Process Analytical Technology Tool Research Articles

In situ real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need. PAT involves the collection of in situ data, thereby providing real time information about the monitored process as well as increasing understanding of that process. Here, three separate PAT tools - optical particle imaging, in situ particle analysis, and Raman spectroscopy - were used to monitor two key steps involved in the formation of a stable emulsion product, emulsification and homogenization, as well as perform a stability assessment. The obtained results provided new insights-particle size decreases during emulsification and homogenization, and molecular changes do not occur during either the emulsification or homogenization steps. Further, the stability assessment indicated that the coarse emulsion product obtained from the emulsification step is stable over the course of 24 hours when mixed. To the best of our knowledge, this is the first report of an analytical methodology for in situ, real time analysis of emulsification and homogenization processes for vaccine adjuvants. Using our proposed analytical methodology, an improved understanding of emulsion-based vaccine adjuvants can now be achieved, ultimately impacting the ability to develop and deliver successful pharmaceuticals.

Monitoring of the Rioja red wine production process by 1 H-NMR spectroscopy.

As an inherently quantitative and unbiased analytical technique, proton nuclear magnetic resonance (1 H-NMR) provides an excellent method to monitor the quality of food and beverages, and a sensitive and informative tool to study the winemaking process. By using NMR, it is possible to monitor quantitative changes in wine metabolites (amino acids, organic acids and some phenolic compounds) during the winemaking process, including wine ageing. This study shows an increase in the concentration of the phenols at the beginning of alcoholic fermentation, as well as a stabilization and slight increase in gallic acid and a slight decrease in resveratrol during the oak barrel ageing step. This study demonstrates the potential of NMR as a process analytical technology (PAT) tool in the wine industry, by monitoring amino acids, organic acids and three polyphenols - gallic acid, catechin and resveratrol - during the winemaking process. This study of the time course evolution of wine has been conducted in a commercial winery rather than an experimental laboratory, demonstrating the capacity of this technique in commercial winemaking production. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A NIR-Based Study of Desorption Kinetics during Continuous Spin Freeze-Drying

The pharmaceutical industry is progressing toward the development of more continuous manufacturing techniques. At the same time, the industry is striving toward more process understanding and improved process control, which requires the implementation of process analytical technology tools (PAT). For the purpose of drying biopharmaceuticals, a continuous spin freeze-drying technology for unit doses was developed, which is based on creating thin layers of product by spinning the solution during the freezing step. Drying is performed under vacuum using infrared heaters to provide energy for the sublimation process. This approach reduces drying times by more than 90% compared to conventional batch freeze-drying. In this work, a new methodology is presented using near-infrared (NIR) spectroscopy to study the desorption kinetics during the secondary drying step of the continuous spin freeze-drying process. An inline PLS-based NIR calibration model to predict the residual moisture content of a standard formulation (i.e., 10% sucrose) was constructed and validated. This model was then used to evaluate the effect of different process parameters on the desorption rate. Product temperature, which was controlled by a PID feedback mechanism of the IR heaters, had the highest positive impact on the drying rate during secondary drying. Using a higher cooling rate during spin freezing was found to significantly increase the desorption rate as well. A higher filling volume had a smaller negative effect on the drying rate while the chamber pressure during drying was found to have no significant effect in the range between 10 and 30 Pa.

A hybrid model for multipoint real time potency observation in continuous direct compression manufacturing operations

The ongoing transition from batch to continuous manufacturing offers both challenges and opportunities in the field of oral solid dosage form production. In turn, Process Analytical Technology (PAT) offers a path towards the successful deployment of continuous tablet manufacturing in rotary tablet presses. One promising PAT tool for this endeavour is the NIR-derived potency measurement. However, the high degree of noise in the data may hamper the extraction of useful information. For this reason, this work focused on the implementation of an adaptive Kalman filter algorithm that incorporates and reconciles the potency prediction given by one or more NIR probes with those of a semi-mechanistic compartmental model developed for the application at hand. This approach allowed for more robust concentration estimations. Furthermore, it was observed that potency levels in multiple locations in the studied tablet press (including those in the finished tablets) could be appropriately inferred using a single in-line measurement data stream. This methodology thus opens the door to advanced process control applications.

Systematic assessment of process analytical technologies for biologics.

The application of process analytical technology (PAT) for biotherapeutic development and manufacturing has been employed owing to technological, economic, and regulatory advantages across the industry. Typically, chromatographic, spectroscopic, and/or mass spectrometric sensors are integrated into upstream and downstream unit operations in in-line, on-line, or at-line fashion to enable real-time monitoring and control of the process. Despite the widespread utility of PAT technologies at various unit operations of the bioprocess, a holistic business value assessment of PAT has not been well addressed in biologics. Thus, in this study, we evaluated PAT technologies based on predefined criteria for their technological attributes such as enablement of better process understanding, control, and high-throughput capabilities; as well as for business attributes such as simplicity of implementation, lead time, and cost reduction. The study involved an industry-wide survey, where input from subject matter industry experts on various PAT tools were collected, assessed, and ranked. The survey results demonstrated on-line liquid Chromatography (LC), in-line Raman, and gas analysis techniques are of high business value especially at the production bioreactor unit operation of upstream processing. In-line variable path-length UV/VIS measurements (VPE), on-line LC, multiangle light scattering (MALS), and automated sampling are of high business value in Protein A purification and polishing steps of the downstream process. We also provide insights, based on our experience in clinical and commercial manufacturing of biologics, into the development and implementation of some of the PAT tools. The results presented in this study are intended to be helpful for the current practitioners of PAT as well as those new to the field to gauge, prioritize and steer their projects for success.

Wet Milling, Seeding, and Ultrasound in the Optimization of the Oiling-Out Crystallization Process

Complicated solution environments in oiling-out crystallization can lead to particle agglomeration with wide size distribution and low purity of the products, due to complex interactions among two liquid phases and one solid phase during the oiling-out crystallization. This research mainly focuses on the optimization of size distribution by controlling particle agglomeration during the oiling-out crystallization process in a model system of propyl paraben–ethanol–water. Nucleation control technologies, wet milling, seeding, and ultrasound were used to limit the agglomeration. Further investigations of wet milling were performed before the nucleation or in the crystal growth stages with different geometries, such as coarse, medium, and fine rotor–stator tooth pairs. An integrated process analytical technology tools (PAT) array, including focused beam reflectance measurement (FBRM), particle visual monitoring (PVM), and attenuated total reflectance ultraviolet/visible (ATR-UV/vis), was used to observe the droplet formation of the dispersed phase, size distributions, and particle shapes during the nucleation and crystal growth. The results demonstrate that wet milling, seeding, and ultrasound technologies can help to optimize the particle size distribution in complex solution environments with different levels of efficiencies.

Non-destructive quantitative analysis of pharmaceutical ointment by transmission Raman spectroscopy

Transmission Raman spectroscopy was used to develop a non-destructive quantitative analytical model for the assay of a crystal dispersion-type ointment containing acyclovir as a model drug with a concentration of 3% w/w. The obtained Raman spectra were pre-processed by applying multiplicative scatter correction, standard normal variate, and first or second derivative by the Savitzky-Golay method to optimize the partial least squares (PLS) regression model. The optimized PLS model showed good prediction performance for 85%, 100%, and 115% label claims, with average recovery values of 100.7%, 99.3%, and 99.8%, respectively. Although the material properties and manufacturing method of acyclovir and white petrolatum were expected to be different from those of the calibration set, the mean recovery value of the commercial product was 104.2%. These results indicate that transmission Raman spectroscopy is a useful process analytical technology tool for product development and quality control of a crystal dispersion-type ointment with low drug concentration.

Utilizing in situ spectroscopic tools to monitor ketal deprotection processes

The use of protection groups to shield a functional group during a synthesis is employed throughout many reactions and organic syntheses. The role of a protection group can be vital to the success of a reaction, as well as increase reaction yield and selectivity. Although much work has been done to investigate the addition of a protection group, the removal of the protection group is just as important – however, there is a lack of methods employed within the literature for monitoring the removal of a protection group in real time. In this work, the process of removing, or deprotecting, a ketal protecting group is investigated. Process analytical technology tools are incorporated for in situ analysis of the deprotection reaction of a small molecule model compound. Specifically, Raman spectroscopy and Fourier transform infrared spectroscopy show that characteristic bands can be used to track the decrease of the reactant and the increase of the expected products over time. To the best of our knowledge, this is the first report of process analytical technology being used to monitor a ketal deprotection reaction in real time. This information can be capitalized on in the future for understanding and optimizing pharmaceutically-relevant deprotection processes and downstream reactions.

Towards real-time release of pharmaceutical tablets: 100% in-line control via near-infrared spatially resolved spectroscopy and 3D microwave resonance technology

In the scope of 100% in-line quality control and real-time release of pharmaceutical tablets, the authors present a flexible inspection module for in-line tablet analysis with integrated multipoint near-infrared (NIR) spectroscopy and 3D microwave resonance technology (3D MRT). Via an industrial case study on Diclofenac Sodium tablets, the abilities of this versatile process analytical technology (PAT) tool are presented. It is demonstrated that the combination of Diclofenac concentration prediction via NIR spectroscopy and mass prediction via 3D MRT allow to estimate the dosage of each individual tablet. Single sample repetition tests were performed on 5 tablets, measured 10 times on three different days. A high accuracy and precision of prediction was shown, with an average standard deviation below 0.5 mg. The inspection run demonstrated the added value of such inspection and sorting strategies based on the calculated dosage of individual tablets.

Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process.

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.

Laser triangulation as a fast and reliable method for determining ribbon solid fraction; focus on accuracy, precision, and measurement time

Roller compaction and dry granulation represent well-established unit operations in the pharmaceutical industry. The ribbon solid fraction is classified as a critical quality attribute, that directly impacts final product quality and performance. The development and evaluation of novel methods measuring ribbon solid fraction represent a subject of current research, since novel analyses strategies need to be established for at-, on–, or in-line process monitoring to overcome limitations of end product testing and to set the course for continuous manufacturing. In this study, a novel analytical device, using the principle of laser triangulation, was investigated to asses its potential being used as at-line process analytical technology tool during a roller compaction process. To this end, the laser triangulation device was compared with X-ray micro-computed tomography and powder based volume displacement measurement techniques using different statistical evaluation methods. Special focus was given to accuracy, precision, and total measurement time. The laser triangulation device was confirmed as highly accurate and precise, enabling the shortest total measurement time compared to the other methods. The findings of this study support the idea of implementing the laser triangulation device as a novel at-line process analytical technology tool into a roller compaction process.

Characterization of cell cycle and apoptosis in Chinese hamster ovary cell culture using flow cytometry for bioprocess monitoring

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.

Process analytical technique (PAT) miniaturization for monoclonal antibody aggregate detection in continuous downstream processing

AbstractThe transition to continuous biomanufacturing is considered the next step to reduce costs and improve process robustness in the biopharmaceutical industry, while also improving productivity and product quality. The platform production process for monoclonal antibodies (mAbs) is eligible for continuous processing to lower manufacturing costs due to patent expiration and subsequent growing competition. One of the critical quality attributes of interest during mAb purification is aggregate formation, with several processing parameters and environmental factors known to influence antibody aggregation. Therefore, a real‐time measurement to monitor aggregate formation is crucial to have immediate feedback and process control and to achieve a continuous downstream processing. Miniaturized biosensors as an in‐line process analytical technology tool could play a pivotal role to facilitate the transition to continuous manufacturing. In this review, miniaturization of already well‐established methods to detect protein aggregation, such as dynamic light scattering, Raman spectroscopy and circular dichroism, will be extensively evaluated for the possibility of providing a real‐time measurement of mAb aggregation. The method evaluation presented in this review shows which limitations of each analytical method still need to be addressed and provides application examples of each technique for mAb aggregate characterization. Additionally, challenges related to miniaturization are also addressed, such as the design of the microfluidic chip and the microfabrication material. The evaluation provided in this review shows why the development of microfluidic biosensors is considered the key for real‐time measurement of mAb aggregates and how it can contribute to the transition to a continuous processing. © 2021 The Authors.Journal of Chemical Technology and Biotechnologypublished by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Real-time particle size analysis using focused beam reflectance measurement as a process analytical technology tool for continuous microencapsulation process

The online real-time particle size analysis of the microencapsules manufacturing process using the continuous solvent evaporation method was performed using focused beam reflectance measurement (FBRM). In this paper, we use FBRM measurements to investigate the effects of polymer type and compare the size distributions to those obtained using other sizing methods such as optical microscope and laser diffraction. FBRM was also utilized to measure the length-weighted chord length distribution (CLD) and particle size distribution (PSD) online during particle solidification, which could not be done with laser diffraction or nested sieve analysis. The chord lengths and CLD data were taken at specific times using an online FBRM probe mounted below the microparticle. The timing of the FBRM determinations was coordinated with the selection of microparticle samples for particle size analysis by optical microscope and laser diffraction calculation as a reference. For all three produced batches tested, FBRM, laser diffraction, and sieve analysis yielded similar results. Hardening time for the transformation of emulsion droplets into solid microparticles occurred within the first 10.5, 19, 25, 30, and 55 min, according to FBRM results. The FBRM CLDs revealed that a larger particle size mean resulted in a longer CLD and a lower peak of particle number. The FBRM data revealed that the polymer type had a significant impact on microparticle CLD and the transformation process.

Multi-wavelength UV-based PAT tool for measuring protein concentration

Process chromatography is commonly used for purification of therapeutic proteins. Most chromatography skids that are used in such operations utilize single ultraviolet (UV) absorbance for monitoring and quantification of protein content. While the signal from such UV measurement is linear with respect to protein concentration at low values of protein concentrations, as the concentration increases across an eluting product peak, it goes manifold over the linear range, resulting in saturation of the UV signal and as a result incomplete quantification of the protein concentration. This can hamper our ability to decide on where to pool the process chromatography peak. It is evident that a simple, fast, and cost‐effective methodology for on-line estimation of protein concentration is the need of the hour. In this paper, a multi-wavelength UV-based approach has been proposed for dilution-free on-line concentration estimation in the range of 0.8–100 g/L. Stable absorbance regions are picked up in the proposed approach from the multi-wavelength UV spectra, thereby offering a solution to the problem of saturation and non-linearity of the UV signal that is otherwise observed at higher concentrations. Further, using chemometrics tools such as principal component analysis (PCA) and partial least squares (PLS), the model has been validated for rapid quantification of protein concentration from the spectra. The predictions from the model were comparable to values measured using an existing UV-based offline method with an R2 of>98%. The proposed process analytical technology (PAT) tool was successfully tested online and exhibited<8% variability and could effectively be used from capture to formulation to enable dilution-free online concentration measurement of IgG. The proposed tool is a simple, low-cost alternative to other methods and could enable integrated/continuous operations throughout the downstream train.

In Situ Quantification of Polyhydroxybutyrate in Photobioreactor Cultivations of Synechocystis sp. Using an Ultrasound-Enhanced ATR-FTIR Spectroscopy Probe.

Polyhydroxybutyrate (PHB) is a very promising alternative to most petroleum-based plastics with the huge advantage of biodegradability. Biotechnological production processes utilizing cyanobacteria as sustainable source of PHB require fast in situ process analytical technology (PAT) tools for sophisticated process monitoring. Spectroscopic probes supported by ultrasound particle traps provide a powerful technology for in-line, nondestructive, and real-time process analytics in photobioreactors. This work shows the great potential of using ultrasound particle manipulation to improve spectroscopic attenuated total reflection Fourier-transformed mid-infrared (ATR-FTIR) spectra as a monitoring tool for PHB production processes in photobioreactors.

Monitoring the hot-air drying process of organically grown apples (cv. Gala) using computer vision

The feasibility of using a computer-vision (CV) system embedded in a hot-air dryer for non-destructive and real-time monitoring of the drying behaviour of organic apples was investigated in the present study. Apple cylinders were subjected to anti-browning treatments with different dipping solutions (water as control, trehalose (4% w/v) or trehalose + ascorbic acid (4% w/v and 1% w/v)) and dipping pressures (atmospheric, 101.3 kPa and sub-atmospheric pressure, 50 kPa) followed by drying at 60 °C to a final dry basis moisture content of 0.18 g g−1. The CV system was used as an in-line process analytical technology (PAT) tool to capture images reflecting the physico-chemical changes during product drying coupled with in-line mass changes and off-line reference analyses. The spatial and colour changes from the image analysis described well the complex and non-homogenous nature of apple drying. The results of spatial changes allowed successful development of accurate linear prediction models for moisture content as a function of area shrinkage (on scaled variables) with excellent prediction capability (|BIAS| < 8.5 10−3, RMSE < 0.04, Adj-R2 ~ = 99%). Also, the CV system identified the differently pre-treated samples, particularly the dipping pressures as reflected by two linear models and their respective parameters. The obtained results demonstrate the versatile advantages of CV systems as an in-line tool for continuous, real-time monitoring of apples during drying. The insights from this study can provide a platform for applications of CV embedded ‘Smart dryers’ as an efficient monitoring and control system for industrial drying processes.

Implementation of spatial filtering technique in monitoring roller compaction process

This study investigates the use of the spatial filtering technique (SFT11spatial filtering technique) to monitor the particle size distribution (PSD22particle size distribution) of granules obtained by roller compaction. In the first part of the study, the influence of the selected process and formulation parameters on the PSD2 of granules is monitored at-line using SFT1. The correlation between the PSD2 obtained by SFT1, sieve analysis, laser diffraction, and dynamic image analysis was satisfactory. The same trend was observed with all methods; however, SFT1 proved to be especially advantageous for monitoring the PSD2 of irregularly shaped granules obtained by roller compaction. Another aim of this study was to investigate the suitability of using the SFT1 method as a potential process analytical technology (PAT33process analytical technology) tool for monitoring and predicting the PSD2 of granules obtained by roller compaction. The SFT1 model for d10 was poor due to less precise detection of smaller particles by SFT; nevertheless, the models for d50 (R2 = 0.93) and d90 (R2 = 0.93) were very good. The at-line models were further tested in real time on samples collected during the milling of ribbons. The correlation between the predicted and achieved values was good; however, it was time and formulation dependent.

SWiVIA – Sliding window variographic image analysis for real-time assessment of heterogeneity indices in blending processes monitored with hyperspectral imaging

Controlling blending processes of solid material using advanced real-time sensing technologies tools is crucial to guarantee the quality attributes of manufactured products from diverse industries. The use of process analytical technology (PAT) tools based on chemical imaging systems are useful to assess heterogeneity information during mixing processes. Recently, a powerful procedure for heterogeneity assessment based on the combination of off-line acquired chemical images and variographic analysis has been proposed to provide specific heterogeneity indices related to global and distributional heterogeneity. This work proposes a novel PAT tool combining in situ chemical imaging and variogram-derived quantitative heterogeneity indices for the real-time monitoring of blending processes. The proposed method, so called sliding window variographic image analysis (SWiVIA), derives heterogeneity indices in real-time associated with a sliding image window that moves continuously until the full blending time interval is covered. The SWiVIA method is thoroughly assessed paying attention at the effect of relevant factors for continuous blending monitoring and heterogeneity description, such as the scale of scrutiny needed for heterogeneity definition or the blending period defined to set the sliding image window. SWiVIA is tested on blending runs of pharmaceutical and food products monitored with an in situ near-infrared chemical imaging system. The results obtained help to detect abnormal mixing phenomena and can be the basis to establish blending process control indicators in the future. SWiVIA is adapted to study blending behaviors of the bulk product or compound-specific blending evolutions.

Online monitoring and control of upstream cell culture process using 1D and 2D-LC with SegFlow interface.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next-generation bioprocessing with the adaptation of continuous bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality-by-design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge-based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next-generation continuous bioprocessing, it is essential to embrace a fundamental shift-in-paradigm from "quality-by-testing" to "quality-by-design," which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D-LC with a post-flow-splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.