This study aims at defining rheological parameters for the characterization of highly concentrated protein solutions. As a basis for comparing rheological behavior with protein solution characteristics the protein phase behavior of Lysozyme from chicken egg white with concentrations up to 225 mg/mL, changing pH values and additive concentrations was studied in a microbatch scale format. The prepared phase diagrams, scored after 40 days (t40) give insights into the kind and kinetics of the phase transitions that occur. Oscillatory frequency sweep measurements of samples with exactly the same conditions were conducted immediately after preparation (t0). The protein solutions behave viscoelastic and show a characteristic curve shape of the storage modulus (G') and the loss modulus (G″). The graphs provide information about the cross-linking degree of the respective sample. The measured rheological parameters were sensitive concerning solution composition, protein concentration and solution inner structure. The rheological moduli G' and G″ and especially the ratio of these parameters over a frequency range from 100 to 40000 rad/sec give information about the aggregation tendency of the protein under tested conditions. We succeeded to correlate protein phase behavior with the defined rheological key parameter ωCO. This point represents the frequency value of the intersection point from G' and G″. In our study Lysozyme expressed a ωCO threshold value of 20000 rad/sec as a lower limit for stable protein solutions. The predictability of lysozyme aggregation tendency and crystallization by means of squeeze flow rheometry is shown.
Nowadays, the performance of experiments in automated microliter scale format is common practice in the biopharmaceutical process development. The increased number of experiments, reduced sample volumes, and usage of robotic platforms require the adjustment of photometric measurements to determine the protein concentration. This work presents the qualification and usage of a disposable measurement device that can be used with conventional microplate photometers. The application of the microfluidic device (μF‐device) allows absorption measurements of protein concentrations from around 0.1 to 100 mg/mL with an accuracy of 99.2% dependent on given protein extinction coefficients. The integrated four measurement chambers of increasing height (100–1500 μm) allow the direct calculation of calibration curves and the determination of protein concentrations independent of used optical path lengths with a sample volume of 36 μL. This study contains the validation of the analytical μF‐device according to ICH Guidelines as well as a representative case study. A salt gradient screening with chromatography columns in microliter scale performed on a liquid handling station presents the usability of the μF‐device. It is shown that an improvement of the repeatability and accuracy of the chromatograms could be achieved by μF‐device implementation in comparison to photometric measurements performed in microtiter plates.
The development of biotechnological processes is challenging due to the diversity of process parameters. For efficient upstream development, parallel cultivation systems have proven to reduce costs and associated timelines successfully while offering excellent process control. However, the degree of automation of such small-scale systems is comparatively low, and necessary sample analysis requires manual steps. Although the subsequent analysis can be performed in a high-throughput manner, the integration of analytical devices remains challenging, especially when cultivation and analysis laboratories are spatially separated. Mobile robots offer a potential solution, but their implementation in research laboratories is not widely adopted. Our approach demonstrates the integration of a small-scale cultivation system into a liquid handling station for an automated cultivation and sample procedure. The samples are transported via a mobile robotic lab assistant and subsequently analyzed by a high-throughput analyzer. The process data are stored in a centralized database. The mobile robotic workflow guarantees a flexible solution for device integration and facilitates automation. Restrictions regarding spatial separation of devices are circumvented, enabling a modular platform throughout different laboratories. The presented cultivation platform is evaluated on the basis of industrially relevant E. coli BW25113 high cell density fed-batch cultivation. The necessary magnesium addition for reaching high cell densities in mineral salt medium is automated via a feedback operation loop between the analysis station located in the adjacent room and the cultivation system. The modular design demonstrates new opportunities for advanced control options and the suitability of the platform for accelerating bioprocess development. This study lays the foundation for a fully integrated facility, where the physical connection of laboratory equipment is achieved through the successful use of a mobile robotic lab assistant, and different cultivation scales can be coupled through the common data infrastructure.
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