The biopharmaceutical industry is moving from a batch to a continuous mode of manufacturing. This shift promises to reduce costs and manufacturing footprint while improving productivity and consistency of the product. This thesis implements miniaturized and automated high-throughput screening techniques alongside a mathematical chromatography model for the development of an integrated continuous chromatography process. The model is used for in-silico optimization of a capture and polishing step of a monoclonal antibody (mAb). The optimization focusses on chromatographic processes that would have to deal with higher titer solutions. The transition to Integrated Continuous Biomanufacturing (ICB) is welcomed by industry and regulatory agencies, which are working together to accomplish this shift. Process development plays a crucial role in defining new processes or adapting existing processes to different modes of operation. High-Throughput Process Development (HTPD) has been used in the biopharmaceutical industry to accelerate and reduce costs of process development, by using miniaturized assays and performing computer-aided studies. However, the industry experiences gaps and sees opportunities for improvement in the HTPD tools that can help the transition to ICB. These gaps, together with a state-of-the-art of HTPD for ICB are presented in Chapter 2. Experts in the field identified microfluidics and modeling to be the most promising technologies to fill in the gaps in process development for ICB. Subsequently, an overview on the state-of-the-art of automation and miniaturization for biopharmaceutical process development is given in Chapter 3. The focus is on different degrees of miniaturization and automation of the technologies for process development, for both Upstream and Downstream processing (USP and DSP, respectively). Liquid-Handling Stations (LHS) are the epitome of automation for process development, and have seen great adoption for the past decades. Examples of the use of this tool for USP and DSP process development are provided. A greater emphasis is placed on the often overlooked microfluidics and how it can also be used as a screening tool, and a SWOT analysis on LHS and microfluidics as potential process development tools is provided. Further comparison between HTS tools for chromatographic process development is needed, since process development efforts for chromatography mostly rely on LHS-based experiments. Three methodologies are selected for this comparison: LHS, microfluidics, and Eppendorf tubes (Chapter 4). To achieve this, protein equilibrium adsorption isotherms are determined with each of the aforementioned methodologies. The microfluidics chip produced in-house provides a platform for resin screening that achieves liquid and resin volume reductions of 15- and up-to 200-fold, respectively. Accurate resin volume determination is ensured with an image analysis software, and resin consumption is as high as 200 nl in the microfluidics system. After validating the HTS methodologies, a cost consideration study aims at fairly comparing the three methodologies for their chromatographic process development potential. Although at a lower Technology Readiness Level, microfluidics can be a viable alternative tool when the protein to be studied is very expensive or scarce (such as in early stages of process development), due to the high degree of miniaturization. Furthermore, it is discussed what would be the possible applications of the different methodologies in chromatographic process development. The HTS methodologies developed paved the way for the implementation of a HTPD approach for the study and optimization of continuous chromatography (Chapters 5 and 6). A large database on the adsorption equilibrium isotherms of mAbs to different protein A (ProA) and Cation-Exchange (CEX) resins is generated from experiments with a LHS. This database is then used to further reduce resin candidates to be used in subsequent experiments. Four resin candidates are used to study the equilibrium adsorption isotherms of mAb to ProA ligands with a clarified cell culture supernatant (harvest). It is shown that pure mAb experiments reflect the same adsorption behavior as harvest experiments for all resin candidates, reducing the need to duplicate experiments in the future. The parameters determined are further used in a mechanistic Lumped Kinetic Model (LKM), used for the in-silico study of column chromatography (Chapter 5). The LKM uses a lumped overall mass transfer parameter that is linearly dependent on feed concentration, in line with mass transfer theory. The hybrid approach to HTPD emphasizes the importance of computational, experimental, and decision-making stages in chromatographic process development. The LKM model described is further developed for the study of continuous chromatography. The continuous model is used for the in-silico optimization of a 3-Column Periodic Counter-current Chromatography (3C-PCC) capture and polishing step, for the purification of mAbs from high-titer solutions (Chapter 6). The model maximizes Productivity and Capacity Utilization (CU) keeping the yield high (99%) and having the flow rate and the percentage of breakthrough achieved in the interconnected phase as constraints. The shape of the breakthrough curve plays an important role in the optimization of continuous chromatography. The optimization results are validated for three different ProA resins, from which the best resin candidate is selected to continuously capture mAb from a harvest solution. The eluates of this operation are pooled and used as input for the continuous CEX step. The experimental results show very good agreement with model’s predictions (lower than 7% deviation) and the proposed methodology helps to develop and optimize a continuous chromatography process in a short amount of time. In summary, this thesis presents the exciting journey of process development for continuous chromatography, from conceptualization and selection of screening techniques until the end result of performing an optimized continuous chromatographic step for the successful capture and polishing of a mAb.@en

The monoclonal antibody (mAb) industry is becoming increasingly digitalized. Digital twins are becoming increasingly important to test or validate processes before manufacturing. High-Throughput Process Development (HTPD) has been progressively used as a tool for process development and innovation. The combination of High-Throughput Screening with fast computational methods allows to study processes in-silico in a fast and efficient manner. This paper presents a hybrid approach for HTPD where equal importance is given to experimental, computational and decision-making stages. Equilibrium adsorption isotherms of 13 protein A and 16 Cation-Exchange resins were determined with pure mAb. The influence of other components in the clarified cell culture supernatant (harvest) has been under-investigated. This work contributes with a methodology for the study of equilibrium adsorption of mAb in harvest to different protein A resins and compares the adsorption behavior with the pure sample experiments. Column chromatography was modelled using a Lumped Kinetic Model, with an overall mass transfer coefficient parameter (k_ov). The screening results showed that the harvest solution had virtually no influence on the adsorption behavior of mAb to the different protein A resins tested. k_ov was found to have a linear correlation with the sample feed concentration, which is in line with mass transfer theory. The hybrid approach for HTPD presented highlights the roles of the computational, experimental, and decision-making stages in process development, and how it can be implemented to develop a chromatographic process. The proposed white-box digital twin helps to accelerate chromatographic process development.

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Biopharmaceuticals are becoming increasingly important in modern healthcare. Monoclonal antibodies (mAb) are one of the most widely used therapeutic proteins and are important for the treatment of cancer and autoimmune diseases, among others. After cell culture there are still large amounts of other impurities (e.g. host cell proteins) in solution. Chromatography is usually the first purification step, allowing to increase purity and reduce volume. This comes associated with high costs and chromatography accounts for a significant portion of total production costs for therapeutic proteins. Chromatographic process development may be time consuming and use large amounts of resins. Therefore, there is increased interest in finding cheaper techniques for chromatographic process development without compromising accuracy. This paper presents a highly sophisticated microfluidic chip approach for efficient adsorption isotherm determinations compared to current chromatographic process development. Implementation of an image analysis software ensures that chromatographic resin volume is accurately determined. The adsorption isotherm performance of microfluidics was compared to the robotic Liquid-handling Station (LHS) and labor intensive Eppendorf tubes. The microfluidic chip allows a 15-fold volume reduction and resin consumptions as low as 100/200 nl (200/100-fold reduction). The microfluidic chip performed comparably to the other miniaturized techniques, using less liquid and resin volume. For process development of expensive products (e.g. monoclonal antibodies), miniaturization (provided by the microfluidic chip) proved to be the most cost effective alternative whereas for less valuable products (e.g. lysozyme) automation (provided by the LHS) was the most cost effective alternative.

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Continuous manufacturing is an indicator of a maturing industry, as can be seen by the example of the petrochemical industry. Patent expiry promotes a price competition between manufacturing companies, and more efficient and cheaper processes are needed to achieve lower production costs. Over the last decade, continuous biomanufacturing has had significant breakthroughs, with regulatory agencies encouraging the industry to implement this processing mode. Process development is resource and time consuming and, although it is increasingly becoming less expensive and faster through high-throughput process development (HTPD) implementation, reliable HTPD technology for integrated and continuous biomanufacturing is still lacking and is considered to be an emerging field. Therefore, this paper aims to illustrate the major gaps in HTPD and to discuss the major needs and possible solutions to achieve an end-to-end Integrated Continuous Biomanufacturing, as discussed in the context of the 2019 Integrated Continuous Biomanufacturing conference. The current HTPD state-of-the-art for several unit operations is discussed, as well as the emerging technologies which will expedite a shift to continuous biomanufacturing.

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Process development in the biotech industry leads to investments around hundred of millions of dollars. It is important to mitigate costs without neglecting the quality of process development. Biopharmaceutical process development is important for companies to develop new processes and be first to market, improve a pre-established process, or start manufacturing a product available by patent expiry (biosimilars). Laboratory automation enables methodical and standardized process development. Miniaturization and parallelization empower laboratories to screen several experimental conditions and define operating windows for purification processes, improving process robustness. Together, they allow for fast and accurate process development in a fraction of the time and cost of nonminiaturized/nonparallel process development approaches. The most widely used High-Throughput Screening technique is a liquid-handling station and microfluidics is taking its first steps in process development. Both are attractive scale-down tools for the characterization of bioprocesses and allow thousands of experiments to be performed per day. High-Throughput Process Development (HTPD) has helped to achieve major breakthroughs in process optimization, both for upstream and downstream processing. Continuous processing is the next step in process development which leads to cost reduction, higher productivity and better quality control; the integration of upstream and downstream processes is seen as a major challenge. In this review, we will focus on the state-of-the-art of miniaturized techniques for process development in the biotechnology industry, and how automation and miniaturization drive process development. A comparison between liquid-handling stations and microfluidics is made and an indication is given of which tools are still lacking for HTPD in the context of Integrated Continuous Biomanufacturing.

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In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.

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