications for each molecule must be set considering available molecule-specific information, the presented information leverages shared learning from multiple companies, to provide guidance for early phase specification setting strategies.

Comparison of helicopter versus ground transport for the interfacility transport of isolated spinal injury.

BACKGROUND CONTEXT: The use and need of helicopter aeromedical transport systems (HEMSs) in health care today is based on the basic belief that early definitive care improves outcomes. Helicopter aeromedical transport system is perceived to be safer than ground transport (GT) for the interfacility transfer of patients who have sustained spinal injury because of the concern for deterioration of neurologic function if there is a delay in reaching a higher level of care. However, the use of HEMS is facing increasing public scrutiny because of its significantly greater cost and unique risk profile. PURPOSE: The aim of the study was to determine whether GT for interfacility transfer of patients with spinal injury resulted in less favorable clinical outcomes compared with HEMS. STUDY DESIGN/SETTING: Retrospective review of all patients transferred to a Level 1 trauma center. PATIENT SAMPLE: Patients identified from the State Trauma Registry who were initially seen at another hospital with an isolated diagnosis of injury to the spine and then transferred to a Level 1 trauma center over a 2-year period. OUTCOME MEASURES: Neurologic deterioration, disposition from the emergency department, in-hospital mortality, interfacility transfer time, hospital length of stay, nonroutine discharge, and radiographic evidence of worsening spinal injury. METHODS: Patients with International Classification of Diseases, Ninth Revision (ICD-9) codes for injury to the spine were selected and records were reviewed for demographics and injury details. All available spine radiographs were reviewed by an orthopedic surgeon blinded to clinical data and transport type. Chi-square and t tests and multivariate linear and logistic regression models were done using STATA version 10. RESULTS: A total of 274 spine injury patients were included in our analysis, 84 (31%) of whom were transported by HEMS and 190 (69%) by GT. None of the GT patients had any deterioration in neurologic examination nor any detectable alteration in the radiographic appearance of their spine injury attributable to the transportation process. Helicopter aeromedical transport system resulted in significantly less transfer time with an average time of 80 minutes compared with 112 minutes with GT (p<.001). Ultimate disposition included 175 (64%) patients discharged to home, 15 (5%) expired patients, and 84 (31%) discharged to extended care facilities. After adjusting for patient age and Injury Severity Score, the use of GT was not a significant predictor of in-hospital mortality (odds ratio, 1.4; 95% confidence interval, 0.3-5), hospital length of stay (11.2+1.3 vs. 9.5+0.8 days, p=.3), or nonroutine discharge (odds ratio, 1.1; 95% confidence interval, 0.5-2.2). CONCLUSIONS: Ground transport for interfacility transfer of patients with spinal injury appears to be safe and suitable for patients who lack other compelling reasons for HEMS. A prospective analysis of transportation mode in a larger cohort of patients is needed to verify our findings.

Analytical characterization of an orally-delivered peptide pharmaceutical product.

The characterization of orally-delivered peptide pharmaceuticals presents several challenges to analytical methods in comparison to characterization of conventional small-molecule drugs. These challenges include the analysis and characterization of difficult-to-separate impurities, secondary structure, the amorphous solid-state form, and the integrity of enteric-coated drug delivery systems. This work presents the multidisciplinary analytical characterization of a parathyroid hormone (PTH) peptide active pharmaceutical ingredient (API) and an oral formulation of this API within enteric-coated sucrose spheres. The analysis of impurities and degradation products in API and formulated drug product was facilitated by the development of an ultrahigh-performance liquid chromatography (UHPLC) method for analysis by high-resolution mass spectrometry (MS). The use of UHPLC allowed for additional resolution needed to detect impurities and degradation products of interest. The secondary structure was probed using a combination of solution-state NMR, infrared, and circular dichroism spectroscopic methods. Solid-state NMR is used to detect amorphous API in a nondestructive manner directly within the coated sucrose sphere formulation. Fluorescence and Raman microscopy were used in conjunction with Raman mapping to show enteric coating integrity and observe the distribution of API beneath the enteric-coating on the sucrose spheres. The methods are combined in a multidisciplinary approach to characterize the quality of the enteric-coated peptide product.

Lower limb immobilization is associated with increased corticospinal excitability.

Temporary immobilization of the leg serves as a useful model for the brain's adaptive responses to casting, long-term confinement to bed rest and possibly to trauma. As part of a larger program using TMS to investigate changes associated with bed rest, we sought to determine whether casting of the leg causes brain excitability changes measurable with TMS, and the time course of resolution of these changes. In this study, eight adults wore a full leg cast for 10 days. TMS measures of motor cortex excitability were gathered before the cast was placed, and then immediately after cast removal, and 24 and 48 h later. A control group did not wear a cast and underwent the same TMS sessions. Significant excitability changes occurred and peaked at 24 h post cast removal in the TMS experimental group but not the non-casted group.

Two toxins from Conus striatus that individually induce tetanic paralysis.

We describe structural properties and biological activities of two related O-glycosylated peptide toxins isolated from injected (milked) venom of Conus striatus, a piscivorous snail that captures prey by injecting a venom that induces a violent, spastic paralysis. One 30 amino acid toxin is identified as kappaA-SIVA (termed s4a here), and another 37 amino acid toxin, s4b, corresponds to a putative peptide encoded by a previously reported cDNA. We confirm the amino acid sequences and carry out structural analyses of both mature toxins using multiple mass spectrometric techniques. These include electrospray ionization ion-trap mass spectrometry and nanoelectrospray techniques for small volume samples, as well as matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis as a complementary method to assist in the determination of posttranslational modifications, including O-linked glycosylation. Physiological experiments indicate that both s4a and s4b induce intense repetitive firing of the frog neuromuscular junction, leading to a tetanic contracture in muscle fiber. These effects apparently involve modification of voltage-gated sodium channels in motor axons. Notably, application of either s4a or s4b alone mimics the biological effects of the whole injected venom on fish prey.

Identification and characterization of homologues of vertebrate beta-thymosin in the marine mollusk Aplysia californica.

The beta-thymosins have been known as actin-sequestering proteins, but now are recognized as molecules with multiple and diverse intracellular and extracellular functions. Two closely related proteins, beta-thymosin(His) and beta-thymosin(Gln), have been de novo sequenced by top-down mass spectrometry in the common neurobiology model, Aplysia californica. As determined by nanoelectrospray quadrupole-enhanced Fourier-Transform mass spectrometry with collisionally activated and electron-capture dissociations, both of these Aplysia beta-thymosins are acetylated and differ by a single residue in the central actin-binding domain. Profiling of individual cells and tissue by matrix-assisted laser desorption/ionization mass spectrometry reveals that these proteins are widely expressed in the Aplysia central nervous system, including in individual identified neurons, neuronal clusters, nerves and connective tissues. Newly identified beta-thymosin(His) and beta-thymosin(Gln) are also detected by mass spectrometry in hemolymph, and in releasates collected from whole ganglia. When applied exogenously, beta-thymosin proteins, purified from nerve cell extract, support the anchoring of neurons, and increase neurite sprouting and total neurite outgrowth in culture. These positive effects on neurite regeneration in cell culture suggest that the beta-thymosin proteins have an extracellular function in the central nervous system of Aplysia californica.

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.

Intraspecific variation of venom injected by fish-hunting Conus snails.

Venom peptides from two species of fish-hunting cone snails (Conus striatus and Conus catus) were characterized using microbore liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and electrospray ionization-ion trap-mass spectrometry. Both crude venom isolated from the venom duct and injected venom obtained by milking were studied. Based on analysis of injected venom samples from individual snails, significant intraspecific variation (i.e. between individuals) in the peptide complement is observed. The mixture of peptides in injected venom is simpler than that in the crude duct venom from the same snail, and the composition of crude venom is more consistent from snail to snail. While there is animal-to-animal variation in the peptides present in the injected venom, the composition of any individual's injected venom remains relatively constant over time in captivity. Most of the Conus striatus individuals tested injected predominantly a combination of two neuroexcitatory peptides (s4a and s4b), while a few individuals had unique injected-venom profiles consisting of a combination of peptides, including several previously characterized from the venom duct of this species. Seven novel peptides were also putatively identified based on matches of their empirically derived masses to those predicted by published cDNA sequences. Profiling injected venom of Conus catus individuals using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry demonstrates that intraspecific variation in the mixture of peptides extends to other species of piscivorous cone snails. The results of this study imply that novel regulatory mechanisms exist to select specific venom peptides for injection into prey.

Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry.

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis.

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.

Characterization of a novel gastropod toxin (6-bromo-2-mercaptotryptamine) that inhibits shaker K channel activity.

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6-6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.

A novel prohormone processing site in Aplysia californica: the Leu-Leu rule.

Neuropeptides are a complex set of signaling molecules produced through enzymatic cleavages from longer prohormone sequences. The most common cleavage sites in prohormones are basic amino acid residues; however, processing is observed at non-basic sites. Cleavage at Leu-Leu sequences has been observed in three Aplysia californica prohormones. To further investigate this unusual event, native and non-native synthetic peptides containing Leu-Leu residues are incubated with homogenates of Aplysia californica ganglia and the resulting products monitored with MALDI MS. Cleavage near and between Leu-Leu residues is observed in the abdominal and buccal ganglia homogenates, confirming the presence of an unidentified peptidase. In addition, fractions from an HPLC separation of buccal ganglia homogenates also produce cleavages at Leu-Leu residues. Products resulting from cleavage at Leu-Leu sites are observed and are produced in larger amounts in acidic and neutral pH ranges, and cleavage is inhibited by the addition of EDTA, suggesting a metal is required for activity.

Anatomical correlates of venom production in Conus californicus.

Like all members of the genus, Conus californicus has a specialized venom apparatus, including a modified radular tooth, with which it injects paralyzing venom into its prey. In this paper the venom duct and its connection to the pharynx, along with the radular sac and teeth, were examined using light and transmission electron microscopy. The general anatomy of the venom apparatus resembles that in other members of the genus, but several features are described that have not been previously reported for other species. The proximal (posterior) quarter of the venom duct is composed of a complex epithelium that may be specialized for active transport rather than secretion. The distal portion of the duct is composed of a different type of epithelium, suggestive of holocrine secretion, and the cells display prominent intracellular granules of at least two types. Similar granules fill the lumen of the duct. The passageway between the lumen of the venom duct and pharynx is a flattened branching channel that narrows to a width of 10 micro m and is lined by a unique cell type of unknown function. Granular material similar to that in the venom duct was also found in the lumen of individual teeth within the radular sac. Mass spectrometry (MALDI-TOF) demonstrated the presence of putative peptides in material derived from the tooth lumen, and all of the more prominent species were also evident in the anterior venom duct. Radular teeth thus appear to be loaded with peptide toxins while they are still in the radular sac.

Orcokinin peptides in developing and adult crustacean stomatogastric nervous systems and pericardial organs.

The orcokinins are a family of neuropeptides recently isolated from several crustacean species. We found orcokinin-like immunoreactivity in the stomatogastric nervous systems and pericardial organs of three decapod crustacean species, Homarus americanus, Cancer borealis, and Panulirus interruptus. The neuropil of the stomatogastric ganglion was stained in adults of all three species as well as in embryonic and larval H. americanus. In H. americanus, the somata giving rise to this projection were found in the inferior ventricular nerve. Matrix-assisted laser desorption/ionization mass spectrometry mass profiling and sequencing with postsource decay led to the identification of six different orcokinin family peptides, including those previously described in other decapods and two novel shorter peptides. Application of exogenous [Ala(13)]orcokinin to the stomatogastric ganglion of H. americanus resulted in changes in the pyloric rhythm. Specifically, the number of lateral pyloric (LP) neuron spikes/burst decreased, and the phase of firing of the pyloric neurons was altered. Together, these data indicate that the orcokinins are likely to function as modulators of the crustacean stomatogastric ganglion.