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Selective protein crystallization is a trending alternative to preparative chromatography in biotechnological downstream processing. To save time and resources in early-stage process development, fast and reliable analytics are required. This work aimed to develop and assess a low-volume, quantitative, analytical tool for faster development of crystallization processes. The analytical tool was based on ultraviolet–visible spectroscopy and partial least-squares modeling and aimed to selectively quantify protein concentrations in heterogeneous supernatants during crystallization process development. For this purpose, a ternary model protein system consisting of hen-egg-white Lysozyme, bovine Ribonuclease A, and equine Cytochrome C was used for model calibration and subsequent crystallization studies for application. In a high-throughput screening, Lysozyme was selectively crystallized varying pH, precipitant concentration, and Lysozyme concentration at 8 °C for 13 days. During a kinetic study, the composition of two selected conditions was monitored over a time range of 7 days. In both studies, the developed tool quantified the different species in the supernatant with high precision. Crystal yield, purity, and selectivity were evaluated with a sensitivity of 96.23% and a short analysis time of 3 min per sample. The studies were carried out in 96-well plates. This said, the methodology could be easily adapted to higher throughput scales, i.e., 384-well or 1536-well plates.

Recording the data necessary to assess the kinetics of a reaction can be labor-intensive. In this technology brief, we show a method to automate this task by utilizing parts of an ÄKTApurifier chromatography system to automatically take samples from a reaction vessel at predefined time intervals and place them in 96-well plates and also enable correlating the samples with in-line spectral data of the reaction solution. Automatic sampling can reduce experimental bottlenecks by enabling overnight reactions or a higher degree of parallelization. To demonstrate the feasibility of the method, we performed batch-PEGylation of lysozyme with varying conditions by changing the molar excess of the PEG reagent. We used analytical cation-exchange chromatography to analyze the samples taken during the batch reaction, determining the concentrations of the individual species present at each time step. Subsequently, we fitted a kinetic model on these data. Fitting the model to four different reaction conditions simultaneously yielded a regression coefficient of R2= 0.871.

Covalent attachment of synthetic polymers to proteins, known as protein–polymer conjugation, is currently one of the main approaches for improving the physicochemical properties of these biomolecules. The most commonly employed polymer is polyethylene glycol (PEG), as evidenced by extensive research and clinical track records for its use in biopharmaceuticals. However, the occurrence of allergic reactions or hypersensitivity and the discovery of PEG antibodies, on the one hand, and the rise of controlled polymerization techniques and novel monomers, on the other hand, have been driving the search for alternative polymers for bioconjugation. The present study describes the synthesis, purification, and properties of conjugates of lysozyme with poly(N-acryloylmorpholine) (PNAM) and poly(oligoethylene glycol methyl ether methacrylate) (POEGMA). Particularly, conjugate species with distinct conjugation degrees are investigated for their residual activity, aggregation behavior, and solubility, by using a high-throughput screening approach. Our study showcases the importance of evaluating conjugates obtained by nonsite-specific modification through isolated species with discrete degrees of conjugation rather than on the batch level. Monovalent conjugates with relatively low molar mass polymers displayed equal or even higher activity than the native protein, while all conjugates showed an improved protein solubility. To achieve a comparable effect on solubility as with PEG, PNAM and POEGMA of higher molar masses were required.

ABSTRACTHigh protein titers are gaining importance in biopharmaceutical industry. A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity. The complexity of the molecule itself, as well as the various molecular interactions, make it difficult to describe their solution behavior. To study the formulation stability, long- and short-range interactions and the formation of complex network structures have to be taken into account. For a better understanding of highly concentrated solutions, we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb formulations. In this study, monoclonal antibody solutions in a concentration range of 50–200 mg/ml at pH 5–9 with and without glycine, PEG4000, and Na2SO4were analyzed. To determine the monomer content, analytical size-exclusion chromatography runs were performed. ζ-potential measurements were conducted to analyze the electrophoretic properties in different solutions. The melting and aggregation temperatures were determined with the help of fluorescence and static light scattering measurements. Additionally, rheological measurements were conducted to study the solution viscosity and viscoelastic behavior of the mAb solutions. The so-determined analytical parameters were scored and merged in an analytical toolbox. The resulting scoring was then successfully correlated with long-term storage (40 d of incubation) experiments. Our results indicate that the sensitivity of complex rheological measurements, in combination with the applied techniques, allows reliable statements to be made with respect to the effect of solution properties, such as protein concentration, ionic strength, and pH shift, on the strength of protein-protein interaction and solution colloidal stability.

In the development of new functional bio-based materials in the field of three-dimensional (3D) printing, visible light-induced dityrosine crosslinking gains increasing interest. In this context, most current bio-based materials and ink formulations rely on previously modified chemical substances with increased tyrosine availability. In contrast, we developed and characterized a photopolymerizable ink formulation for extrusion printing based on the unmodified and naturally occurring protein casein. Manufacturability of formulations containing protein, photoinitiating system, buffer and a thickening agent turned out to be a key factor for the ink development. In total, eight different thickening agents were assessed regarding their suitability to increase the viscosity of the ink formulation to expand the fabrication window for extrusion-based 3D printing. The mechanical properties of the ink formulation and hydrogel in presence of sodium alginate were further characterized and the macroscopic fabrication of auxetic structures consisting of up to 30 layers was achieved by applying extrusion-based printing.

Extrusion-based printing relying on pneumatic dispensing systems is the most widely employed tool in bioprinting. However, standardized and reliable methods for process development, monitoring and control are still not established. Suitable printing parameters are often determined in a trial-and-error approach and neither process monitoring nor real-time adjustments of extrusion pressure to environmental and process-related changes are commonly employed. The present study evaluates an approach to introduce flow rate as a main process parameter to monitor and control extrusion-based bioprinting. An experimental setup was established by integrating a liquid flow meter between the cartridge and nozzle of a pneumatically driven bioprinter to measure the actual flow of dispensed ink in real-time. The measured flow rate was fed to a Python-based software tool implementing a proportional-integral-derivative (PID) feedback loop that automatically and dynamically adapted the extrusion pressure of the bioprinter to meet a specified target flow rate. The performance of the employed experimental setup was evaluated with three different model inks in three application examples. a) Continuous dispensing: Several runs of continuous dispensing showed that the PID-based pressure control was able to generate a steady flow rate more consistently and precisely than constant pressure settings. b) Adaptation to ink inhomogeneities: Deliberately created ink inhomogeneities were successfully compensated for by real-time pressure adjustments which profoundly enhanced the printing quality compared to printing without adaptive pressure. c) Process transfer to other nozzle types: Experiments with different nozzle types demonstrated the potential of the established setup to facilitate and accelerate process transfer and development. The present study provides an alternative approach for process design, monitoring and control by introducing flow rate as a main process parameter. We propose bioprinting processes to be based on flow rate specifications instead of constant pressure settings. This approach has the potential to save time by avoiding tedious parameter screenings and to introduce an active, real-time control over the printing process. Subjective influences by individual users during process development can be reduced and the process transfer between different devices and experimental setups can be facilitated and accelerated.

A new experimental set-up for on-line visualization of the intra-particle uptake kinetics during packed bed chromatography has been designed and tested. Confocal laser scanning microscopy was used to analyze the dynamics of protein adsorption to porous stationary phases. In combination with this, a flow cell was developed that could be packed with chromatography media and operated as a fully functional mini-scale chromatography column. Adsorption profiles of single- and two-component mixtures containing BSA and IgG 2a during packed bed cation-exchange chromatography were investigated. The two proteins appear to exhibit different transport characteristics. For BSA a classical “shrinking core” behavior could be detected. The profiles obtained during IgG 2a adsorption point toward a different transport mode, which deviates from the classical pore-diffusion picture. For the two-component system, a superposition of the single-component profiles combined with a classical displacement of the weaker bound protein species was found. The results indicate that depending on the adsorbed protein the uptake can vary tremendously, even for adsorption to the same chromatographic support. It is clearly shown that the new microcolumn allows in situ quantitative investigations of protein adsorption dynamics within a single particle, which adds a new tool to the available methods for characterizing and optimizing protein adsorption chromatography. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 359–368, 2002.

We introduce a robust and scale-flexible approach to macromolecule purification employing tailor-made magnetic adsorbents and high-gradient magnetic separation technology adapted from the mineral processing industries. Detailed procedures for the synthesis of large quantities of low-cost defined submicron-sized magnetic supports are presented. These support materials exhibit unique features, which facilitate their large-scale processing using high magnetic field gradients, namely sufficiently high magnetization, a relatively narrow particle size distribution and ideal superparamagnetism. Following systematic optimization with respect to activation chemistry, spacer length and ligand density, conditions for preparation of effective high capacity (Qmax = 120 mg g⁻¹) strongly interacting (Kd < 0.3 μm) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies ≈95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of ≈4.1 at the expense of only a 4% loss in α-amylase activity. Efficient recovery of trypsin from the same feedstock was demonstrated at a vastly increased scale using a high-gradient magnetic separation system to capture loaded benzamidine-linked adsorbents following batch adsorption. With the aid of a simple recycle loop over 80% of the initially adsorbed trypsin was recovered in-line with an overall purification factor of ≈3.5. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 301–313, 2002.

A new fluid distribution system designed for expanded bed adsorption was introduced and studied in a 150-cm diameter column. Based on fluid application through a rotating distributor, it eradicates the need for perforated plates, meshes, or local mixers. The effect of rotation rate on column performance was examined by fluidizing a 30-cm high bed of supports with tap water and introducing pulses of dye or acetone tracer. Linear bed expansion was seen as the superficial fluid velocity was raised from 170 cm · h⁻¹ to 450 cm · h⁻¹ (3000 L · h⁻¹ to 8000 L · h⁻¹), and there was little change in expansion characteristics as distributor rotation rate was increased from 2.5 to 10 rpm. The distributor was observed to generate a flow pattern suitable for expanded bed adsorption when the supports were fluidized at a superficial fluid velocity of 283 cm · h⁻¹ and dye pulses introduced. At a rotation rate of 2.5 rpm, no significant dead zones were observed, and a discrete band was formed that moved up through the bed. Furthermore, the pattern of dye movement could be used to calculate interstitial linear fluid velocities of 460 cm · h⁻¹ and 572 cm · h⁻¹ at the column wall and center, respectively, indicating a parabolic flow profile. The distributor rotation rate giving the best operating conditions was found to be 2.5 rpm when the bed was fluidized at a flow velocity of 283 cm · h⁻¹ and the residence time distribution of acetone tracer examined. Under these conditions, the coefficient of axial dispersion was 6.1 × 10⁻⁶ m² · s⁻¹ and 29 theoretical plates were measured. When the rotation rate was raised to 10 rpm, the coefficient of axial dispersion increased to 8.08 × 10⁻⁶ m² · s⁻¹ and the number of theoretical plates decreased to 22. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 35–43, 2002; DOI 10.1002/bit.10170

The suitability of ion‐selective electrodes (ISE) for the determination of residence time distribution (RTD) in turbid, cell‐containing fluids was examined. The electrodes were found to give reproducible signals in biomass‐containing feedstock with up to 20% wet weight of solids. The enhanced feedstock compatibility of IES, when compared to other tracer sensing devices, allows the study of expanded bed system hydrodynamics under relevant operating conditions. Within the linear range of the corresponding ISE‐tracer pair, both examined ISE (Li+‐ or Br−‐selective) showed to be insensitive against the range of flow rate and pH normally employed during expanded bed adsorption (EBA) of proteins. Analyzing the RTD obtained after a perfect ion tracer pulse in terms of the PDE model (PDE, axially dispersed plug‐flow exchanging mass with stagnant zones) gave a quantitative description of the underlying hydrodynamic situation during EBA processing. These data provided a powerful tool to make predictions on the adsorptive global process performance with a defined feedstock type and composition. The link between the hydrodynamic events during feedstock application and the actual process performance was shown when applying intact yeast cell suspensions at different biomass content (up to 7.5% wet weight) and buffer conductivity (5−12 mS) onto an EBA column filled with the adsorbent Streamline Q XL as fluidized phase. On the basis of our experimental results, a guideline for the successful application of the ISE/RTD method to EBA process design is presented.

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the ‘loaded’ adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h−1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h−1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h−1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption. [ABSTRACT FROM AUTHOR]

The technology of bioprinting is arousing a growing interest in biopharmaceutical research and industry. In order to accelerate process development in the field of bioprinting, image-based analysis methods are non-invasive, time- and cost-saving tools which are useable for printer characterization, bioink printability evaluation, and process optimization. Image processing can also be used for the study of reproducibility, since reliable production is important in the transition from research to industrial application, and more precisely to clinical studies. This study revolves around the establishment of an automated and image-based line analysis method for bioprinting applications which enables an easy comparison of 3D-printed lines. Diverse rheological properties of bioinks and the printing process affect the geometry of the resulting object. The line represents a simple geometry, where the influence of the rheological properties and printing parameters is directly apparent. Therefore, a method for line analysis was developed on the basis of image recognition. At first, the method is tested for several substances such as Nivea®, pure and colored Kolliphor solutions, and two commercially available hydrogel formulations which can be used as bioinks. These are Biogelx™-ink-RGD by Biogelx and Cellink® Bioink by Cellink. The examination of limitations showed that transparent materials such as Kolliphor-based solutions cannot be analyzed with the developed method whereas opaque materials such as Nivea® and both bioinks can be analyzed. In the course of process characterization, the method was used to investigate the shrinkage behavior for both bionks. With the help of the line analysis tool, a shrinkage behavior of both bioinks was demonstrated and thus, process time could be identified as a critical process parameter.

The importance of 3D printing technologies increased significantly over the recent years. They are considered to have a huge impact in regenerative medicine and tissue engineering, since 3D bioprinting enables the production of cell-laden 3D scaffolds. Transition from academic research to pharmaceutical industry or clinical applications, however, is highly dependent on developing a robust and well-known process, while maintaining critical cell characteristics. Hence, a directed and systematic approach to 3D bioprinting process development is required, which also allows for the monitoring of these cell characteristics. This work presents the development of a flow cytometry-based analytical strategy as a tool for 3D bioprinting research.