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Multi-attribute PAT for UF/DF of Proteins—Monitoring Concentration, particle sizes, and Buffer Exchange

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Abstract

Ultrafiltration/diafiltration (UF/DF) plays an important role in the manufacturing of biopharmaceuticals. Monitoring critical process parameters and quality attributes by process analytical technology (PAT) during those steps can facilitate process development and assure consistent quality in production processes. In this study, a lab-scale cross-flow filtration (CFF) device was equipped with a variable pathlength (VP) ultraviolet and visible (UV/Vis) spectrometer, a light scattering photometer, and a liquid density sensor (microLDS). Based on the measured signals, the protein concentration, buffer exchange, apparent molecular weight, and hydrodynamic radius were monitored. The setup was tested in three case studies. First, lysozyme was used in an UF/DF run to show the comparability of on-line and off-line measurements. The corresponding correlation coefficients exceeded 0.97. Next, urea-induced changes in protein size of glucose oxidase (GOx) were monitored during two DF steps. Here, correlation coefficients were ≥ 0.92 for static light scattering (SLS) and dynamic light scattering (DLS). The correlation coefficient for the protein concentration was 0.82, possibly due to time-dependent protein precipitation. Finally, a case study was conducted with a monoclonal antibody (mAb) to show the full potential of this setup. Again, off-line and on-line measurements were in good agreement with all correlation coefficients exceeding 0.92. The protein concentration could be monitored in-line in a large range from 3 to 120 g L− 1. A buffer-dependent increase in apparent molecular weight of the mAb was observed during DF, providing interesting supplemental information for process development and stability assessment. In summary, the developed setup provides a powerful testing system for evaluating different UF/DF processes and may be a good starting point to develop process control strategies.

Piping and instrumentation diagram of the experimental setup and data generated by the different sensors. A VP UV/Vis spectrometer (FlowVPE, yellow) measures the protein concentration. From the data of the light scattering photometer (Zetasizer, green) in the on-line measurement loop, the apparant molecular weight and z-average are calculated. The density sensor (microLDS) measures density and viscosity of the fluid in the on-line loop

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Authors and Affiliations

  1. Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Fritz-Haber-Weg 2, 76131, Karlsruhe, Germany

    Laura Rolinger, Matthias Rüdt, Juliane Diehm & Jürgen Hubbuch

  2. Sanofi-Aventis Deutschland GmbH, Industriepark Höchst, 65926, Frankfurt am Main, Germany

    Jessica Chow-Hubbertz, Martin Heitmann & Stefan Schleper

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  1. Laura Rolinger
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  2. Matthias Rüdt
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Correspondence to Jürgen Hubbuch.

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Published in the topical collection Advances in Process Analytics and Control Technology with guest editor Christoph Herwig.

Laura Rolinger and Matthias Rüdt contributed equally to this work.

Appendix: Debye plots

Appendix: Debye plots

Figure 10 shows the regressions curves for the calculation of the second virial coefficient and Table 2 shows the obtained values. Every measurement of the calibration points consists of the average of three runs. The standard deviation of those three runs is not shown in the graph because they were consistently less than 1%. As mentioned in the “Off-line analytics by light scattering” section, five concentration series were measured for GOx and mAb to obtain different A2 for each buffer composition. Due to the instability of GOx in urea buffer and the relatively low concentration, buffer related changes in the A2 were neglected. Instead the A2 of GOx in the feed buffer was used for the whole process. For the mAb concentrations of up to 120 g L− 1 were reached during the second UF. Therefore, an extra A2 for the DF buffer was used because with higher concentrations the A2 value becomes more important for the calculation of the apparent molecular weight.

Fig. 10
figure 10

For the calculation of the second virial coefficient A2, concentration series for lysozyme (a), GOx (b), and mAb (c) were measured by SLS (circles). The gradient of the regression line was divided by two and used as A2. For lysozyme and GOx, the concentration series was only measured with feed buffer (blue). For the mAb, a concentration in both the feed (orange) and DF buffer (blue) was measured

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Table 2 Second virial coefficients A2 from Debye plots
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Rolinger, L., Rüdt, M., Diehm, J. et al. Multi-attribute PAT for UF/DF of Proteins—Monitoring Concentration, particle sizes, and Buffer Exchange. Anal Bioanal Chem 412, 2123–2136 (2020). https://doi.org/10.1007/s00216-019-02318-8

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