Abstract

This work details the transformation of a conventional HPLC system to a low back pressure liquid chromatography set-up for automated serum/plasma depletion and fractionation. A Dionex U3000 HPLC was converted to low back pressure operation (125 psi max) by replacing all narrow-bore lines to larger inner-diameter tubing. The system was configured to use two immunoaffinity columns, first for depletion of the top 14 most abundant proteins (Seppro IgY14), then for the next 200-300 proteins (Seppro SuperMix). The autosampler was dual-purposed for both injection and fraction collection. Both the flow-through and SuperMix bound proteins were collected in an automated fashion. Three samples could be depleted consecutively before the system required user intervention, and up to nine samples could be depleted within a 24 h period. This study documents the validation of the instrument performance with a 90-patient sample set, demonstrating overall CVs for 86 of the 90 samples to be within the 95% confidence intervals. Additionally, there was excellent reproducibility within the same patient (biological replicates) across days.

Links

Publisher Full Text

Authors

Janecki DJ, Pomerantz SC, Beil EJ, Nemeth JF

Institution

Janssen Research and Development, Pharmaceutical Companies of Johnson&Johnson, USA. djanecki@its.jnj.com

Source

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 902: 2012 Aug 1 pg 35-41

MeSH

Area Under Curve
Blood Proteins
Chromatography, High Pressure Liquid
Humans
Immunochromatography
Reproducibility of Results

Pub Type(s)

Journal Article

Language

eng

PubMed ID

22771237