(360f) Confocal Imaging of Protein Gel Layer Formation during Tangential Flow Filtration to Inform Process Conditions Reducing Protein Losses and Increasing Protein Concentration

There are about 570 antibody therapeutics at various phases of clinical trials across the biopharmaceutical industry with 51 mAbs and 11 biosimilars in late-stage trials (Kaplan and Reichart, 2019). Of these 93% are produced in the US and Europe, and half are based on fully human genetic sequences (Grilo and Mantalaris, 2019). Therapeutic levels of monoclonal antibodies (mAbs) lie between 5 to 1000 mg per patient per treatment (Garidel et al., 2017; Maruthamuthu, 2020). Hence, mAbs must be concentrated to high levels (generally in the range of 150 to 200 mg/mL) to achieve therapeutic concentrations and/or reduce dosage volume (Garidel et al., 2017).

Tangential flow filtration (TFF) is the major unit operation in biopharma manufacturing for concentration of proteins. TFF uses shear forces along the membrane surface to limit protein accumulation on the membrane during the ultrafiltration process that occurs at pressures of up to 60 psig. Decreases in flux through the membrane correlate to increases in viscosity and osmotic pressure that occur when bulk protein concentration increases (Baek and Zydney, 2018). In addition, protein accumulation on the membrane’s surface results in a gel layer that causes resistance to flux. Mechanisms of gel layer formation are complex and may depend on flow patterns, cross-membrane velocities, and biophysical properties that are a function of protein concentration, molecular weight, and pI.

We report a prototype confocal membrane chamber that enables observation of protein layer development on the surface of the membrane and along the length of the ultrafiltration module. This optical flow cassette was fabricated from a commercially available TFF cassette (Ultracel 30 kD_a pellicon 3) to expose the feed screen and the membrane underneath. A thin, optically transparent plexiglass cover placed over the surface of the feed screen forms a viewable feed channel enabling confocal imaging within a 4mm, optical path length. Flow passes across the membrane through a 1 mm high channel (distance from window to membrane surface). The cover was glued in place along the edges to form a pressure tight seal resulting in an operational replica of a bench-top TFF cassette with a membrane surface area of 44 cm².

A solution of Bovine IgG (50 mg/mL; in 10 mM citrate buffer with 50 mM NaCl, pH 5.5) was doped with a fluorescently labeled protein and concentrated in this prototype optical flow cell. This protein was used since its molecular weight of 150 kD is in the same range of molecular weights of many therapeutic mAbs presented in the literature, with a pI of 7.2 that lies in the mid-range of therapeutic mAbs whose pI’s range from 6 to 9. The accumulation of protein on the membrane surface was captured with z-stack imaging. Together with separate measurements of flux, retentate concentration, transmembrane pressure, and flow rates, these images are helping to inform development of a computational model for predicting conditions that maximize protein concentration and recovery (Rossi et al., 2021). In this paper, we report visualization and characteristics of a gel layer at TFF membrane surfaces for bulk protein concentrations ranging from 50 to 200 mg/mL for process conditions using confocal microscopy.

References

Baek, Y. and A. Zydney. (2018). Intermolecular interactions in highly concentrated formulations of recombinant therapeutic proteins, Current Opinion in Biotechnology, 53, 59-64.

Garidel, P., Kuhn, A. B., Schäfer, L. V., A. R., Karow-Zwick, & M. Blech. (2017). High-concentration protein formulations: how high is high? European Journal of Pharmaceutics and Biopharmaceutics, 119, 353-360.

Grilo, A. L., A. Mantalaris. (2019). The increasingly human and profitable monoclonal antibody market, Trends Biotechnol., 37, 9-15.

Kaplon, H., J. M. Reichart. (2019). Antibodies to watch in 2019, Mabs, 11, 219-238.

Maruthamutu, M. K., S. R. Rudge, A. Ardekani, M. R. Ladisch, and M. S. Verma. (2020). Process Analytical Technologies and Data Analytics for the Manufacture of Monoclonal Antibodies, Trends in Biotechnology, 38(10), 1169-1186.

Rossi, F., J. Zuponcic, E. Ximenes, S. Geng, Y. Taa, V. Corvari, M. Ladisch, R. Reklaitis. (2021). “Dynamic Optimization of an Ultrafiltration System for the Concentration of Monoclonal Antibody Solutions under Uncertainty,” Abstract submitted to Annual AIChE meeting.