Impact of micro and macroporous TFF membranes on product sieving and chromatography loading for perfusion cell culture

Abstract

Bioprocess intensification can be achieved through high cell density perfusion cell culture with continuous protein capture integration. Protein passage and cell retention are commonly accomplished using tangential flow filtration systems consisting of microporous membranes. Significant challenges, including low efficiency and decaying product sieving over time, are commonly observed in these cell retention devices. Here, we demonstrate that a macroporous membrane overcomes the product sieving challenges when comparing to several other membrane chemistries and pore sizes within the microporous range. This way, variable chromatography column loading is avoided. The macroporous membrane yielded a 13,000 L/m2 volumetric throughput. The membrane's cut‐off size results in an increased permeate turbidity due to particles passage, such as cell debris, through pores ranging from 1 to 4 µm. In addition, successful chromatography column plugging mitigation was achieved by employing depth filtration before the chromatographic step. Depth filtration volumetric throughputs were between 600 and 1,000 L/m2. Combing a macroporous cell retention device with a depth filter not only provided an alternative to address the challenge of undesired long protein residence times in the bioreactor due to product sieving decay, but also exhibited a throughput increase, making the integration of multicolumn capture chromatography with a perfusion cell culture a more robust process.

Wide‐surface pore microfiltration membrane drastically improves sieving decay in TFF‐based perfusion cell culture and streamline chromatography integration for continuous bioprocessing

“…Here, both Antibody and DNA average sieving coefficients were both reduced only when employing the 0.65-µm membrane (Figure 3). This finding confirms our previous (Pinto et al, 2020) With the membrane hydraulic permeability (shown in Table 1) and assuming uniform cylindrical pores, the mean pore radius (r p ) was determined using the equation as follows (Zeman & Zydney, 1996):…”

“…Our previous study (Pinto, Napoli, & Brower, 2020) showed an integrated continuous bioprocess with practically nonproduct retention when employing macroporous (>1 µm pore size) perfusion membranes. This observation, corroborated by Wang et al (2017) and Wang et al (2019), highlights that microfiltration membranes retain particles within its pore size range, which leads to product sieving.…”

“…This way, molecules can pass through the membrane pores even with several fold greater lengths than the membrane's nominal retention rating. Although a typical perfusion cell culture operates at permeate fluxes that are theoretically favorable of inducing DNA transmission (Latulippe et al, 2007), severe DNA sieving decay was reported (Pinto et al, 2020). In a potential pertinent study for perfusion cell culture, Li, Borujeni, and Zydney (2015) showed that DNA transmission is enhanced in asymmetric hollow fibers.…”

Performance of an acoustic settler versus a hollow fiber–based ATF technology for influenza virus production in perfusion

“…Disadvantages regarding the use of ATF systems in viral vaccine production due to an accumulation and eventual degradation of virions inside of the bioreactor may be alleviated by selection of membranes that are better suited for this type of application or specifically designed for virus production processes. Similarly to what was tested for membrane-based perfusion culture in recombinant protein production (Esclade et al 1991;Mercille et al 1994;Pinto et al 2019), different membrane chemistries, pore sizes, and properties such as hydrophobicity and surface charge should be characterized regarding product sieving and membrane fouling. For example, Genzel et al (2014) tested various polysulfone and polyether sulfone membranes for influenza virus production in perfusion mode.…”

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

“…Hence, the use of PS membranes can be equally important for related perfusion processes where expressed proteins are considered as product, but retained by PES membranes with a nominal cutoff of 0.2 μm (Karst, Serra, Villiger, Soos, & Morbidelli, 2016;Kelly et al, 2014). Alternatively, other studies identified the use of large-pore membranes of 2 μm and larger as a solution for production retention (Pinto, Napoli, & Brower, 2019;S. B. Wang, Godfrey, Radoniqi, Lin, & Coffman, 2019).…”