Mechanistic modeling of empty-full separation in recombinant adeno-associated virus production using anion-exchange membrane chromatography.

Recombinant adeno-associated viral vectors (rAAVs) have become an industry-standard technology in the field of gene therapy, but there are still challenges to be addressed in their biomanufacturing. One of the biggest challenges is the removal of capsid species other than that which contains the gene of interest. In this work, we develop a mechanistic model for the removal of empty capsids-those that contain no genetic material-and enrichment of full rAAV using anion-exchange membrane chromatography. The mechanistic model was calibrated using linear gradient experiments, resulting in good agreement with the experimental data. The model was then applied to optimize the purification process through maximization of yield studying the impact of mobile phase salt concentration and pH, isocratic wash and elution length, flow rate, percent full (purity) requirement, loading density (challenge), and the use of single-step or two-step elution modes. A solution from the optimization with purity of 90% and recovery yield of 84% was selected and successfully validated, as the model could predict the recovery yield with remarkable fidelity and was able to find process conditions that led to significant enrichment. This is, to the best of our knowledge, the first case study of the application of de novo mechanistic modeling for the enrichment of full capsids in rAAV manufacturing, and it serves as demonstration of the potential of mechanistic modeling in rAAV process development.

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode.

Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps and size-based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7->3.4 and 2.5-3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.

Effect of protein fouling on filtrate flux and virus breakthrough behaviors during virus filtration process.

Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log10 ). However, it is still constrained by protein fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.

Integrated continuous downstream process of monoclonal antibody developed by converting the batch platform process based on the process characterization.

A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic-counter current chromatography (PCCC) with two protein A columns was used for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow-through chromatography (FTC) with two connected columns of different separation modes (anion-mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected-column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5-10 L/day) and feed concentrations (1-3.2 g/L) were performed with protein A columns of 1-5 mL and FTC columns of 3-10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank.

Using artificial neural networks to accelerate flowsheet optimization for downstream process development.

An optimal purification process for biopharmaceutical products is important to meet strict safety regulations, and for economic benefits. To find the global optimum, it is desirable to screen the overall design space. Advanced model-based approaches enable to screen a broad range of the design-space, in contrast to traditional statistical or heuristic-based approaches. Though, chromatographic mechanistic modeling (MM), one of the advanced model-based approaches, can be speed-limiting for flowsheet optimization, which evaluates every purification possibility (e.g., type and order of purification techniques, and their operating conditions). Therefore, we propose to use artificial neural networks (ANNs) during global optimization to select the most optimal flowsheets. So, the number of flowsheets for final local optimization is reduced and consequently the overall optimization time. Employing ANNs during global optimization proved to reduce the number of flowsheets from 15 to only 3. From these three, one flowsheet was optimized locally and similar final results were found when using the global outcome of either the ANN or MM as starting condition. Moreover, the overall flowsheet optimization time was reduced by 50% when using ANNs during global optimization. This approach accelerates the early purification process design; moreover, it is generic, flexible, and regardless of sample material's type.

Production of polyhydroxyalkanoates from renewable resources: a review on prospects, challenges and applications.

Bioplastics replace synthetic plastics of petrochemical origin, which contributes challenge to both polymer quality and economics. Novel polyhydroxyalkanoates (PHA)-composite materials, with desirable product quality, could be developed, thus targeting the global plastics market, in the coming years. It is possible that PHA can be a greener substitute for their petroleum-based competitors since they are simply decomposed, which may lessen the pressure on municipal and industrial waste management systems. PHA production has proven to be the bottleneck in industrial application and commercialization because of the high price of carbon substrates and downstream processes required to achieve reliability. Bacterial PHA production by these municipal and industrial wastes, which act as a cheap, renewable carbon substrate, eliminates waste management hassles and acts as an efficient substitute for synthetic plastics. In the present review, challenges and opportunities related to the commercialization of polyhydroxyalkanoates are discussed and presented. Moreover, it discusses critical steps of their production process, feedstock evaluation, optimization strategies, and downstream processes. This information may provide us the complete utilization of bacterial PHA during possible applications in packaging, nutrition, medicine, and pharmaceuticals.

Large-scale crystallization as an intermediate processing step in insulin downstream process: explored advantages and identified tool for process intensification.

The rising global prevalence of diabetes and increasing demand for insulin, calls for an increase in accessibility and affordability of insulin drugs through efficient and cost-effective manufacturing processes. Often downstream operations become manufacturing bottlenecks while processing a high volume of product. Thus, process integration and intensification play an important role in reducing process steps and time, volume reduction, and lower equipment footprints, which brings additional process efficiencies and lowers the production cost. Manufacturers thrive to optimize existing unit operation to maximize its benefit replacing with simple but different efficient technologies. In this manuscript, the typical property of insulin in forming the pH-dependent zinc-insulin complex is explored. The benefit of zinc chloride precipitation/crystallization has been shown to increase the in-process product purity by reducing the product and process-related impurities. Incorporation of such unit operation in the insulin process has also a clear potential for replacing the high cost involved capture chromatography step. Same time, the reduction in volume of operation, buffer consumption, equipment footprint, and capabilities of product long time storage brings manufacturing flexibility and efficiencies. The data and capabilities of simple operation captured here would be significantly helpful for insulins and other biosimilar manufacturer to make progresses on cost-effective productions.

Sugaring-out extraction of erythromycin from fermentation broth.

This study reports the sugaring-out extraction of erythromycin from fermentation broth using acetonitrile (ACN) as solvent and glucose as a mass separating agent. Different process parameters-glucose concentration, temperature, ACN/water ratio and pH-were optimized to achieve maximum extraction of erythromycin. 88% (w/w) of erythromycin was extracted from the model system with following optimized conditions: glucose 156.3 g/L; temperature 4 °C; ACN/water ratio 1 and pH 8.3. Further, the effect of typical fermentation media components (starch, soybean flour, CaCO3, NaCl and (NH4)2SO4) on sugaring out extraction of erythromycin was also investigated. Starch, soybean flour and CaCO3 were observed to affect erythromycin extraction only at higher concentration. Removal of suspended solids from simulated as well as real broth prior to extraction enhanced the extraction efficiency (from 72% to 87%). Sugaring out extraction of erythromycin was found to be more effective than salting out extraction. Also, higher partition coefficient was achieved in the present work than other reported methods using carbohydrates as mass separating agent. Further, it was found that the antimicrobial activity of erythromycin was preserved during sugaring out extraction of erythromycin.

The impact of technical failures during cultivation of an inclusion body process.

In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical impact of technical failures to understand and mitigate their impact accurately and reduce economic damage. We investigated the impact in the upstream and downstream of a recombinant antibody fragment inclusion body production process chain to provide integrated empirical data and knowledge. First, we provided a reproducible process chain that yielded high inclusion body content, high specific product titer, and a refolding yield of 30%. The inclusion body downstream proved to be of high reproducibility. Through the intended introduction of technical failures, we were not only able to shed more light on the empirical responses in the upstream and downstream, but also on process-boosting parameters that would have been neglected. Herein, a short increase in temperature during the cultivation clearly increased the refolding yield.

New approaches for itaconic acid production: bottlenecks and possible remedies.

Currently, growing attention is being devoted to the conversion of biomass into value-added products, such as itaconic acid (IA), which is considered as the cleanest alternative to petroleum-based acrylic acid. IA is an unsaturated dicarboxylic acid that is used as a building block chemical for the production of several value-added products such as poly-itaconic acid. IA and its derivatives have a wide range of potential applications in textile, paint, pharmaceutical and chemical industries. Presently, industries are producing IA on the large scale by fermentation from glucose. However, due to the primary utility of glucose as a food, it cannot meet the global demand for IA production in an economical way. The main challenge, so far, has been the production technology, which does not support cost-effective and competitive production of IA. This review discusses the various bottlenecks faced during each step of IA production, along with possible remedies to deal with these problems. Furthermore, it reviews the recent progress in fermentative IA production and sheds light on different microorganisms used, potential substrates and fermentation conditions. The review also covers market potential for IA, which indicates that IA can be produced cost-effectively from sustainable substrates, and it has the potential to replace petrochemicals in the near future.

Microbial ribonucleases (RNases): production and application potential.

Ribonuclease (RNase) is hydrolytic enzyme that catalyzes the cleavage of phosphodiester bonds in RNA. RNases play an important role in the metabolism of cellular RNAs, such as mRNA and rRNA or tRNA maturation. Besides their cellular roles, RNases possess biological activity, cell stimulating properties, cytotoxicity and genotoxicity. Cytotoxic effect of particular microbial RNases was comparable to that of animal derived counterparts. In this respect, microbial RNases have a therapeutic potential as anti-tumor drugs. The significant development of DNA vaccines and the progress of gene therapy trials increased the need for RNases in downstream processes. In addition, RNases are used in different fields, such as food industry for single cell protein preparations, and in some molecular biological studies for the synthesis of specific nucleotides, identifying RNA metabolism and the relationship between protein structure and function. In some cases, the use of bovine or other animal-derived RNases have increased the difficulties due to the safety and regulatory issues. Microbial RNases have promising potential mainly for pharmaceutical purposes as well as downstream processing. Therefore, an effort has been given to determination of optimum fermentation conditions to maximize RNase production from different bacterial and fungal producers. Also immobilization or strain development experiments have been carried out.

Continuous and autonomous-flow separation of laccase enzyme utilizing functionalized aqueous two-phase system with computer vision control.

Downstream processing of biomolecules, particularly therapeutic proteins and enzymes, presents a formidable challenge due to intricate unit operations and high costs. This study introduces a novel cysteine (cys) functionalized aqueous two-phase system (ATPS) utilizing polyethylene glycol (PEG) and potassium phosphate, referred as PEG-K3PO4/cys, for selective extraction of laccase from complex protein mixtures. A 3D-baffle micro-mixer and phase separator was meticulously designed and equipped with computer vision controller, to enable precise mixing and continuous phase separation under automated-flow. Microfluidic-assisted ATPS exhibits substantial increase in partition coefficient (Kflow = 16.3) and extraction efficiency (EEflow = 88 %) for laccase compared to conventional batch process. Integrated and continuous-flow process efficiently partitioned laccase, even in low concentrations and complex crude extracts. Circular dichroism spectra of laccase confirm structural stability of enzyme throughout the purification process. Eventually, continuous-flow microfluidic bioseparation is highly useful for seamless downstream processing of target biopharmaceuticals in integrated and autonomous manner.

Physicochemical cell disruption of Bacillus sp. for recovery of polyhydroxyalkanoates: future bioplastic for sustainability.

Polyhydroxybutyrate (PHB) is known for wide applications, biocompatibility, and degradability; however, it cannot be commercialized due to conventional recovery using solvents. The present study employed mechanical cell-disruption methods, such as Pestle and mortar, sonication, and glass bead vortexing, for solvent-free extraction of PHA from Bacillus sp. Different time intervals were set for grinding (5, 10, 15 min), sonicating (1, 3 and 5 min), and vortexing (2, 5 and 8 g glass beads with 5, 10 and 15 min each) hence studying their effect on cell lysis to release PHA. Tris buffer containing phenylmethyl sulfonyl fluoride (PMSF) (20 mM Tris-HCl, pH 8.0, 1 mM PMSF) was employed as a lysis buffer to study its action over Bacillus cells. Its presence was checked with the above methods in cell lysis. Sonicating cells for 5 min in the presence of lysis buffer achieved a maximum PHA yield of 45%. Cell lysis using lysis buffer yielded 35% PHA when vortexing with 5 g glass beads for 15 min. Grinding cells for 15 min showed a maximum yield of 34% but lacked a lysis buffer. The overall results indicated that the action of lysis buffer and physical extraction methods improved PHA yield by %. Therefore, the study sought to evaluate the feasibility of applying laboratory methods for cell disruption. These methods can showcase possible opportunities in large-scale applications. The polymer yield results were compared with standard sodium hypochlorite extraction. Confirmation of obtained polymers as polyhydroxy butyrate (PHB) was made through FTIR and 1HNMR characterization.

The outlooks and key challenges in renewable biomass feedstock utilization for value-added platform chemical via bioprocesses.

The conversion of renewable biomass feedstock into value-added products via bioprocessing platforms has become attractive because of environmental and health concerns. Process performance and cost competitiveness are major factors in the bioprocess design to produce desirable products from biomass feedstock. Proper pretreatment allows delignification and hemicellulose removal from the liquid fraction, allowing cellulose to be readily hydrolyzed to monomeric sugars. Several industrial products are produced via sugar fermentation using either naturally isolated or genetically modified microbes. Microbial platforms play an important role in the synthesis of several products, including drop-in chemicals, as-in products, and novel compounds. The key elements in developing a fermentation platform are medium formulation, sterilization, and active cells for inoculation. Downstream bioproduct recovery may seem like a straightforward chemical process, but is more complex, wherein cost competitiveness versus recovery performance becomes a challenge. This review summarizes the prospects for utilizing renewable biomass for bioprocessing.

Host cell proteins in monoclonal antibody processing: Control, detection, and removal.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Evaluation of the Robustness Verification of Downstream Production Process for Inactivated SARS-CoV-2 Vaccine and Different Chromatography Medium Purification Effects.

BACKGROUND: Large-scale vaccine production requires downstream processing that focuses on robustness, efficiency, and cost-effectiveness. METHODS: To assess the robustness of the current vaccine production process, three batches of COVID-19 Omicron BA.1 strain hydrolytic concentrated solutions were selected. Four gel filtration chromatography media (Chromstar 6FF, Singarose FF, Bestarose 6B, and Focurose 6FF) and four ion exchange chromatography media (Maxtar Q, Q Singarose, Diamond Q, and Q Focurose) were used to evaluate their impact on vaccine purification. The quality of the vaccine was assessed by analyzing total protein content, antigen content, residual Vero cell DNA, residual Vero cell protein, and residual bovine serum albumin (BSA). Antigen recovery rate and specific activity were also calculated. Statistical analysis was conducted to evaluate process robustness and the purification effects of the chromatography media. RESULTS: The statistical analysis revealed no significant differences in antigen recovery (p = 0.10), Vero HCP residue (p = 0.59), Vero DNA residue (p = 0.28), and BSA residue (p = 0.97) among the three batches of hydrolytic concentrated solutions processed according to the current method. However, a significant difference (p < 0.001) was observed in antigen content. CONCLUSIONS: The study demonstrated the remarkable robustness of the current downstream process for producing WIBP-CorV vaccines. This process can adapt to different batches of hydrolytic concentrated solutions and various chromatography media. The research is crucial for the production of inactivated SARS-CoV-2 vaccines and provides a potential template for purifying other viruses.

The challenge of downstream processing of spray dried amorphous solid dispersions into minitablets designed for the paediatric population - A sustainable product development approach.

Poorly water-soluble drugs present a significant challenge in the development of oral solid dosage forms (OSDs). In formulation development the appropriate use of excipients to adjust solubility, and the choice of manufacturing method and pharmaceutical processes to obtain a dosage form to meet the needs of the patient group, is crucial. Preparing an amorphous solid dispersion (ASD) is a well-established method for solubility enhancement, and spray drying (SD) a common manufacturing method. However, the poor flowability of spray dried materials poses a significant challenge for downstream processing. Promoting sustainability in OSD development involves embracing a versatile formulation design, which enables a broader spectrum of patients to use the product, as opposed to altering existing dosage forms retrospectively. The objective of the current study was to develop a formulation of spray dried indomethacin ASD suited to the production, by direct compression, of instant release paediatric minitablets. Excipients evaluated were PVP or HPMCAS in solid dispersions at the preformulation phase, and MCC and lactose as a filler in direct compression. From the studied formulations, a 3:1 ratio blend of Vivapur 200/Pharmatose 200 M (MCC/lactose) with 0.5% (w/w) magnesium stearate was found to be the most promising in tableting, and minitablets containing a 6.22% content of spray-dried ASD of indomethacin/PVP K 29-32 could be obtained with desired tablet hardness and pharmaceutical quality, complying with tests of weight variation and fast disintegration in an aqueous environment. As a case example, this study provides a good foundation for further studies in harnessing a sustainable approach to the development of pharmaceutical formulations that can appropriately serve different patient sub-populations.

Proteomics-based method to comprehensively model the removal of host cell protein impurities.

Mechanistic models mostly focus on the target protein and some selected process- or product-related impurities. For a better process understanding, however, it is advantageous to describe also reoccurring host cell protein impurities. Within the purification of biopharmaceuticals, the binding of host cell proteins to a chromatographic resin is far from being described comprehensively. For a broader coverage of the binding characteristics, large-scale proteomic data and systems level knowledge on protein interactions are key. However, a method for determining binding parameters of the entire host cell proteome to selected chromatography resins is still lacking. In this work, we have developed a method to determine binding parameters of all detected individual host cell proteins in an Escherichia coli harvest sample from large-scale proteomics experiments. The developed method was demonstrated to model abundant and problematic proteins, which are crucial impurities to be removed. For these 15 proteins covering varying concentration ranges, the model predicts the independently measured retention time during the validation gradient well. Finally, we optimized the anion exchange chromatography capture step in silico using the determined isotherm parameters of the persistent host cell protein contaminants. From these results, strategies can be developed to separate abundant and problematic impurities from the target antigen.

Combining eutectic solvents and food-grade silica to recover and stabilize anthocyanins from grape pomace.

Concentrated in the skins of red grapes are the anthocyanins, the primary colorants responsible for the fruits' reddish-purple color. These colorants are recognized for their significant antioxidant properties and potent nutraceutical and pharmaceutical ingredients. Nevertheless, their widespread use is compromised by the (i) need for more efficient yet sustainable downstream processes for their recovery and (ii) by the challenges imposed by their poor stability. In this work, these drawbacks were overcome by applying eutectic solvents and stabilizing agents. Besides, the anthocyanins were successfully loaded into a solid host material (approved in both food and pharmaceutical sectors) based on silicon dioxide (SiO2, loading capacity: 1extract:7silica m/m). Summing up, with the process developed, the extraction yield (21 mganthocyanins.gbiomass-1) and the stability (under 55, 75, and 95 °C) of the recovered anthocyanins were over three times better than with the conventional process. Finally, the raw materials and solvents were recycled, allowing an economical and environmentally friendly downstream process.

Understanding adsorption behavior of antiviral labyrinthopeptin peptides in anion exchange chromatography.

Lantipeptides from bacterial sources are increasingly important as biopharmaceuticals because of their broad range of applications. However, the availability of most lantipeptides is low, and systematic approaches for downstream processing of this group of peptides is still lacking. Model-based development for chromatographic separations has proven to be a useful tool for developing reliable purification processes. One important compound of such a model is the adsorption behavior of the components of interest. In ion-exchange chromatography, the adsorption equilibrium between salt and proteins can be described using the steric mass action (SMA) formalism. Beyond, the model parameters may be related to the lanthipeptides physico-chemical properties. In this study, the antiviral lantipeptides labyrinthopeptin A1 and A2, purified from Actinomadura namibiensis culture broth, were characterized for their adsorption behavior in anion-exchange chromatography in the range from pH 5.0-7.4. The experiments necessary to determine the three SMA parameters were chosen in a way to limit the amount of peptides needed. Linear gradient elution was applied successfully to separate A1 and A2 and to determine the characteristic charge νi and the equilibrium constant [Formula: see text] . Batch adsorption experiments using a robotic workstation for high throughput and accuracy provided non-linear adsorption isotherms and the steric factor σi. Labyrinthopeptin A1 and A2 show a very different adsorption behavior even though the fundamental structure of the two peptides is similar. keq of A1 ranging from 0.18 to 0.88 are approximately one order of magnitude smaller than that of A2 ranging from 3.44 to 9.73 indicating the higher affinity of A2 to the stationary phase. At pH 7.0 σ was 1.12 and 0.60 for A1 and A2, respectively which was expected based on the molecular weight of the peptides. The characteristic charge for both peptides was also theoretically estimated from the amino acids involved in electrostatic interactions which was in good agreement with experimental data. Thereby, this work provides an useful approach to estimate SMA parameters based on simple structural information that can be applied early in chromatographic ion-exchange process development for peptides and may help adapting the processes for future designed lanthipeptides.