Comparing Container Closure Integrity Test Methods - Performance of Headspace Carbon Dioxide Analysis versus Helium Leakage Using Positive Controls.

As described in USP Chapter <1207>, the deterministic leak test methods using laser-based gas headspace analysis and helium leakage are those with the highest sensitivities. As stated in the chapter, ″no single package leak test or package seal quality test method is applicable to all product-package systems″; therefore, knowing the advantages and disadvantages of both of these techniques, and the extent to which they can be substituted for each other, is valuable. In an effort to begin addressing this issue, a systematic study using these two techniques has been performed. This study used the same well-defined positive controls prepared with microcapillaries for both measurement techniques. For the headspace gas analysis technique, the headspace carbon dioxide content was measured at multiple timepoints during three separate conditioning cycles using either a 0.5, 1.0, or 2.0 bar CO2 overpressure; the observed change in headspace carbon dioxide was then used to determine an ingress rate for each positive control. For the helium leakage technique, the positive controls were measured with a standard helium leak detector with 100% helium atmosphere on the atmospheric pressure side of the artificial defects. The resulting leakage rates from both techniques were compared for ingress into both ISO 2R and ISO 10R vials. The obtained correlation between helium and carbon dioxide leakage rates resulted in a minimum R2 coefficient of 0.98 across all 12 runs. Additionally, both setups met the acceptance criteria for accuracy with their respective calibrated standards.

Yellow droopy eyes: A case of obstructive jaundice from Burkitt's lymphoma with involvement of bilateral cavernous sinus.

Burkitt's lymphoma follows a lymphogenous spread early in the disease. The central nervous system can be involved via a hematogenous route but involvement of the cavernous sinus (CS) is rare and can be misdiagnosed as other pathology of primary neoplastic, infective, or vascular origin. We present a case of a 73-year-old gentleman with painless jaundice and subjective heaviness to his eyes that progressed to partial ptosis of the left eye, complete ptosis of the right eye with diplopia, found to have disseminated Burkitt's lymphoma with bilateral deposits to the CS. Early recognition of Burkitt's lymphoma with CS involvement is important as it often signifies disseminated disease with implications on chemotherapy regimen, treatment outcomes, and survival.

Container Closure Integrity Test Method Development on Vials Stored at -80°C Using Headspace Carbon Dioxide Analysis.

Maintaining container closure integrity (CCI) is challenging for vials that are packaged at room temperature and stored and/or transported at a colder temperature, such as -80°C. Cold temperatures can affect the sealing mechanisms of the package because of the glass transition temperature (-50°C to -70°C) of the rubber stopper used to seal the vial and the different coefficients of thermal expansion of each of the primary packaging components. Most CCI tests are conducted at room temperature and detect leaks from permanent defects that always exist under all storage conditions. However, previous research shows that temporary leaks that develop during cold storage can reseal when the vials are brought back up to room temperature and, therefore, can no longer be detected. The following study demonstrates two methods for CCI testing that can be performed with product-filled, frozen vials, packaged with unmodified headspace conditions using carbon dioxide ingress as a leak indicator. The two methods utilize different gas flow depending on the storage conditions. The first method generates effusive flow through a leaking defect, whereas the second generates diffusive flow. Experimental data revealed the effusive procedure detected laser-drilled defects ≥2 µm in empty glass vials and microwire defects in empty glass and plastic vials with effective defect sizes ≥0.4 and 0.6 µm, respectively. The diffusive procedure detected laser-drilled defects ≥2 µm in empty glass vials and microwire defects in empty glass and plastic vials with effective defect sizes ≥ 0.8 and 2.6 µm, respectively. Liquid product interactions with the defect as well as length of the storage period were also explored.

Patient-induced progressive facial subcutaneous emphysema masquerading as odontogenic abscess.

Facial subcutaneous emphysema (SE) is an uncommon sequelae of dental procedures and often attributed to the use of high-speed air-driven handpieces during surgical extractions, forcing air through fascial spaces. Rarely have there been documented cases of patient-induced SE. In this case report, we present an 18-year-old woman who was referred to the emergency department with a 5-day history of progressive swelling and pain to her right cheek, following a prolonged, but simple extraction of tooth 18. While the dentist and emergency physicians were concerned about an infectious aetiology, history taking, clinical review and imaging corroborated the diagnosis of patient-induced SE secondary to habitual straw use. This report highlights the need for routine postextraction counselling of sinus precautions irrespective of extraction complexity. Additionally, emergency physicians should be aware of SE masquerading as other pathology, including odontogenic abscesses, allergic reactions, angioedema and gas-forming bacterial infections, such as necrotising fasciitis, to ensure appropriate treatment is provided to patients.

TMJ pain as a presentation of metastatic breast cancer to the right mandibular condyle.

Cancer metastasis to the oral and maxillofacial region is uncommon, and metastasis to the mandibular condyle is considered rare. We present a case of a 56-year-old woman with a history of invasive ductal cell carcinoma of the right breast, 10 years in remission, presenting with a 6-month history of symptoms typical of temporomandibular joint (TMJ) dysfunction. Imaging revealed an osteolytic lesion of her right TMJ and subsequent open biopsy confirmed the diagnosis of metastatic breast cancer. Despite the rarity of metastatic cancer to the head and neck region, it is still important for clinicians from both medical and dental backgrounds to consider this differential diagnosis, particularly in patients with a history of hormonal positive subtype of breast cancer. Given that bony metastasis can manifest even 10 years after initial diagnosis, surveillance which includes examination of the head and neck region is important, and may include routine plain-film imaging surveillance with an orthopantomogram (OPG).

Container Closure Integrity Test Using Frequency Modulation Spectroscopy Headspace Analysis with Carbon Dioxide as a Tracer Gas.

As described in USP <1207>, the container closure integrity (CCI) of a pharmaceutical package must be maintained throughout the product lifecycle to ensure sterility and stability. Current CCI test methods can be time-consuming, destructive, and lack the required sensitivity. This study presents a novel, fast, and nondestructive method for CCI testing that uses carbon dioxide as a tracer gas under effusive pressure conditions. Two types of defects were tested: laser-drilled defects located in the glass body (2, 5, and 10 µm nominal diameter) and tungsten wires inserted between the stopper and the landing seal of the vial (41, 64, and 80 µm outer diameter). During each test session, vials were placed in a pressure vessel, isolated from ambient conditions, and pressure-cycled by first pulling a vacuum and then applying an overpressure of pure carbon dioxide gas. After being exposed to 20 psig (34.7 psia) of carbon dioxide for 30 min, the overpressure was released and the vials were measured on an FMS-Carbon Dioxide Headspace Analyzer. This headspace gas analyzer utilizes a tunable diode laser absorption spectroscopy technique that employs frequency modulation to enhance measurement sensitivity. An increase of ≥1 torr in the headspace carbon dioxide content after completion of the pressure cycling procedure was intended to serve as confirmation of leak detection. All empty vials with either a 2 µm laser-drilled defect or 41 µm wire (effective defect size ∼2 µm), or greater, at the stopper-seal interface were detected by this method. Furthermore, vials filled with 1 mg/mL bovine serum albumin in phosphate-buffered saline containing a 5 µm laser-drilled defect below the liquid level or a 64 µm wire (effective defect size ∼6.1 µm), or greater, at the stopper-seal interface (defect above the liquid level) were detected. This test can be used for a wide variety of vial types and headspace compositions.

Proteomic identification of synaptic caspase substrates.

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapse-specific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses. Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states.

Method Development for Container Closure Integrity Evaluation via Headspace Gas Ingress by Using Frequency Modulation Spectroscopy.

USP <1207.1> Section 3.5 states that "A deterministic leak test method having the ability to detect leaks at the product's maximum allowable leakage limit is preferred when establishing the inherent integrity of a container-closure system." Ideally, container closure integrity of parenteral packaging would be evaluated by measuring a physical property that is sensitive to the presence of any package defect that breaches package integrity by increasing its leakage above its maximum allowable leakage limit. The primary goals of the work presented herein were to demonstrate the viability of the nondestructive, deterministic method known as laser-based gas headspace analysis for evaluating container closure integrity and to provide a physical model for predicting leak rates for a variety of container volumes, headspace conditions, and defect sizes. The results demonstrate that laser-based headspace analysis provides sensitive, accurate, and reproducible measurements of the gas ingress into glass vial-stopper package assemblies that are under either diffusive or effusive leak conditions. Two different types of positive controls were examined. First, laser-drilled micro-holes in thin metal disks that were crimped on top of 15R glass vials served as positive controls with a well-characterized defect geometry. For these, a strong correlation was observed between the measured ingress parameter and the size of the defect for both diffusive and effusive conditions. Second, laser-drilled holes in the wall of glass vials served as controls that more closely simulate real-world defects. Due to their complex defect geometries, their diffusive and effusive ingress parameters did not necessarily correlate; this is an important observation that has significant implications for standardizing the characterization of container defects. Regardless, laser-based headspace analysis could readily differentiate positive and negative controls for all leak conditions, and the results provide a guide for method development of container closure integrity tests.LAY ABSTRACT: The new USP 39 <1207>, "Package Integrity Evaluation-Sterile Products", states in section 3.4.1: "tracer gas tests performed using … laser-based gas headspace analysis [have] been shown to be sensitive enough to quantitatively analyze leakage through the smallest leak paths found to pose the smallest chance of liquid leakage or microbial ingress in rigid packaging." In addition, USP <1207> also states that "for such methods, the limit of detection can be mathematically predicted on the basis of gas flow kinetics." Using the above statements as a foundation, this paper presents a theoretical basis for predicting the gas ingress through well-defined defects in product vials sealed under a variety of headspace conditions. These calculated predictions were experimentally validated by comparing them to measurements of changes in the headspace oxygen content or total pressure for several different positive controls using laser-based headspace analysis. The results demonstrated that laser-based headspace analysis can, by readily differentiating between negative controls and positive controls with a range of defect sizes on the micron scale, be used to assess container closure integrity. The work also demontrated that caution must be used when attempting to correlate a leak rate to an idealized defect-size parameter.

Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.

OBJECTIVES: Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. DESIGN AND METHODS: For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. RESULTS: Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. CONCLUSION: Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.

Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

Translational dynamics of water at the phospholipid interface.

The residual water-proton magnetic relaxation dispersion profile obtained from suspensions of phospholipid vesicles in deuterium oxide was found to be a logarithmic function of the proton Larmor frequency at high magnetic field strengths, and independent of Larmor frequency at low magnetic field strengths. The residual proton relaxation is caused by dipole-dipole coupling between the residual water proton in otherwise deuterated water and the phospholipid protons. The logarithmic dependence on magnetic field strength is the signature of water-proton diffusive exploration on the interface that is approximately two-dimensionally constrained. Application of relaxation theory for two-dimensional diffusion to the spin-lattice relaxation data yields a translational correlation time of approximately 70 ps for water diffusing in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles.

Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins.

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.

PICquant: a quantitative platform to measure differential peptide abundance using dual-isotopic labeling with 12C6- and 13C6-phenyl isocyanate.

We have developed a complete system for the isotopic labeling, fractionation, and automated quantification of differentially expressed peptides that significantly facilitates candidate biomarker discovery. We describe a new stable mass tagging reagent pair, (12)C(6)- and (13)C(6)-phenyl isocyanate (PIC), that offers significant advantages over currently available tags. Peptides are labeled predominantly at their amino termini and exhibit elution profiles that are independent of label isotope. Importantly, PIC-labeled peptides have unique neutral-mass losses upon CID fragmentation that enable charge state and label isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. To exploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS(2) data acquisition strategy and a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach, dubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwise confound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the system allows rapid identification of peptides that are differentially expressed between control and patient groups.

MAZIE: a mass and charge inference engine to enhance database searching of tandem mass spectra.

Peptide sequence identification using tandem mass spectroscopy remains a major challenge for complex proteomic studies. Peptide matching algorithms require the accurate determination of both the mass and charge of the precursor ion and accommodate uncertainties in these properties by using a wide precursor mass tolerance and by testing, for each spectrum, several possible candidate charges. Using a data acquisition strategy that includes obtaining narrow mass-range MS(1) "zoom" scans, we describe here a post-acquisition algorithm dubbed mass and charge (Z) inference engine (MAZIE), which accurately determines the charge and monoisotopic mass of precursor ions on a low-resolution Thermo LTQ-XL mass spectrometer. This is achieved by examining the isotopic distribution obtained in the preceding MS(1) zoom spectrum and comparing to theoretical distributions for candidate charge states from +1 to +4. MAZIE then writes modified data files with the corrected monoisotopic mass and charge. We have validated MAZIE results by comparing the sequence search results obtained with the MAZIE-generated data files to results using the unmodified data files. Using two different search algorithms and a false discovery rate filter, we found that MAZIE-interpreted data resulted in 80% (using SEQUEST) and 30% (using OMSSA) more high-confidence sequence identifications. Analyses of these results indicate that the accurate determination of the precursor ion mass greatly facilitates the ability to differentiate between true and false positive matches, while the determination of the precursor ion charge reduces the overall search time but does not significantly reduce the ambiguity of interpreting the search results. MAZIE is distributed as an open-source PERL script.

Chromium(III) complexes as intermolecular probes.

Metal ion complexes provide flexible paramagnetic centers that may be used to define intermolecular contacts in a variety of solution phase environments because both the charge and electronic relaxation properties of the complex may be varied. For most complex ions, there are several proton equilibria that may change the effective charge on the complex as a function of pH which in turn affects the efficacy of application for defining the electrostatic surfaces of co-solute molecules. We report here spectrophotometric and nuclear spin relaxation studies on aqueous solutions of chromium(III) complexes of EDTA, DTPA, and bis-amides of both. The effective charges available from these paramagnetic centers range from -3 to +1 and we report the pH ranges over which the effective charge is defined with confidence for application in magnetic relaxation experiments.

High frequency dynamics in hemoglobin measured by magnetic relaxation dispersion.

The magnetic relaxation dispersion profiles for formate, acetate, and water protons are reported for aqueous solutions of hemoglobin singly and doubly labeled with a nitroxide and mercury(II) ion at cysteines at beta-93. Using two spin labels, one nuclear and one electron spin, a long intramolecular vector is defined between the two beta-93 positions in the protein. The paramagnetic contributions to the observed 1H spin-lattice relaxation rate constant are isolated from the magnetic relaxation dispersion profiles obtained on a dual-magnet apparatus that provides spectral density functions characterizing fluctuations sensed by intermoment dipolar interactions in the time range from the tens of microseconds to approximately 1 ps. Both formate and acetate ions are found to bind specifically within 5 angstroms of the beta-93 spin-label position and the relaxation dispersion has inflection points corresponding to correlation times of 30 ps and 4 ns for both ions. The 4-ns motion is identified with exchange of the anions from the site, whereas the 30-ps correlation time is identified with relative motions of the spin label and the bound anion in the protein environment close to beta-93. The magnetic field dependence of the paramagnetic contributions in both cases is well described by a simple Lorentzian spectral density function; no peaks in the spectral density function are observed. Therefore, the high frequency motions of the protein monitored by the intramolecular vector defined by the electron and nuclear spin are well characterized by a stationary random function of time. Attempts to examine long vector fluctuations by employing electron spin and nuclear spin double-labeling techniques did not yield unambiguous characterization of the high frequency motions of the vector between beta-93 positions on different chains.

Magnetic relaxation dispersion probe.

The magnetic field dependence of nuclear spin-lattice relaxation rates provides a powerful approach to characterizing intra and intermolecular dynamics. NMR spectrometers that provide extensive magnetic relaxation dispersion profiles may switch magnetic field strengths rapidly by either moving the sample or by changing the current in an electromagnet. If the sample is moved, the polarization and detection fields may be very high, which provides both high sensitivity and resolution. This report summarizes the design of a pneumatic sample transport system for glass sample containers that may be used in either a dual magnet spectrometer or in a single magnet system that exploits the fringe field as the secondary magnetic field.

Magnetic relaxation dispersion of lithium ion in solutions of DNA.

The magnetic field dependence of the nuclear spin-lattice relaxation rate constant defines the magnetic relaxation dispersion (MRD) and provides a direct characterization of the molecular dynamics that cause fluctuations in the magnetic couplings in the system and may also indicate the dimensional constraints on the motion. The counterion cloud surrounding a linear polyelectrolyte ion, such as DNA in solution, provides an interesting opportunity for ion confinement that helps in understanding the thermodynamics and the dynamics of the interactions between the polyion and other solutes. The MRD profiles of lithium ion and tetramethylammonium ion were recorded in dilute aqueous solutions of native calf thymus DNA, which provides a long, charged rod that reorients slowly. The 7Li ion relaxes through the nuclear electric quadrupole coupling and the proton-lithium dipole-dipole coupling; the protons of the tetramethylammonium ion relax by dipole-dipole coupling. MRD profiles of the 7Li+ ion are dominated by transient interactions with the DNA that yield a linear dependence of the spin-lattice relaxation rate constant on the logarithm of the Larmor frequency. This magnetic field dependence is consistent with diffusive ion motions that modulate two spatial coordinates that characterize the relaxation couplings in the vicinity of the polyion. Motions around the rod and fluctuations in the ion distance from the rod are consistent with these constraints for lithium. The magnetic field dependence of the tetramethylammonium ion proton relaxation rate constant is weak, but also approximately a linear function of the logarithm of the Larmor frequency, which implies that the field dependence is caused in part by local order in the DNA solution.