Glen Reed Bolton

Normal‐flow virus filtration: detection and assessment of the endpoint in bioprocessing

The breakthrough of a model virus, bacteriophage ΦX‐174, through normal‐flow virus filters was studied using both commercial process fluids and model feedstreams. The results indicate that (i) ΦX‐174 is a reasonable model for a mammalian parvovirus [MMV (murine minute virus)] in virus filtration studies; (ii) ΦX‐174 LRV [log(reduction value)] shows a better correlation with percentage flow decline compared with volume processed under a variety of conditions; (iii) although the extent of decline in virus LRV is dependent on the mechanism of filter fouling, the fouling mechanisms operative in a viral validation study are representative of those likely to be found under actual production conditions. The mechanism of LRV decline by many process streams was proposed to be due to selective plugging of small pores. A theoretical model as well as a predictive equation for LRV decline versus flow decay was derived; experimental results from filtration studies using pore‐plugging feedstocks were consistent with the equation. As protein solutions may vary in their adsorptive versus plugging behaviour during filtration, an evaluation of the LRV‐versus‐flow‐decay relationship on a biopharmaceutical‐product‐specific basis may be warranted.

High-performance tangential flow filtration: a highly selective membrane separation process☆

Abstract High-performance tangential flow filtration (HPTFF) is a new technology for protein and nucleotide purification. Conventional ultrafiltration (UF) is limited to separation of solutes that differ by tenfold in size. HPTFF is a two-dimensional purification method that exploits differences in both size and charge characteristics of biomolecules. It is hence possible to separate biomolecules with the same molecular weight. It is even possible to retain one biomolecule while passing a larger molecular weight species through the membrane. Current processes often use ion-exchange chromatography, UF and size exclusion chromatography (SEC) in three separate steps for purification, concentration and buffer exchange. HPTFF makes it possible to perform all of these steps in a single-unit operation, thereby reducing production costs. HPTFF uses the same linear scale-up principles already established for UF. Since HPTFF builds on existing UF technology, there is already a well-established industrial infrastructure in place for implementation of HPTFF processes. HPTFF can provide high-resolution purification while maintaining the inherent high throughput and high yield characteristics of conventional UF. HPTFF can therefore be used in initial, intermediate, and final purification stages. This presentation overviews the key aspects of this technology in terms of operation and optimization and reviews several real-world separations in biotechapplications such as IgG and monoclonal antibodies.

Increasing the capacity of parvovirus-retentive membranes : performance of the Viresolve Prefilter

Removal of small parvoviruses from highly purified proteins can be performed using normal‐flow filters. The entrapment of protein aggregates, denatured proteins and other impurities can cause plugging and a decrease in filter capacity. In the present study a variety of prefilters were investigated for their ability to remove the species that foul Viresolve™ NFP (normal‐flow parvovirus) filters. The Viresolve™ Prefilter, which utilizes entrapped diatomaceous earth to hydrophobically bind fouling species, provided a dramatic increase in virus filter capacity for solutions containing human IgG or a variety of monoclonal antibodies. We found that the component of the human IgG stream that bound to the Prefilter, when analysed using SDS/PAGE, isoelectric‐focusing, size‐exclusion chromatography, CD and ANS (1‐anilinonaphthalene‐8‐sulphonate) titration, consisted of monomeric IgG variants containing more exposed hydrophobic surfaces. The bound component may represent oxidized or otherwise degraded IgG species or a subset of IgG molecules with more hydrophobic antigen‐binding surfaces. The results indicate that NFP membranes do not foul solely as a result of entrapment of protein aggregates in the pore structure. The Viresolve™ Prefilter has a high permeability, did not diminish protein yield and provided consistent performance between different media lots, device lots and device scales.