Methods for removing suspended particles from soluble protein solutions

Abstract

The present invention provides soluble protein solutions, free of suspended particles in high yield. More particularly, the current invention provides a method for removing suspended particles from soluble protein solutions by filtering the soluble protein solution through highly purified diatomaceous earth.

Description

[0001] This application claims the benefit under 35 U.S.C. § 119 (e) of U.S. Provisional Application No. 60 / 241,967, filed Oct. 19, 2000, which is incorporated herein by reference in its entirety.1. FIELD OF THE INVENTION [0002] The present invention relates to methods for removing suspended particles from soluble protein solutions. In particular, the methods of the invention are useful for removing suspended particles from secreted protein solutions and lysates, including bacterial lysates containing a heterologous protein. 2. BACKGROUND OF THE INVENTION [0003] Proteins play critical roles in functions such as metabolism, gene expression, signal transduction, cellular and extracellular structures, which are essential to the survival and / or reproduction of any living organism. Many proteins may be used in therapeutic and / or diagnostic applications, particularly when available in pure form. Contaminants often prevent realization of therapeutic and / or diagnostic goals and may endanger...

AI Technical Summary

Benefits of technology

[0011] The present invention addresses this need by providing rapid, efficient and inexpensive methods for removing suspended particles from soluble protein solutions. The present invention provides soluble protein solutions, free of suspended particles in high yield, while avoiding the use of expensive capital equipment or chemicals that require expensive disposal.

[0014] Preferably, the amount of DNA and endotoxins in a bacterial lysate is reduced. Then, the lysate is filtered through highly purified diatomaceous earth to remove suspended particles, which dramatically reduces lysate turbidity. In one embodiment, the highly purified diatomaceous earth is packed in a filter press.

[0015] In a preferred embodiment, flocculation with polyethyleneimine at between about pH 7.3 and about pH 7.7 reduces the amount of DNA and endotoxins in the lysate. Preferably, the amount of DNA in the lysate is reduced by between about 100-fold and about 150-fold. In one embodiment, the amount of endotoxins in the lysate is reduced by between about 1,000-fold and about 10,000-fold. In another embodiment, the turbidity of the lysate is reduced by between about 200-fold and about 300-fold.

Problems solved by technology

Contaminants often prevent realization of therapeutic and / or diagnostic goals and may endanger the health of a patient.

Protein purification is often a significant challenge, especially when large amounts of protein are required for therapeutic or diagnostic purposes.

Removing suspended particles from soluble protein solutions is often an important practical problem in purifying proteins of therapeutic or diagnostic significance, particularly when heterologous proteins are expressed in either eukaryotic or procaryotic cells.

However, large scale centrifugation equipment is very expensive capital equipment and is often a limiting factor in removing suspended particles from soluble protein solutions on a process scale.

Major problems with centrifugation include low yields and air entrapment in the supernatant that can lead to substantial protein denaturation.

This incompatibility typically results in the formation of two separate phases of very different compositions.

However, aqueous two-phase extraction is time consuming, expensive and requires large amounts of chemicals, which must be properly disposed in compliance with environmental regulations.

Further, the chemicals used in extraction must be removed from the protein of interest and the two-phase distribution of protein may limit product yield.

Finally, two-phase extraction lacks generality since only a limited number of proteins can be purified by this method.

However, microfiltration on a process scale is a complicated operation that requires precise optimization of a number of variables such as transmembrane pressure, shear force, flow rate, concentration, pH, ionic strength, etc.

Thus, process scale microfiltration frequently requires considerable development time.

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