Closing Plenary Talk - Versatile macromolecules and their biomedical applications

Polymer reaction and colloidal engineering have reached a level of maturity allowing us to synthesize macromolecules and small particles covering very wide and often even overlapping ranges of sizes and compositions. On the other hand, the number of potential applications in various areas is very large, and the limiting step is probably mostly in our capability to imagine a “certain” structure to solve a “certain” problem. In this presentation we discuss through a number of examples, how to define the properties that a material should have to solve a certain problem, and then how to synthesize the corresponding macromolecule. In particular, we focus on a class of polymers exhibiting a comb-like structure, where the pendants have a chemical composition, length and order along the backbone that can be accurately controlled through controlled free radical polymerization techniques. Each pendant is prepared before hand through living techniques, including ring-opening polymerization, which allows preparing macromonomers with very different properties in terms of hydrophilicity, hydrophobicity, biodegradability, etcetera. The result is a very versatile macromolecule that can be adapted to provide the specific functionalities, including self-assembly and nanoparticle stabilization, needed to solve a particular problem. Specific applications will be discussed mainly in the area of drug delivery and tissue engineering. Starting from the specific problem to be solved, the desired properties of the appropriate macromolecules are discussed and the corresponding synthesis process is described. Finally, an outlook on different application areas is provided

The Role of Digitalization in the Continuous Integrated Manufacturing of Therapeutic Proteins

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Theory of activated-rate processes under shear with application to shear-induced aggregation of colloids

Using a novel approximation scheme within the convective diffusion (two body Smoluchowski) equation framework, we unveil the shear-driven aggregation mechanism at the origin of structure-formation in sheared colloidal systems. The theory, verified against numerics and experiments, explains the induction time followed by explosive (irreversible) rise of viscosity observed in charge-stabilized colloidal and protein systems under steady shear. The Arrhenius-type equation with shear derived here, extending Kramers theory in the presence of shear, is the first analytical result clearly showing the important role of shear-drive in activated-rate processes as they are encountered in soft condensed matter

A systematic approach for process development and quality control in continuous perfusion cultures

Biopharmaceutical industry is facing numerous challenges. Increased cost pressure, changing markets, and the need for more manufacturing flexibility are main drivers for a gently mindset change: the switch from batch to continuous production of biopharmaceuticals. Mammalian cell perfusion cultures represent not only a valuable tool for the production of therapeutics, but also for process intensification of state-of-the art fed-batch processes. Considering N-stage perfusion processes, the knowledge on time and cost effective development and scale-up procedures to achieve a reliable reactor operation and desired product characteristics is still limited. However, a robust and optimized reactor operation is a prerequisite for a successful end-to-end integration production facility. In this study a comprehensive optimization framework for mammalian cell perfusion cultures was defined. In a first step, rapid evaluation of suitable operating conditions in small scale perfusion cultures allowed to identify key process parameters to facilitate steady state operation and process control with a given media formulation. A further refining was performed in a stirred tank perfusion bioreactor setup by sequential screening of different steady state set points while targeting improved product yields and desired product quality characteristics. Minimizing cell specific perfusion rate, as well as varying perfusion rate and viable cell density set point at a suitable CSPR were evaluated targeting optimal and robust operation and product quality control. The tuning of key cell culture parameters led to an improved performance and control of the perfusion culture. Especially, the decrease of the cell specific perfusion rate prevented excessive cellular growth and reduced significantly the loss of product in the bleed stream. Constant patterns of product quality attributes such as N-linked glycosylation and charge isoforms were observed within each steady state. Overall, product quality distributions remained quite stable and did not vary between different operating set points. In a last step, the use of additional media supplements might provide an additional tool to effectively control and tune product quality towards desired distributions. Overall, this study presents a systematic approach for the development of intensified continuous cultures and underlines the potential of perfusion cultures to simultaneously achieve high productivities while tuning towards desired characteristics of consistently expressed therapeutic proteins

Mammalian cell perfusion cultures: “Intensification by growth inhibition”

Mammalian cell perfusion processes have regained the focus of attraction for the production of therapeutic proteins, especially as a part of an end-to-end integrated production facility. Besides reduced equipment size, higher volumetric productivities, and enhanced cost efficiency, continuous processes enable stable and steady state operation giving more homogeneous and even improved product quality. However, the knowledge on time and cost effective development and scale-up procedures to obtain a stable process operation and desired product quality control is still limited. The combination of long process duration and the lack of automated high-throughput systems for perfusion demand for an optimized strategy regarding the development and control of these processes. In this study, mammalian cell perfusion cultures have been performed targeting high volumetric productivities and enhanced process and product quality control. Shake tube cultures revealed suitable starting conditions (cell specific perfusion rate, media composition, flow rates) for process design and control. Different strategies for minimizing cellular growth were tested: a) minimizing cell specific perfusion rate, b) chemical growth inhibition c) environmental growth inhibition by lowering the culture temperature. Active growth inhibition targeted cell cycle arrest to reduce cellular growth and to trigger higher productivities. The loss of product in the bleed was effectively reduced by all strategies. Especially, lower culture temperatures were an effective tool for process intensification leading to increased cellular productivities and reduced cellular growth, while the use of a chemical inhibitor did not lead to higher protein quantities. Flow cytometry revealed that more cells were arrested in the G1 phase, leading to lower cell proliferation. Resulting product quality patterns remained constant within each operation set point and indicated the possibility of enhanced product quality during perfusion cell cultures. A variety of tools can be used to intensify continuous cultures. A temperature down-shift at the start of the production phase represents the most promising tool for process intensification. The different steps present a systematic and efficient procedure for the development of perfusion cell cultures targeting optimal process performance and tunable product quality control

Dynamic process control of twin-column periodic countercurrent chromatography processes

Twin-column periodic countercurrent chromatography has become a promising solution for continuous downstream processes as chromatography equipment for both process development and GMP manufacturing has become available. Twin-column periodic countercurrent processes have been utilized successfully in many applications including purification of biologics, such as monoclonal antibodies (mAbs), bispecific antibodies and fusion proteins, the purification of peptides and also small molecules such as antibiotics and fatty acid ethyl esters. This presentation deals with the online UV-based control of two twin-column periodic countercurrent processes, 2C-PCC and MCSGP, covering applications in chromatographic capture and polishing. 2C-PCC is a capture process significantly improving the process performance (productivity, resin utilization, buffer consumption, product concentration) of affinity capture, e.g. the capture of mAbs, in comparison to traditional single column chromatography. MCSGP is a polishing process to solve difficult ternary separation challenges, allowing purification with high product yield and purity in situations where traditional single column chromatography faces a yield-purity trade-off. For robust operation in view of commercial manufacturing using these two cyclic processes, UV-based dynamic control strategies have been developed and tested. In this presentation a UV-based control strategy for 2C-PCC based on online-determination of breakthrough curve signals is introduced and case studies for its application in protein A chromatography are shown. The control strategy accounts for changes in resin capacity and, in case of continuous upstream, for changes in titer occurring over time, and adjusts the operating parameters such that capacity utilization and yield are kept constant. A second control strategy for MCSGP based on the online evaluation of the elution peak signal is presented based on a case study. The method accounts for shifts of the product peak e.g. due to changes in temperature and buffer preparation (e.g. during buffer refill). An application of the control strategy in protein purification is presented. The presented methods represent important tools for robust manufacturing using twin column processe

Aggregation Mechanism of an IgG2 and two IgG1 Monoclonal Antibodies at low pH: From Oligomers to Larger Aggregates

Purpose: To identify the aggregation mechanism and the stability characteristics of three different monoclonal antibodies under acidic conditions. Methods: The aggregation kinetics is analyzed by a combination of light scattering, size exclusion chromatography and fluorescence techniques and the aggregation data are correlated to protein structure, hydrophobicity, charge and antibody subclass. Results: In the investigated conditions, the antibody aggregation follows a mechanism consisting of two-steps: reversible monomer oligomerization followed by irreversible cluster-cluster aggregation. The kinetics of the two steps is differently affected by the operating conditions: mild destabilizing conditions induce formation of oligomers which are stable within weeks, while stronger denaturing conditions promote aggregation of oligomers to larger aggregates which eventually precipitate. For different antibodies significant differences in both oligomerization and growth rates are found, even for antibodies belonging to the same subclass. For all antibodies the aggregate formation is accompanied by a structure re-organization with an increase in the ordered β-sheet structures. At low pH the aggregation propensity of the investigated antibodies does not correlate with antibody subclass, surface net charge and hydrophobicity of the non-native state. Conclusions: The aggregation mechanism of three antibodies in acidic conditions as well as differences and analogies in their stability behavior has been characterize

Effects of temperature and concentration on mechanism and kinetics of thermally induced deposition from coffee extracts

Production of soluble (instant) coffee powders typically involves extraction of roasted coffee by water followed by evaporation in order to concentrate extracts before spray or freeze drying to produce dry coffee powder. In the course of evaporation, deposition of dissolved material from coffee extracts is a major cause of fouling at the heat exchange surfaces of evaporators. Therefore, in order to improve the design and optimization of evaporation processes of coffee extracts, better understanding of the deposition mechanism and kinetics is needed. In this study, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the initial formation of nanometer scale deposits on surfaces exposed to coffee extracts. OWLS measurements were complemented by light scattering from extract solutions, gravimetry of macroscopic deposits, and scanning electron microscopy imaging of deposited layers. Primary molecular-scale layers of about 1 mg m^−2 were rapidly formed in the first stage of deposition, even at ambient temperature, followed by the secondary deposition with kinetics strongly dependent on temperature. Secondary deposition rates were low and largely independent of the extract concentration at ambient temperature, but became strongly dependent on the extract concentration at elevated temperatures. In particular, activation energies for the deposition between 25◦C and 70◦C were much higher for the original extract (13.3 mass %, solids) than for diluted extracts (up to 1.3 mass %, solids). Furthermore, heating of the original extracts above 60◦C resulted in rapid aggregation of suspended macromolecules into large clusters, while only gradual aggregation was observed in diluted extracts

Continuous chromatographic processes with a small number of columns: Comparison of simulated moving bed with Varicol, PowerFeed, and ModiCon

The Simulated Moving Bed process and its recent extensions called Varicol, PowerFeed and ModiCon are studied, in the case where a small number of columns are used, i.e. from three to five. A multiobjective optimization approach, using genetic algorithms and a detailed model of the multicolumn chromatographic process, is applied to optimize each process separately, and allow for comparison of the different operating modes. The non-standard SMB processes achieve better performance than SMB, due to the availability of more degrees of freedom in the operating conditions of the process, namely the way to carry out asynchronous switches for Varicol, and the different flow rates and feed concentration during the switching interval for PowerFeed and for ModiCon, respectively. We also consider the possibility of combining two non-standard operating modes in a new hybrid process, and evaluate also in this case the possible performance. Finally, a critical assessment of the results obtained and of the potential for practical implementation of the different techniques is reporte

Integrated continuous processing for the manufacture of monoclonal antibodies

Continuous manufacturing is currently being considered by the Biopharmaceutical Industry not only for the classical reasons which make continuous operation preferred over the batch one, but also for recent initiatives of the regulatory agencies. We discuss here a series of experiments where a perfusion reactor with CHO cells for the production of a monoclonal antibody has been operated in the continuous mode and connected to a two column continuous protein A chromatographic unit for product capture. A few steady states are examined and the use of simulation models for process design and control is illustrated