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).

Synthesis, characterization, and in-vitro cytocompatibility of amorphous ß-tri-calcium magnesium phosphate ceramics.

Biphasic mixtures of crystalline ß-tricalcium magnesium phosphate (ß-TCMP) and an amorphous calcium magnesium phosphate have been synthesized and reported to support enhanced hMSC differentiation in comparison to ß-tricalcium phosphate (ß-TCP) due to the release of increased amounts of bioactive ions. In the current study, completely amorphous ß-TCMP has been synthesized which is capable of releasing increased amounts of Mg(2+) and PO4(3-) ions, rather than a biphasic mixture as earlier reported. The amorphous phase formed was observed to crystallize between temperatures of 400-600°C. The scaffolds prepared with amorphous ß-TCMP were capable of supporting enhanced hMSC proliferation and differentiation in comparison to commercially available ß-TCP. However, a similar gene expression of mature osteoblast markers, OCN and COL-1, in comparison to biphasic ß-TCMP was observed. To further study the role of Mg(2+) and PO4(3-) ions in regulating hMSC osteogenic differentiation, the capability of hMSCs to mineralize in growth media supplemented with Mg(2+) and PO4(3-) ions was studied. Interestingly, 5mM PO4(3-) supported mineralization while the addition of 5mM Mg(2+) to 5mM PO4(3-) inhibited mineralization. It was therefore concluded that the release of Ca(2+) ions from ß-TCMP scaffolds also plays a role in regulating osteogenic differentiation on these scaffolds and it is noted that further work is required to more accurately determine the exact role of Mg(2+) in regulating hMSC osteogenic differentiation.

Subvisible (2-100 µm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Biotherapeutic formulation factors affecting metal leachables from stainless steel studied by design of experiments.

Trace amounts of metals are inevitably present in biotherapeutic products. They can arise from various sources. The impact of common formulation factors such as protein concentration, antioxidant, metal chelator concentration and type, surfactant, pH, and contact time with stainless steel on metal leachables was investigated by a design of experiments approach. Three major metal leachables, iron, chromium, and nickel were monitored by inductively coupled plasma-mass spectrometry. It was observed that among all the tested factors, contact time, metal chelator concentration, and protein concentration were statistically significant factors with higher temperature resulting in higher levels of leached metals. Within a pH range of 5.5-6.5, solution pH played a minor role for chromium leaching at 25°C. No statistically significant difference was observed due to type of chelator, presence of antioxidant, or surfactant. In order to optimize a biotherapeutic formulation to achieve a target drug product shelf life with acceptable quality, each formulation component must be evaluated for its impact.

Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.

The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Šwith the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.

Comparative evaluation of disodium edetate and diethylenetriaminepentaacetic acid as iron chelators to prevent metal-catalyzed destabilization of a therapeutic monoclonal antibody.

Understanding the effect of metal chelators with respect to their ability to inhibit metal-catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na(2)EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation, and fragmentation demonstrated that DTPA was equivalent to Na(2)EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na(2)EDTA and DTPA exhibited the capacity to reduce protein degradation. However, substoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na(2)EDTA in biologic protein formulations.