Evaluation of mild pH elution protein A resins for antibodies and Fc-fusion proteins.

Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. However, the use of low pH (3.0-4.0) to elute the molecule and achieve acceptable yield (>85 %) can lead to product degradation (e.g. fragmentation, aggregation) for molecules sensitive to low pH. In this paper, we describe a comprehensive evaluation of two protein A resins with ligands designed to elute at a milder pH as a result of modified sequences in their Fc and VH3 binding regions. One of the evaluated resins has been made commercially available by Purolite and named Praesto Jetted A50 HipH. Results demonstrated that Jetted A50 HipH could elute the Fc-fusion protein and most mAbs evaluated with an elution pH at or above 4.6. Elution and wash optimization determined run conditions for high recovery (>90 % monomer yield), reduction of high molecular weight (HMW) species (>50 %), and significant host cell protein (HCP) clearance at the mildest elution pH possible. For a pH-stable mAb and a pH-sensitive fusion protein, cell culture material was purified with optimized conditions and demonstrated the mild elution pH resins' ability to purify product with acceptable yield, comparable or better impurity clearance, and significantly milder native eluate pH compared to traditional resins. The benefits of the mild elution pH resins were clearly exemplified for the pH-sensitive protein, where a milder elution buffer and native eluate pH resulted in only 2 % HMW in the eluate that remained stable over 48 h. In contrast, a traditional protein A resin requiring low pH elution led to eluate HMW levels of 8 %, which increased to 16 % over the same hold time. Additionally, these resins have high dynamic binding capacity and allow the use of traditional HCP washes. Therefore, Jetted A50 HipH is an ideal candidate for a platform protein A resin and provides flexibility for pH-sensitive proteins and stable mAbs, while preserving product quality, recovery, and seamless integration into a downstream process.

Scale-up of Sterilizing-grade Membrane Filters from Discs to Pleated Cartridges: Effects of Operating Parameters and Solution Properties.

UNLABELLED: For direct flow sterilizing-grade filtration, a linear scale-up between the performance of discs and pleated filter cartridges has traditionally been assumed. Linear scale-up assumes that the filtration performances, defined here as filter flux and capacity, scale linearly with the membrane area and remains independent of the selected device formats. However, experimental results show that the later assumption does not hold in all cases. In this article, we investigated the effect of solution properties and operating parameters on scale-up with both fouling and non-fouling feeds. For non-fouling solutions, such as buffers, the flux ratio, defined as α, between pleated filter cartridges and disc filters range from 0.5 to 0.85. For complex fouling feeds, such as protein or cell culture media solutions, the ratio of initial flux between pleated filter cartridges and discs was the same as the flux ratio, α. For fouling solutions, the ratio of filtration capacity between pleated cartridges and discs, referred to as capacity ratio, ß, was variable. We found that ß was sensitive to the particle size distribution of the challenge solution and the mode of filtration operation (constant pressure or constant flux), whereas it was less sensitive to the magnitude of the operating pressure or flux and concentration of the fouling species. For most conditions tested, ß among pleated cartridges and discs was within ±20% variation of unity. At the end, we present a modified standard model that accounts for both variations in flux ratio, α, as well as capacity ratios, ß, for estimating the requirement for membrane area at manufacturing scale with proteinacious fouling and non-protein/non-fouling feeds. The data show that for cases where filtration is capacity controlled, flux ratios between the pleated filter and disc are not critical. For such cases, the use of a high-area laid-over pleated cartridge construction allows for reducing the number of 10 inch pleated filter cartridges required to process the batch volume. LAY ABSTRACT: Scale-up remains at the core of a process development. For direct flow sterilizing-grade filtration, a linear scale-up between the performance of discs and pleated filter cartridges has traditionally been assumed. Linear scale-up assumes that the filtration performances, defined here as filter flux and capacity, scale linearly with the membrane area and remains independent of the selected device formats. However, experimental results show that the later assumption does not hold in all cases. We investigated the effect of solution properties and operating parameters on scale-up from membrane disc to pleated filter cartridges. Typical values of flux and capacity ratios and the guidelines on scale-up of direct flow filters from bench-scale to manufacturing-scale are presented. Specifically, we found that the flux ratio for pleated filter and small-scale disc range from 0.5 to 0.85, and capacity ratio for most cases is within ±20% variation of unity, with some exceptions.

Internal virus polarization model for virus retention by the Ultipor(®) VF Grade DV20 membrane.

Several recent studies have reported a decline in virus retention during virus challenge filtration experiments, although the mechanism(s) governing this phenomenon for different filters remains uncertain. Experiments were performed to evaluate the retention of PP7 and PR772 bacteriophage through Ultipor VF Grade DV20 virus filters during constant pressure filtration. While the larger PR772 phage was fully retained under all conditions, a 2-log decline in retention of the small PP7 phage was observed at high throughputs, even under conditions where there was no decline in filtrate flux. In addition, prefouling the membrane with an immunoglobulin G solution had no effect on phage retention. An internal polarization model was developed to describe the decline in phage retention arising from the accumulation of phage in the upper (reservoir) layer within the filter which increases the challenge to the lower (rejection) layer. Independent support for this internal polarization phenomenon was provided by confocal microscopy of fluorescently labeled phage within the membrane. The model was in good agreement with phage retention data over a wide range of phage titers, confirming that virus retention is throughput dependent and supporting current recommendations for virus retention validation studies. These results provide important insights into the factors governing virus retention by membrane filters and their dependence on the underlying structure of the virus filter membrane.