Large scale purification of viruses and viral vectors for gene therapy applications and viral vaccines is a major separation challenge. Here tangential flow microfiltration and ultrafiltration using flat sheet membranes has been investigated for concentration of human influenza A virus. Ultrafiltration membranes with molecular weight cutoffs of 100 and 300 kDa as well as 0.1, 0.2 and 0.45 microm microfiltration membranes have been tested. The results indicate that use of 300 kDa membranes not only concentrate the virus particles but also lead to a significant removal of host cell proteins and DNA in the permeate. Tangential flow filtration may be used to fractionate virus particles. Human influenza A virus particles are spherical with an average size of 100 nm. Use of a 0.1 microm membrane leads to passage of virus particles less than 100 nm into the permeate and an increase of larger particles in the retentate. These results suggest that control of the transmembrane pressure, membrane pore size and pore size distribution could enable isolation of intact virus particles from damaged virions. Isolation of the virus particles of interest from viral fragments and other particulate matter could result in simplification of subsequent purification steps. Larger pore size membranes such as 0.45 microm that allow the passage of all virus particles may be used to remove host cell fragments. In addition virus particles attached to these fragments will be removed. Careful selection of membrane morphology and operating conditions will be essential in order to maximize the benefit of tangential flow filtration steps in the purification of viral products from cell cultures.

A new general approach for improving polymer substratum biocompatibility is proposed. In a first example, polysulfone (PSf) film was modified by covalent end-on grafting of poly(ethylene glycol) (PEG) (2, 5, and 10 kDa) using well-defined, photoreactive α-4-azidobenzoyl β-methoxy PEG conjugates (ABMPEG). After adsorption from aqueous solution, ABMPEG was photografted under wet conditions onto PSf, where the degree of surface functionalization could be controlled through the applied ABMPEG concentration during adsorption. Attained surface characteristics, after changing systematically ABMPEG concentration, molecular weight, and the ratio of binary ABMPEG mixtures, were monitored by air−water contact angles (CA, captive bubble method) and partially also by X-ray photon spectroscopy (XPS). For ABMPEG 10 kDa adsorption kinetics and grafting efficiency as a function of applied concentration were evaluated by both CAs and fibronectin (FN) adsorption (in situ ellipsometry) to surfaces modified at different degrees of functionalization. CAs attained equilibrium values only after about 1−2 h, suggesting that surface organization processes retard ABMPEG adsorption. FN adsorption decreased monotonically as the degree of surface functionalization increased. Human skin fibroblast interaction with ABMPEG 10 kDa functionalized PSf films was studied, and a clear optimum of fibroblast−material interaction on mildly modified surfaces could be found based on the number of adhering cells, but also on morphological criteria including overall cell morphology, cell spreading, and formation of focal adhesion contacts, visualized by fluorescent staining of vinculin. The results suggest that adhesive proteins such as FN are adsorbed in a biologically active state yielding enhanced cell−substratum interaction when a hydrophobic substratum is surface modified at an intermediate degree with hydrophilic, flexible, sterically demanding, and possibly “self-assembled” macromolecules, e.g., PEG. Presumably, those macromolecules exert a lateral pressure upon neighboring adsorbed adhesive proteins, yielding surface bound but in their active conformation stabilized proteins with high biological activity.

A novel approach described earlier for improving polymer substratum biocompatibility(1) is further elucidated. Polysulfone (PSf) spin-coating films were modified by covalent end-on grafting of hydrophilic and sterically demanding photo-reactive poly(ethylene glycol) (PEG) conjugates (ABMPEG; 10 kDa). The degree of grafting density was varied systematically, yielding a wide spectrum of attained surface characteristics monitored by air-water contact angles (captive bubble method). Fibronectin (FN) adsorption was studied by in situ ellipsometry and found to decrease monotonically as ABMPEG grafting density increased. The adhesive interaction of human skin fibroblasts with these substrata and, in particular, the effect of FN precoating were investigated in detail. A clear optimum of cell-substratum interactions was found for mildly modified substrata, employing well established microscopic and immunofluorescence techniques, namely the monitoring of cell adhesion and spreading, overall cell morphology, organization of FN receptors, and focal adhesions as well as FN matrix formation. The results suggest that cell interactions with hydrophobic polymer substrata are enhanced considerably when modified with hydrophilic and sterically demanding PEG moieties at a low surface coverage due to enhanced biologic activity of adsorbed and intercalated adhesive proteins such as FN.