Quantifying the Vial-Capping Process: Reexamination Using Micro-Computed Tomography.

A vial-capping process for lyophilization stopper configurations was previously quantified using residual seal force (RSF). A correlation between RSF and container closure integrity (CCI) was established, and component positional offsets were identified to be the primary source of variability in RSF measurements.To gain insight into the effects of stopper geometry on CCI, serum stoppers with the same rubber formulation were investigated in this study. Unlike lyophilization stoppers that passed CCI (per helium leak testing) even with RSF of 0 N owing to their excellent valve seal, serum stoppers consistently failed CCI when RSF was <15.8 N. When the plug was removed, both types of stoppers exhibited a comparable critical lower RSF limit (19-20 N), below which CCI could not be maintained. When CCI was retested at later time points (up to 6 mo), some previously failed vials passed CCI, suggesting that CCI improvement might be related to rubber relaxation (viscous flow), which can fill minor imperfections on the vial finish.To confirm component positional offsets are the primary sources of RSF variability, a novel quantification tool-micro-computed tomography (micro-CT)-was used in this study. Micro-CT provided images for quantification of positional offsets of the cap and stopper that directly correlated with RSF fluctuations. Serum stoppers and lyophilization stoppers are comparable in RSF variations, although lyophilization stoppers are more robust in CCI. The use of micro-CT provides a nondestructive and innovative tool in quantitatively analyzing component features of capped vials that would otherwise be difficult to investigate.

Quantifying the Vial Capping Process: Residual Seal Force and Container Closure Integrity.

Capping completes the closure of parenteral drug products in the final packaging container and is critical in maintaining an integral seal to ensure product quality. Residual seal force (RSF) is considered the sole quantifiable attribute for measuring seal "goodness" and potentially enables nonsubjective, consistent setting of cappers across manufacturing sites. However, the consistency and reliability of RSF measurement and data have been scarcely reported, and the relationship between RSF and container closure integrity (CCI) remains poorly understood.Here, we present a large data set generated from a commercial capper and the results from a laboratory capper of glass vials and rubber stoppers with aluminum caps. All RSF values exhibited significant variability. We evaluated four potential sources of variability: the capper, the RSF tester, the time-dependent nature of RSF, and the components. We determined that the capper, the tester, and the time-dependent nature are not main sources. Dimensional tolerances of the packaging components were the root cause for the container closure system (CCS) configurations tested in this study.This study correlated RSF with CCI (via helium leakage), although CCI is not sensitive to RSF; CCI was maintained even for loosely capped vials with no measurable RSF. This was attributed to the stopper's two sealing surfaces: the valve seal and the land seal. A methodology capable of differentiating the two seals' functions demonstrated that vials with only the valve seal always passed leakage testing for a selected CCS configuration in this study, while vials with only the land seal failed CCI at low RSF values. This observation allows proposal of a low RSF limit that is safe even when the valve seal is defective. Simplified statistical analysis of commercial capping data, with the input of sample size, allowed the relationship between RSF's low limit and an allowable failing rate to be established. Overall, despite the inherent variability of RSF, this study shows that it is a feasible parameter for capping process quantification and demonstrates the potential of RSF measurement in capper setup.LAY ABSTRACT: Pharmaceutical vials are typically closed off with an elastomeric stopper that is secured onto the vial with an aluminum crimp cap (or seal) such that the entire assembly is meant to protect the vial's contents from external contamination. Therefore, the capping process is critical for ensuring container closure integrity. Characterizing the effectiveness of a seal in a nonsubjective and quantifiable manner is challenging. In this communication, we report the evaluation of residual seal force measurements (the compression force that the crimp cap exerts on the stopper) to evaluate capping for a large set of samples generated on both an at-scale commercial capper and a benchtop laboratory capper. We propose a test methodology, based on a statistical approach, for establishing permissible lower residual force limits that would provide a high degree of confidence to the capping process. This is a useful tool for consistent capper setup and capping process quantification.

Processing Impact on Monoclonal Antibody Drug Products: Protein Subvisible Particulate Formation Induced by Grinding Stress.

Subvisible particle formation in monoclonal antibody drug product resulting from mixing and filling operations represents a significant processing risk that can lead to filter fouling and thereby lead to process delays or failures. Several previous studies from our lab and others demonstrated the formation of subvisible particulates in mAb formulations resulting from mixing operations using some bottom-mounted mixers or stirrer bars. It was hypothesized that the stress (e.g., shear/cavitation) derived from tight clearance and/or close contact between the impeller and shaft was responsible for protein subvisible particulate generation. These studies, however, could not distinguish between the two surfaces without contact (tight clearance) or between two contacting surfaces (close contact). In the present study we expand on those findings and utilize small-scale mixing models that are able to, for the first time, distinguish between tight clearances and tight contact. In this study we evaluated different mixer types including a top-mounted mixer, several impeller-based bottom-mounted mixers, and a rotary piston pump. The impact of tight clearance/close contact on subvisible particle formation in at-scale mixing platforms was demonstrated in the gap between the impeller and drive unit as well as between the piston and the housing of the pump. Furthermore, small-scale mixing models based on different designs of magnetic stir bars that mimic the tight clearance/close contact of the manufacturing-scale mixers also induced subvisible particles in mAb formulations. Additional small-scale models that feature tight clearance but no close contact (grinding) suggested that it is the repeated grinding/contacting of the moving parts and not the presence of tight clearance in the processing equipment that is the root cause of protein subvisible particulate formation. When multiple mAbs, Fabs (fragment antigen binding), or non-antibody related proteins were mixed in the small-scale mixing model, for molecules investigated, it was observed that mAbs and Fabs appear to be more susceptible to particle formation than non-antibody-related proteins. In the grinding zone, mAb/Fab molecules aggregated into insoluble particles with neither detectable soluble aggregates nor fragmented species. This investigation represents a step closer to the understanding of the underlying stress mechanism leading to mAb subvisible particulate formation as the result of drug product processing.LAY ABSTRACT: Mixing and fill finish are important unit operations in drug product manufacturing for compounding (dilution, pooling, homogenization, etc.) and filling into primary packaging containers (vials, pre-filled syringes, etc.), respectively. The current trend in adopting bottom-mounted mixers as well as low fill-volume filling systems has raised concerns about their impact on drug product quality and process performance. However, investigations into the effects of their use for biopharmaceutical products, particularly monoclonal antibody formulations, are rarely published. The purpose of this study is three-fold: (1) to revisit the impact of bottom-mounted mixer design on monoclonal antibody subvisible particle formation; (2) to identify the root cause for subvisible particle formation; and (3) to fully utilize available particle analysis tools to demonstrate the correlation between particle count in the solution and filter fouling during sterile filtration. The outcomes of this study will benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product process challenges.

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.

Quantitative analysis of TALE-DNA interactions suggests polarity effects.

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.