An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Characterization of Polymeric Syringes Used for Intravitreal Injection.

Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g. 50 µL) and high injection forces. DPs in vials are typically transferred and injected using disposable polymer syringes, which can feature a silicone oil (SO) coating. In our syringe in-use study, we compared dead volume, total SO content and SO layer distributions of three IVT transfer injection syringes. We assessed multiple potential impact factors such as protein concentration, needle gauge, injection speed, surfactant type and the impact of the in-use hold time on sub-visible particle (SvP) formation and injection forces. Pronounced differences were observed between the syringes regarding SvP generation. Siliconized syringes showed higher SvP counts as compared to non-siliconized syringes. In some cases injection forces exceeded 20 N, which caused needles to burst off during injection. The syringes also showed relevant differences in total SO content and dead volume. In conclusion, specific consideration in the selection of an adequate transfer injection syringe are required. This includes extensive testing and characterization under intended and potential in-use conditions and the development of in-use handling procedures.

Method to Predict Glass Vial Fogging in Lyophilized Drug Products.

Glass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging's expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry. For that reason, we evaluated a simple method ("simulated fogging") applicable to drug product formulations in a specific container closure system. Two different vial types with different surface hydrophilicity were tested using 3 model protein formulations, comparing the simulated fogging test and the degree of fogging after actual lyophilization. The simulated fogging method could predict fogging and showed a correlation to fogging in lyophilized drug product glass vials. We observed that all formulations showed fogging in the hydrophilic vials. By contrast, hydrophobic vials prevented fogging, however, interestingly with remaining defects of so-called droplet formation. Other than extent of fogging, no additional differences of lyophilized cake properties or other product quality attributes were observed between products using the different glass vial types tested.

Phase-Appropriate Application of Analytical Methods to Monitor Subvisible Particles Across the Biotherapeutic Drug Product Life Cycle.

The phase-appropriate application of analytical methods to characterize, monitor, and control particles is an important aspect of the development of safe and efficacious biotherapeutics. The AAPS Product Attribute and Biological Consequences (PABC) focus group (which has since transformed into an AAPS community) conducted a survey where participating labs rated their method of choice to analyze protein aggregation/particle formation during the different stages of the product life cycle. The survey confirmed that pharmacopeial methods and SEC are the primary methods currently applied in earlier phases of the development to ensure that a product entering clinical trials is safe and efficacious. In later phases, additional techniques are added including those for non-GMP extended characterization for product and process characterization. Finally, only robust, globally-accepted, and stability-indicating methods are used for GMP quality control purposes. This was also consistent with the feedback during a webinar hosted by the group to discuss the survey results. In this white paper, the team shares the results of the survey and provides guidance on selecting phase-appropriate analytical methods and developing a robust particle control strategy.

Variance Between Different Light Obscuration and Flow Imaging Microscopy Instruments and the Impact of Instrument Calibration.

Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the sizing accuracy of LO analysis was evaluated by using a calibration suspension of lower refractive index beads made of silica suspended in sucrose solution. It was demonstrated that the sizing accuracy was strongly dependent on the reference material's properties used for calibration. It was also demonstrated that flow imaging microscopy suffered from the same artifact, albeit to a smaller extent. We further tested different LO sensors and instruments. Interestingly, our results show that the sizing accuracy varied from instrument to instrument, strongly depending on the properties of the sensor. To summarize, sizing and counting accuracies were dependent on the material used for calibration and its optical properties as well as the calibration curve, the sensor, and the instrument supplier. Closer match of optical properties between calibration system and test system seems to improve the sensitivity of the measurement. The results of this study raise the following major practical implications: (1) LO and flow imaging microscopy are not truly orthogonal analytical methods, (2) while matching optimal properties of material used for calibration and test items increased sensitivity, this is of poor practical applicability given that analytes contain multiple particles, and (3) setting product-specific limits for SVPs require special considerations with regard to the data sets used.

Comparing Physical Container Closure Integrity Test Methods and Artificial Leak Methodologies.

The sterility of drug products intended for parenteral administration is a critical quality attribute (CQA) because it serves to ensure patient safety and is thus a key requirement by health authorities. While sterility testing is a probabilistic test, the assurance of sterility is a holistic concept including adequate design of manufacturing facilities, process performance, and product design. Container closure integrity testing (CCIT) is necessary to confirm the integrity of a container closure system (CCS), until the end of a product's shelf life. The new and revised United States Pharmacopeia (USP) General Chapter <1207> is a comprehensive guidance on CCI. Nevertheless, practical considerations including the choice of CCIT methods, the acceptance criteria, or the positive control samples (artificial leaks) must be addressed by the pharmaceutical manufacturer.This study is the first to provide a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA), and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).The results from these experiments provide comprehensive data to allow a direct comparison of the capabilities of the individual methods. The results confirmed that the He leak detection method, which is considered the "gold-standard" for pCCIT regarding method sensitivity, indeed demonstrates the highest detection sensitivity (lowest detection limit). In comparison to the dye ingress method, HSA and vacuum decay also demonstrated better detection sensitivity in our study.Capillary leaks with orifice diameter (capillary leak with flow according to an ideal orifice) and micro holes yielded similar leak rates, whereas capillaries with nominal diameters yielded significantly lower leak rates. In conclusion, method sensitivity cannot be compared by means of a leak diameter, but requires the consideration of multiple impacting factors (e.g., path length, uniformity).LAY ABSTRACT: Sterility of drug products intended for parenteral administration is a critical quality attribute to ensure patient's safety and is thus a key requirement by health authorities. The absence of microbial contamination must be demonstrated by container closure integrity (CCI) of the container closure system (CCS). Currently, the revised United States Pharmacopeia (USP) General Chapter <1207> provides the most extensive guidance on how CCI should be assessed. Nevertheless, practical considerations on the choice of an appropriate CCIT method, artificial leaks or the choice of an acceptance criteria are lacking and must be addressed by the pharmaceutical manufacturer.This study provides a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA) and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).

Container Closure Integrity Testing of Prefilled Syringes.

Prefilled syringes (PFSs) are increasingly preferred over vials as container closure systems (CCSs) for injectable drug products when facilitated or self-administration is required. However, PFSs are more complex compared to CCSs consisting of vial, rubber stopper, and crimp cap. Container closure integrity (CCI) assurance and verification has been a specific challenge for PFSs as they feature several sealing areas. A comprehensive understanding of the CCS is necessary for an appropriate CCI assessment as well as for packaging development and qualification. A comprehensive CCI assessment of 6 different PFSs from 3 different manufacturers (including 1 polymeric PFS) was conducted using helium leak testing. PFS components were manipulated to systematically assess the contribution of the different sealing areas to CCI, namely rigid needle shield (RNS)/needle, RNS/tip cone, and the individual ribs of a syringe plunger. The polymeric PFS required an equilibrium measurement for accurate container closure integrity testing. The different sealing areas and a single plunger rib were shown to provide adequate CCI. Acceptable tip cap movement until the point of CCI failure was estimated. The assessment of acceptable tip cap movement demonstrated the importance of considering the RNS/tip cone seal design to ensure CCI of the PFS upon post assembly possesses and shipment.

Mutations in PIH1D3 Cause X-Linked Primary Ciliary Dyskinesia with Outer and Inner Dynein Arm Defects.

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance. Here we report maternally inherited and de novo mutations in PIH1D3 in four men affected with PCD. PIH1D3 is located on the X chromosome and is involved in the preassembly of both outer (ODA) and inner (IDA) dynein arms of cilia and sperm flagella. Loss-of-function mutations in PIH1D3 lead to absent ODAs and reduced to absent IDAs, causing ciliary and flagellar immotility. Further, PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4. This result has clinical and genetic counseling implications for genetically unsolved male case subjects with a classic PCD phenotype that lack additional phenotypes such as intellectual disability or retinitis pigmentosa.

Loss of histone H3 methylation at lysine 4 triggers apoptosis in Saccharomyces cerevisiae.

Monoubiquitination of histone H2B lysine 123 regulates methylation of histone H3 lysine 4 (H3K4) and 79 (H3K79) and the lack of H2B ubiquitination in Saccharomyces cerevisiae coincides with metacaspase-dependent apoptosis. Here, we discovered that loss of H3K4 methylation due to depletion of the methyltransferase Set1p (or the two COMPASS subunits Spp1p and Bre2p, respectively) leads to enhanced cell death during chronological aging and increased sensitivity to apoptosis induction. In contrast, loss of H3K79 methylation due to DOT1 disruption only slightly affects yeast survival. SET1 depleted cells accumulate DNA damage and co-disruption of Dot1p, the DNA damage adaptor protein Rad9p, the endonuclease Nuc1p, and the metacaspase Yca1p, respectively, impedes their early death. Furthermore, aged and dying wild-type cells lose H3K4 methylation, whereas depletion of the H3K4 demethylase Jhd2p improves survival, indicating that loss of H3K4 methylation is an important trigger for cell death in S. cerevisiae. Given the evolutionary conservation of H3K4 methylation this likely plays a role in apoptosis regulation in a wide range of organisms.

Size fractionation of microscopic protein aggregates using a preparative fluorescence-activated cell sorter.

Protein aggregation, which takes place both in vivo and in vitro, is an important degradative pathway for all proteins. Protein aggregates have distinct physicochemical and biological properties that are important to study and characterize from the perspective of both fundamental and applied sciences. The size of protein aggregates varies across a huge range, spanning several orders of magnitude. Currently, protein aggregates larger than hundreds of nanometers in diameter are impossible to physically fractionate. Here, we present a new method to fractionate microscopic proteinaceous particles using preparative fluorescence-activated cell sorting technology.

Bre1p-mediated histone H2B ubiquitylation regulates apoptosis in Saccharomyces cerevisiae.

BRE1 encodes an E3 ubiquitin protein ligase that is required for the ubiquitylation of histone H2B at lysine 123 (K123). Ubiquitylation of this histone residue is involved in a variety of cellular processes including gene activation and gene silencing. Abolishing histone H2B ubiquitylation also confers X-ray sensitivity and abrogates checkpoint activation after DNA damage. Here we show that Saccharomyces cerevisiae Bre1p exhibits anti-apoptotic activity in yeast and that this is linked to histone H2B ubiquitylation. We found that enhanced levels of Bre1p protect from hydrogen-peroxide-induced cell death, whereas deletion of BRE1 enhances cell death. Moreover, cells lacking Bre1p show reduced lifespan during chronological ageing, a physiological apoptotic condition in yeast. Importantly, the resistance against apoptosis is conferred by histone H2B ubiquitylation mediated by the E3 ligase activity of Bre1p. Furthermore, we found that the death of Deltabre1 cells depends on the yeast caspase Yca1p, because Deltabre1 cells exhibit increased caspase activity when compared with wild-type cells, and deletion of YCA1 leads to reduced apoptosis sensitivity of cells lacking Bre1p.

Increasing the number of diagnostic mutations in malignant hyperthermia.

Malignant hyperthermia (MH) is an autosomal dominant disorder characterized by abnormal calcium homeostasis in skeletal muscle in response to triggering agents. Today, genetic investigations on ryanodine receptor type 1 (RYR1) gene and alpha1 subunit of the dihydropyridine receptor (DHPR) (CACNA1S) gene have improved the procedures associated with MH diagnosis. In approximately 50% of MH cases a causative RYR1 mutation was found. Molecular genetic testing based on RYR1 mutations for MH diagnosis is challenging, because the causative mutations, most of which are private, are distributed throughout the RYR1 gene. A more comprehensive genetic testing procedure is needed. Therefore, we aim to expand the genetic information related to MH and to evaluate the effect of mutations on the MH phenotype. Performing an in-depth mutation screening of the RYR1 transcript sequence in 36 unrelated MH susceptible (MHS) patients, we identified 17 novel, five rare, and eight non-disease-causing variants in 23 patients. The 13 remaining MHS patients presented no known variants, neither in RYR1 nor in the CACNA1S binding regions to RYR1. The 17 novel variants were found to affect highly conserved amino acids and were absent in 100 controls. Excellent genotype-phenotype correlations were found by investigating 21 MHS families-a total of 186 individuals. Epstein-Barr virus (EBV) lymphoblastoid cells carrying four of these novel mutations showed abnormal calcium homeostasis. The results of this study contribute to the establishment of a robust genetic testing procedure for MH diagnosis.