Evaluation of loading characteristics and IgG binding performance of Staphylococcal protein A on polypropylene capillary-channeled polymer fibers

Abstract

The loading characteristics of recombinant Staphyloccocus aureus protein A (rSPA) on polypropylene (PP) capillary-channeled polymer (C-CP) fibers were investigated through breakthrough curves and frontal analysis. The dynamic adsorption data was fit to various isotherm models to assess the possible mode of rSPA-PP fiber adsorption. Among them, the Langmuir-linear model fit the experimental data best, suggesting a two-stage mechanism of adsorption. The first stage involves the formation of a monolayer coverage, which follows the Langmuir isotherm. When the adsorbate concentration increases, solute starts to adsorb onto the already adsorbed layer, following a linear adsorption response. The relationship between the rSPA loading and flow rate and column length was also investigated. These two parameters are related through the residence time of rSPA in the column. It was determined that loading at the flow rate of 0.5 mL min(-1) (∼28 mm s(-1)) with a 1×10(-5) M (0.5 mg mL(-1)) rSPA feed concentration on a 30-cm (0.762 mm i.d.) column could conveniently produce a reasonable binding capacity of rSPA on PP surface within only 6 min. Under those conditions, the rSPA binding at 50% breakthrough was found to be ∼2.1 mg g(-1) fiber. Operation of the rSPA-modified columns across ten complete processing cycles using clean-in-place conditions (including urea, guanidine HCl, and NaOH) commonly used in the bioprocessing industry allows assessment of the robustness of the rSPA capture layers. In all cases, the robustness was quite good, with the relative responses providing insights to the rSPA/PP surface structure.

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

“…The process of packing C-CP fibers into microbore columns has been described [13,14]. Two rotations of PP fibers (equaling 480 single fibers) are stretched on to a dye fork.…”

“…Breakthrough experiments and frontal analysis are the most common means of assessing the solute loading of chromatographic media [14]. Absorbance rSPA-modified C-CP fiber columns were eventually prepared by loading rSPA at a concentration of 500 µg mL -1 (~1 x 10 -5 M) at a flow rate of 0.5 mL min -1 for ~6 minutes.…”

“…As a result, affinity chromatography employing the protein A ligand has been a method of choice to quantify the mAb or to fraction it for further downstream aggregate and charge variant determinations [12]. This laboratory has developed polypropylene (PP) capillary-channeled polymer (C-CP) fibers as supports for protein A affinity separations [13,14]. Characterized by eight axial channels running along their length, C-CP fibers inter-digitate in column format to form micrometer-sized channels that facilitate highly efficient protein separations at linear velocities of up to 100 mm s -1 with very low back pressure (<0.14 MPa cm −1 ) [15,16].…”

“…Loading rSPA at the flow rate of 0.5 mL min −1 (∼28 mm s −1 ) with a 1 × 10 −5 M (0.5 mg mL −1 ) feed concentration on a 30-cm (0.762 mm i.d.) column results in a complete coverage of rSPA on the PP surface within only 6 minutes [14]. The present study investigates the potential application of rSPA-modified C-CP PP columns to the quantification of IgG in complex biological matrices.…”

A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and quantum dots

“…This protein can specifically combine with the Fc region of mammalian immunoglobulin, including inhuman beings, rabbits, sheep, etc. [20]. However, SPA cannot combine with bird immunoglobulins.…”