Risk Mitigation of Drug Shortages-A New Concept for Vials Designed to Improve Fill and Finish Performance.

Drug shortages are a severe threat to human health and life. The situation in the U.S. became so critical, that the U.S. Food and Drug Administration formed a task force in 2018 in order to identify root causes and potential solutions. Manufacturing issues, including rejects and disruptions during fill and finish processes, are a main root cause for inefficient drug manufacturing and resulting delays within the supply chain. This is of particular relevance as the standard pharmaceutical vial filling process can introduce various damages to containers starting from cosmetic defects, such as scratches or abrasions, to fatal events like glass breakage. To overcome the occurrence of undesirable effects (e.g., breakage from introduced damages), SCHOTT developed the EVERIC smooth vial. It is characterized by an outer surface coated in such a way that the excellent pristine properties of the produced glass surface are preserved. In addition, the glass-to-glass friction is reduced and sustainable to different environmental conditions caused during processing. A comparative filling campaign was performed on a commercial line at Hoffmann-La Roche to analyze the efficiency of such a surface modification. In this case, SCHOTT's silicone-free coating option was used, possibly being the preferred solution for certain highly silicone-sensitive biological drugs. Container strength was analyzed before and after line processing of coated as well as uncoated vials. The results demonstrated the strength-preserving effect of the coating in comparison to uncoated vials. Furthermore, the 100% cosmetic inspection proved that the number of rejections could be significantly reduced. Thus, the preserving effect of the outer surface modification of the glass containers was demonstrated impressively. In addition, analyses of the filled media and washing water revealed no residues of the coating material. The vial quality obtained from the surface modification greatly improved the line performance by nearly eliminating all undesirable effects affecting the machinability.

Evaluation of Container Closure System Integrity for Storage of Frozen Drug Products: Impact of Capping Force and Transportation.

Frozen-state storage and cold-chain transport are key operations in the development and commercialization of biopharmaceuticals. Today, several marketed drug products are stored (and/or shipped) under frozen conditions to ensure sufficient stability, particularly for live viral vaccines. When these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial at the target's lowest temperature to ensure container closure integrity and thus both sterility and safety of the drug product. The container closure integrity assessment in the frozen state (e.g., -20°C, -80°C) should include container closure integrity (CCI) of the container closure system (CCS) itself, impact of processing (e.g., capping process on CCI), and impact of shipment and movement on CCI in the frozen state. The objective of this work was to evaluate the impact of processing and shipment on CCI of a CCS in the frozen state. The impact on other quality attributes was not investigated. In this light, the ThermCCI method was applied to evaluate the impact of shipping stress and variable capping force on CCI of frozen vials and to evaluate the temperature limits of rubber stoppers. In conclusion, retaining CCI during cold storage is mostly a function of vial-stopper combination, and temperatures below -40°C may pose a risk to the CCI of a frozen drug product. Variable capping force may have an influence on the CCI of a frozen drug product if not appropriately assessed. Regarding the impact of shipment on the CCI of glass vials, no indication was given at room temperature, -20°C, or -75°C when compared with static storage at such temperatures.LAY ABSTRACT: Today, several marketed products are stored (and/or shipped) under frozen conditions to ensure sufficient stability. When these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial and ensure container closure integrity and thus both sterility and safety of the drug product. The impact of processing and shipment on the container closure integrity (CCI) of a container closure system (vial, stopper, and flip-off cap) in the frozen state is assessed. A helium-leakage test at low temperature (ThermCCI) was used to evaluate the impact of shipping stress and variable capping force on CCI of frozen vials as well as the temperature limits of rubber stoppers. In conclusion, it was found that retaining CCI during cold storage is mostly a function of vial-stopper combination and that temperatures below -40°C may pose a risk to the CCI of a frozen drug product. Variable capping force may have an influence on the CCI of a frozen drug product if not appropriately assessed. Additionally, it was observed that the shipment of the frozen glass vials did not affect the CCI.

Delamination Propensity of Glass Containers for Pharmaceutical Use: A Round Robin Activity Looking for a Predictive Test.

Delamination, which is the formation of flakes in drug products owing to specific and localized corrosion of glass vials, is a rare but serious problems, on which the FDA (U.S. Food and Drug Administration) put a warning to the pharma industry in 2011. The Technical Committee (TC) TC12 of the International Commission on Glass (ICG) was created in 2012 with the aim to study the problems related to pharma packaging. The first task of TC12 was to address the problem of predicting the propensity of glass vials to delamination, leaving the study of the mechanism(s) of flake formation as a possible future activity. This paper reports on the results obtained in a round robin test, which involved all the labs of the companies represented in the TC.Five types of vials with different expected delamination propensities were tested using a protocol that includes autoclaving at 121°C of vials filled with NaCl solution adjusted to pH 8 with NaOH solution, a coloration test, and ICP-OES determination of Si, B, and Al.Although there was no flake formation, the results showed that the combination of strong coloration at the bottom of the vials and high silicon concentration in the solution is correlated to an observable morphological modification/corrosion of the inner surface of vials in the bottom region. The test protocol is therefore useful for checking the quality of the vials with respect to the propensity to corrosion. Regarding delamination, no direct correlation with the testing results could be obtained yet. The method allows catching differences in the corrosion behavior, mainly between sets of vials with comparable surface:volume.LAY ABSTRACT: The U.S. Food and Drug Administration (FDA) warned the pharma industry about glass delamination inside primary packaging containers. Delamination is a type of glass corrosion that produces glass flakes, which could seriously affect patient health.Fortunately, delamination is a very rare event. On the other hand, it is very difficult to predict its occurrence. In 2012, the International Commission on Glass (ICG) created a Technical Committee (TC) on pharma packaging-with the initial goal to study an easy and reliable test for predicting the propensity of vials to delamination-involving the most important glass vial producers and pharma companies. This paper reports on the results obtained in a round robin test on different types of vials with different expected propensities to delamination. A specific testing protocol was adopted. In none of the vials, including those with an expected high propensity, glass flakes were observed, demonstrating that delamination is a rare event. However, the test is able to predict the occurrence of morphological modification/corrosion of the inner surface of vials in the bottom region. Therefore, the testing protocol is proposed as a method to evaluate differences in the corrosion behavior mainly between sets of vials with comparable surface:volume.

Impact of Vial Washing and Depyrogenation on Surface Properties and Delamination Risk of Glass Vials.

PURPOSE: The proper understanding of glass delamination is important to glass manufacturers, pharmaceutical companies, and health authorities to mitigate the occurrence of glass flakes from the vial when in contact with specific drug product solutions. The surface of glass vials is altered during glass cane- and vial forming processes and is exposed to different stress conditions during drug product processing before coming in contact with the drug product solution. In this study, the impact of vial washing and depyrogenation including an evaluation of various residual water volumes on surface properties of glass vials was investigated for a defined set of vials. METHODS: 3D laser scanning microscopy was established as a new method for topographic analysis of curved surfaces of glass vials operating in high-throughput mode. A subset of vials was subsequently exposed to delamination stress testing and both the stressed solution and inner vial surface were analyzed by a panel of conventional and advanced analytical techniques including 3D laser scanning microscopy. RESULTS: The data showed that vial washing and depyrogenation strongly influenced surface properties, in particular those of uncoated vials. Surface characteristics such as pits increased depending on the process conditions, which especially applies to Expansion 33 vials. Even low residual water volumes of 50 µL after vial washing were sufficient to change the surface properties of the glass and weaken the surface in those positions prone to glass delamination. An increase in pits was related to a greater risk for glass delamination. CONCLUSIONS: Vial processing conditions need to be assessed when aiming at minimizing the glass delamination risk during parenteral product storage.

Sealing Behaviour of Container Closure Systems under Frozen Storage Conditions: Nonlinear Finite Element Simulation of Serum Rubber Stoppers.

There has been a growing interest in recent years in the assessment of suitable vial/stopper combinations for storage and shipment of frozen drug products. Considering that the glass transition temperature (Tg) of butyl rubber stoppers used in container closure systems (CCSs) is between -55 °C to -65 °C, a storage or shipment temperature of a frozen product below the Tg of the rubber stopper may require special attention because below the Tg the rubber becomes more plastic like and loses its elastic (sealing) characteristics. Thus, they risk not maintaining container closure integrity (CCI). Given that the rubber regains its elastic properties and reseals after rewarming to ambient temperature, leaks during frozen temperature storage and transportation are transient and the CCI methods used at room temperature conditions are unable to confirm CCI in the frozen state. Hence, several experimental methods have been developed in recent years in order to evaluate CCI at low temperatures. Finite element (FE) simulations were applied in order to investigate the sealing behaviour of rubber stoppers for the drug product CCS under frozen storage conditions. FE analysis can help in reducing the experimental design space and thus the number of measurements needed, as they can be used as an add-on to experimental testing. Several scenarios have been simulated including the effect of thermal history, rubber type, storage time, worst-case CCS geometric tolerances, and capping pressure. The results of these calculations have been validated with experimental data derived from laboratory experiments (CCI at low temperatures), and a concept for tightness has been developed. It has been concluded that FE simulations have the potential to become a powerful predictive tool toward a better understanding of the influence of cold storage on the rubber sealing properties (and hence on CCI) when dealing with frozen drug products.LAY ABSTRACT: The growing interest in the assessment of suitable vial/stopper combinations for storage and shipment of frozen drug products has led to the development of a number of experimental methods to evaluate container closure integrity at low temperatures. The application of finite element simulations could aid in the investigation of the sealing behaviour of rubber stoppers for drug product container closure systems under frozen storage conditions by simplifying the experimental design space and the number of experimental measurements needed. In this work several scenarios have been simulated including the effect of thermal history, rubber type, storage time, worst-case container closure system geometric tolerances, and capping pressure. The results have been further validated with experimental data derived from laboratory experiments and a concept for tightness was developed. In conclusion, finite element simulations have shown the potential to become a powerful predictive tool toward a better understanding of the influence of cold storage on the rubber sealing properties (and hence on container closure integrity) when dealing with frozen drug products.

Characterization of surface properties of glass vials used as primary packaging material for parenterals.

The appropriate selection of adequate primary packaging, such as the glass vial, rubber stopper, and crimp cap for parenteral products is of high importance to ensure product stability, microbiological quality (integrity) during storage as well as patient safety. A number of issues can arise when inadequate vial material is chosen, and sole compliance to hydrolytic class I is sometimes not sufficient when choosing a glass vial. Using an appropriate pre-treatment, such as surface modification or coating of the inner vial surface after the vial forming process the glass container quality is often improved and interactions of the formulation with the surface of glass may be minimized. This study aimed to characterize the inner surface of different type I glass vials (Exp33, Exp51, Siliconized, TopLyo™ and Type I plus®) at the nanoscale level. All vials were investigated topographically by colorimetric staining and Scanning Electron Microscopy (SEM). Glass composition of the surface was studied by Time-of-Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) and X-ray Photoelectron Spectroscopy (XPS), and hydrophobicity/hydrophilicity of the inner surface was assessed by dye tests and surface energy measurements. All containers were studied unprocessed, as received from the vendor, i.e. in unwashed and non-depyrogenized condition. Clear differences were found between the different vial types studied. Especially glass vials without further surface modifications, like Exp33 and Exp51 vials, showed significant (I) vial-to-vial variations within one vial lot as well as (II) variations along the vertical axis of a single vial when studying topography and chemical composition. In addition, differences and heterogeneity in surface energy were found within a given tranche (circumferential direction) of Exp51 as well as Type I plus® vials. Most consistent quality was achieved with TopLyo™ vials. The present comprehensive characterization of surface properties of the different vial types may serve as basis to further guide the selection of adequate primary packaging based on the desired quality target product profile and to support studies of glass surface interactions with formulations. The proposed analytical method panel can be used for characterization of future glass vials either before delivery to the manufacturer or drug product manufacturing.

Evaluation of Glass Delamination Risk in Pharmaceutical 10 mL/10R Vials.

Glass delamination is characterized by the dissociation of glass flakes from the glass surface. Since glass delamination is time dependent, 5 vial types were investigated to assess delamination under accelerated stress conditions published as quick tests in literature and compared to stress testing recommended per United States Pharmacopoeia <1660>. A broad panel of analytical techniques was employed to test the solution for visible/subvisible particles and leachables and characterize topography and composition of the surface. The vial types showed significant differences in surface durability when applying the same stress conditions. An increase in glass leachables and change in topography were shown for uncoated vials. An indication for an elevated delamination risk was confirmed for Expansion 33 vials only by the compiled analytical data set including particle assessment and change in elemental composition of the near glass surface investigated by dynamic secondary ion mass spectrometry. The delamination test protocols differ in test solution, handling, and time. Before choosing the most appropriate protocol to predict delamination propensity and mimic real-time conditions, long-term storage data are needed. A combination of analytical techniques to study the risk for long-term corrosion of glass is highly recommended covering the 3 aspects: visible/subvisible particle assessment, solution analysis, and surface characterization.

Container Closure Integrity Testing-Practical Aspects and Approaches in the Pharmaceutical Industry.

The assurance of sterility of a parenteral drug product, prior to any human use, is a regulatory requirement. Hence, all strategies related to container closure integrity (CCI) must demonstrate absence of microbial contamination through leaks as part of the container closure system (CCS) qualification, during manufacturing, for quality control purposes and to ensure microbiological integrity (sterility) during storage and shipment up to the end of product shelf life. Current regulatory guidances, which differ between countries and regions, provide limited detail on how to assess CCI. The new revision of USP <1207> aims to provide extensive and detailed guidance for CCI assessments for sterile products. However, practical questions and considerations are yet to be addressed by the pharmaceutical industry. These may include: (1) choice of method, for example whether a deterministic CCI method (e.g., helium leak) is preferable over the probabilistic CCI method (e.g., microbial ingress), (2) the type of primary packaging (e.g., vial, syringe, device), (3) dosage form (e.g., liquid versus lyophilisate), (4) suitable acceptance criteria, (5) appropriate sample size, (6) the most appropriate way to introduce artificial leaks into the CCS, (7) ensuring suitable assurance of CCI during drug product manufacturing, and (8) evaluating CCI under intended shipment and storage conditions (e.g., in the frozen state).A group of European industry peers have met to discuss these and other related questions in order to provide their viewpoint and best practice on practical approaches to CCI. Their perspective is provided in this white paper. Through these discussions, it became clear that there is currently no gold standard for CCI test methods or for the generation of artificial leaks; therefore flexibility toward CCI approaches is required. Although there should be flexibility, any CCI approach must consider the intended use (e.g., CCS qualification, routine manufacturing, or quality control) and product design (e.g., primary packaging, liquid versus dried product).LAY ABSTRACT: The assurance of sterility of a parenteral drug product prior to any human use is a regulatory requirement. Hence, all strategies related to container closure integrity (CCI) must demonstrate absence of microbial contamination through leaks as part of the container closure system (CCS) qualification, during manufacturing, for quality control purposes and to ensure microbiological integrity (sterility) during storage and shipment up to the end of shelf life. Current regulatory guidances, which differ between countries and regions, provide limited detail on how to assess CCI. The new revision of USP <1207> aims to provide extensive and detailed guidance for CCI assessments for sterile products. However, practical questions and considerations are yet to be addressed by the pharmaceutical industry.A group of European industry peers have met to discuss these and other related questions in order to provide their viewpoint and best practice on practical approaches to CCI. Their perspective is provided in this white paper. Through these discussions, it became clear that there is currently no gold standard for CCI test methods or for the generation of artificial leaks; therefore flexibility toward CCI approaches is required. Although there should be flexibility, any CCI approach must consider the intended use (e.g., CCS qualification, routine manufacturing, or quality control) and product design (e.g., primary packaging, liquid versus dried product).

Artificial Leaks in Container Closure Integrity Testing: Nonlinear Finite Element Simulation of Aperture Size Originated by a Copper Wire Sandwiched between the Stopper and the Glass Vial.

Container closure integrity (CCI) testing is required by different regulatory authorities in order to provide assurance of tightness of the container closure system against possible contamination, for example, by microorganisms. Microbial ingress CCI testing is performed by incubation of the container closure system with microorganisms under specified testing conditions. Physical CCI uses surrogate endpoints, such as coloration by dye solution ingress or gas flow (helium leakage testing). In order to correlate microbial CCI and physical CCI test methods and to evaluate the methods' capability to detect a given leak, artificial leaks are being introduced into the container closure system in a variety of different ways. In our study, artificial leaks were generated using inserted copper wires between the glass vial opening and rubber stopper. However, the insertion of copper wires introduces leaks of unknown size and shape. With nonlinear finite element simulations, the aperture size between the rubber stopper and the glass vial was calculated, depending on wire diameter and capping force. The dependency of the aperture size on the copper wire diameter was quadratic. With the data obtained, we were able to calculate the leak size and model leak shape. Our results suggest that the size as well as the shape of the artificial leaks should be taken into account when evaluating critical leak sizes, as flow rate does not, independently, correlate to hole size. Capping force also affected leak size. An increase in the capping force from 30 to 70 N resulted in a reduction of the aperture (leak size) by approximately 50% for all wire diameters. From 30 to 50 N, the reduction was approximately 33%. LAY ABSTRACT: Container closure integrity (CCI) testing is required by different regulatory authorities in order to provide assurance of tightness of the container closure system against contamination, for example, by microorganisms. Microbial ingress CCI testing is performed by incubation of the container closure system with microorganisms under specified testing conditions. Physical CCI uses surrogate endpoints, such as coloration by dye solution ingress or gas flow. In order to correlate microbial ingress CCI and physical CCI test methods and to evaluate the methods' capability to detect a given leak, artificially created defects (artificial leaks) are being introduced into the container closure system in a variety of different ways. In our study, artificial leaks were generated using inserted copper wires between the glass vial opening and rubber stopper. Up to date, the insertion of copper wires introduced leaks of unknown size and shape. With nonlinear finite element simulations, the effective aperture size between the rubber stopper and the glass vial was calculated, depending on wire diameter and capping force, and the leak shape was modelled. Our results suggest that the size as well as the shape of the artificial leaks should be taken into account when evaluating critical leak sizes, as flow rate does not, independently, correlate to the hole size.

Impact of Vial Capping on Residual Seal Force and Container Closure Integrity.

UNLABELLED: The vial capping process is a critical unit operation during drug product manufacturing, as it could possibly generate cosmetic defects or even affect container closure integrity. Yet there is significant variability in capping equipment and processes, and their relation to potential defects or container closure integrity has not been thoroughly studied. In this study we applied several methods-residual seal force tester, a self-developed system of a piezo force sensor measurement, and computed tomography-to characterize different container closure system combinations that had been sealed using different capping process parameter settings. Additionally, container closure integrity of these samples was measured using helium leakage (physical container closure integrity) and compared to characterization data. The different capping equipment settings lead to residual seal force values from 7 to 115 N. High residual seal force values were achieved with high capping pre-compression force and a short distance between the capping plate and plunge. The choice of container closure system influenced the obtained residual seal force values. The residual seal force tester and piezoelectric measurements showed similar trends. All vials passed physical container closure integrity testing, and no stopper rupture was seen with any of the settings applied, suggesting that container closure integrity was warranted for the studied container closure system with the chosen capping setting ranges. LAY ABSTRACT: The vial capping process is a critical unit operation during drug product manufacturing, as it could possibly generate cosmetic defects or even affect container closure integrity. Yet there is significant variability in capping equipment and processes, and their relation to potential defects or container closure integrity has not been thoroughly studied. In this study we applied several methods-residual seal force tester, a self-developed system of a piezo force sensor measurement, and computed tomography-to characterize different container closure system combinations that had been sealed using different capping process parameter settings. The residual seal force tester can analyze a variety of different container closure systems independent of the capping equipment. An adequate and safe residual seal force range for each container closure system configuration can be established with the residual seal force tester and additional methods like computed tomography scans and leak testing. In the residual seal force range studied, the physical container closure integrity of the container closure system was warranted.

The Pharmaceutical Capping Process-Correlation between Residual Seal Force, Torque Moment, and Flip-off Removal Force.

The majority of parenteral drug products are manufactured in glass vials with an elastomeric rubber stopper and a crimp cap. The vial sealing process is a critical process step during fill-and-finish operations, as it defines the seal quality of the final product. Different critical capping process parameters can affect rubber stopper defects, rubber stopper compression, container closure integrity, and also crimp cap quality. A sufficiently high force to remove the flip-off button prior to usage is required to ensure quality of the drug product unit by the flip-off button during storage, transportation, and until opening and use. Therefore, the final product is 100% visually inspected for lose or defective crimp caps, which is subjective as well as time- and labor-intensive. In this study, we sealed several container closure system configurations with different capping equipment settings (with corresponding residual seal force values) to investigate the torque moment required to turn the crimp cap. A correlation between torque moment and residual seal force has been established. The torque moment was found to be influenced by several parameters, including diameter of the vial head, type of rubber stopper (serum or lyophilized) and type of crimp cap (West(®) or Datwyler(®)). In addition, we measured the force required to remove the flip-off button of a sealed container closure system. The capping process had no influence on measured forces; however, it was possible to detect partially crimped vials. In conclusion, a controlled capping process with a defined target residual seal force range leads to a tight crimp cap on a sealed container closure system and can ensure product quality. LAY ABSTRACT: The majority of parenteral drug products are manufactured in a glass vials with an elastomeric rubber stopper and a crimp cap. The vial sealing process is a critical process step during fill-and-finish operations, as it defines the seal quality of the final product. An adequate force to remove the flip-off button prior to usage is required to ensure product quality during storage and transportation until use. In addition, the complete crimp cap needs to be fixed in a tight position on the vial. In this study, we investigated the torque moment required to turn the crimp cap and the force required to remove the flip-off button of container closure system sealed with different capping equipment process parameters (having different residual seal force values).

Evaluation of Container Closure System Integrity for Frozen Storage Drug Products.

Sometimes, drug product for parenteral administration is stored in a frozen state (e.g., -20 °C or -80 °C), particularly during early stages of development of some biotech molecules in order to provide sufficient stability. Shipment of frozen product could potentially be performed in the frozen state, yet possibly at different temperatures, for example, using dry ice (-80 °C). Container closure systems of drug products usually consist of a glass vial, rubber stopper, and an aluminum crimped cap. In the frozen state, the glass transition temperature (Tg) of commonly used rubber stoppers is between -55 and -65 °C. Below their Tg, rubber stoppers are known to lose their elastic properties and become brittle, and thus potentially fail to maintain container closure integrity in the frozen state. Leaks during frozen temperature storage and transportation are likely to be transient, yet, can possibly risk container closure integrity and lead to microbial contamination. After thawing, the rubber stopper is supposed to re-seal the container closure system. Given the transient nature of the possible impact on container closure integrity in the frozen state, typical container closure integrity testing methods (used at room temperature conditions) are unable to evaluate and thus confirm container closure integrity in the frozen state. Here we present the development of a novel method (thermal physical container closure integrity) for direct assessment of container closure integrity by a physical method (physical container closure integrity) at frozen conditions, using a modified He leakage test. In this study, different container closure systems were evaluated with regard to physical container closure integrity in the frozen state to assess the suitability of vial/stopper combinations and were compared to a gas headspace method. In summary, the thermal physical container closure integrity He leakage method was more sensitive in detecting physical container closure integrity impact than gas headspace and aided identification of an unsuitable container closure system. LAY ABSTRACT: Sometimes, drug product for parenteral administration is stored in a frozen state (e.g., -20 °C or -80 °C), particularly during early stages of development of some biotech molecules in order to provide sufficient stability. Container closure systems for drug products usually consist of a glass vial, rubber stopper, and an aluminum crimped cap. In the frozen state, the glass transition temperature (Tg) of commonly used rubber stoppers is between -55 and -65 °C. Leaks during frozen temperature storage and transportation are likely to be transient, yet they can possibly risk container closure integrity and lead to microbial contamination and sterility breach. After thawing, the rubber stopper is expected to re-seal the container closure system. Given the transient nature of the possible impact on container closure integrity in the frozen state, typical container closure integrity testing methods (used at room temperature conditions) are unable to evaluate and thus confirm container closure integrity in the frozen state. Here we present the development of a novel method (thermal container closure integrity) for direct measurement of container closure integrity by a physical method (physical container closure integrity) at frozen conditions, using a modified He leakage test. In this study, we found that the thermal container closure integrity He leakage method was more sensitive in detecting physical container closure integrity impact than gas headspace and aided identification of an unsuitable container closure system.

The pharmaceutical vial capping process: Container closure systems, capping equipment, regulatory framework, and seal quality tests.

Parenteral drug products are protected by appropriate primary packaging to protect against environmental factors, including potential microbial contamination during shelf life duration. The most commonly used CCS configuration for parenteral drug products is the glass vial, sealed with a rubber stopper and an aluminum crimp cap. In combination with an adequately designed and controlled aseptic fill/finish processes, a well-designed and characterized capping process is indispensable to ensure product quality and integrity and to minimize rejections during the manufacturing process. In this review, the health authority requirements and expectations related to container closure system quality and container closure integrity are summarized. The pharmaceutical vial, the rubber stopper, and the crimp cap are described. Different capping techniques are critically compared: The most common capping equipment with a rotating capping plate produces the lowest amount of particle. The strength and challenges of methods to control the capping process are discussed. The residual seal force method can characterize the capping process independent of the used capping equipment or CCS. We analyze the root causes of several cosmetic defects associated with the vial capping process.

Investigating factors leading to fogging of glass vials in lyophilized drug products.

Vial "Fogging" is a phenomenon observed after lyophilization due to drug product creeping upwards along the inner vial surface. After the freeze-drying process, a haze of dried powder is visible inside the drug product vial, making it barely acceptable for commercial distribution from a cosmetic point of view. Development studies were performed to identify the root cause for fogging during manufacturing of a lyophilized monoclonal antibody drug product. The results of the studies indicate that drug product creeping occurs during the filling process, leading to vial fogging after lyophilization. Glass quality/inner surface, glass conversion/vial processing (vial "history") and formulation excipients, e.g., surfactants (three different surfactants were tested), all affect glass fogging to a certain degree. Results showed that the main factor to control fogging is primarily the inner vial surface hydrophilicity/hydrophobicity. While Duran vials were not capable of reliably improving the level of fogging, hydrophobic containers provided reliable means to improve the cosmetic appearance due to reduction in fogging. Varying vial depyrogenation treatment conditions did not lead to satisfying results in removal of the fogging effect. Processing conditions of the vial after filling with drug product had a strong impact on reducing but not eliminating fogging.

Product-enhanced reverse transcriptase assay for replication-competent retrovirus and lentivirus detection.

The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes.

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

Phase 1 trial of FVIII gene transfer for severe hemophilia A using a retroviral construct administered by peripheral intravenous infusion.

In a phase 1 dose escalation study, 13 subjects with hemophilia A received by peripheral intravenous infusion a retroviral vector carrying a B-domain-deleted human factor VIII (hFVIII) gene. Infusions were well tolerated. Tests for replication competent retrovirus have been negative. Polymerase chain reaction (PCR) analyses demonstrate the persistence of vector gene sequences in peripheral blood mononuclear cells in 3 of 3 subjects tested. Factor VIII was measured in serial samples using both a one-stage clotting assay and a chromogenic assay. While no subject had sustained FVIII increases, 9 subjects had FVIII higher than 1% on at least 2 occasions 5 or more days after infusion of exogenous FVIII, with isolated levels that ranged from 2.3% to 19%. Pharmacokinetic parameters of exogenous FVIII infused into subjects 13 weeks after vector infusion showed an increased half-life (T1/2; P <.02) and area under the curve (AUC, P <.04) compared with prestudy values. Bleeding frequency decreased in 5 subjects compared with historical rates. These results demonstrate that this retroviral vector (hFVIII(V)) is safe and, in some subjects, persists more than a year in peripheral blood mononuclear cells, with measurable factor VIII levels and with increased available FVIII activity (increased T1/2 and AUC) after infusion of exogenous FVIII concentrate.