Development and Comparison of Alternative Methods for the Purification of Adalimumab Directly from Harvested Cell Culture Fluid.

Research background: Protein A affinity chromatography is a well-established method currently used in the pharmaceutical industry. However, the high costs usually associated with chromatographic separation of protein A and the difficulties in continuous operation make the investigation of alternative purification methods very important. Experimental approach: In this study, extraction/back-extraction and precipitation/dissolution methods were developed and optimised. They were compared with protein A and cation exchange chromatography separations in terms of yield of monoclonal antibody (mAb) and amount of residual impurities, such as DNA and host cell proteins, and amount of mAb aggregates. For a comprehensive comparison of the different methods, experiments were carried out with the same cell-free fermentation broth containing adalimumab. Results and conclusions: Protein A and cation exchange chromatographic separations resulted in high yield and purity of adalimumab. The precipitation-based process resulted in high yield but with lower purity. The extraction-based purification resulted in low yield and purity. Thus, the precipitation-based method proved to be more promising than the extraction-based method for direct purification of adalimumab from harvested cell culture fluid. Novelty and scientific contribution: Although alternative purification methods may offer the advantages of simplicity and low-cost operation, further significant improvements are required to compete with the performance of chromatographic separations of adalimumab from true fermentation broth.

Comparison of Amorphous Solid Dispersions of Spironolactone Prepared by Spray Drying and Electrospinning: The Influence of the Preparation Method on the Dissolution Properties.

This research aimed to compare two solvent-based methods for the preparation of amorphous solid dispersions (ASDs) made up of poorly soluble spironolactone and poly(vinylpyrrolidone-co-vinyl acetate). The same apparatus was used to produce, in continuous mode, drug-loaded electrospun (ES) and spray-dried (SD) materials from dichloromethane and ethanol-containing solutions. The main differences between the two preparation methods were the concentration of the solution and application of high voltage. During electrospinning, a solution with a higher concentration and high voltage was used to form a fibrous product. In contrast, a dilute solution and no electrostatic force were applied during spray drying. Both ASD products showed an amorphous structure according to differential scanning calorimetry and X-ray powder diffraction results. However, the dissolution of the SD sample was not complete, while the ES sample exhibited close to 100% dissolution. The polarized microscopy images and Raman microscopy mapping of the samples highlighted that the SD particles contained crystalline traces, which can initiate precipitation during dissolution. Investigation of the dissolution media with a borescope made the precipitated particles visible while Raman spectroscopy measurements confirmed the appearance of the crystalline active pharmaceutical ingredient. To explain the micro-morphological differences, the shape and size of the prepared samples, the evaporation rate of residual solvents, and the influence of the electrostatic field during the preparation of ASDs had to be considered. This study demonstrated that the investigated factors have a great influence on the dissolution of the ASDs. Consequently, it is worth focusing on the selection of the appropriate ASD preparation method to avoid the deterioration of dissolution properties due to the presence of crystalline traces.

Electrospun Solid Formulation of Anaerobic Gut Microbiome Bacteria.

A model anaerobic bacterium strain from the gut microbiome (Clostridium butyricum) producing anti-inflammatory molecules was incorporated into polymer-free fibers of a water-soluble cyclodextrin matrix (HP-ß-CD) using a promising scaled-up nanotechnology, high-speed electrospinning. A long-term stability study was also carried out on the bacteria in the fibers. Effect of storage conditions (temperature, presence of oxygen) and growth conditions on the bacterial viability in the fibers was investigated. The viability of the sporulated anaerobic bacteria in the fibers was maintained during 12 months of room temperature storage in the presence of oxygen. Direct compression was used to prepare tablets from the produced bacteria-containing fibers after milling (using an oscillating mill) and mixing with tableting excipients, making easy oral administration of the bacteria possible. No significant decrease was observed in bacterial viability following the processing of the fibers (milling and tableting).

Development of Bioepoxy Resin Microencapsulated Ammonium-Polyphosphate for Flame Retardancy of Polylactic Acid.

Ammonium-polyphosphate (APP) was modified by microencapsulation with a bio-based sorbitol polyglycidyl ether (SPE)-type epoxy resin and used as a flame retardant additive in polylactic acid (PLA) matrix. The bioresin-encapsulated APP (MCAPP) particles were characterized using Fourier transform infrared (FTIR) spectroscopy and Raman mapping, particle size distribution was determined by processing of scanning electron microscopic (SEM) images. Interaction between the APP core and the bioresin shell was revealed by combined thermogravimetric analysis (TGA)­FTIR spectroscopy. The APP to SPE mass ratio of 10 to 2 was found to be optimal in terms of thermal, flammability, and mechanical properties of 15 wt% additive containing biocomposites. The bioresin shell effectively promotes the charring of the APP-loaded PLA composites, as found using TGA and cone calorimetry, and eliminates the flammable dripping of the specimens during the UL-94 vertical burning tests. Thus, the V-0 rating, the increased limiting oxygen index, and the 20% reduced peak of the heat release rate was reached compared to the effects of neat APP. Furthermore, better interfacial interaction of the MCAPP with PLA was indicated by differential scanning calorimetry and SEM observation. The stiff interphase resulted in increased modulus of these composites. Besides, microencapsulation provided improved water resistance to the flame retardant biopolymer system.

Raman Spectroscopy for Process Analytical Technologies of Pharmaceutical Secondary Manufacturing.

As the process analytical technology (PAT) mindset is progressively introduced and adopted by the pharmaceutical companies, there is an increasing demand for effective and versatile real-time analyzers to address the quality assurance challenges of drug manufacturing. In the last decades, Raman spectroscopy has emerged as one of the most promising tools for non-destructive and fast characterization of the pharmaceutical processes. This review summarizes the achieved results of the real-time application of Raman spectroscopy in the field of the secondary manufacturing of pharmaceutical solid dosage forms, covering the most common secondary process steps of a tablet production line. In addition, the feasibility of Raman spectroscopy for real-time control is critically reviewed, and challenges and possible approaches to moving from real-time monitoring to process analytically controlled technologies (PACT) are discussed.

Oral bioavailability enhancement of flubendazole by developing nanofibrous solid dosage forms.

The bioavailability of the anthelminthic flubendazole was remarkably enhanced in comparison with the pure crystalline drug by developing completely amorphous electrospun nanofibres with a matrix consisting of hydroxypropyl-ß-cyclodextrin and polyvinylpyrrolidone. The thus produced formulations can potentially be active against macrofilariae parasites causing tropical diseases, for example, river blindness and elephantiasis, which affect altogether more than a hundred million people worldwide. The bioavailability enhancement was based on the considerably improved dissolution. The release of a dose of 40 mg could be achieved within 15 min. Accordingly, administration of the nanofibrous system ensured an increased plasma concentration profile in rats in contrast to the practically non-absorbable crystalline flubendazole. Furthermore, easy-to-grind fibers could be developed, which enabled compression of easily administrable immediate release tablets.

Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases.

Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).

Quantitative and qualitative analysis of cell culture media powders for mammalian cells by Raman microscopy.

Cell culture media are essential for large-scale recombinant protein production using mammalian cell cultures. The composition and quality of media significantly impact cell growth and product formation. Analyzing media poses challenges due to complex compositions and undisclosed exact compositions. Traditional methods like NMR and chromatography offer sensitivity but require time-consuming sample preparation and lack spatial information. Raman chemical mapping characterizes solids, but its use in cell culture media analysis is limited so far. We present a chemometric evaluation for Raman maps to qualify and quantify media components, evaluate powder homogeneity, and perform lot-to-lot comparisons. Three lots of a marketed cell culture media powder were measured with Raman mapping technique. Chemometrics techniques have outlined a strategy to extract information from complex data. First, a spectral library has been structured. In addition to the 23 spectra for presumed ingredients, we obtained another 9 pure components with Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS). Then the Spectral Angle Mapper-Orthogonal Projection (SAM-OP) algorithm revealed whether references actually occur in the mapped media powders. Finally, a quantification was provided by Classical Least Squares (CLS) modelling. Quantities of 18 significant amino acids mostly correlated with the reference method. The proposed method can be generally applied even for such complicated samples. Leveraging Raman mapping and innovative chemometric methods enhance recombinant protein production by improving the understanding of the spatial distribution and composition of cell culture media in mammalian cell cultivations.

Raman and NIR spectroscopy-based real-time monitoring of the membrane filtration process of a recombinant protein for the diagnosis of SARS-CoV-2.

This research shows the detailed comparison of Raman and near-infrared (NIR) spectroscopy as Process Analytical Technology tools for the real-time monitoring of a protein purification process. A comprehensive investigation of the application and model development of Raman and NIR spectroscopy was carried out for the real-time monitoring of a process-related impurity, imidazole, during the tangential flow filtration of Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein. The fast development of Raman and NIR spectroscopy-based calibration models was achieved using offline calibration data, resulting in low calibration and cross-validation errors. Raman model had an RMSEC of 1.53 mM, and an RMSECV of 1.78 mM, and the NIR model had an RMSEC of 1.87 mM and an RMSECV of 2.97 mM. Furthermore, Raman models had good robustness when applied in an inline measurement system, but on the contrary NIR spectroscopy was sensitive to the changes in the measurement environment. By utilizing the developed models, inline Raman and NIR spectroscopy were successfully applied for the real-time monitoring of a process-related impurity during the membrane filtration of a recombinant protein. The results enhance the importance of implementing real-time monitoring approaches for the broader field of diagnostic and therapeutic protein purification and underscore its potential to revolutionize the rapid development of biological products.

Effects of omitting titanium dioxide from the film coating of a pharmaceutical tablet - An industrial case study of attempting to comply with EU regulation 2022/63.

Recently, concerns have been raised about the safety of titanium dioxide (TiO2), a commonly used component of pharmaceutical film coatings. The European Union has recently prohibited the application of this material in the food industry, and it is anticipated that the same will happen in the pharmaceutical industry. For this reason, pharmaceutical manufacturers have to consider the possible impact of removing TiO2 from the film coating of tablets. In this paper, we present a case study of a commercially produced tablet where the film coating containing TiO2 was replaced with a coating using calcium carbonate (CaCO3) or with a transparent coating. The performance of the coatings was compared by measuring the moisture absorption rate and the dissolution profile of the tablets. In these regards, there were negligible differences between the coating types. The tablets contained a highly photosensitive drug, the ability of the coatings to protect the drug was evaluated through environmental stability and photostability measurements. The HPLC results showed that the inclusion of TiO2 does not provide additional benefits, when humidity and thermal stress is applied, however its role was vital in protecting the drug from external light. There were several decomposition products which appeared in large quantities when TiO2 was missing from the coating. These results imply that photosensitivity is an issue, replacing TiO2 will be challenging, though its absence can be tolerated when the drug does not need to be protected from light.

Challenges in detecting magnesium stearate distribution in tablets.

Magnesium stearate (MS) is the most commonly used lubricant in pharmaceutical industry. During blending, MS particles form a thin layer on the surfaces of the excipient and drug particles prohibiting the bonding from forming between the particles. This hydrophobic layer decreases the tensile strength of tablets and prevents water from penetrating into the tablet restraining the disintegration and dissolution of the tablets. Although overlubrication of the powder mass during MS blending is a well-known problem, the lubricant distribution in tablets has traditionally been challenging to measure. There is currently no adequate analytical method to investigate this phenomenon. In this study, the distribution of MS in microcrystalline cellulose (MCC) tablets was investigated using three different blending scales. The crushing strength of the tablets was used as a secondary response, as its decrease is known to result from the overlubrication. In addition, coating of the MCC particles by MS in intact tablets was detected using Raman microscopic mapping. MS blending was more efficient in larger scales. Raman imaging was successfully applied to characterize MS distribution in MCC tablets despite low concentration of MS. The Raman method can provide highly valuable visual information about the proceeding of the MS blending process. However, the measuring set-up has to be carefully planned to establish reliable and reproducible results.

Improvement of drug processability in a connected continuous crystallizer system using formulation additive.

Continuous crystallization in the presence of polymer additives is a promising method to omit some drug formulation steps by improving the technological and also pharmacological properties of crystalline active ingredients. Accordingly, this study focuses on developing an additive-assisted continuous crystallization process using polyvinylpyrrolidone in a connected ultrasonicated plug flow crystallizer and an overflow mixed suspension mixed product removal (MSMPR) crystallizer system. We aimed to improve the flowability characteristics of small, columnar primary plug flow crystallizer-produced acetylsalicylic acid crystals as a model drug by promoting their agglomeration in MSMPR crystallizer with polyvinylpyrrolidone. The impact of the cooling antisolvent crystallization process parameters (temperature, polymer amount, total flow rate) on product quality and quantity was investigated. Finally, a spatially segmented antisolvent dosing method was also evaluated. The developed technology enabled the manufacture of purified, constant quality products in a short startup period, even with an 85% yield. We found that a higher polymer amount (7.5-14%) could facilitate agglomeration resulting in "good" flowability without altering the favorable dissolution characteristics of the primary particles.

Interpretable artificial neural networks for retrospective QbD of pharmaceutical tablet manufacturing based on a pilot-scale developmental dataset.

As the pharmaceutical industry increasingly adopts the Pharma 4.0. concept, there is a growing need to effectively predict the product quality based on manufacturing or in-process data. Although artificial neural networks (ANNs) have emerged as powerful tools in data-rich environments, their implementation in pharmaceutical manufacturing is hindered by their black-box nature. In this work, ANNs were developed and interpreted to demonstrate their applicability to increase process understanding by retrospective analysis of developmental or manufacturing data. The in vitro dissolution and hardness of extended-release, directly compressed tablets were predicted from manufacturing and spectroscopic data of pilot-scale development. The ANNs using material attributes and operational parameters provided better results than using NIR or Raman spectra as predictors. ANNs were interpreted by sensitivity analysis, helping to identify the root cause of the batch-to-batch variability, e.g., the variability in particle size, grade, or substitution of the hydroxypropyl methylcellulose excipient. An ANN-based control strategy was also successfully utilized to mitigate the batch-to-batch variability by flexibly operating the tableting process. The presented methodology can be adapted to arbitrary data-rich manufacturing steps from active substance synthesis to formulation to predict the quality from manufacturing or development data and gain process understanding and consistent product quality.

Oligonucleotide Formulations Prepared by High-Speed Electrospinning: Maximizing Loading and Exploring Downstream Processability.

The aim of this study was to develop antisense oligonucleotide tablet formulations using high-speed electrospinning. Hydroxypropyl-beta-cyclodextrin (HPßCD) was used as a stabilizer and as an electrospinning matrix. In order to optimize the morphology of the fibers, electrospinning of various formulations was carried out using water, methanol/water (1:1), and methanol as solvents. The results showed that using methanol could be advantageous due to the lower viscosity threshold for fiber formation enabling higher potential drug loadings by using less excipient. To increase the productivity of electrospinning, high-speed electrospinning technology was utilized and HPßCD fibers containing 9.1% antisense oligonucleotide were prepared at a rate of ~330 g/h. Furthermore, to increase the drug content of the fibers, a formulation with a 50% drug loading was developed. The fibers had excellent grindability but poor flowability. The ground fibrous powder was mixed with excipients to improve its flowability, which enabled the automatic tableting of the mixture by direct compression. The fibrous HPßCD-antisense oligonucleotide formulations showed no sign of physical or chemical degradation over the 1-year stability study, which also shows the suitability of the HPßCD matrix for the formulation of biopharmaceuticals. The obtained results demonstrate possible solutions for the challenges of electrospinning such as scale-up and downstream processing of the fibers.

Development of fast-dissolving dosage forms of curcuminoids by electrospinning for potential tumor therapy application.

Curcuminoids (CUs) of antitumor and various other potential biological activities have extremely low water solubility therefore special formulation was elaborated. New fast dissolving reconstitution dosage forms of four CUs were prepared as fibrous form of 2-hydroxypropyl-ß-cyclodextin (HP-ß-CD). In the electrospinning process HP-ß-CD could act both as solubilizer and fiber-forming agent. The solubilization efficiency of the CU-HP-ß-CD systems was determined with phase-solubility measurements. The electrospun CUs were amorphous and uniformly distributed in the fibers according to XRD analysis and Raman mappings. The fibrous final products had fast (<5 min) and complete dissolution. In typical iv. infusion reconstitution volume (20 mL) fibers containing 40-80 mg of CU could be dissolved, which is similar to the currently proposed dose (<120 mg/m2). The in vitro cytostatic effect data showed that the antitumor activity of the CU-HP-ß-CD complexes was similar or better compared to the free APIs.

Evaluation of Different Thermoanalytical Methods for the Analysis of the Stability of Naproxen-Loaded Amorphous Solid Dispersions.

The aim of this research was to investigate three thermoanalytical techniques from the glass transition temperature (Tg) determination point of view. In addition, the examination of the correlation between the measured Tg values and the stability of the amorphous solid dispersions (ASDs) was also an important part of the work. The results showed that a similar tendency of the Tg can be observed in the case of the applied methods. However, Tg values measured by thermally stimulated depolarization currents showed higher deviation from the theoretical calculations than the values measured by modulated differential scanning calorimetry, referring better to the drug-polymer interactions. Indeed, the investigations after the stress stability tests revealed that micro-thermal analysis can indicate the most sensitive changes in the Tg values, better indicating the instability of the samples. In addition to confirming that the active pharmaceutical ingredient content is a crucial factor in the stability of ASDs containing naproxen and poly(vinylpyrrolidone-co-vinyl acetate), it is worthwhile applying orthogonal techniques to better understand the behavior of ASDs. The development of stable ASDs can be facilitated via mapping the molecular mobilities with suitable thermoanalytical methods.

In-line particle size measurement based on image analysis in a fully continuous granule manufacturing line for rapid process understanding and development.

The present paper serves as a demonstration how an in-line PAT tool can be used for rapid and efficient process development in a fully continuous powder to granule line consisting of an interconnected twin-screw wet granulator, vibrational fluid bed dryer, and a regranulating mill. A new method was investigated for the periodic in-line particle size measurement of high mass flow materials to obtain real-time particle size data of the regranulated product. The system utilises a vibratory feeder with periodically altered feeding intensity in order to temporarily reduce the mass flow of the material passing in front of the camera. This results in the drastic reduction of particle overlapping in the images, making image analysis a viable tool for the in-line particle size measurement of high mass-flow materials. To evaluate the performance of the imaging system, the effect of several milling settings and the liquid-to-solid ratio was investigated on the product's particle size in the span of a few hours. The particle sizes measured with the in-line system were in accordance with the expected trends as well as with the results of the off-line reference particle size measurements. Based on the results, the in-line imaging system can serve as a PAT tool to obtain valuable real-time information for rapid process development or quality assurance.

Real-time amino acid and glucose monitoring system for the automatic control of nutrient feeding in CHO cell culture using Raman spectroscopy.

An innovative, Raman spectroscopy-based monitoring and control system is introduced in this paper for designing dynamic feeding strategies that allow the maintenance of key cellular nutrients at an ideal level in Chinese hamster ovary cell culture. The Partial Least Squares calibration models built for glucose, lactate and 16 (out of 20) individual amino acids had very good predictive power with low root mean square errors values and high square correlation coefficients. The developed models used for real-time measurement of nutrient and by-product concentrations allowed us to gain better insight into the metabolic behavior and nutritional consumption of cells. To establish a more beneficial nutritional environment for the cells, two types of dynamic feeding strategies were used to control the delivery of two-part multi-component feed media according to the prediction of Raman models (glucose or arginine). As a result, instead of high fluctuations, the nutrients (glucose together with amino acids) were maintained at the desired level providing a more balanced environment for the cells. Moreover, the use of amino acid-based feeding control enabled to prevent the excessive nutrient replenishment and was economically beneficial by significantly reducing the amount of supplied feed medium compared to the glucose-based dynamic fed culture.

Powder filling of electrospun material in vials: A proof-of-concept study.

The present paper reports the powder filling of milled electrospun materials in vials, which contained voriconazole and sulfobutylether-ß-cyclodextrin. High-speed electrospinning was used for the production of the fibrous sample, which was divided into 6 parts. Each portion was milled using different milling methods and sizes of sieves to investigate whether the milling influences the powder and filling properties. Bulk and tapped density tests, laser diffraction and angle of repose measurements were applied to characterize the milled powders, while a vibratory feeder was used for the feeding experiments. The correlation between the material property descriptors and the feeding responses was investigated by multivariate data analysis. Based on the results, three samples were chosen for the vial filling, which was accomplished with 3400 mg electrospun material containing 200 mg voriconazole, representative of the commercial product. The feed rate was set to fit the 240 g/h production rate of the electrospinning and the relative standard deviation of three repeated vial filling was determined to see the accuracy of the process. This research shows that by applying a suitable milling method it is possible to process electrospun fibers to a powder, which can be filled into vials and used as reconstitution dosage forms.

[Real-time monitoring of drug crystallization with and without additives]. / Gyógyszerhatóanyag-kristályosodás valós ideju vizsgálata és módosítása segédanyaggal.

Crystallization processes can be evaluated from both kinetic and thermodinamic point of view with real-time analitical methods, effects of production parameters on the final quality can be estimated as well. Consequently there is an increasing emphasis on analytical devices being applicable for real-time detection. Among these techniques Raman spectrometry is advantageously utilizable for real-time monitoring of crystallizations. Impurities can dramatically change the nucleation and crystal growth, thus they can alter the physical and chemical properties of the final product. The use of different additives (polymers;surface active ingredients) in the crystallization step in order to modify the product morphology methodically is a new direction in the scientific literature. This study provides an overview of crystallization processes in the presence of additives as well as a summary concerning the monitoring of the drug crystallizations by real-time Raman spectrometry. Furthermore the effect of polyvinyl-pyrrolidone was examined in the course of cooling crystallization of Donepezil HCl, while the process was monitored by in-line Raman spectrometry.