Purification of butyrylcholinesterase using membrane adsorption

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The present invention relates to purification of butyrylcholinesterase using anion exchange material, where the butyrylcholinesterase content is enriched at least 10 fold per total protein in the composition.

This application claims priority to U.S. Provisional Application Serial

BACKGROUND OF THE INVENTION

Toxic organophosphorous (OP) agents pose a risk in both civilian and military contexts. OP agents include nerve gases (e.g., soman, sarin, tabun, VX), pesticides, and cocaine. These agents are believed to act by irreversibly inhibiting acetylcholinesterase, which can result in broncho-constriction, respiratory failure, and death. The cholinesterase polypeptides acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) have been successfully applied following exposure to these agents, as well as prophylactically (Doctor et al. (2001) Chemical Warfare Agents: Toxicity at low levels, pp 191-214). hi particular, human BuChE has been shown to protect against a wide range of agents. Human BuChE can be used prophylactically without additional post-exposure treatment, and it has a long half- life in humans, rodents, and primates (Ostergaard et al. (1988) Acta Anaesth. Scand., 32:266- 69; Raveh et al. (1993) Biochem. Pharmacol. 45:2465-74; Raveh et al. (1997) Toxicol. Appl. Pharmacol. 145:43-53; Allon et α/. (1998) Toxicol. ScL 43:121-28). Because the enzyme comes from a human source, the risk of an adverse immune response is minimized.

Previous efforts at purifying cholinesterase enzymes have relied on anion column chromatography from plasma or Cohn Fraction IV paste (see e.g., Granwald et al. (1997) J. Biochem. Biophys. Methods 34:123-35; Lockridge et al. (2005) J Med Chem Biol Radiol. 3:nihms5095). For large-scale production these methods would require employing cumbersome chromatography column packing procedures and large amounts of buffers.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the finding that large amounts of cholinesterase proteins can be purified with high efficiency using anion exchange membranes. Membranes offer a number of advantages over column chromatography for large-scale protein purification. Membranes can tolerate faster flow rates, reducing the process time for purification. Membranes have higher dynamic binding capacity, so that smaller adsoption media volumes are used for the same amount of total protein loaded. In addition, smaller volumes of buffers will be required per lot produced. Membranes are easier to scale up than columns, making pilot scale developments and optimization efforts more predictable and relevant for large scale manufacturing processes. Membranes are also easier to use as they do not require cumbersome packing procedures associated with large production scale columns.

In some embodiments, the invention provides methods of making an enriched butyrylcholinesterase (BChE) composition from a biological source having BChE, comprising the steps of applying the biological source having BChE to an anion exchange material; washing the material; and eluting BChE from the anion exchange material, wherein the BChE is enriched after anion exchange at least 10 fold per total protein in the composition as measured by activity per total protein. In some embodiments, BChE is enriched at least 20, 40, 60, 70, 80, 90, 100, 150, 200, or more per total protein, as measured by activity per total protein. In some embodiments, the method is applied to large-scale production of BChE.

In some embodiments, the biological source is a biological fluid, for example, blood or a blood fraction. In some embodiments, the biological fluid is selected from the group consisting of plasma, serum, and Cohn fraction IV or subfractions thereof. In some embodiments, the biological source is milk (e.g., from a transgenic animal), a transgenic plant or plant cell, or a recombinant cell. In some embodiments, the recombinant cell is a HEK, COS, C 127, or CHO cell. In some embodiments, the biological source is an organ, e.g., liver or kidney. In some embodiments, the biological source is a mammal, e.g. , human, rabbit, horse, monkey, cow, goat, sheep, rat, or mouse.

In some embodiments, the BChE is affinity purified after the step of eluting from the anion exchange material. In some embodiments, the affinity ligand is selected from the group consisting of a monoclonal antibody, a cocaine analog, and procainamide.

In some embodiments, the biological source is in liquid form and filtered before application to the anion exchange material. In some embodiments, the biological source material is subjected to solvent-detergent treatment. In some embodiments, the biological source is Cohn fraction IV, wherein the Cohn fraction IV is contacted with a fumed silica compound, adjusted to pH 4.0-4.5, and filtered through a filter media. In some embodiments, the anion exchange group (i.e. , the functional group) is attached to a membrane or a resin. In some embodiments, the anion exchange group is attached to a membrane. In some embodiments, the anion exchange group is a quaternary amine (Q) or diethylaminoethane (DEAE). In some embodiments, two or more anion exchange membranes are connected in series.