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The egg white protein avidin is known for its strong binding to biotin. Because of various applications in bioanalytics and biopharmaceutical industries, there is a great interest in the manufacturing of highly purified avidin. For the first time, the distribution behavior of avidin and the major contaminants occurring in egg white, ovalbumin, ovotransferrin (conalbumin), ovomucoid (ovotrypsin-inhibitor) and lysozyme in aqueous two-phase systems (ATPS) are presented. The results revealed a high capability of PEG/phosphate systems for the intermediate purification of the highly valuable protein avidin from chicken egg white solutions. Several PEG/phosphate systems varying in PEG molecular weight, pH and NaCl concentration were investigated in an automated small scale screening performed on a liquid handling station. The high-throughput screening was used to determine binodal curve and tie-lines as well as protein distribution and recovery. Tandem Reversed Phase HPLC was applied for very fast quantification of both impurities and active (biotin binding) avidin within 4.25 min per sample. The main finding was that avidin partitioned in almost all ATPSs to the salt-rich bottom phase while distribution of impurities depended strongly on chosen experimental conditions. Starting from a pre-purified avidin solution containing 11% (c/c) avidin, in the most effective small-scale ATPS, a purification factor of 5.7 was obtained with an avidin yield of 92% in the bottom phase. In this particular system, a removal of ovalbumin of 65%, ovomucoid of 99.4%, lysozyme of 99.7% and ovotransferrin of 95.4% was achieved. The screening results were confirmed in common lab scale experiments representing a scale up by factor 15.

Photoinitiated polymerization is employed to produce submicron polymer particles in aerosols. By ultraviolet (UV) irradiation of the aerosol monomer solution droplets, produced with the help of an atomizer, polymerization is initiated by free radical generation. The aerosol process allows the production of surfactant-free polymer particles without any solvent required, while photochemistry results in instantaneous formation of free radicals by cleavage of excited photoinitiator molecules. Furthermore, the initiation can take place independent of temperature, which provides polymerization at ambient temperature. A continuous experimental setup with a flow-through photoreactor is developed, which is characterized by a sub-minute aerosol residence time. The experiments reveal that spherical nanopolymers can be formed as 1-to-1 copy from the monomer droplets.

Product titers have steadily increased in the biopharmaceutical industry over the past decades adding new challenges to downstream purification processes. For that reason, unit operations are needed that can be operated in a continuous fashion offering high productivity, one such alternative being aqueous two phase systems. These systems were shown to have high selectivity while offering mild working conditions ensuring the integrity of biomolecules. Nevertheless, driving forces for aqueous–aqueous partitioning of proteins are still not well understood and process development is thus based on empirical screenings. Here we investigated the use of molecular dynamics (MD) simulations to determine phase system properties of polyethylenglycol (PEG)—phosphate systems that guide protein distribution. MD simulations were accurate enough to resolve differences in certain properties of upper and bottom phases (e.g. the relative hydrophobicity of PEG as well as H- H-bonding) over a wide range of PEG and salt concentrations (0–35% w/w) as well as different PEG molecular weights (282–1471 kDa). We successfully correlated these properties with phase formation resulting in a model that was capable of predicting binodals and tie lines for the systems investigated. We were also able to extract parameters necessary for the use of the Abraham equation which was shown to be capable of predicting partitioning coefficients of proteins. These properties were compared to experimentally determined values measured by using solvatochromic dyes. Both datasets, experimentally and in silico determined phase system properties showed good correlation with partitioning coefficients of lysozyme in these systems.

A new concept for chromatography process development based on high-through put data and mechanistic modeling will be presented in this paper. The concept is established in close cooperation between experimentation, modeling and model-based experimental design and allows for robustness analyses and upscale predictions. It will be demonstrated based on a case study: the optimization of a multicomponent separation (lysozyme, ribonuclease A and cytochrome c on SP Sepharose FFTM), subject to pH conditions and optimal settings for the shape of the elution gradient. Peak resolution and a precise prediction of retention times were chosen as performance variables in the case study to demonstrate the flexibility of the concept. It was shown that the concept of model-integrated process development is simple to perform from miniaturized scale on. The data, derived from model-based optimally designed experiments, provided sufficient information for process development, the model was calibrated and predictions for optimal separation setups as well as for the upscale showed a high precision. Consequently, the accumulation of data from high-throughput screenings can be used profitably for model-based process optimization and upscale predictions.

Protein solubility is a key parameter affecting the performance of every biotechnological downstream processing operation. Current methods used to determine protein solubility data, however, are often characterized by the need of pure protein crystals and long experimental time frames lying in the range of weeks to months, while other methods often fail to reach the solubility limits due to a too high dilution during the generation of the respective experimental solutions. This article presents an efficient, high throughput compatible method—based on photometric turbidity measurements—which allows a gentle and controlled concentration of the solution while constantly analyzing the solution state. All experimental tools were developed using the robotic liquid handling station Tecan Freedom Evo 200. The ability to combine sample concentration and solution state analysis acts as a basis for the determination of process relevant solubility phase diagrams and analysis of relevant kinetic effects during precipitation. Effects of several buffers on protein solubility and solubility curves of different insulin variants are shown as examples of relevant screening parameters.

Merging aqueous two-phase systems (ATPS) with high throughput screening techniques is a promising approach which will certainly lead to a more frequent use of ATPS both in industry and in research. Despite the diverse possible applications of polymer–polymer or polymer–salt based extraction systems for biotechnological downstream processes the use of these systems has so far been limited. This is mostly due to the poor predictability and the large number of parameters influencing the partitioning result. Especially the large number of possible parameter combinations makes robotic high throughput screening an interesting alternative to manual experiments. Methods using the robotic pipetting station Tecan Freedom Evo 200 for the development of ATPS processes are described in this paper. Characteristic values such as tie lines, binodal curve and volumetric measurement are essential for the characterization of this kind of systems and can successfully be determined by the robotic system. Most important, the measurement of protein concentrations, partition coefficients and yields could also be automated and parallelized offering the possibility to design ATPS processes while screening a broad range of parameters with a high sample throughput.

In preparative protein chromatography, broad dynamic ranges of protein concentrations as well as co-elution of product and impurities are common. Despite being the standard in biopharmaceutical production, monitoring of preparative chromatography is generally limited to surrogate signals, e.g. UV absorbance at 280 nm. To address this problem, variable pathlength (VP) spectroscopy in conjunction with Partial Least Squares regression (PLS) was used to monitor preparative chromatography. While VP spectroscopy enabled the acquisition of absorbance data for a broad concentration range, PLS modelling allowed for the differentiation between the protein species. The approach was first implemented for monitoring the separation of lysozyme from cytochrome c at an overall loading density of 92 g/l. The same method was then applied to the polishing step of a monoclonal antibody (mAb) at 40 g/l loading density. For PLS model prediction of the mAb monomer and the high molecular weight variants (HMWs), the root mean square error (RMSE) was 1.07 g/l and 0.42 g/l respectively. To demonstrate the usability of the approach for in-line control, pooling decisions for both separation problems were subsequently taken based on the computed concentrations or thereof derived purities. In summary, VP spectroscopy in conjunction with PLS modelling is a promising option for in-line monitoring and control of future chromatography steps at large scale.