Methods for removing suspended particles from soluble protein solutions

a technology of suspended particles and soluble protein, which is applied in the direction of solid separation, peptides, water/sewage treatment by ion exchange, etc., can solve the problems of high cost of large-scale centrifugation equipment, protein purification is often a significant challenge, and endanger the health of patients, etc., to avoid the use of expensive capital equipment or chemicals, the effect of rapid and efficient operation and low cos

Inactive Publication Date: 2006-02-02
NV ORGANON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

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  • Methods for removing suspended particles from soluble protein solutions
  • Methods for removing suspended particles from soluble protein solutions

Examples

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example 1

6. EXAMPLE 1

Removing Suspended Particles from E. Coli Lysate Containing SY161

[0055] 6.1. Lysis of E. Coli Cells Expressing SY161

[0056] SY161 may be produced in E. coli strain TG1 transformed with plasmid pMc5-SY161-S3C. This clone represents 13 mutations from the original Staphylokinase gene subcloned from Staphylococcus aureus.

[0057] The E. coli cells expressing SY161 were harvested by centrifugation and stored at −70° C. prior to use. The frozen cell paste was broken into pieces and suspended in about 7.0 volumes (weight / volume) of lysate buffer (50 mM sodium phosphate, pH 9.5 containing 5 mM EDTA) using, an overhead mixer set at between about 500 RPM to about 1000 RPM. Mixing was continued until the cell paste was completely suspended in the lysate buffer. A microfluidizer unit was assembled by connecting the required air pressure lines, coolant lines and hoses. The microfluidizer was then purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to a...

example 2

7. EXAMPLE 2

Removing Suspended Particles from E. Coli Lysate

[0065] 7.1 Lysis of E. Coli Cells that do not Express a Heterologous Protein

[0066]E. coli null cells (E. coli TG1, pMc5-8 (Δ clone)) for expression of SY161 were harvested by centrifugation and stored at −70° C. prior to use. The frozen cell paste was suspended in about 7.0 volumes (weight / volume) of lysate buffer (50 mM sodium phosphate buffer, pH 9.5, containing 5 mM EDTA). The frozen cell paste was stirred for about 0.5 hour with a Silverson Lab Mixer Emulsifier (Model L4R) at about 3,000 rpm to resuspend the cells. A microfluidizer (Model 110Y) was connected to compressed air and the cooling chamber was filled with ice. The homogenizer was purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to about 14,000 psi. The suspended cells were fed into a homogenizer and lysed under the operational pressure of between about 13,000 psi to about 14,000 psi; The once-lysed cell suspension was colle...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B01D15/04
CPCC07K1/34B01J20/14
Inventor WAN, MINRABIDEAU, SUSAN
Owner NV ORGANON
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