Capillary-Channeled Polymer (C-CP) Fibers as a Stationary Phase for Sample Clean-Up of Protein Solutions for Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry

2014

J. Sep. Science

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A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary-channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary-channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.

Section: C-cp Fiber Tip Construction and Preparationmentioning

confidence: 99%

Initial evaluation of protein A modified capillary-channeled polymer fibers for the capture and recovery of immunoglobulin G

Schadock-Hewitt

2014

J. Sep. Science

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“…The polymer fiber stationary phase discussed here is the capillary-channeled polymer (C-CP) fiber [6,7]. C-CP fibers have been investigated over the past decade as a stationary phase in HPLC [8][9][10][11][12][13] and SPE [14,15] applications, particularly of proteins. C-CP fibers are unique in shape, having average diameters of 35-50 m, with eight channels running the entire length of the fiber.…”

Section: Introductionmentioning

confidence: 99%

Loading characteristics and chemical stability of headgroup‐functionalized poly(ethylene glycol)‐lipid ligand tethers on polypropylene capillary‐channeled polymer fibers

Schadock-Hewitt

J of Separation Science

2014

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Polypropylene capillary-channeled polymer fibers have been modified by adsorption of headgroup-functionalized poly(ethylene glycol)-lipids to generate a species-specific stationary phase. In order to study ligand binding characteristics, a fluorescein-labeled poly(ethylene glycol)-lipid was used as a model system. Breakthrough curves and frontal analysis were employed to characterize the surface loading characteristics across a range of lipid concentrations and mobile phase flow rates. Efficient mass transfer and fluid transport yield a linear adsorption isotherm up to the maximum loading concentration of 3 mg/mL, at a linear velocity of 57.1 mm/s. Under these conditions, the dynamic binding capacity was found to be 1.52 mg/g of fiber support. Variation of the linear velocity from 8.6 to 57.1 mm/s showed only small changes in breakthrough volume. The maximum capacity of 1.8 mg/g is found under conditions of a load velocity of 34.2 mm/s and a concentration of 3 mg/mL lipid. Exposure of the lipid modified fibers to several challenge solvents reveals a chemically robust system, with only 50% acetonitrile and hexanes able to disrupt the lipid adsorption. The straightforward capillary-channeled polymer fiber surface modification with headgroup-functionalized lipids provides both a diverse yet practically robust ligand tethering system.

“…Marcus and co-workers have investigated the merits of capillary-channeled polymer (C-CP) fiber stationary phases 13,14,40 for protein separations under RP-LC, ion exchange chromatography and affinity chromatography [41][42][43][44][45][46][47] conditions. These 30-to 50-μm diameter polymer fibers are made in a melt-spin process by extrusion from PP, PET, and nylon 6 base materials through an orifice with the desired shape.…”

Section: Introductionmentioning

confidence: 99%

Overload Effects in Reversed Phase Protein Separations using Capillary‐Channeled Polymer Fiber Columns

Wang

2018

Biotechnology Progress

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In preparative chromatography, overloading effects are a fact of life. However, peak distortion is problematic in analytical scale chromatography, especially in the case of bio-macromolecule separations. Capillary-channeled polymer fibers have been employed for fast protein separations in reversed phase, ion exchange, and hydrophobic interaction liquid chromatography. Although the primary advantage of the phase is operation at high linear velocities (~100 mm s ) without van Deemter C-term limitations, the limited specific surface area (<5 m g ) suggests that a thorough understanding of overloading effects is needed. We evaluate important factors (injected mass and volume) affecting overload in terms of peak height, width and shape (asymmetry). Overload conditions readily apparent as the peak shape changes from a Gaussian distribution to a left-triangle with slight tailing; more-or-less classical overload behavior. Three methods were used to compute column efficiency in terms of plate counts (N), including the area height, half-height, and the Dorsey-Foley approaches. The half-height method is best-suited for describing column overload, accounting for peak distortion and achieving high levels of consistency. The limiting plate count (N ) and the column sample loading capacity (ω ) were key metrics to characterize overall column performance. Peak capacity (P) was used to assess gradient elution separation performance. Increased flow rates and column length result in enhanced loading capacity. pH mismatch between the sample matrix and the mobile phase, a common cause of protein overload with other phases, was not responsible for distorted peak shapes unless the solvent pH is close to the pI of protein samples.