Development and Validation of Reversed Phase HPLC Method for Analysis of Immunotherapeutic Peptide PADRE and its Applications

Order of Publishing in Issue: 
4
Volume :6
Issue :1
January, 2012 - March, 2012
Page No: 
35-43
Authors: 
Srinivas Poondru[1,2], Venugopal Marasanapalle [1,3], Poonam Saraf [1] and Bhaskara Jasti [1*]
Address: 
[1] Thomas J. Long School of Pharmacy & Health Sciences, University of the Paciûc, 3601 Paciûc Avenue, Stockton, CA 95211, United States
Address: 
[2] OSI Pharmaceuticals, Inc., Deerfield, IL, USA
Address: 
[3] Forest Laboratories, New York, NY, USA
Email-ID: 
bjasti@pacific.edu

Abstract

A reversed phase high performance chromatography (RP-HPLC)method was developed and validated for the quantification of PADRE, a peptide with proven immunogenic activity in vitro.The developed method was used to determine PADRE stability within biomatrices like plasma, intact tumor and tumor homogenate in vitro and to determine PADRE release from its PLGA base dmicroparticle formulation.A reversed phase C8 column was used and the elution was performed using a concave gradient flow. Mobile phase consisted of acetonitirile and 0.01M phosphate buffer at 10:90 ratio containing 0.01% trifluoroacetic acid, adjusted to pH 3, was increased using a concave gradient to 40:60 v/v in 15 minutes. The retention time of PADRE was found to be 10.4 minutes. The method was linear in the range of 0.5-100 ìg/ml and the regression coefficient was obtained as 0.9997. The method was found to be accurate with recovery in the range of 97.80 and 104.64%. Precision ofthe proposed method was established by determination of intra day and inter day variability using standard solutions. The percentage relative standard deviation values were all within acceptable range. The detection and quantitation limit for the method were found to be 0.156ìg/ml and 0.5 ìg/ml. Utilizing the proposed method, degradation profiles of PADRE within biomatrices were obtained. PADRE exhibited rapid and maximum degradation within plasma incomparison to intact tumor or tumor homogenates. PADRE was encapsulated within PLGA microparticles as a means to improve the stability in vivo. The proposed HPLC method was also used to characterize PADRE encapsulation within its PLGA based microparticle dosage form. The encapsulation efficiency was found to be 27.2±6.3% and the surface associated peptide was quantified as 21.6±2.2%. The release of PADRE from the dosage form was found to be biphasic. The method may further be utilized to quantify PADRE at tumor site and in plasma upon systemic injection and also to study improvement in PADRE stability using enzyme inhibitors and various drug delivery systems.

Keywords: 
PADRE, Reversed phase HPLC, Stability, Release
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