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

Trang, Hung K.; Marcus, R. Kenneth

doi:10.1016/j.jpba.2017.04.028

Cited by 17 publications

(8 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Due to the potential of antibody analytics, several different formats besides traditional chromatography material using staphylococcal protein A ligands have been successfully developed. Protein A ligands have been immobilized on capillaries filled with monoliths , monolithic discs wide pore material such as Poros used for perfusion chromatography , dextran grafted agarose media and capillary‐channeled polymerpolypropylene fibers . Also conjoint LC originally proposed by Tennikova has been used to determine antibodies and other proteins in a single step .…”

Section: Introductionmentioning

confidence: 99%

High‐capacity protein A affinity chromatography for the fast quantification of antibodies: Two‐wavelength detection expands linear range

Satzer

Jungbauer

2018

J of Separation Science

View full text Add to dashboard Cite

The high‐throughput analysis of antibodies from processes can be enhanced when the linear range is expanded and sample preparation is kept to a minimum. We developed a fast chromatography method based on a hexameric variant of staphylococcal protein A immobilized on Toyopearl matrix, TSK 5 PW using two wavelengths. A protocol with 5 min runtime and a single‐wavelength detection at 280 nm yielded an upper limit of quantification of 2.10 mg/mL and a lower limit of quantification of 0.06 mg/mL. The optimized method with a runtime of 2 min and two‐wavelength detection at 280 and 300 nm allowed us to span a valid concentration range of 0.01–5.20 mg/mL using two calibration curves. Sample selectivity was tested using mock supernatant mixed with antibody concentrations of 0.1–2.1 mg/mL, sample stability in the autosampler was shown for at least 24 h. We also tested the capabilities of the method to determine purity of an antibody sample by calculating the ratio of peak area of elution to peak area of flow‐through, which correlated well with the expected purity. The method will be very useful for process development and in‐process control, spanning concentrations from seed fermentation to harvest and purification.

show abstract

Section: Introductionmentioning

confidence: 99%

High‐capacity protein A affinity chromatography for the fast quantification of antibodies: Two‐wavelength detection expands linear range

Satzer

Jungbauer

2018

J of Separation Science

View full text Add to dashboard Cite

show abstract

“…C-CP fibers are extruded from polypropylene (PP), polyester (PET), and nylon 6-based polymers in the form of 30−50 μm diameter fibers having eight channels running along their periphery [29,30]. This diversity of base materials, along with facile surface modifications, provides platforms for RP [29,31], IEC [32,33], and hydrophobic interaction chromatography [27,34], as well as a variety of immunoaffinity mode protein separations [35,36]. Importantly, the 1−4 μm spacing between the fiber channels provides free flow of proteins in viscous media without significant fouling [30,37].…”

Section: Introductionmentioning

confidence: 99%

Comparison of the separation of proteins of wide‐ranging molecular weight via trilobal polypropylene capillary‐channeled polymer fiber, commercial superficiously porous, and commercial size exclusion columns

Huang

McClain

Marcus

2022

J of Separation Science

View full text Add to dashboard Cite

Reversed phase and size‐exclusion chromatography methods are commonly used for protein separations, although they are based on distinctly different principles. Reversed phase methods yield hydrophobicity‐based (loosely‐termed) separation of proteins on porous supports, but tend to be limited to proteins with modest molecular weights based on mass transfer limitations. Alternatively, size‐exclusion provides complementary benefits in the separation of higher mass proteins based on entropic, not enthalpic, processes, but tend to yield limited peak capacities. In this study, microbore columns packed with a novel trilobal polypropylene capillary‐channeled polymer fiber were used in a reversed phase modality for the separation of polypeptides and proteins of molecular weights ranging from 1.4 to 660 kDa. Chromatographic parameters including gradient times, flow rates, and trifluoroacetic acid concentrations in the mobile phase were optimized to maximize resolution and throughput. Following optimization, the performance of the trilobal fiber column was compared to two commercial‐sourced columns, a superficially porous C4‐derivatized silica and size exclusion, both of which are sold specifically for protein separations and operated according to the manufacturer‐specified conditions. In comparison to the commercial columns, the fiber‐based column yielded better separation performance across the entirety of the suite, at much lower cost and shorter separation times.

show abstract

“…Marcus and co-workers have described the use of capillary-channeled polymer (C-CP) fibers as stationary phases for liquid chromatography (LC) separations of proteins via reversed-phase (RP), ion exchange (IEC), hydrophobic interaction (HIC), and affinity modalities [30][31][32][33][34][35][36]. The combination of high column permeability and low surface porosity provides high throughput and yield macromolecule separations [37][38][39].…”

Section: Introductionmentioning

confidence: 99%

Solid-phase extraction of exosomes from diverse matrices via a polyester capillary-channeled polymer (C-CP) fiber stationary phase in a spin-down tip format

et al. 2020

Self Cite

View full text Add to dashboard Cite

Exosomes, a subset of the extracellular vesicle (EV) group of organelles, hold great potential for biomarker detection, therapeutics, disease diagnosis, and personalized medicine applications. The promise and potential of these applications are hindered by the lack of an efficient means of isolation, characterization, and quantitation. Current methods for exosome and EV isolation (including ultracentrifugation, microfiltration, and affinity-based techniques) result in impure recoveries with regard to remnant matrix species (e.g., proteins, genetic material) and are performed on clinically irrelevant time and volume scales. To address these issues, a polyethylene terephthalate (PET) capillary-channeled polymer (C-CP) fiber stationary phase is employed for the solid-phase extraction (SPE) of EVs from various matrices using a micropipette tip-based format. The hydrophobic interaction chromatography (HIC) processing and a spin-down workflow are carried out using a table-top centrifuge. Capture and subsequent elution of intact, biologically active exosomes are verified via electron microscopy and bioassays. The performance of this method was evaluated by capture and elution of exosome standards from buffer solution and three biologically relevant matrices: mock urine, reconstituted non-fat milk, and exosome-depleted fetal bovine serum (FBS). Recoveries were evaluated using UV-Vis absorbance spectrophotometry and ELISA assay. The dynamic binding capacity (50%) for the 1-cm-long (~ 5 μL bed volume) tips was determined using a commercial exosome product, yielding a value of ~ 7 × 10 11 particles. The novel C-CP fiber spin-down tip approach holds promise for the isolation of exosomes and other EVs from various matrices with high throughput, low cost, and high efficiency. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02728-z) contains supplementary material, which is available to authorized users.

show abstract

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

Cited by 17 publications

References 20 publications

High‐capacity protein A affinity chromatography for the fast quantification of antibodies: Two‐wavelength detection expands linear range

High‐capacity protein A affinity chromatography for the fast quantification of antibodies: Two‐wavelength detection expands linear range

Comparison of the separation of proteins of wide‐ranging molecular weight via trilobal polypropylene capillary‐channeled polymer fiber, commercial superficiously porous, and commercial size exclusion columns

Solid-phase extraction of exosomes from diverse matrices via a polyester capillary-channeled polymer (C-CP) fiber stationary phase in a spin-down tip format

Contact Info

Product

Resources

About