The stability of pharmaceutical solutions containing proteins is dependent on various factors and especially on the type of pretreatment to which it has been subjected. It is very important that various types of contamination be removed from these solutions, as regulatory authorities require numerous mandatory controls for the removal of contaminants. Contamination with bacteria or fungi can be easily prevented when the pharmaceutical solution is filtered with a sterile ultrafiltration membrane having for instance a nominal pore diameter of 0.2 μm. Viruses can be extracted by a chemical treatment or by the use of a strongly basic ion exchanger. Endotoxins can be also removed with a basic ion exchanger or by ultrafiltration.
Proteases are enzymes which break up proteins and polypeptides by hydrolytically splitting the amino acids which are the building blocks of the proteins. When proteases are present in a pharmaceutical formulation containing a protein such as an antibody, there is a loss of the desired antibody and decomposition products are produced, which cause undesirable side effects in patients who are treated with such a pharmaceutical formulation. During the processing (Down Stream Processing) of a target protein, produced, for example, by gene technology, antibodies can accumulate in the cultured cells and must be separated before further processing. Intrinsic cell proteases are also simultaneously released during lysing of the cells, which can immediately break up the target protein.
In order to prevent or at least delay the deleterious effects of proteases, it is known that small synthetic molecules can be employed which have an inhibitory effect and a very high affinity for the active center of the proteases. A disadvantage in this case is the potential danger presented by such synthetic protease inhibitors, as well as their limited solubility and low stability in aqueous media. That is why a quick and efficient distribution of such protease inhibitors in large volumes is complicated. It is also known to those skilled in the art that chromatographic carriers, such as spherical gels, may be used to immobilize protease inhibitors. Since the removal should occur as far as possible “up stream” in the purification sequence in order to keep the production loss low, large diameter chromatographic columns are required for the processing, making such processing costly and labor-intensive.
It is known from U.S. Pat. No. 6,248,238 that a cationic protease inhibitor can be deployed by means of bulk adsorption onto the surface of a semipermeable membrane comprising a negatively-charged polymer. A disadvantage of such a deployment is that the membrane used is not electrically neutral, but instead, is negatively charged with the monomer used, often causing irreversible binding.
DE 44 32 628 A1 discloses a dead-end filtration module for the selective separation of substances from fluids by filtration on porous membrane adsorbers. The individual substances to be separated are retained on the filter cassettes or membranes in accordance with a specific adsorption provided by ion exchange or by membranes carrying pigment ligands. The adsorbed substances are selectively desorbed, eluted and absorbed with suitable elution fluids. However, it is difficult to bind all classes of known proteases to such membrane adsorbers.
Accordingly, the principal object of the present invention is to provide membranes for the quick, efficient and inexpensive removal of a broad spectrum of proteases including acid proteases, metalloproteases, cystein proteases and serine proteases from biological fluids and pharmaceutical liquids so that their deleterious effects may be prevented or at least delayed.