The COVID-19 pandemic stressed the need for accelerating the development of novel vaccines. Over the past decades, the bottleneck in the biopharmaceutical process development shifted from optimizing fermentation processes to developing suitable purification strategies. Thereby, improving process understanding can significantly accelerate the development of purification processes. Unlike other biopharmaceutical products, vaccines are often more complex products containing molecules from different origins. The manufacturing process is therefore also more demanding. Consequently, no platform process is available for protein subunit vaccine purification. In the case of expressing a novel antigen in a host cell system, knowledge of the possible impurities – in this case host cell proteins (HCPs) – allows for rational and systematic process development. Therefore, this thesis focuses on developing characterization strategies of recurrent HCP impurities (from E. coli host cells) and on the integration of this information into modeling tools that advance removal strategies of these proteins, with a focus on protein-based antigen vaccines.

Firstly, the complete host cell proteome from antigen expressing E. coli host cells (BLR(DE3) and HMS174(DE3)) were characterized in chapter 2. Around 2000 HCPs were identified from the E. coli harvest sample using mass spectrometry based proteomics. Furthermore, an extensive HCP database including their expression levels, and physicochemical properties was constructed. Additionally, the profiles of an antigen expressing and null plasmid strain were compared. From a downstream processing perspective, the differences may be minor and the findings from the BLR(DE3) null strain can be applied to determine a purification strategy for the BLR(DE3) antigen-producing strain and HMS174(DE3) strain. The dataset of identified proteins was connected to databases describing the physicochemical properties of HCPs. Finally, protein property maps that help to identify a suitable downstream processing (DSP) strategy in comparison with the physicochemical properties of the target antigen, were generated.

Preparative chromatography based on differences in physicochemical properties is one of the main techniques for purification of vaccines. As follow-up to chapter 2, an experimental retention map of the host cell proteome during a salt gradient on hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEX) was constructed and reported and described in chapter 3. Furthermore, this study identified patterns in the retention behavior of HCPs based on their protein-protein interactions, molecular function, and cell location. To be able to predict the retention behavior of yet uncharacterized proteins, a quantitative structure-property relationship (QSPR) model was constructed using IEX retention data. Subsets of proteins, identified according to retention patterns, were used to build additional QSPR models, with monomer subsets yielding the most accurate predictions.

To achieve a higher level of process understanding, mechanistic models (MM) of chromatography columns are used in process development. These models primarily describe behavior of the target protein and selected process- or product-related impurities. However, it is beneficial to also include recurring HCP impurities in MMs. Hereby, critical HCPs causing issues when remaining in the product, are not necessarily abundant in the cell lysate and are often not individually described. A method for determining binding parameters of the entire host cell proteome including low abundant proteins to selected chromatography resins is still lacking.

Chapter 4 introduces a method to determine the above mentioned isotherm parameters of individual HCPs in a comprehensive manner. Fractions obtained from linear gradient elution experiments with different gradient lengths are analyzed by shotgun proteomics in order to extract the retention times of the individual HCPs. From the extracted retention volumes per gradient, isotherm parameters for all individual HCPs detected in the harvest were regressed. This method was exemplified using the BLR E. coli harvest, validated, and subsequently employed to optimize a capture step in silico.

Finally, chapter 5 gives an overview of the additionally investigated high-throughput sample preparation and analysis methods. This involved packing filter plates with resin for batch adsorption, which was explored to determine isotherm parameters instead of low gradient elution (LGE) experiments. Additionally, ion exchange high-performance liquid chromatography (IEX-HPLC) was investigated as an analytical technique instead of mass spectrometry (MS).

In summary, this thesis presents a comprehensive, large-scale characterization of HCPs from widely employed E. coli host cell strains for the production of protein vaccines. Moreover, a validated approach to determine isotherm parameters of all detectable HCPs in the harvest sample is presented.

Vaccination is a strong and effective way to prevent spreading of infectious diseases and promotes global health. In the future, the importance of vaccines is expected only to increase, driven by factors such as increased international traveling, higher healthcare expenditures, and a growing population. To meet the growing demands, it is necessary to shorten process development timelines for the production of new vaccines without compromising on safety, efficacy, consistency, and stability of the product. Therefore, it is necessary to advance process development approaches of vaccines to respond quickly and in event of an emerging infectious disease. The work in this thesis employed mathematical modeling and simulation techniques to accelerate this process development. The developed methods are particularly valuable for early phase process development, aiming to enhance process knowledge and minimize the consumption of valuable resources and material. While the project has a focus on vaccine production processes, the modeling tools and methods developed are also applicable to other (bio)pharmaceutical processes. In Chapter 2, we discussed the present and future process development approaches in (bio)pharmaceutical purification with an emphasis on vaccines. The primary needs are to establish standardized processes and to improve understanding of both production processes and host cell impurities. Modeling, when combined with high throughput experimentation, can play a crucial role in achieving these goals...

Continuous biomanufacturing is considered the future phase for the optimization of production processes in the biopharmaceutical industry. Productivity, product quality and consistency are greatly improved while production costs and environmental footprint are drastically reduced. The manufacturing of monoclonal antibodies (mAbs), an important biopharmaceutical in the treatment of cancers, autoimmune disorders, and, more recently, COVID-19, is eligible for this continuous processing due to patent expiration and the subsequent need to lower manufacturing costs...

Targeting to improve the utilization of lignocellulosic residues in the ethanol processing industry, this work aimed to test if the product inhibition of the enzymatic hydrolysis could be relieved by extractive reaction using aqueous two-phase systems (ATPS). The performance of enzymatic hydrolysis in ATPS is not well defined in literature. In this thesis, this extractive reaction was tested in terms of experimental conversion of sugarcane bagasse, simulations through conceptual process design and economic feasibility. A thermodynamic framework was developed in order to predict ATPS formation. The screening of ATPS and partition coefficient of the solutes were performed in a high throughput station. The ATPS were composed by polymer and salt. The enzymes were represented by the enzymatic cocktail Cellic CTec (Novozymes). The development of this platform consisted of two main parts: determination of phase diagrams (binodal curves and tie lines) and quantification of the solutes (sugar and proteins) in both top and bottom phases. The most promising ATPS were experimentally explored for enzymatic hydrolysis of sugarcane bagasse. Process design simulated two scenarios: hydrolysis occurring in the bottom phase and in the top phase. Topics such as the adsorption of phase forming components to the bagasse fibers and the influence of enzyme load on the hydrolysis were explored. The sugarcane bagasse hydrolysis in ATPS was conceptually assessed through the implementation of a model composed by two parts: hydrolysis and ATPS multi-batch separation. The designed case characterized by the ATPS hydrolysis was compared to the base case defined as conventional hydrolysis. Regarding the thermodynamic modelling of ATPS, the application of Flory-Huggins (FH) model to predict phase separation in polymer-salt systems was assessed. The implementation and analysis of FH theory involved the estimation of interchange energy (푤푖푗) and the calculation of phase diagrams. There were no statistical differences in determining the phase diagram in HTP platforms and bench-scale, verifying the reliability of methods and equipment suggested in this work. Moreover, tailored approaches to quantify the solutes were 8 presented, taking into account the limitations of techniques that can be applied with ATPS due to the interference of phase forming components with the analytics. This fast methodology proposes to screen up to six different polymer-salt systems in eight days and supplies the results to understand the influence of sugar and protein concentrations on their partition coefficients. Exploring experimentally the ATPS hydrolysis provided strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. In the conceptual design of extractive enzymatic hydrolysis, one of the major bottlenecks identified was the partitioning of glucose to both phases. The resultant conceptual process design operates as a tool to evaluate ATPS hydrolysis and compare it to conventional one. On the other hand, the thermodynamic model could not quantitatively describe the data. This occurs mainly because of the strong influence of random experimental errors on the estimation of interchange energy, systematic errors when translating the observed data to calculated partition concentrations, and FH not being an exact description of phase separation in salt based ATPS. The high throughput screening methodology indicated ATPS able to partition sugar and enzymes. The selected ATPS presented no significant improvements to perform the enzymatic conversion of sugarcane bagasse compared to the conventional hydrolysis. The main reasons were the influence of phase forming components on the enzymatic activity and the low selectivity of sugars in the ATPS. To disclose the application of ATPS in the ethanol processing industry, the recovery and reuse of the phase forming components are imperative for economic feasibility. Moreover, the developed high throughput platform could be further employed to exhaustively screen systems to design effective ATPS for the partition of sugars and proteins in polymer-salt systems.

The growing group of health-conscious consumers drives the development and sales in the market of low- or non-alcoholic beverages. In particular, sales in alcohol-free beer (AFB) has drastically gained in recent years, giving the incentive to further advance alcohol-free products. This thesis comprises the development of a new technology to produce an alcohol-free beer with an improved flavour, that is, an AFB with a significantly lower concentration in Strecker aldehydes. Despite the fact that Strecker aldehydes are present at trace concentrations of a few micrograms per litre, these compounds are majorly associated with the characteristic wort offflavour in alcohol-free beer. In the first stage, the origin of wort flavours is elucidated, and furthermore, the impact of the changed production process in comparison to regular beer and its resulting changes in the product is discussed. The most significant pathways of origin are the Maillard reactions and Strecker degradation, however, many other formation pathways have been suggested. While alcohol-free beers produced through regular fermentation and subsequent dealcoholisation are generally low in wort off-flavour, biologically produced AFBs exhibit a rather strong wortiness. This is because yeast’s activity during the fermentation is restricted to prevent alcohol formation, having the side effect that other compounds such as aldehydes are not converted to neutral or characteristic beer flavours. As it is impossible to prevent the formation of wort flavour, selective removal is required to produce an alcohol-free beer with improved flavour profile (balance). Subsequently, technologies applied to remove aldehydes from aqueous streams are investigated. Mechanisms that can be exploited for such removal are various – from separation based on size or volatility (distillation, pervaporation or membrane filtration) through sorption or conversion – past research has nearly studied all removal techniques available for aldehydes. However, most of the available techniques are not selective enough for a complex matrix such as beer, or not suitable for the application in the food industry. Nonetheless, adsorption is identified as the most promising technique to facilitate selective aldehyde removal. As a benchmark system, a dealcoholisation plant (spinning cone column) is studied with special focus on wort flavour removal. While volatile aldehydes such as 3-methylbutanal can relatively easily be reduced, methional, having a high boiling point, is not sufficiently separated from the end-product. Furthermore, due to the impact of heat, and ergo, the continuous formation and evaporation of these flavours, a plateau in their concentration is observed, where no further removal seems possible. This theory is proven by spiking a reactive sugar into the base and forming the mass balance over the system. Hence, in order to achieve a high reduction on high boiling wort flavours, another technology, such as adsorption, is required. To test, whether a suitable adsorbent can be identified, a wide screening experiment covering three adsorbent types (amine-functionalized polymers, hydrophobic resins and zeolites) and a total of 21 materials is performed. ZSM-5 type zeolites prove to have a superior selectivity as well as a relatively high capacity compared to other adsorbents. The choice of the right pore size and hydrophobicity is crucial for the separation success. Adsorption of the compounds of interest is non-competitive within the design space and robust against small changes in the beer matrix. The process is transferred to pilot scale, where it is shown that the zeolite effectively reduces wort off-flavour while maintaining the original character of the beverage. The reduced wort flavour concentration is also maintained over a period of 4 months ageing. Interestingly, the formation rates of aldehydes related to ageing are similar for the treated and reference product, with the exception of trans-2-nonenal, which is manifold lower in the treated AFB. A trained sensory panel confirms the difference in taste for both, the fresh and the aged product. In order to upscale this process to industrial scale, also the dynamics of the adsorption process on granular, binderless ZSM-5 zeolite is studied. The homogenous surface diffusion model is employed to regress the intraparticle diffusivity. The so obtained parameters indicate strong intraparticle mass transfer limitations as well as an inverse correlation of the effective diffusion coefficient to the molecules’ hydrophobicity. To avoid this process bottleneck, material improvements such as smaller crystal size and smaller granules are recommended. Finally, all obtained parameters are translated into a unit operation design. With a simple packed-bed operation of crushed particles, the production costs can be as much as 40 % lower than thermal dealcoholisation unit. Nonetheless, several assumptions still need to be verified, such as the material regenerability as well as other non- process-related factors such as material stability and food safety. Concluding, the newly developed technology for selective wort flavour removal in AFB in combination with restricted fermentation represents a great alternative to conventionally produced AFB.

Flavor-active components are key contributors to the profile of the final produced beer product. Their preservation and control during different stages of processing is crucial, since they might be lost during processing due to their volatile nature. In order to produce a final beer product with balanced flavor profile, which is acceptable by the consumer, the level of these components in the beer matrix should be adjusted and controlled. Various techniques can be applied for flavor control and recovery, such as distillation/stripping, pervaporation, supercritical extraction, and adsorption. Chapter two of this thesis discusses the recent advances in various techniques, which are applied for flavor recovery among which adsorption is a technique, which showed potential for selective removal and recovery of flavor/non-flavor-active components. This technique can be combined with heat processing, distillation/stripping, or can be used as a standalone technique. The focus of the work presented in chapter three of this thesis is on method development for selective removal and recovery of flavoractive volatile components mainly belonging to the group of esters, higher alcohols, and diketones, through adsorption technique. In order to investigate the single and competitive adsorption behavior of flavor-active components and their synergistic effects, high throughput experimentation technique is applied, improved for volatile components and isotherms are obtained using batch uptake experimentation. The competitive adsorption behavior of flavor-active components is investigated on various food-grade hydrophobic adsorbents, in order to study the influence of physical and chemical nature of the components and adsorbent properties on selectivity for each tested adsorbate over ethanol. Based on the results obtained from thermodynamic studies through various isotherm models, the appropriate adsorbent material is selected for further studies in the design stage. In the next step, deeper study is conducted on flavor-active esters, presented in chapters four, five, and six, which contribute to beer with a fruity taste and aroma. With adjusting their level in the final beer product and their fractionation, various products can be produced with fruity taste. Further investigation is performed on their competitive adsorption behavior both through batch uptake experimentation, and dynamic breakthrough analysis tests, discussed in chapters four and six respectively. Since ester components are present at low concentration level together with ethanol, which is present at higher concentration in comparison in various process streams, the influence of ethanol and temperature on their competitive adsorption is further investigated, discussed in chapter four. Physical properties such as isosteric heat, entropy, and Gibbs energy of adsorption, are calculated from performed thermodynamic studies, which contribute to our deeper understanding of the adsorption phenomena on the selected adsorbents. Considering the time-consuming steps, which are required to be followed for constructing the adsorption isotherms through batch uptake experimentation, the application of predictive models developed based on adsorbed solution theory, is evaluated in chapter five, for prediction of multicomponent adsorption isotherms for flavor-active esters from single-component adsorption isotherms, when experimental data for multicomponent behavior is not available. The predictive model developed based on IAST, was capable to predict the multicomponent adsorption behavior for the tested condition with accuracy and can be used as a tool for prediction of isotherms, when data on multicomponent adsorption is not available. Possibility for separation of flavor-active esters is further investigated in a fixed-bed column in lab-scale, discussed in chapter six, to study their breakthrough behavior and their separation under various process conditions (ethanol concentration and temperature). The results of the experimental tests obtained through breakthrough analysis and fractionation, are used for validation of simulations. Based on the results obtained in lab-scale, separation of flavor-active components is further investigated in a large-scale column, through simulation of various scenarios for separation. The performed experiments in the lab-scale and results of the simulations at large-scale give an insight on competitive adsorption behavior of these components, when present in a mixture. For a more detailed prediction of the adsorption behavior, future outlooks are discussed in chapter seven, to study the optimized condition considering the process conditions, and integration of the adsorption with other alternatives such as distillation/stripping.