(161a) Dynamic Single-Pass Tangential Flow Filtration Technique for Concentration and Purification of Precipitated Antibodies

Continuous precipitation has recently emerged as a promising downstream alternative to Protein A chromatography for capturing highly concentrated monoclonal antibodies (mAbs) with initial titers up to 20 g/L [1]. The precipitated mAbs can then be dewatered and washed (to remove host cell proteins) using single pass tangential flow filtration (SPTFF), which can be easily integrated in a continuous, intensified downstream purification process. However, SPTFF for precipitated mAbs often exhibits low filtrate flux, and thus low single pass conversion, due to severe concentration polarization and fouling [1], limiting the achievable purification factor and requiring the use of multiple membrane stages.

In this study, a dynamic vibration assisted SPTFF system (SANI Membranes, Farum, Denmark) was used to enhance filtration performance of precipitated mAbs. Precipitation conditions were adopted from previous studies by Behboudi et al. [2] using human serum immunoglobulin G (hIgG) as a model protein with zinc chloride and polyethylene glycol used as precipitating agents. Data were obtained with the protein dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) with sodium malonate added to improve the filteratbility of the precipitate [2]. Flux-stepping experiments [3,4] were performed under both vibrating and non-vibrating conditions to evaluate the critical flux and conversion for different concentrations of precipitated hIgG. Our results demonstrate that vibration reduces the extent of concentration polarization and fouling, thereby enabling operation at higher conversion. For example, experiments performed using a 5 g/L hIgG as initial feed gave a critical flux in vibration-assisted SPTFF that was more than 2X higher compared to that obtained in conventional SPTFF (without vibration) using the same membrane module. Long term filtration experiments under vibration showed stable SPTFF performance with a critical flux 70 LMH (feed flux 85 LMH) and concentration factor higher that 6.5 for 24 hours of continuous operation. These results clearly demonstrate the potential of dynamic vibratory filtration to significantly increase the critical flux and conversion during SPTFF of precipitated proteins for low-cost continuous bio-manufacturing.

Reference

[1] M. Minervini, A.L. Zydney, Effect of module geometry on the sustainable flux during microfiltration of precipitated IgG, J. Memb. Sci. 660 (2022). https://doi.org/10.1016/j.memsci.2022.120834.

[2] A. Behboudi, M. Minervini, Z.S. Badinger, W.W. Haddad, A.L. Zydney, Addition of sodium malonate alters the morphology and increases the critical flux during tangential flow filtration of precipitated immunoglobulins, Protein Sci. 33 (2024). https://doi.org/10.1002/pro.5010.

[3] A.S. Chaubal, A.L. Zydney, Single-Pass Tangential Flow Filtration (SPTFF) of Nanoparticles: Achieving Sustainable Operation with Dilute Colloidal Suspensions for Gene Therapy Applications, Membranes (Basel). 13 (2023). https://doi.org/10.3390/membranes13040433.

[4] Z. Li, A.L. Zydney, Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates, Biotechnol. Prog. 33 (2017). https://doi.org/10.1002/btpr.2545.