Application of polydopamine-coated nylon capillary-channeled polymer fibers as a stationary phase for mass spectrometric phosphopeptide analysis.

Capillary-channeled polymer (C-CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC-MS. Taking advantage of the oxidative self-polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C-CP fibers. Confirmation of the polydopamine coating on the fibers (nylon-PDA) was made through attenuated total reflection-FTIR (ATR-FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon-PDA. Subsequent loading of Fe3+ to the nylon-PDA matrix was confirmed by SEM/energy dispersive X-ray spectroscopy (SEM/EDX). The Fe3+ -bound nylon-PDA fibers packed in a microbore column format were tested in the off-line preconcentration of phosphopeptides from a 1:100 mixture of ß-casein/BSA digests for MALDI-TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean-up and enrichment of phosphopeptides from a 1:1000 mixture of ß-casein/BSA protein digests that were determined by subsequent ESI-MS analysis.

Fiber-based HIC capture loop for coupling of protein A and size exclusion chromatography in a two-dimensional separation of monoclonal antibodies.

During process development and manufacturing of monoclonal antibodies (mAbs), it is critical to characterize structure-function relationships to properly control the levels of mAb aggregation and potency of the protein product. With two-dimensional high performance liquid chromatography (2D-HPLC) technology, protein A (ProA) affinity chromatography can be used in the first dimension to isolate and measure the concentration of mAb, with the effluent transferred to a second dimension of size exclusion chromatography (SEC) to measure purity (i.e. aggregation). Described here is a 2D-HPLC method for characterizing mAb concentration and aggregation level, combining ProA purification, a novel injector-loop capture step, and aggregation determination by SEC. Unique here, polyester capillary-channeled polymer (C-CP) fibers are packed into PEEK tubing and used as the inter-column injection loop, capturing and releasing the mAbs via a hydrophobic interaction chromatography (HIC) process. The applicability of the HIC capture method was investigated on three ProA columns, including a homemade C-CP fiber format, and commercial POROS® A 20 µm and TSKgel Protein A-5PW columns. The HIC fiber capture loop was compared with standard, open sample loops in terms of solute recoveries, degree of affected aggregation, and chromatographic resolution. Advantages are seen in terms of limiting in-system mAb aggregation due to reduced low-pH solvent exposure, improved 2D chromatographic resolution, better monomer/aggregate ratio fidelity, and enhanced quantitative figures of merit.

Grafting polymerization of glycidyl methacrylate onto capillary-channeled polymer (C-CP) fibers as a ligand binding platform: Applications in immobilized metal-ion affinity chromatography (IMAC) protein separations.

Immobilized metal-ion affinity chromatography (IMAC) is a valuable method for preparative and analytical-scale protein separations. Nylon 6 capillary-channeled polymer (C-CP) fibers were grafted with glycidyl methacrylate (GMA) as a monomer with ceric ammonium nitrate (in dilute nitric acid) used as the initiator. The polymerization reaction occurs rapidly (15 min) in a residential microwave. Iminodiacetic acid (IDA) is then attached to the grafted GMA polymers by reacting with the reactive terminal epoxide groups. Different parameters regarding the grafting time, initiator concentration and conversion time were investigated to find the optimal conditions for the entire modification process. The resulting nylon-IDA fibers were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). The resulting carboxyl density and copper binding capacity were determined to be 612 ±â€¯21 µmol g-1 and 375 ±â€¯12 µmol g-1, respectively. When charged with Cu2+ ions and packed in a column format, the nylon-IDA fibers can be applied as an IMAC stationary phase for the separation of histidine rich proteins. The performance of this novel phase was evaluated through the separation of a mixture of model proteins (cytochrome C, α-chymotrypsinogen A and lysozyme) and a recombinant histidine-tagged protein (his-tagged ubiquitin). Despite multi-step modifications, columns of the modified fibers still maintain the anticipated high levels of throughput and efficiency, with binding capacities of 6.89 ±â€¯0.56 mg lysozyme g-1 fiber and 6.32 ±â€¯0.12 mg His-tagged ubiquitin g-1 fiber.

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-1 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.

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

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.