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Cell death is one of the failure modes of mammalian cell culture. Apoptosis is a regulated cell death process mainly observed in cell culture. Timely detection of apoptosis onset allows opportunities for preventive controls that ensure high productivity and consistent product quality. Capacitance spectroscopy captures the apoptosis-related cellular properties changes and thus quantifies the percentage of dying cells. This study demonstrated a quantification model that measures the percentage of apoptotic cells using a capacitance spectrometer in an at-line setup. When predicting the independent test set collected from bench-scale bioreactors, the root-mean-squared error of prediction was 8.8% (equivalent to 9.9% of the prediction range). The predicted culture evolution trajectory aligned with measured values from the flow cytometer. Furthermore, this method alarms cell death onset earlier than the traditional viability test, that is, the trypan blue exclusion test. Compared to flow cytometry (the traditional early cell death detection method), this method is rapid, simple, and less labor-intensive. In addition, this at-line setup can be easily transferred between scales (e.g., lab-scale for development to manufacturing scale), which benefits process transfers between facilities, scale-up, and other process transitions.

Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls.Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range and offering similar spectral shape across all probes was first constructed, improving prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales in the model, reducing prediction bias. External parameter orthogonalization, a preprocessing method, also mitigated the interference from feeding, which further improved model performance. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an RThe culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. Additionally, the incorporation of at-line spectra following orthogonal design into the calibration set was shown to generate calibration models that are more robust than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectral collection is easier, faster, and more material-sparing than in-line spectra collection. This article is protected by copyright. All rights reserved.

Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls. Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range that had similar spectra shape across all probes was first constructed, which improved the prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales into the model, reducing the prediction bias. External parameter orthogonalization also mitigated the interference from feeding, which further improved the model performance. The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. In addition, incorporation of at-line spectra following orthogonal design into the calibration set is more likely to generate robust calibration models than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectra collection is easier, faster, and more material-sparing than in-line spectra collection. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R2 of 92.4%.

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.

The purpose of the present study was to investigate the feasibility of developing a fast non-destructive at-line transmission Raman spectroscopy (TRS) method for core tablet potency and content uniformity (CU) as part of a real-time release testing (RTRt) control strategy. The effects of tablet hardness and weight (thickness), API particle size, and concentration were studied by using a novel experimental design called generalized subset designs (GSDs). A subset of 28 experiments plus three replicate center points were selected for a total of 31 experiments. Matrix effects included tablets with active pharmaceutical ingredient (API) at seven concentration levels (14 to 26%w/w), and API particle size (17 to 71 μm), tablet weight (275 to 328 mg), and tablet hardness (8 to 16 SCU) at two levels. Three calibration models were developed, by using partial least squares (PLS) and different preprocessing conditions (model nos. 1 to 3). In model no. 1, all matrix effects were excluded. This model showed high potency prediction errors (RMSEP of 10.0%). When all matrix variations were included in the multivariate calibration according to the GSD as shown in model nos. 2 and 3, the prediction accuracy was greatly improved (RMSEP 2.56 and 1.74% respectively). The statistical significance of the tablet weight, hardness, and API particle size in the %Recovery (TRS vs. the reference HPLC method) was investigated by using MODDE Pro (Sartorius Stedim Data Analytics). Statistically significant effects were identified if the calculated p value was ≤ 0.05. The main effect hardness, the cross-term hardness×particle size, and the quadratic term cal level×cal level showed to be statistically significant. However, these effects had a very small impact on tablet prediction accuracy (± 1.0%w/w) well within the intermediate precision of the method. A non-destructive TRS method for core tablet potency and CU was fully validated, following ICH Q2 and EMEA NIR guidelines. The applicability of the method to process development batches was demonstrated and compared to a previously developed and validated NIR method.

The force exerted by wet mass in a granulator on a thin cylindrical probe called the drag force flow (DFF) sensor reflects wet mass consistency and is indicative of the granule densification. Comparison of flow force during wet granulation of batches with different binder concentration indicated good correlation with the granulation's resistance to flow and interparticulate interactions measured by the FT4 Powder Rheometer. This indicated that the force pulse magnitude (FPM) measured by the DFF sensor was indicative of fundamental material properties (e.g., shear viscosity and granule size/density). In addition, the time-profile of the FPM signal from the DFF sensor compared well with that of the impeller amperage, with consistent differentiation between different formulations.

In this work, an example of analytical method quality by design (AQbD) principles applied to an in-line NIR method to monitor blend potency and uniformity in the manufacturing of an oral solid dosage product is presented. An integrated process analytical technology (PAT) method was developed following the AQbD workflow, including risk assessment, design of experiments (DoE), method control strategy, and method maintenance. Several aspects particular to partial least square (PLS) chemometric method development and validation (such as the combined use of DoE, optimization, and multivariate data analysis) are addressed. Results from the risk assessment showed that the active pharmaceutical ingredient (API) particle size was the most significant risk factor due to the presence of aggregates. This risk was reduced by introducing changes in the API crystallization procedure and by including a wide range of calibration blends within the expected range of particle size. Other potential risks related to the instrumentation, the API and excipient properties, and the environment were further studied and minimized. System suitability tests based on Mahalanobis distance and discriminant analysis (DA) techniques were developed to ensure the quality of the spectral data before blend potency calculation. The use of AQbD provided a robust and rugged method that has the potential to be used as part of the product real-time release (RTR) control strategy.

In this paper we demonstrate the feasibility of replacing KF for water content testing in bulk powders and tablets with at-line near infrared (NIR) or microwave resonance (MR) methods. Accurate NIR and MR prediction models were developed with a minimalistic approach to calibration. The NIR method can accurately predict water content in bulk powders in the range of 0.5-5% w/w. Results from this method were compared to a MR method. We demonstrated excellent agreement of both NIR and MR methods for powders vs. the reference KF method. These methods are applicable to in-process control or quality control environments. One of the aims of this study was to determine if a calibration developed for a particular product could be used to predict the water content of another product (with related composition) but containing a different active pharmaceutical ingredient (API). We demonstrated that, contrary to the NIR method, a general MR method can be used to predict water content in two different types of blends. Finally, we demonstrated that a MR method can be developed for at-line moisture determination in tablets.

Fully automated dissolution test systems have become common throughout the pharmaceutical industry, and they are frequently utilized in laboratories that have a high sample throughput such as stability testing and quality control laboratories. Although these systems were introduced approximately 20 to 30 years ago, very few “new” automated dissolution systems have been introduced in the last 10 years. Thus, there is a unique demand for a novel approach in designing a dissolution system which incorporates many of the technological advances that have been made in the last decade. This case study describes the entire process of creating a novel fully automated dissolution system. The introduction of the Cetus 840® automated micro-sampling dissolution system, developed by Hatch Science® in collaboration with Bristol-Myers Squibb, integrates several new technologies, such as micro-sampling, cameras to record the dissolution test, a robust vessel washing routine, on-line high-performance liquid chromatography, vibration sensor, and a more hydrodynamic sampling system.

NIR imaging and Raman mapping of the dissolution of model pharmaceutical formulations containing the HCl salt of a developmental compound, were carried out using a custom designed flow through cell. The results of this work have shown that NIR imaging and Raman mapping are capable of monitoring the distribution of the components in a formulation during dissolution while also revealing any form changes which may occur in real time. The NIR and Raman data revealed that the drug underwent conversion to the free base when water was used as the dissolution medium. However, in 0.1M HCl this conversion was no longer seen as the medium was below the pHmax (the pH of saturation of both unionised and ionised species and above which the free base can form) of the drug. The data from both approaches broadly agreed demonstrating the applicability of these methods to studying and enhancing our understanding of the complex physical and chemical processes which occur during dissolution in real time.

Hydrochlorothiazide (HCTZ) 60 mg strength tablets containing commonly used excipients and hydroxypropyl cellulose, marketed as either Klucel-EF (HPC, NF from Hercules, USA) or HPC-L (HPC, NF from Nippon Soda, Japan), as a binder were manufactured using identical aqueous wet granulation process. The tablets containing Klucel-EF as a binder exhibited higher dissolution rates than those manufactured using HPC-L. The granulations containing Klucel-EF or HPC-L showed no significant differences in compressibility and compactibility based on analysis performed using the Instron-Stress-Strain Analyzer. Both HPC grades met NF specifications and there were no differences for the NF test results in the certificates of analysis by their respective vendors. Further evaluation of both HPC grades indicated that the cloud point values for Klucel-EF and HPC-L in water were 39(+/- 1) and 48 degrees C, respectively. The differences in cloud points of Klucel-EF and HPC-L were correlated to the differences in the percent hydroxypropoxy content and the degree of molecular substitution, which were higher for Klucel-EF than for HPC-L. These structural features make Klucel-EF less hydrophilic. Since the cloud point of Klucel-EF was similar to the dissolution medium temperature of 37(+/- 2) degrees C, it may present a less viscous layer surrounding the HCTZ granules enabling faster dissolution of the drug.

A headspace capillary gas chromatographic method was developed to determine trifluoroacetic acid (TFA) in a bulk substance (ceronapril). The bulk sample is reacted in a sealed headspace vial with dimethyl sulfate in concentrated sulfuric acid to convert TFA to methyl trifluoroacetate (MTFA). A portion of the headspace of the vial containing the MTFA is chromatographed on a PoraPLOT Q 0.32 mm I.D. capillary column in the split injection mode with flame ionization detection. The limit of detection is less than 10 ppm (w/w).

A capillary gas chromatographic method has been developed to quantitate the methylboronic acid in a lyophilized formulation. The formulation is first dissolved in 1 M hydrochloric acid. Next a tetrahydrofuran solution of the internal standard, 1-butaneboronic acid, and a methylene chloride solution of the derivatizing reagent, pinacol (2,3-dimethyl-2,3-butanediol), are added. The two-phase mixture is shaken for 3 h at 60°C during which time the methylboronic acid is extracted into the methylene chloride phase and reacted with pinacol to form the cyclic pinacol-boronate ester. The product formed in methylene chloride is injected in the split mode onto a capillary gas chromatographic system equipped with a flame ionization detector. Quantitation is achieved by an external standard method.

An automated capillary column gas chromatography electron-impact mass selective detection method has been developed for the determination of a major tranquilizing agent, fluphenazine. Using a new purification method and incorporating a deuterated internal reference, a limit of quantitation of 50 pg per ml of plasma was obtained.