Application of PAT Probes in Aluminum Phosphate Adjuvant Manufacturing.

PURPOSE: This study is focused on monitoring process parameters and quality attributes of aluminum phosphate (AlPO4) using multiple in-line probes incorporated into an industrial-scale adjuvant suspension manufacturing unit. METHODS: The manufacturing of aluminum adjuvant suspension was monitored at manufacturing scale using conductivity, turbidity, infrared, and particle sizing and count probes to follow the continuous evolution of particle formation and size distribution, and the reaction kinetics during the synthesis of AlPO4. RESULTS: The data showed that AlPO4 forms large particles at the early stages of mixing, followed by a decrease in size and then stabilization towards the later stages of mixing and pH adjustment. The results provided a complementary view of process events and assisted in optimizing several parameters, e.g., flow rate of reactants AlCl3 and Na3PO4 solutions, mixing rate, pH, and conductivity of AlPO4, as well as adjuvant quality attribute such as particle size, thus streamlining and shortening the process development stage. CONCLUSION: The results of this study showed the usefulness of the in-line probes to automate continuous assessment of AlPO4 batch-to-batch consistency during in-house adjuvant production at the industrial scale.

Selective anomer crystallization from aqueous solution: Monitoring lactose recovery under mutarotation limitation via attenuated total reflection Fourier-transform spectroscopy and theoretical rate analysis.

Lactose is typically produced via cooling crystallization either from whey or whey permeate (edible grade) or from aqueous solution (pharmaceutical grade). While in solution, lactose is present in 2 anomeric forms, α- and ß-lactose. During cooling crystallization under standard process conditions, only α-lactose crystallizes, depleting the solution of α-anomer. In practice, mutarotation kinetics are often assumed to be much faster than crystallization. However, some literature reports limitation of crystallization by mutarotation. In the present research, we investigate the influence of operating conditions on mutarotation in lactose crystallization and explore the existence of an operation regimen where mutarotation can be disregarded in the crystallization process. Therefore, we study crystallization from aqueous lactose solutions by inline monitoring of concentrations of α- and ß-lactose via attenuated total reflection Fourier-transform spectroscopy. By implementing a linear cooling profile of 9 K/h to a minimum temperature of 10°C, we measured a remarkable increase in ß/α ratio, reaching a maximum of 2.19. This ratio exceeds the equilibrium level by 36%. However, when the same cooling profile was applied to a minimum temperature of 25°C, the deviation was significantly lower, with a maximum ß/α ratio of 1.72, representing only an 8% deviation from equilibrium. We also performed a theoretical assessment of the influence of process parameters on crystallization kinetics. We conclude that mutarotation needs to be taken into consideration for efficient crystallization control if the crystal surface area and supersaturation are sufficiently high.

Application of front-face fluorescence spectroscopy as a tool for monitoring changes in milk protein concentrate powders during storage.

This study investigated the feasibility of front-face fluorescence spectroscopy (FFFS) to predict the solubility index and relative dissolution index (RDI) of milk protein concentrate (MPC) powders during storage. Twenty MPC powders with varying protein contents from 4 different commercial manufacturers were used in this study. The MPC powders were stored at 2 temperatures (25 and 40°C) for 0, 1, 2, 4, 8, and 12 wk. The front-face fluorescence spectra of tryptophan and Maillard products were recorded and analyzed with chemometrics to predict solubility of MPC powders. The similarity maps showed clear discrimination of the MPC samples stored at 25 and 40°C. Partial least squares regression models were developed using the fluorescence spectra of tryptophan and Maillard products to predict the solubility index and RDI measurements of MPC powders, and the prediction models were validated using an independent test set. Coefficients of determination (R2) of 0.76, 0.84, and 0.68 were obtained between fluorescence spectra (tryptophan emission, Maillard emission, and Maillard excitation, respectively) and solubility index. The R2 values for the RDI predictions were 0.58 and 0.60 for the data set of tryptophan emission and Maillard emission, respectively. The ratio of prediction error to standard deviation was >2 for Maillard emission fluorescence spectra and solubility index measurements, indicating good practical utility of the partial least squares regression prediction models. The results indicated that the solubility and dissolution behavior of MPC powders were related to their protein content and storage conditions that could be measured using FFFS. Hence, FFFS can be used as a rapid nondestructive analytical technique to predict the solubility and dissolution characteristics of MPC powders.

Evaluating the crystallization of lactose at different cooling rates from milk and whey permeates in terms of crystal yield and purity.

The cooling rate of supersaturated lactose solution is one of the important parameters determining the yield and size distribution of lactose crystals. The influence of increasing cooling rate on lactose crystallization and quality of lactose crystals was evaluated in concentrated solutions prepared from deproteinized whey powder (DPW) and milk permeate powder (MPP). Concentrated permeates (DPW and MPP) with 60% (wt/wt) total solids were prepared by reconstituting permeate powders in water at 80°C for 2 h for lactose dissolution. Three cooling rates, 0.04°C/min (slow), 0.06°C/min (medium), and 0.08°C/min (fast) were studied in duplicate. A common rapid cooling step (80 to 60°C at 0.5°C/min) followed by slow, medium, and fast cooling rates were applied as per the experimental design from 60 to 20°C. After crystallization, the crystal slurry was centrifuged, washed with cold water, and dried. The dried lactose crystals were weighed to calculate the lactose yield. Final mean particle chord lengths were measured at the end of crystallization using focused beam reflectance measurement for slow, medium, and fast cooling rates, and observed to be not significantly different for DPW (27-33 µm) and MPP (31-34 µm) concentrates. Similarly, the lactose yield for slow, medium, and fast cooling rates in the DPW and MPP concentrates were in the range of 71 to 73% and 76 to 81%, respectively, and no significant difference between the 3 cooling rates was found. Qualitative analysis of dried lactose crystals exhibited no noticeable differences in the crystal purity with increasing cooling rate. This study evaluated the possibility of reducing the crystallization times by 8 h compared with current industrial practice without compromising the crystal yield and quality.

Process analytical approaches for the coil-to-globule transition of poly(N-isopropylacrylamide) in a concentrated aqueous suspension.

The coil-to-globule transition of poly(N-isopropylacrylamide) (PNIPAM) microgel particles suspended in water has been investigated in situ as a function of heating and cooling rate with four optical process analytical technologies (PAT), sensitive to structural changes of the polymer. Photon Density Wave (PDW) spectroscopy, Focused Beam Reflectance Measurements (FBRM), turbidity measurements, and Particle Vision Microscope (PVM) measurements are found to be powerful tools for the monitoring of the temperature-dependent transition of such thermo-responsive polymers. These in-line technologies allow for monitoring of either the reduced scattering coefficient and the absorption coefficient, the chord length distribution, the reflected intensities, or the relative backscatter index via in-process imaging, respectively. Varying heating and cooling rates result in rate-dependent lower critical solution temperatures (LCST), with different impact of cooling and heating. Particularly, the data obtained by PDW spectroscopy can be used to estimate the thermodynamic transition temperature of PNIPAM for infinitesimal heating or cooling rates. In addition, an inverse hysteresis and a reversible building of micrometer-sized agglomerates are observed for the PNIPAM transition process.

Novel methods to study the effect of protein content and dissolution temperature on the solubility of milk protein concentrate: Focused beam reflectance and ultrasonic flaw detector-based methods.

Processing, storage, dissolution conditions, and the composition of milk protein concentrates (MPC) affect the solubility of high-protein dairy powders. Increasing the storage temperature and time decrease the solubility of MPC and milk protein isolates (MPI). The MPC and MPI are popular ingredients in high-protein food products and have a variety of protein contents. In addition, the dissolution temperature has been shown to affect the solubility of the powders. This study focused on determining how protein content and dissolution temperature affect the solubility of MPC and MPI. For this study, 11 powders were obtained from a commercial manufacturer. The powders were classified as A, B, C, and D, and they had a mean protein content of 85, 87, 88, and 90%, respectively. A 5% (wt/wt) concentration of powder was dissolved in water at 40 and 48°C. The solubility of the MPC and MPI samples were characterized using an ultrasonic flaw detector (UFD) and focused beam reflectance measurement (FBRM). The UFD and FBRM data were collected every 15 and 10 s, respectively, for 1,800 s. At both dissolution temperatures, the UFD and FBRM data showed that the solubility decreased as the protein content increased. Powders A and B were found to be more soluble because they had a lower relative velocity standard deviation, high area under the attenuation curve, high peak height, and low peak time. With the FBRM, the fine and medium particle count decreased and large particle count increased as the protein content increased. Powders dissolved at 48°C typically had a lower relative velocity standard deviation, higher area under the attenuation curve, higher peak height, and lower peak time than the powders dissolved at 40°C. The FBRM showed that powders dissolved at 48°C reached a stable counts before the powders dissolved at 40°C. Overall, the study showed that increasing the protein content led to a reduction in solubility and increasing the dissolution temperature improved the solubility of the powders.

Application of percolation threshold to disintegration and dissolution of ibuprofen tablets with different microcrystalline cellulose grades.

The study presented was conducted to determine whether a percolation threshold value, previously determined for ibuprofen/microcrystalline cellulose (MCC) blends using percolation theory and compression data (Queiroz et al., 2019), could translate to tablet disintegration and dissolution data. The influence of MCC grade (air stream dried versus spray dried) on tablet disintegration and dissolution was also investigated. Complementary to conventional disintegration and dissolution testing, Raman imaging determined drug distribution within tablets, and in-line particle video microscopy (PVM) and focused-beam reflectance measurement (FBRM) monitored tablet disintegration. Tablets were prepared containing 0-30% w/w ibuprofen. Raman imaging confirmed the percolation threshold by quantifying the number and equivalent circular diameters of ibuprofen domains on tablet surfaces. Across the percolation threshold, a step change in dissolution behaviour occurred, and tablets containing air stream dried MCC showed slower disintegration rates compared to tablets containing spray dried MCC. Dissolution measurements confirmed experimentally a percolation threshold in agreement with that determined using percolation theory and compression data. An increase in drug domains, due to cluster formation, and less efficient tablet disintegration contributed to slower ibuprofen dissolution above the percolation threshold. Slower dissolution was measured for tablets containing air stream dried compared to spray dried MCC.

Understanding the Efficiency of Aluminum Coagulants Used in Dissolved Air Flotation (DAF).

This paper reports on the efficiency of five aluminum coagulants for the treatment of a paper mill wastewater by dissolved air flotation (DAF). The coagulants studied were: alum, a polyaluminum chloride coagulant of high aluminum content and intermediate basicity (PAC-MB), another with intermediate aluminum content and high basicity (PAC-HB), a polyaluminum nitrate sulfate of intermediate aluminum content and basicity (PANS) and one hybrid coagulant formed by the combination of PANS and a mixture of polyamines (PANS-PA). The influence of Al speciation on contaminants removal and the main flocculation mechanisms involved have been analyzed. High removal of suspended solids together with significant removal of dissolved and colloidal material (COD and silica) were obtained, which is required for extended reuse of this process water. PAC-HB was the best product for removing suspended solids (85%) and soluble silica (50%) with a rather limited COD removal (5%), while PANS-PA obtained high turbidity (90%) and silica removal (45%) together with a significant soluble COD removal (15%). Monomeric Al (Ala, Alm) was more efficient in removing suspended solids and soluble COD than polymeric or colloidal Al (Alc, Alu), but the latter was more efficient in removing soluble silica. Results demonstrated that the main flocculation mechanism varies with the aluminum dosage, being predominantly charge neutralization at low dosages and sweep flocculation at high dosages. The floc strength factor however, was very high and similar for all the coagulants and dosages tested (85-90%), as it was mainly determined by the behavior of the pre-flocculated suspended solids present in wastewater. The reflocculation factor varied from 45 to 75% at the lowest dosages to almost zero at the highest dosages, confirming the transition from charge neutralization to sweep flocculation. The flocs formed by PANS-PA had lower strength than the others and it decreased with the dosage while its reflocculation factor was almost zero, even at low dosages. Due to the polyamines present in this coagulant, its flocculation mechanism is through both charge neutralization and patch formation, especially at low dosages, and sweep flocculation and interparticle bridge formation at high dosages.

Insights on role of polymers in precipitation of celecoxib from supersaturated solutions as assessed by focused beam reflectance measurement (FBRM).

Supersaturating drug delivery systems (SDDS) have dominated the commercial and academic spheres owing to their potential in overcoming the solubility issue of poorly soluble drugs. Precipitation inhibitors are used as excipients in such formulations which has necessitated the development of supersaturation assays that evaluate their precipitation-inhibition efficacy. Such assays are able to give relative estimates of polymer efficacy ceteris paribus within a given set-up. However, the estimates of different laboratories cannot be compared with each other owing to high variability in procedure. Microarray plate method allows comprehensive replicates and decent statistics that make the method an edge over the other exploratory assays. In the current study, the precipitation-inhibition performance of three polymers on the precipitation of a model BCS class II drug was evaluated using the microarray plate method. Quantitative estimations were made through application of Poisson equation for nucleation rates and area under curve. Insights of the precipitation process at particle level were obtained through focused beam reflectance measurement (FBRM) technique coupled with end-process PVM imaging. Through real-time particle size analysis, FBRM technique demonstrated the potential for discerning the role of polymer as nucleation-inhibitor or crystal growth inhibitor. The events observed in the scaled-up FBRM analysis could be correlated with the events observed visually and spectrophotometrically. Powder X-ray diffraction and scanning electron microscopy were performed to capture the influence of polymers on the precipitates formed. This study was able to demonstrate the applicability of microarray plate method for quantitative estimations of precipitation kinetics that can be utilized for excipient screening for poorly soluble drugs having intra-luminal precipitation as a problem. FBRM analysis is highly valuable to gain mechanistic insights and put to rest the prevalent conjecture-based role attribution for polymers.

PAT-based batch statistical process control of a manufacturing process for a pharmaceutical ointment.

In this study, a process analytical technology (PAT)-based batch statistical process control (BSPC) model was developed for the laboratory-scale manufacturing process of a commercially available pharmaceutical ointment. The multivariate BSPC model was developed based on the in-line measured viscosity (viscometer), product temperature (viscometer), particle size distribution (PSD) (focused beam reflectance measurement (FBRM)) and active pharmaceutical ingredient (API) concentration (Raman spectroscopy) of four reference batches using a partial least squares (PLS) approach. From this in-line collected data, the characteristic trajectory of the batch process under normal operating conditions was acquired. To assess the capability of the process analyzers and BSPC model to detect deviations from the expected batch trajectory, two test batches with induced process and formulation disturbances were monitored in-line. The elevated process temperature in test batch 1 resulted in a deviating viscosity, product temperature and number of small particles (<100 µm). After correcting the process temperature, the viscosity and product temperature were within the control interval, while the particle size was smaller compared to the reference batches. For test batch 2, API was added at three different time points, whereas the same amount of API was added in one step during manufacturing of the reference batches. The induced disturbance was reflected in the in-line measured viscosity, PSD and API concentration. The combination of process analyzers and multivariate batch modelling enabled early fault detection and real-time process adjustments, thereby preventing batch loss or reprocessing. In addition, the feasibility of the investigated process analyzers to measure certain quality attributes in-line during manufacturing of an ointment was demonstrated.

Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools.

PURPOSE: Aluminum-based adjuvants including aluminum phosphate (AlPO4) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO4, such as particle size and chemical composition. Control of the vaccine adjuvant throughout the manufacturing process, including raw materials and the intermediate and final product stages, can be effectively achieved through monitoring of such key product attributes to help ensure product quality. METHODS: This study focuses on the compositional analysis of AlPO4 adjuvant at the intermediate and final manufacturing stages using the off-line methods Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and the in-line method Attenuated Total Reflectance (ATR). Particle size distribution of AlPO4 was measured off-line using Laser diffraction (LD) and in-line using Focused Beam Reflectance Measurement (FBRM®). RESULTS: There was no observable difference in size distribution between the intermediate and final stage AlPO4 by off-line and in-line analysis, in both small- or large-scale production samples. Consistent peak shifts were observed in off-line and in-line infrared (IR) spectroscopy as well as off-line XPS for both small- and large-scale AlPO4 manufacturing runs. Additionally, IR spectroscopy and FBRM® for size distribution were used as in-line process analytical technology (PAT) to monitor reaction progress in real-time during small-scale AlPO4 manufacturing from raw materials. The small-scale adsorption process of a model protein antigen (Tetanus toxoid) to AlPO4 adjuvant was also monitored by in-line ReactIR probe. CONCLUSION: This study demonstrated that in-line PAT can be used to monitor particle size and chemical composition for the various stages of adjuvant manufacturing from raw materials through intermediate to final adjuvant product stage. Similar approaches can be utilized to help assess lot-to-lot consistency during adjuvant manufacturing and vaccine product development. Moreover, the use of in-line PAT is highly conductive to advanced manufacturing strategies such as real-time product release testing and automated processes of the future.

Application of In-line Focused Beam Reflectance Measurement to Brivanib Alaninate Wet Granulation Process to Enable Scale-up and Attribute-based Monitoring and Control Strategies.

Application of in-line real-time process monitoring using a process analytical technology for granule size distribution can enable quality-by-design development of a drug product and enable attribute-based monitoring and control strategies. In this study, an in-line laser focused beam reflectance measurement (FBRM) C35 probe was used to investigate the effect of formulation and process parameters on the granule growth profile over time during the high shear wet granulation of a high drug load formulation of brivanib alaninate. The probe quantitatively captured changes in the granule chord length distribution (CLD) with the progress of granulation and delineated the impact of water concentration used during granulation. The results correlated well with offline particle size distribution measured by nested sieve analyses. An end point indication algorithm was developed that was able to successfully track the process time needed to reach the target CLD. Testing of the brivanib alaninate granulation through 25-fold scale-up of the batch process indicated that the FBRM CLD profile can provide a scale-independent granule attribute-based process fingerprint. These studies highlight the ability of FBRM to quantitate a granule attribute of interest during wet granulation that can be used as an attribute-based scale-up and process monitoring and control parameter.

Estimation of Chlamydomonas reinhardtii biomass concentration from chord length distribution data.

A novel method to estimate the concentration of Chlamydomonas reinhardtii biomass was developed. The method employs the chord length distribution information gathered by means of a focused beam reflectance probe immersed in the culture sample and processes the data through a feedforward multilayer perceptron. The multilayer perceptron architecture was systematically optimised through the application of a simulated annealing algorithm. The method developed can predict the concentration of microalgae with acceptable accuracy and, with further development, it could be implemented online to monitor the aggregation status and biomass concentration of microalgal cultures.

Resolution and Sensitivity of Inline Focused Beam Reflectance Measurement During Wet Granulation in Pharmaceutically Relevant Particle Size Ranges.

Real-time process monitoring using a process analytical technology for granule size distribution can enable quality-by-design in drug product manufacturing. In this study, the resolution and sensitivity of chord length distribution (CLD) measured inline inside a high shear granulator using focused beam reflectance measurement (FBRM) C35 probe was investigated using different particle size grades of microcrystalline cellulose (MCC). In addition, the impact of water and impeller tip speed on the measurement accuracy as well as correlation with offline particle sizing techniques (FBRM, laser diffraction [Malvern Mastersizer®], microscopy [Sympatec QicPic®], and nested sieve analysis) was studied. Inline FBRM resolved size differences between different MCC grades, and the data correlated well with offline analyses. Impeller tip speed changed the number density of inline CLD measurements while addition of water reduced the CLD of dry MCC, likely due to deagglomeration of primary particles. In summary, inline FBRM CLD measurement in high shear granulator provides adequate resolution and reproducible measurements in the pharmaceutically relevant size range both in the presence and in the absence of water. Therefore, inline FBRM can be a valuable tool for the monitoring of high shear wet granulation.

Real-time assessment of granule densification in high shear wet granulation and application to scale-up of a placebo and a brivanib alaninate formulation.

Real-time monitoring and control of high shear wet granulation (HSWG) using process analytical technologies is crucial to process design, scale-up, and reproducible manufacture. Although significant progress has been made in real-time measurement of granule size distribution using focused beam reflectance measurement (FBRM), real-time in-line assessment of granule densification remains challenging. In this study, a drag force flow (DFF) sensor was developed and used to probe wet mass consistency in real-time. In addition, responses from FBRM and DFF sensors were compared to assess complementarity of information on granulation progress from the two probes. A placebo and a brivanib alaninate formulation were granulated with different concentrations of binder or water, respectively, while measuring granule size growth, densification, and DFF sensor response. The DFF sensor was able to quantitatively characterize with high resolution a response of wet mass consistency distinct from granule size distribution. The wet mass consistency parameter correlated well with granule densification, which was shown as a critical material attribute that correlated with tablet dissolution. In addition, application of DFF sensor to scale-up of granulation was demonstrated. These results showed the value of wet mass consistency measurement using DFF for WG monitoring and control.

PDADMAC flocculation of Chinese hamster ovary cells: enabling a centrifuge-less harvest process for monoclonal antibodies.

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.

Demonstration of FBRM as process analytical technology tool for dewatering processes via CST correlation.

The current challenges associated with the design and operation of net-energy positive wastewater treatment plants demand sophisticated approaches for the monitoring of polymer-induced flocculation. In anaerobic digestion (AD) processes, the dewaterability of the sludge is typically assessed from off-line lab-bench tests - the capillary suction time (CST) test is one of the most common. Focused beam reflectance measurement (FBRM) is a promising technique for real-time monitoring of critical performance attributes in large scale processes and is ideally suited for dewatering applications. The flocculation performance of twenty-four cationic polymers, that spanned a range of polymer size and charge properties, was measured using both the FBRM and CST tests. Analysis of the data revealed a decreasing monotonic trend; the samples that had the highest percent removal of particles less than 50 microns in size as determined by FBRM had the lowest CST values. A subset of the best performing polymers was used to evaluate the effects of dosage amount and digestate sources on dewatering performance. The results from this work show that FBRM is a powerful tool that can be used for optimization and on-line monitoring of dewatering processes.

Focused beam reflectance measurement to monitor nimodipine precipitation process.

Crystallization of nimodipine in liquid-filled soft gelatin capsule during storage was reported for some commercial products, resulting in product recalls due to product quality and more importantly safety concerns. In this study, a real time particle monitoring tool, focused beam reflectance measurement, was used to evaluate the precipitation conditions of nimodipine in co-solvents. Upon water addition, two particle populations were discovered, appearing at different percentage of water content. Two transitions (i.e. sudden increase in particle counts) were observed, possibility related to nucleation and crystal growth of nimodipine. Furthermore, lowering storage temperature increased the tendency of nimodipine precipitation. Most critically, it was determined that with certain excipient, the drug precipitation occurred at approximately 7% (w/w) water content. Considering that all the orally administered liquid filled soft gelatin capsule shells contain residue water content as plasticizer, moisture transfer from the shell to the formulation may occur during long term storage, resulting in drug precipitation, particularly under cold temperature conditions.