Pepsin, Puried

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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-A white or yellowish-white powder, spongy mass, or translucent scales or granules. Freely soluble in water, producing more or less opalescence; practically insoluble in alcohol, in chloroform, and in ether. Purified Pepsin used in the second tier of the Dissolution test has an activity that is determined by the following method.

1 Activity

1.1 Pepsin solution

Transfer about 2.5 mg of Purified Pepsin, accurately measured, to a 100-mL volumetric flask, dilute with 10 mM hydrochloric acid to volume, and mix.

[NOTE-Prepare immediately before use.]

1.2 2.0% Hemoglobin solution

Dissolve and dilute 2.5 g of bovine hemoglobin with water to 100 mL. Dilute 80 mL of this solution with 0.3 M hydrochloric acid to a volume of 100 mL.

1.3 Trichloroacetic acid solution

Dilute 5 g of trichloroacetic acid with water to 100 mL.

1.4 Test Solution

Transfer 5.0 mL of 2.0% Hemoglobin solution to a suitable container equilibrated at 37°. Add 1.0 mL of Pepsin solution, mix by swirling, and incubate at 37° for 10 minutes. Immediately add 10.0 mL of Trichloroacetic acid solution, mix by swirling, and incubate at 37° for 5 minutes. Pass through a filter having a 0.8-µm or finer porosity.

1.5 Control solution

Transfer 5.0 mL of 2.0% Hemoglobin solution to a suitable container equilibrated at 37°. Mix by swirling, and incubate at 37° for 10 minutes. Immediately add 10.0 mL of Trichloroacetic acid solution and 1.0 mL of Pepsin solution, mix by swirling, and incubate at 37° for 5 minutes. Pass through a filter having a 0.8-µm or finer porosity.

1.6 Procedure

Determine the absorbances of the Test solution and Control solution, in 1-cm cells, at a wavelength of about 280 nm, using water as the reference. Calculate the activity of the portion of Purified Pepsin taken by the formula:

10,000(A_U - A_C)

in which A_U  and A_C are the absorbances of the Test solution and the Control solution, respectively.