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Evaluation of the performance of a pilot-scale membrane system to separate and concentrate arabinoxylans, hydroxycinnamic acids and fermentable sugars from nejayote. Total suspended solids (TSS) from nejayote were removed by a novel enzymatic process to avoid UF-membrane fouling (Asaff and Reyes 2015; modified to pilot-scale). The free-TSS nejayote was submitted to ultrafiltration and diafiltration processes (5 kDa MWCO polyethersulfone membrane) to separate and concentrate arabinoxylans. Reverse osmosis (cross-linked polyamide membrane) was performed to concentrate hydroxycinnamic acids and fermentable sugars (MW

Virus-like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo-assembled VLPs are typically dis- and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high-pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material-consuming disassembly experiments. To this end, we developed a low-volume and high-resolution disassembly screening that provides time-resolved insight into the VLP disassembly progress. In this study, two variants of C-terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high-throughput screening results, a diafiltration-based disassembly process step was developed. Compared with mixing-based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration-based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high-molecular-weight species.

The object of research is the secondary material resources of processing plant and animal raw materials, namely fruit pomace and milk whey. One of the most problematic areas is that the waste of these industries has high rates of biological and chemical oxygen consumption of wastewater, which significantly impairs the operation of local and city treatment facilities. In addition, the parameters of extraction and determination of the quality indicators of the obtained product are not well defined. The processing of whey and pomace of fruit crops can reduce the environmental burden on the environment and increase the efficiency of technological processes through resource conservation and obtaining a surplus product. The problem is solved, in particular, by using the process of extracting plant waste using milk whey and the process of energy-saving membrane concentration. In the course of the study, let’s use pectin-containing plant waste from juice production, namely watermelon, pumpkin, quince, beetroot, apple and a mixture of orange and tangerine pomace. The results obtained indicate that the process of extracting apple pomace with milk whey is promising, since the highest pectin content in the extract is established for apple pomace. The main amount of pectin substances passes into the extract starting from 75 minutes to 90 minutes. Extraction-hydrolysis for 2 hours at a temperature of 85°C, pH=2–2.5 units determines the best results. Ultrafiltration of the obtained extract makes it possible to increase the pectin content up to 3.0% with a protein content of more than 6.0%. The membranes used in the experiment are characterized by high selectivity for protein and pectin. The fact is established that diafiltration makes it possible to effectively purify whey-pectin concentrates from ballast impurities with simultaneous enrichment of the concentrate with high-molecular components of whey. A technological scheme for the production of pectin-whey concentrate with high organoleptic and detoxification characteristics is proposed. The proposed technological process has a number of positive features, in particular, a high content of pectin substances, high value indicators, a significant reduction in the burden on the environment, economic benefits through energy conservation and obtaining a surplus product.

Background: This study assesses the feasibility of producing hyperimmune anti-COVID-19 intravenously administrable immunoglobulin (C-IVIG) from pooled convalescent plasma (PCP) to provide a safe and effective passive immunization treatment option for COVID-19. Materials & methods: PCP was fractionated by modified caprylic acid precipitation followed by ultrafiltration/diafiltration to produce hyperimmune C-IVIG. Results: In C-IVIG, the mean SARS-CoV-2 antibody level was found to be threefold (104 ± 30 cut-off index) that of the PCP (36 ± 8.5 cut-off index) and mean protein concentration was found to be 46 ± 3.7 g/l, comprised of 89.5% immunoglobulins. Conclusion: The current method of producing C-IVIG is feasible as it uses locally available PCP and simpler technology and yields a high titer of SARS-CoV-2 antibody. The safety and efficacy of C-IVIG will be evaluated in a registered clinical trial (NCT 04521309).

In this study, milk protein isolate (MPI) and micellar casein concentrate (MCC) were obtained with ultrafiltration (UF) and microfiltration (MF), respectively. The objective of this work was to evaluate the effect of diafiltration (DF), the addition of water during filtration, on the colloidal dissociation of casein micelles. Two levels of DF were applied after an initial concentration step to 2x the volume fraction, at a total protein concentration of about 7% (w/v). Under these conditions, while permeation of protein was still measured in the permeates, calcium ion transmission reached a plateau. The molecular details of the serum phases were studied throughout the filtration process. A comparison of serum phases with and without whey protein (UF vs MF) was carried out by examining the two processes in parallel, applying similar filtration conditions. Final 4x concentrates (based on initial volume of milk) were also compared. Viscosity was higher for UF than the corresponding MF concentrates, is spite of the higher level of colloidal protein present in the MF concentrates, which would suggest a change in the hydration or the volume/weight ratio (voluminosity) of casein micelles in MCC retentates. Centrifugal supernatants contained casein proteins both in MPC and MCC retentates. There were no statistically significant changes in the apparent hydrodynamic diameter of the casein micelles when measured by dynamic light scattering, in the original milk serum. A two dimensional chromatography was carried out on the soluble aggregates to determine differences in the amount of individual caseins. The aggregates showed only traces of whey proteins. The composition of the final concentrates, albeit similar in total proteins, showed profound differences in ionic strength and soluble proteins, suggesting that these parameters may be used as an indication of ingredient quality and functionality.

The field of extracellular vesicles (EVs) is relatively new and the methods for EV isolation and quantification are still maturing. For example, there is no consensus on how to separate free stain from labelled EVs. Here we report a comparison of the recovery of labelled EVs following separation from free stain using ultracentrifugation, diafiltration with different devices and a charged size exclusion chromatography column. Of the methods we tested, the charged size exclusion column provided the greatest recovery of labelled EVs.

The Bio separation is stringent to the area of engineering for biochemistry. All bio separation operation should be suits to detach, purifying, or regain the coveted product. The bio separation depends on, essential property involved thermo constancy, solubility, diffusion, shipment, isoelectric pH, and other things. The purifying of biological products is get by a bio separations operations include a lengthy series of procedures, and all procedure needs the employ single or more unity operations, like filtrate, extraction, chromatography and desiccation. The Filtering isolate a components depending to volume while a liquid solute flux during a pored midst down a compressing differential. Super filtration of a protein solute at fixed size is completed with the addendum of H2O or buffer to the feed in an process named diafiltration. Fluid chromatography is a form of absorption operation, which is, depends on the affinity of different solution for specified form of solid. The generality efficiency chromatography process employ each the adsorption sites ready. While chromatography to purifying a protein is scaled up from the laboratory level, knowing of the laboratory elution profile is useful in interpreting the great level elution profile.

The high protein content of Arthrospira platensis makes this cyanobacterium a great component of food. Thus, the protein obtained should guarantee the nutrient characteristic, in this case, as bioactivity and techno-functional properties. Membrane separation processes (MSPs) have been highlighted by their capacity to separate proteins and keep their properties. Therefore, MSP was used to concentrate and separate the proteins from A. platensis, which were characterized by their physicochemical and techno-functional properties. Different ultrafiltration (UF) membranes and operational conditions (10- and 50-kDa pore size, conventional and tangential flow, hollow fiber type and pressures of 1.0, 1.5, and 2.0 bar) were tested and associated with diafiltration (DF). The maximized condition of UF/DF (hollow fiber, 50 kDa, tangential flow at 1.5 bar, associated with one DF) was up scaled (pilot scale) and allowed concentrate and permeate fractions with 81.75% and 61.20% of protein, respectively, to be obtained. A protein concentrate with 142 mg L−1 of phycocyanin, 90% of antioxidant activity, with proteins more soluble at high pH (50% soluble protein at pH 7.0) and molecular weight among 5–15 and 100 kDa, was obtained. Protein thermal and emulsifying properties were maintained by the MSP, and the concentrate fraction presented results similar as A. platensis biomass, which confirms the advantage of this method in guaranteeing the protein quality.

A membrane process for the recovery of ≥ 90 % of the soluble peptides (with antioxidant activity) found in concentrated lecithin-free egg yolk hydrolysates (LFEYH) was established. Hydrolysis of concentrated free lecithin egg yolk protein dispersions (10 and 20 %) was conducted with Alcalase 2.4 L, an endoprotease from Bacillus Licheniformis. The obtained hydrolysates were fractionated by ultrafiltration (UF), where the effect of the membrane material and molecular weight cut-off (MWCO) was studied. Two flat sheet regenerated cellulose membranes and three tubular ceramic membranes with different MWCOs (50, 10 and 5 kDa) were tested. Different hydrodynamic operating conditions were established in all membranes to work under sustainable flux during UF. The 50 kDa ceramic membrane was found to be the most efficient in terms of flux (78.45 and 47.84 kg m−2 h-1 for 10 and 20 % protein dispersions, respectively) and showed lower fouling. The use of diafiltration (DF), allowed recovering more than 90 % of soluble and heat-stable peptides, which also showed high antioxidant activity, from hydrolysates produced in both concentrated (10 and 20 %) egg yolk protein dispersions.

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

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O(2) affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to haptoglobin and unimoleular O(2) offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

Using membrane filtration it is possible to selectively concentrate proteins and, in the case of microfiltration, concentrate casein micelles. During filtration, water is often added and this practice, called diafiltration, causes further release of permeable components and maintains filtration efficiency. Filtration causes changes in composition of the protein as well as the soluble phase, including soluble calcium, which is a critical factor controlling the gelation properties of the casein micelles in milk. It was hypothesized that concentrates obtained using membrane filtration with or without diafiltration would have different gelation behavior. To test this hypothesis, two concentrates of similar casein micelle volume fraction were prepared, using spiral wound polymeric microfiltration membranes with a 800 kDa molecular weight cutoff, with or without diafiltration. The concentrates showed a gelation behavior comparable to that of skim milk, with a similar gelation time and with a higher firmness, due to the higher number of protein linkages in the network. In contrast, the hydrolysis of κ-casein by chymosin and casein aggregation were inhibited in diafiltered casein micelle suspensions. When the concentrates were recombined with the original skim milk to a final concentration of 5% protein, which re-established a similar soluble phase composition, differences in gelation behavior were no longer observed: both treatments showed similar gelation time and gel firmness. These results confirmed that membrane filtration can result in concentrates with different functionality, and that ionic environmental conditions are critical to the aggregation behavior of casein micelles. This is of particular significance in industrial settings where these fractions are used as a way to standardize proteins in cheese making.

Introduction: In continuous renal replacement therapy, conduction and convection are controlled allowing prescribing dosage regimen improving survival. In contrast, adsorption is an uncontrolled property altering drug disposition. Whether adsorption depends on flowrates is unknown. We hypothesized an in vitro model may provide information in conditions mimicking continuous renal replacement therapy in humans. Methods: ST150®-AN69 filter and Prismaflex dialyzer, Baxter-Gambro were used. Simulated blood flowrate was set at 200 mL/min. The flowrates in the filtration (continuous filtration), dialysis (continuous dialysis), and diafiltration (continuous diafiltration) were 1500, 2500, and 4000 mL/h, respectively. Routes of elimination were assessed using NeckEpur® analysis. Results: The percentages of the total amount eliminated by continuous filtration, continuous dialysis, and continuous diafiltration were 82%, 86%, and 94%, respectively. Elimination by effluents and adsorption accounted for 42% ± 7% and 58% ± 5%, 57% ± 7% and 43% ± 6%, and 84% ± 6% and 16% ± 6% of amikacin elimination, respectively. There was a linear regression between flowrates and amikacin clearance: Y = 0.6 X ± 1.7 (R2 = 0.9782). Conversely, there was a linear inverse correlation between the magnitude of amikacin adsorption and flowrate: Y = –16.9 X ± 84.1 (R2 = 0.9976). Conclusion: Low flowrates resulted in predominant elimination by adsorption, accounting for 58% of the elimination of amikacin from the central compartment in the continuous filtration mode at 1500 mL/h of flowrate. Thereafter, the greater the flowrate, the lower the adsorption of amikacin in a linear manner. Flowrate is a major determinant of adsorption of amikacin. There was an about 17% decrease in the rate of adsorption per increase in the flowrate of 1 L/min.

Lignin is a renewable and abundant source for production of bio-based chemicals and is a valuable alternative to crude oil to obtain aromatic building blocks. It is built from aromatic units with strong chemical linkages, which need to be cleaved to enable the use of the aromatic compounds in industrial applications. In addition to depolymerizing lignin, efficient fractionation and conversion of the resulting complex mixtures is an essential step in the valorization of lignin derivatives for different applications. In this work, we studied the separation of a lignin oil obtained by catalytic cleavage in supercritical ethanol (scEtOH) of technical lignin produced by means of soda pulping of wheat straw. The use of six commercial polymeric nanofiltration (NF) membranes and one in-house developed Grignard-functionalized ceramic membrane was investigated for the fractionation of a mixture of lignin derivatives. Separation by molecular weight (MW) was observed with the polymeric NP030 membrane but not with the other membranes tested. The development of a protocol using this membrane in the diafiltration mode for fractionation of crude lignin oils (CLOs) is discussed.

Ultrafiltration/diafiltration (UF/DF) plays an important role in the manufacturing of biopharmaceuticals. Monitoring critical process parameters and quality attributes by process analytical technology (PAT) during those steps can facilitate process development and assure consistent quality in production processes. In this study, a lab-scale cross-flow filtration (CFF) device was equipped with a variable pathlength (VP) ultraviolet and visible (UV/Vis) spectrometer, a light scattering photometer, and a liquid density sensor (microLDS). Based on the measured signals, the protein concentration, buffer exchange, apparent molecular weight, and hydrodynamic radius were monitored. The setup was tested in three case studies. First, lysozyme was used in an UF/DF run to show the comparability of on-line and off-line measurements. The corresponding correlation coefficients exceeded 0.97. Next, urea-induced changes in protein size of glucose oxidase (GOx) were monitored during two DF steps. Here, correlation coefficients were ≥ 0.92 for static light scattering (SLS) and dynamic light scattering (DLS). The correlation coefficient for the protein concentration was 0.82, possibly due to time-dependent protein precipitation. Finally, a case study was conducted with a monoclonal antibody (mAb) to show the full potential of this setup. Again, off-line and on-line measurements were in good agreement with all correlation coefficients exceeding 0.92. The protein concentration could be monitored in-line in a large range from 3 to 120 g L− 1. A buffer-dependent increase in apparent molecular weight of the mAb was observed during DF, providing interesting supplemental information for process development and stability assessment. In summary, the developed setup provides a powerful testing system for evaluating different UF/DF processes and may be a good starting point to develop process control strategies.

The main goal of this study was to select micro- and ultrafiltration membranes that can be used for the purification of mushroom tyrosinase, replacing salting-out dual-step processes followed by centrifugations. In experiments, a raw extract from white mushrooms was used with high level of ballast proteins and brownish impurities. Four microfiltration membranes for the removal of undesired high molecular weight compounds were screened and that made of nitrocellulose was selected due to high recovery of enzymatic activity. Then diafiltration and concentration on the membrane made of polyethersulphone (300 kDa) was selected to recover 8% of proteins and 58% of tyrosinase activity with five- to seven purification fold, 10% of proteases, and 8% of brown impurities. It was shown that tyrosinase can be pre-purified by selected membranes yielding the enzyme quality at least comparable to that after double salting-out method but in one device. In both cases, subsequent purification by ion-exchange chromatography slightly increased purification degree of the enzyme and brown impurity removal. The surplus of membrane pre-purification is substantially higher thermal stability of the enzyme, enlarged after the chromatographic step, due to very low content of proteolytic enzymes.

Phycocyanin (PC), a natural blue pigment found in the cyanobacterium Arthrospira platensis, has anticancer, anti-inflammatory, and antioxidant properties. The purity of PC is the key factor for its application in the food, pharmaceutical and medical industries; however, the purification processes needed to achieve high purity can impact its bioactivity. This study evaluates whether microfiltration (MF) or ultrafiltration (UF) can be used to produce PC with food-grade purity. MF and UF following freeze-thaw extraction of PC from A. platensis biomass resulted in a low product purity, with other cellular constituents forming a cake on the membrane surface during the filtration process and preventing the concentration of PC. Phosphate buffer extraction resulted in more PC (65.5 mg g−1) with purity of 0.53. When the UF was followed by one step of diafiltration, purity was further enhanced to 0.76. However, the purification resulted in a 30% loss of antioxidant activity. This study demonstrates that phosphate buffer extraction followed by UF and one step of DF is suitable to produce food-grade PC from A. platensis, with the advantage of only requiring a single piece of equipment to perform the purification.

Efficient separation of serum from colloidal proteins in bovine milk in their native form can be achieved by microfiltration (MF). This study assessed partitioning of serum proteins (SP) in skim milk by a MF process using 0.1 μm graded permeability ceramic membranes with two diafiltration (DF) steps, from a mass balance and energy utilisation perspective. A mass balance focused on dry matter and true protein yielded recoveries of 99.5 and 95.3%, respectively. However, an accurate mass balance relative to colloidal and SP contents in the retentate and permeate streams was not achieved, linked to errors surrounding quantification of nitrogen fractions in processed streams using standard methodologies designed for raw milk. Additionally the energy required for each MF/DF step was 13.1, 13.7 and 20.6 kW h kg−1 of SP removed, respectively, demonstrating the dynamic relationship between SP partition relative to diafiltrant utilisation and its impact on energy consumption.

Membrane ultra- and microfiltration is a chemical unit operation used to purify and concentrate liquid suspensions of colloids and particles. Typical industrial applications are removing or concentrating colloids, proteins, or cells in water treatment, food processing, and bioprocessing. The filtration performance suffers from the agglomeration of the filtrated matter on the membrane surface as a filter cake. This filter cake adds hydrodynamic resistance on top of the membrane's resistance and decreases the process's overall efficiency. Traditional applications measure the resistances for a specific filtrate at the point of operation and adapt the process design and the operating conditions accordingly. This thesis aims to provide mechanistic insights into the filtration process by developing experimental methods for microscopic filter cake visualization and applying these methods to analyze colloid and particle interactions in the filter cake during filtration. Finally, the thesis presents a novel ultrafiltration-based lab-scale microgel purification process and validates the process compared to state-of-the-art technologies. This thesis developed two devices for visually observing filter cakes: The first is a microfluidic system that withholds the filtrated particles at a membrane-mimicking structure and enables a visual observation of the filter cake's cross-section. Filtering soft spherical polyethyleneglycol particles provoked avalanche-like compaction of the filter cake during cake built up. The filtration of additive manufactured non-isotropic any-shape particles showed the dependency of the hydraulic resistance and the cake morphology from the particle shape and material. These microscopic particle scale results give essential insights into the particle interactions occurring in filter cakes and permeated assemblies. The second observation device developed and applied in this thesis is a flat sheet membrane filtration cell that visually observes the filter cake using Confocal Laser Scanning Microscopy. During cross-flow cleaning procedures, microgel filter cakes unravel instability-driven 3D patterns on the cake surface, accelerating cake removal. Finally, the microscopic filtration results are supplemented with developing a tangential flow microgel lab-scale ultrafiltration device. The thesis validates the applicability of the process for purifying microgels and reports operational procedures, process quantification, and the purification's challenges compared to state-of-the-art technologies. This thesis's findings provide one step towards bridging the gap between microscopic particle interactions and filtration cake layer properties. The presented results need to be incorporated into filtration models to translate the fundamental phenomena into applicable separation processes in future works. Finally, the findings are not only relevant for filter cakes, but additionally give insights in various applications of permeated micro-particle assemblies. The properties of self-assembling microparticle scaffolds, e.g., as tissue in cell culture, depend strongly on the particle-particle interactions, the assembly's morphology, and its hydraulic resistance. This interdisciplinary perspective will transfer the methods presented here to drive future developments.

In the framework of modern bioprocessing continuous ultrafiltration/diafiltration (UF/DF) is getting increasingly popular. However, while continuous UF can be easily implemented using a so-called single pass tangential flow filtration (SPTFF) module, continuous DF requires a more complicated setup including several SPTFF modules and intermittent dilution steps. Recently, we introduced a novel module design for continuous DF allowing simultaneous delivery of fresh buffer while withdrawing the permeate, thus achieving high degrees of buffer exchange within a single unit. In addition, the system allows to cyclically switch the flow direction of DF buffer through the membranes. Those uncommon features, however, also make it more difficult to determine an operation optimum experimentally by means of trial and error. Therefore, here a detailed finite element model of the physical processes within the module is presented, predicting key figures such as the obtained diafiltration efficiency and the resulting pressures. Because within the module all flow channels are filled by a 3D-printed porous grid supporting the membranes from both sides, the modified Brinkman equation was used to simulate the hydrodynamics, while common mass balance differential equations including accumulation, convection, and an anisotropic dispersion term were used for the simulation of concentration profiles of dissolved species. The predicted key figures are in good agreement with experimental results, obtained for feed solutions including up to 50 g/L of protein and being operated with and without switching the flow direction of the diafiltration buffer. A thorough parameter study reveals that the module shows the best performance for unidirectional flow of the diafiltration buffer, reaching diafiltration efficiencies independence to the applied diavolumes which are comparable to the ones of a conventional multi-stage setup using three SPTFF modules. Therefore, the simulation-based evaluation of optimum operation conditions reveals that the new module design has the potential to realize truly continuous diafiltration setups with high efficiency, requiring only one unit and no extra external piping for returning diafiltration in counterflow. Such simplified setups should be especially useful in small, flexible processing plants as they are increasingly demanded in the biopharmaceutical industry.

Das Virus SARS-CoV-2 (=severe acute respiratory syndrome coronavirus type 2) wurde zum ersten Mal in der chinesischen Stadt Wuhan entdeckt, als es zum Ausbruch der Infektionskrankheit Covid-19 kam. Mittlerweile sind schon mehrere Impfstoffe gegen Covid-19 auf dem Markt. Nach einer Impfung, sei es mit einem mRNA Impfstoff, ein Vektorimpfstoff oder einem Proteinimpfstoff, bildet der menschliche Körper Antikörper gegen das SARS-CoV-2 Spike Protein, welches ihn vor einem schweren Verlauf der Krankheit schützt. Im Zuge dieser Bachelorarbeit wurde ein Herstellungsprozess für einen Proteinimpfstoff für das SARS-CoV-2 Virus ausgearbeitet. Mittels HEK293 Zellen soll das SARS-CoV-2 Spike Protein hergestellt werden. Nach der Aufreinigung muss die Proteinlösung formuliert und dann abgefüllt werden. Das Projekt bestand aus 10 Personen, die jeweils einen Teil des Prozesses ausarbeiten sollten. Jedes Gruppenmitglied sollte über den jeweiligen Abschnitt ein R&I Schema (=Rohrleitungs- und Instrumentenfließschema), ein Lastenheft, zwei SOPs (=Standard Operating Procedure) und einen Master Batch Record erstellen. Die vorliegende Bachelorarbeit beschreibt die Formulierungsanlage mit einer Diafiltration. Nach der Chromatographie sind die Spike Proteine in einem Natriumdihydrogenphosphat Puffer, welcher jedoch nicht für die Injektion in den Menschen gedacht ist. Deshalb muss ein spezieller Formulierungspuffer, bestehend aus einer Kochsalzlösung mit Stabilisatoren und Adjuvantien, hergestellt werden. Dann erfolgt der Pufferwechsel mittels Diafiltration. Nach erfolgreicher Prüfung der Qualität und Proteinkonzentration, kann der Impfstoff abgefüllt und verwendet werden. The SARS-CoV-2 virus (=severe acute respiratory syndrome coronavirus type 2) was first discovered in Wuhan, China, when the infectious disease Covid-19 broke out. Several vaccines have been on the market since then. After a vaccination, the human body produces antibodies against the SARS-CoV-2 spike protein. These antibodies protect us from a severe course of the disease. In the course of this bachelor thesis, a manufacturing process for a protein vaccine for the SARS-CoV-2 virus was developed. By cultivating HEK293 cells, which were modified to produce the SARS-CoV-2 spike protein, the vaccine could be produced. After harvesting the cells, the spike protein was purified, formulated and then bottled. The project consisted of 10 people, who had to develop one part of the whole process. Each group member should create a piping and instrumentation diagram (P&ID), one user requirement specification (URS), two standard operating procedures (SOP) and one master batch record (MBR). This bachelor thesis describes the formulation process with a following diafiltration step. After the chromatography step the spike proteins are within a sodium dihydrogen phosphate buffer, which is not intended for human injection. Therefore a specific formulation buffer must be prepared and then has to be exchanged with the chromatography buffer by diafiltration. After a successful quality check the vaccine can be bottled, stored and eventually used.

The aim of this study was to develop an extraction method to produce highly functional oat protein concentrates. We investigated the possibility of combining enzyme-aided slightly alkaline (pH 8.0) extraction with ultrafiltration and subsequent diafiltration for concentration of the extracted oat proteins. A further aim was to study how the deamidation of oat proteins with protein-glutaminase (PG) improves the solubility of proteins as a function of the following parameters: pH (6.0–9.0), enzyme dosage (4–20 U/g protein), and incubation time (1–4 h) with response surface methodology (RSM). Furthermore, we investigated selected functional properties, such as heat-induced gelation and solubility, of the oat protein concentrates. The chosen parameters for the enzymatic deamidation pre-treatment process by PG were as follows: pH 8.0, dosage 11.0 U/g protein, and an incubation time of 4 h (1 h at native pH and 3 h at pH 8.0). Two oat protein concentrates were produced, non-deamidated and ultrafiltered, and deamidated and ultrafiltered, with protein concentrations of 45.0 and 52.4%, respectively. The solubility of both oat protein concentrates was significantly improved at neutral and slightly alkaline pH compared to the solubility of proteins extracted from the starting material. Additionally, both oat protein concentrates produced equally strong heat-induced gel-like structures at a protein concentration of 10%.

The polyelectrolyte poly(sodium 4-styrenesulfonate) undergoes aromatic–aromatic interaction with the drug chlorpheniramine, which acts as an aromatic counterion. In this work, we show that an increase in the concentration in the dilute and semidilute regimes of a complex polyelectrolyte/drug 2:1 produces the increasing confinement of the drug in hydrophobic domains, with implications in single chain thermodynamic behavior. Diafiltration analysis at polymer concentrations between 0.5 and 2.5 mM show an increase in the fraction of the aromatic counterion irreversibly bound to the polyelectrolyte, as well as a decrease in the electrostatic reversible interaction forces with the remaining fraction of drug molecules as the total concentration of the system increases. Synchrotron-SAXS results performed in the semidilute regimes show a fractal chain conformation pattern with a fractal dimension of 1.7, similar to uncharged polymers. Interestingly, static and fractal correlation lengths increase with increasing complex concentration, due to the increase in the amount of the confined drug. Nanoprecipitates are found in the range of 30–40 mM, and macroprecipitates are found at a higher system concentration. A model of molecular complexation between the two species is proposed as the total concentration increases, which involves ion pair formation and aggregation, producing increasingly confined aromatic counterions in hydrophobic domains, as well as a decreasing number of charged polymer segments at the hydrophobic/hydrophilic interphase. All of these features are of pivotal importance to the general knowledge of polyelectrolytes, with implications both in fundamental knowledge and potential technological applications considering aromatic-aromatic binding between aromatic polyelectrolytes and aromatic counterions, such as in the production of pharmaceutical formulations.

Sugar excipients such as sucrose and maltose are widely used for biopharmaceutical formulation to improve protein stability and to ensure isotonicity for administration. However, according to recent literature, pharmaceutical-grade sucrose contained nanoparticulate impurities (NPIs) that result in protein aggregation and degradation. The objective of this study was to evaluate the filtrate flux behavior of sugar solution during ultrafiltration (UF) and diafiltration (DF). Filtrate flux data were obtained using either a tangential flow filtration (TFF) system for DF experiments or a normal flow filtration system for UF experiments. In diafiltration experiments, which were performed using 7 g/L of human immunoglobulin G in a 20 mM histidine buffer with the 100 mM sucrose or maltose, the filtrate flux with sucrose solution decreased significantly. In contrast, the one with maltose solution was in good correspondence with the calculated filtrate flux accounting for the effects of solution viscosity. This large decline in the flux was also observed during UF experiments, in which the presence of NPIs was identified by dynamic light scattering analysis and by capturing an SEM image of the membrane surface after filtration. In addition, highly purified sucrose resulted in a much lower flux decline in TFF in the absence of NPIs. These results provide important insights into the factors governing the optimization of the UF/DF process using appropriate excipients for biopharmaceutical formulation.