Evaluation of a nnU-Net type automated clinical volumetric tumor segmentation tool for diffuse low-grade glioma follow-up.

BACKGROUND AND PURPOSE: Diffuse low-grade gliomas (DLGG) are characterized by a slow and continuous growth and always evolve towards an aggressive grade. Accurate prediction of the malignant transformation is essential as it requires immediate therapeutic intervention. One of its most precise predictors is the velocity of diameter expansion (VDE). Currently, the VDE is estimated either by linear measurements or by manual delineation of the DLGG on T2 FLAIR acquisitions. However, because of the DLGG's infiltrative nature and its blurred contours, manual measures are challenging and variable, even for experts. Therefore we propose an automated segmentation algorithm using a 2D nnU-Net, to 1) gain time and 2) standardize VDE assessment. MATERIALS AND METHODS: The 2D nnU-Net was trained on 318 acquisitions (T2 FLAIR & 3DT1 longitudinal follow-up of 30 patients, including pre- & post-surgery acquisitions, different scanners, vendors, imaging parameters…). Automated vs. manual segmentation performance was evaluated on 167 acquisitions, and its clinical interest was validated by quantifying the amount of manual correction required after automated segmentation of 98 novel acquisitions. RESULTS: Automated segmentation showed a good performance with a mean Dice Similarity Coefficient (DSC) of 0.82±0.13 with manual segmentation and a substantial concordance between VDE calculations. Major manual corrections (i.e., DSC<0.7) were necessary only in 3/98 cases and 81% of the cases had a DSC>0.9. CONCLUSION: The proposed automated segmentation algorithm can successfully segment DLGG on highly variable MRI data. Although manual corrections are sometimes necessary, it provides a reliable, standardized and time-winning support for VDE extraction to asses DLGG growth.

Understanding Loss of Soluble High Molecular Weight Species during Filtration of Low Concentration Therapeutic Monoclonal Antibodies.

Sterile filtration is an integral step in the manufacturing process of biological therapeutics. Protein adsorption to the surface of the filter is an unfortunate, common occurrence that can result in manufacturing difficulties, such as filter fouling or product loss. Although many filters have surface modifications to minimize adsorption, under certain conditions binding can still occur. We observed the loss of high molecular weight species (HMWS) during sterile filtration of eight different therapeutic monoclonal antibodies formulated at low protein concentrations across a commonly used hydrophilic polyvinylidene fluoride or polyvinylidene difluoride (PVDF) filter membrane. The protein absorption was specific to HMWS, and each antibody exhibited different degrees of filter adsorption. Debye screening length parameters of the solution (e.g. ionic strength) were adjusted, and influenced the amount of HMWS lost during filtration. Additionally, HMWS of a representative antibody (mAb1) were observed to be more positively charged than other size variants by ion-exchange chromatography. From these results, it is concluded that this HMWS loss is due to electrostatic interactions between HMWS and the filter surface. This adsorption can be reduced by increasing the ionic strength of the buffer matrix, demonstrating the influence of the Debye screening length in the filtration of low concentration proteins.

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.

Split-Cakes, Still Delicious.

"Elegant" lyophilized pharmaceutical product cakes are expected to appear as uniform foamy plugs with little shrinkage and minimal cracking. While studying internal cake structures, we have on occasion observed some cakes that were very sharply split horizontally, roughly in halves, with foamy top and lamellar bottom regions. After many years and numerous experiments, we can finally propose a mechanism for the formation of these cakes with unusual internal structures. This phenomenon involves a complex interplay of momentum, heat, mass transfer, and phase equilibria.LAY ABSTRACT: Freeze drying (lyophilization) is a common unit operation in the manufacturing of pharmaceutical drugs. The typical final lyophilized product is expected to look like a uniform porous plug, or cake, that has foamy (sponge-like) morphology. However, we have occasionally observed cakes that were split horizontally, with the top and bottom layers exhibiting very distinctive and totally different structures. This intriguing phenomenon has not been discussed in the literature. In this report, we present experimental results that lead us to a mechanism by which split-cakes form.

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.

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).

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.

Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature.

This study was designed to assess the effects of cooling rate, storage temperature, and formulation composition on trehalose phase distribution and protein stability in frozen solutions. The data demonstrate that faster cooling rates (>100°C/min) result in trehalose crystallization and protein aggregation as determined by Fourier Transform Near-Infrared (FT-NIR) spectroscopy and size-exclusion chromatography, respectively. Conversely, at slower cooling rates (≤1°C/min), trehalose remains predominantly amorphous and there is no effect on protein stability. Evaluation of storage temperatures demonstrates that aggregation increases more rapidly at -14°C compared with higher (-8°C) and lower (-20°C) storage temperatures; however, a relatively higher amount of cumulative aggregation was observed at lower (-20°C) temperature compared with higher storage temperatures (-14°C and -8°C). Further evaluation of the effects of formulation composition suggests that the phase distribution of amorphous and crystallized trehalose dihydrate in frozen solutions depends on the ratio of trehalose to mAb. The results identify an optimal range of trehalose-mAb (w/w) ratio, 0.2-2.4, capable of physically stabilizing mAb formulations during long-term frozen storage-even for fast cooled (>100°C/min) formulations.

An improved method for visualizing the morphology of lyophilized product cakes.

Due to low optical contrast, the morphology of lyophilized product cakes is difficult to observe and photograph. Furthermore, internal structures are normally not visible unless the cake is fractured. Because most lyophilized substances are hygroscopic and quite fragile, the product cake, once removed from the vial, will rapidly degrade. We propose herein a technique that allows a lyophilized product cake to be preserved, manipulated, and easily observed outside the vial. This technique yields high-quality, cross sectional images that reveal intricate fine structures without the use of expensive specialized equipment.

Visualization Techniques for Assessing Design Factors That Affect the Interaction between Pharmaceutical Vials and Stoppers.

We developed several techniques for visualizing the fit between a stopper and a vial in the critical flange area, a location typically hidden from view. Using these tools, it is possible to identify surfaces involved in forming the initial seal immediately after stopper insertion. We present examples illustrating important design elements that can contribute to forming a robust primary package. These techniques can also be used for component screening by facilitating the identification of combinations that do not fit well together so that they can be eliminated early in the selection process.