Application of protein A-modified capillary-channeled polymer polypropylene fibers to the quantitation of IgG in complex matrices.

Polypropylene (PP) capillary-channeled polymer (C-CP) fibers loaded with recombinant Staphyloccocus aureus protein A (rSPA) were used as an affinity chromatography stationary phase for the quantitation of immunoglobulin G (IgG) in complex biological matrices. Optimization of the chromatographic method regarding mobile phase components and load/elution conditions was performed. The six-minute analysis, including a load step with 12mM phosphate at pH 7.4, an elution step with 0.025% phosphoric acid and a re-equilibration step, was employed for quantitation of IgG1 from 0.075 to 3.00mgmL in an IgG-free CHO cell supernatant matrix. Quantification of IgG1 content in a different CHO cell line was accomplished using the external calibration curve as well as using a standard addition approach. The high level of agreement between the two approaches suggests that the protein A-modified C-CP fiber phase is immune from matrix effects due to concomitant species such as host cell proteins (HCPs), host cell DNA, media components and other leachables and extractables. The inter-day and intra-day precision of the method were 3.1 and 3.5%RSD respectively for a single column. Column-to-column variability was 1.31 and 6.62%RSD for elution time and peak area, respectively, across columns prepared in different batches. The method reported here is well-suited for IgG analysis in complex harvest cell culture media in both the development and production environments.