High-sensitivity proteolysis assay

Abstract

The present invention includes a highly sensitive method for detecting the presence of proteases in a sample which are present at very low levels.

Description

[0001]This application claims the benefit of U.S. provisional patent application No. 60 / 874,191; filed Dec. 11, 2006, which is herein incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates, inter alia, to assays and compositions for detecting proteolytic activity in a sample.BACKGROUND OF THE INVENTION[0003]When recombinantly expressing peptides such as antibodies, vaccines or other therapeutic polypeptides (e.g., interferon or erythropoietin), plant-derived hydrolysates are often added to the cell culture media in order to increase the titer. The small peptide chains in the hydrolysates are derived from protease-driven degradation of soy and wheat by-products of the food industry which results in the unintended introduction of proteases to the culture. The presence of the proteases leads to in culture degradation of the antibodies, particularly if engineered to be secreted into the culture. Later purification is also complicated if the p...

AI Technical Summary

Benefits of technology

[0007]The present invention provides a method for determining the presence of proteolytic activity (e.g., a protease) in a sample comprising incubating the sample with a substrate for said protease 8 or more hours and determining proteolysis of said substrate. In an embodiment of the invention, the method comprising the steps of: (a) combining the sample with a peptide substrate and, optionally, with a reducing agent; (b) incubating the sample for at least 8 hours at room temperature (e.g., about 22° C. or 28° C. (e.g., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C.)); and (c) determining proteolysis of the substrate (e.g., by heating the sample, substrate and buffer to at least 90° C. for at least 5 minutes, electrophoresing the sample, substrate and buffer on a SDS-polyacrylamide gel and staining the gel with a protein indicator stain. In an embodiment of the invention, the gel is a 4-12% or 4-20% discontinuous SDS-polyacrylamide gel. In an embodiment of the invention, about 1

Problems solved by technology

The small peptide chains in the hydrolysates are derived from protease-driven degradation of soy and wheat by-products of the food industry which results in the unintended introduction of proteases to the culture.

The presence of the proteases leads to in culture degradation of the antibodies, particularly if engineered to be secreted into the culture.

Later purification is also complicated if the proteases are not sufficiently inactivated or removed and are, instead, carried over, in an active form, to the purified product.

The variability makes removal or inactivation of the proteases more difficult.

For this reason, complete inactivation is not always successful.

Some enzymes are not removed during this step due to inefficiency associated with ultrafiltration when used for removal of polypeptides from a sample.

The presence of even very low levels of proteases in a peptide product can cause significant problems with respect to the quality and stability of the product over time.

Slow proteolytic degradation of the product is a problem that must be monitored in order to both track product quality and stability and to develop purification procedures for complete removal of protease contamination from the product.

There are known methods for detecting proteases in a sample, however, the sensitivity of such assays is relatively low.

Since the quantity of protease present in a sample of purified antibody product is, generally, very low, the known protease assays are insufficient.

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