title: Evaluation of charged aerosol detector for purity assessment of protein.
abstract: Commercially available protein reference standard materials are widely used for the quantitation of intact proteins in biopharmaceuticals, food, and consumer products. However, the purity of protein reference standard materials are often assumed to be 100% or they may be assigned an inaccurate value because the methods used to determine protein purity often lack specificity and accuracy. In this study, a high performance liquid chromatography system equipped with a charged aerosol detector (HPLC-CAD) was used for universal response detection to provide a practical, specific, accurate, and robust method for the determination of the purity of protein reference standard materials. This work demonstrates the near uniform CAD responses for six proteins with different molecular weights and different structures. Flow injection analysis (FIA) was used to compare protein responses under various mobile phase and diluent compositions. Similar CAD responses for all six proteins were observed when the mobile phase composition included trifluoroacetic acid (TFA) and acetonitrile. These are typical conditions regularly applied to the separation of proteins by reversed phase (RP) chromatography. The universal response feature of the CAD was employed to determine the purity of Bowman-Birk inhibitor (BBI) reference standard material. This protein is an important ingredient in soybean products and has various therapy applications. Three major components were observed in the commercially available reference standard material by reversed phase gradient HPLC, BBI-Native, BBI-Isoform 1, and BBI-Isoform 2 at relative compositions of 60.0%, 34.2%, and 5.8%, respectively.
authors: Johnson & Johnson, Product and Process Scientific Solutions, Analytical Research & Development, Raritan, NJ 08869, USA.
authors: Rui
authors: R
authors: Wang
authors: Xiande
authors: X
authors: Wang
authors: Jose
authors: J
authors: Paulino
authors: Lori
authors: L
authors: Alquier
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000336:Aerosols / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008956:Models, Chemical; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D000336:Aerosols; D011506:Proteins; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014269:Trifluoroacetic Acid; C032159:acetonitrile