Methods of protein fractionation using high performance tangential flow filtration

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The present invention is directed to an improved method of fractionation of molecules of interest from a given feedstream. It should be noted that the production of large quantities of relatively pure, biologically active molecules is important economically for the manufacture of human and animal pharmaceutical formulations, proteins, enzymes, antibodies and other specialty chemicals. In the production of many polypeptides, antibodies and proteins, various recombinant DNA techniques have become the method of choice since these methods allow the large scale production of such proteins. The various "platforms" that can used for such production includes bacteria, yeast, insect or mammalian cell cultures and transgenic animals. For transgenic animal systems, the preferred animal type is production in mammals, but this platform production method also contemplates the use of avians or even transgenic plants to produce exogenous proteins, antibodies, or fragments or fusions thereof. Producing recombinant protein involves transfecting host cells with DNA encoding the protein and growing the host cells, transgenic animals or plants under conditions favoring expression of the recombinant protein or other molecule of interest. The prokaryote - E. coli has been a favored host system because it can be made to produce recombinant proteins in high yields. However, numerous U.S. patents on the general expression of DNA encoding proteins exist, for a variety of expression platforms from E. coli to cattle have been developed.With improvements in the production of exogenous proteins or other molecules of interest from biological systems there has been increasing pressure on industry to develop new techniques to enhance and make more efficient the purification and recovery processes for the biologies and pharmaceuticals so produced. That is, with an increased pipeline of new products, there is substantial interest in devising methods to bring these therapeutics, in commercial volumes, to market quickly. At the same time the industry is facing new challenges in terms of developing novel processes for the recovery of transgenic proteins and antibodies from various bodily fluids including milk, blood and urine. The larger the scale of production the more complex these problems often become. In addition, there are further challenges imposed in terms of meeting product purity and safety, notably in terms of virus safety and residual contaminants, such as DNA and host cell proteins that might be required to be met by the various governmental agencies that oversee the production of biologically useful pharmaceuticals. Several methods are currently available to separate molecules of biological interest, such as proteins, from mixtures thereof. One important such technique is affinity chromatography, which separates molecules on the basis of specific and selective binding of the desired molecules to an affinity matrix or gel, while the undesirable molecule remains unbound and can then be moved out of the system. Affinity gels typically consist of a ligand-binding moiety immobilized on a gel support. For example, GB 2,178,742 utilizes an affinity chromatography method to purify hemoglobin and its chemically modified derivatives based on the fact that native hemoglobin binds specifically to a specific family of poly-anionic moieties. For capture these moieties are immobilized on the gel itself. In this process, unmodified hemoglobin is retained by the affinity gel, while modified hemoglobin, which cannot bind to the gel because its poly-anion binding site is covalently occupied by the modifying agent, is removed from the system. Affinity chromatography columns are highly specific and thus yield very pure products; however, affinity chromatography is a relatively expensive process and therefore very difficult to put in place for commercial operations. Typically, genetically engineered biopharmaceuticals are purified from a supernatant containing a variety of diverse host cell contaminants. Reversed-phase high-performance liquid chromatography (RP-HPLC) can be used for protein purification because it can efficiently separate molecular species that are exceptionally similar to one another in terms of structure or weight. Moreover, in another industry that faces some of the same challenges new answers are needed. The dairy industry has been one of the greatest advocates of using membrane systems for fractionation, clarification and purification using the technology since its beginning to concentrate and fractionate whey, as well as treat wastewater, In the 1980s, researchers in the dairy industry began using membranes to concentrate milk for use in the production of non-standardized cheese. In recent years, improved technologies are making membrane-concentrated milk more attractive than ever. At the same time, technological advancements in membrane materials, process engineering and functionality of milk constituents have made membrane separation processes practical and useful at nearly every stage of milk treatment. Though these practices cannot yet be applied to all facets of the dairy industry, their potential is immense.For example, membrane separation may be particularly attractive to fluid milk processors in the future because it demands little energy and does not destroy any product during treatment. Four basic types of membrane filtration present potential applications for the dairy industry- reverse osmosis (PC), nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF) - each serving a different purpose. Some application processes involve only a single membrane; however, advanced approaches are using two or more membrane processes in a given application. However, these processes, though useful, are still limiting with regard to some aspects of the dairy industry, food preparation industry and biopharmaceutical production in transgenic animals. In both the biotech industry and in the dairy industry ultrafiltration has traditionally been used for size-based separation of protein mixtures where the ratio of the protein molecular masses have to be at least around 10 to 1. This has been a limiting factor in many industrial applications throughout industry and in particular in the recovery of biopharmaceuticals in the milk of transgenic mammals. Significant research has taken place in the optimization of ultrafiltration systems by altering the physiochemical conditions (i.e. pH and ionic strength) to achieve higher selectivities (Van Reis et al. (1997)). According to the methods of the current invention improvements have been made to optimize conditions more in the direction of pH and ionic strength to make possible the development of high-performance tangential flow filtration (HPTFF) in various feedstreams including milk.HPTFF exploits multiple phenomena to maximize separation performance. These include the manipulation of solution pH and ionic strength to maximize differences in solute effective volumes as well as the use of membranes with controlled pore size.As mentioned, current industrial and biopharmaceutical processes often use ion-exchange chromatography, UF and size exclusion chromatography (SEC) in three separate steps for purification, concentration and buffer exchange. However, even in conjunction with one another, these processes are limited in terms of what they can separate. Even ultrafiltration (UF) is generally limited to separation of solutes that differ by at least tenfold in size. In addition, molecular species that are similar in charge can also be very difficult to separate. 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.Molecules that differ less than threefold in size can he separated through the use of highly selective charged membranes and careful optimization of buffer and fluid dynamics. Knowledge of the isoelectric point (pi) of the desired molecule of interest is the main factor in HPTFF. This will then dictate membrane setup and the intrinsic charge profile of the membrane, pore size, and flow characteristics. Moreover, HPTFF makes it possible to perform all of these steps in a single-unit operation, thereby reducing production costs. In addition, HPTFF uses the same linear scale-up principles already established for UF. HPTFF is also assisted by optimizing the trans-membrane pressure.Depending on membrane type, it can be classified as microfiltration or ultrafiltration. Microfiltration membranes, with a pore size between 0.1 and 10 μm, are typically used for clarification, sterilization, removal of microparticulates, or for cell harvests. Ultrafiltration membranes, with much smaller pore sizes between 0.001 and 0.1 μm, are used for separating out and concentrating dissolved molecules (protein, peptides, nucleic acids, carbohydrates, and other biomolecules), for exchange buffers, and for gross fractionation. However, limitations exist on the degree of protein purification achievable in ultrafiltration. These limits are due mainly to the phenomena of concentration polarization, fouling, and the wide distribution in the pore size of most membranes.