Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification

Bibi, Noor Shad; Singh, Naveen Kumar; Dsouza, Roy N.; Aasım, Muhammad; Fernández‐Lahore, Marcelo

doi:10.1016/j.chroma.2012.11.036

Cited by 37 publications

(29 citation statements)

References 37 publications

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“…In the present study, GMA was graft-polymerized onto monolithic cryogels using both chemical as well as gamma irradiation initiation techniques. Compared to previous reports [16,17], the scope of this work is to compare the effects of using different graft-initiation procedures to develop efficient weak anion-exchange adsorbents. Their physico-chemical properties were characterized by various methods, while their chromatographic properties were assessed in terms of the binding capacities of BSA at various operational flows rates.…”

Section: A N U S C R I P Tmentioning

confidence: 99%

“…A 0.4 g of dried adsorbents was first grafted with 2.5 ml GMA and 50 mg CAN as an initiator containing 31.25 mL of nitrogen purged water (0.1 M nitric acid) for 3 hours at 40 °C. The grafted monolithic cryogels was extensively washed with tap water until a neutral pH was achieved and then dried at 50 °C as described previously [17].…”

Section: Chemical Grafting (Cg) Proceduresmentioning

confidence: 99%

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High capacity cryogel-type adsorbents for protein purification

Singh

Dsouza

Grasselli³

et al. 2014

Journal of Chromatography A

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Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties.

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Section: A N U S C R I P Tmentioning

confidence: 99%

Section: Chemical Grafting (Cg) Proceduresmentioning

confidence: 99%

High capacity cryogel-type adsorbents for protein purification

Singh

Dsouza

Grasselli³

et al. 2014

Journal of Chromatography A

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“…The surface area of these matrices, however, is lower than that of conventional fixed beds, having a lower adsorption capacity, which reduces the yield in purification processes. To overcome this difficulty, it is common chemical or physical modifications on its surface for specific applications . An alternative is to transform such structures into adsorbents of specific affinity by grafting desirable radicals .…”

Section: Introductionmentioning

confidence: 99%

Enhancements in sugar immobilization in polymeric macroporous matrices for affinity capture

Silva

Nascimento

et al. 2019

J of Applied Polymer Sci

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The use of macroporous monolithic matrices in the purification of biocompounds is constantly growing and developing. In this work, the objective was to optimize the quantity of N-acetyl-D-glucosamine (D-GlcNAc) immobilized on the surface of macroporous polymeric cryogels for capture of lectins from less clarified solutions. Surface response methodology was applied and it was observed that the immobilization temperature of the glutaraldehyde (GLU) and the D-GlcNAc concentration influenced the amount of sugar immobilized. The matrices produced with 1.1% of allyl glycidyl ether were functionalized by GLU. Optimal maximum condition was obtained with mean value of 160.39 AE 26.38 mg of D-GlcNAc immobilized per gram of dry cryogel. Characterization analyses of the matrices showed that the activation process was effective, maintaining the macroporous structure and physical characteristics. The adsorbents produced were tested for capture of lectins from a crude protein solution of barley. At tested conditions, adsorbent capture around 11% of protein in solution but reduce the hemaglutinating capacity in 40%, demonstrating its selectivity. The cryogels functionalized with D-GlcNAc present potential for use in capture compounds by affinity with carbohydrates, such as lectins.

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“…Commercial resin‐based anion‐exchangers suffer from poor binding capacities for plasmid DNA due to small pore sizes (<30 nm in diameter), and were initially designed and optimized for the purification of proteins, which exclude pDNA (with a typical hydrodynamic radius around 150–250 nm) from entering the inner pores of resins. Therefore, high capacity adsorbents with convective ‘superpores’ are being developed for the efficient purification of plasmid DNA . Several RNase‐free bioprocesses based on anion‐exchange chromatography have consequently been developed, however, they are rather complex and time‐consuming, and need an additional operational unit to accommodate the resulting burden of RNA impurity .…”

Section: Introductionmentioning

confidence: 99%

pDNA capture using grafted adsorbents

Singh

Dsouza

Yelemane

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND: 'Expanded' composite materials are of interest as an alternative, or as a supplement, to packed-bed chromatography during bioproduct recovery and purification. Functionalized non-woven fabrics and mega-porous bodies are examples of systems that showed promise. However, there is scarce information on their suitability to capture and release plasmid DNA (pDNA), an important type of product employed in gene therapy. RESULTS: Composite adsorbents were prepared using either chemical (CG-DEAE-NW) or gamma-irradiated graft-polymerization (GIR-DEAE-MP), and subsequently modified to have diethylamino ethanol (DEAE) functionality. Capture experiments showed that pDNA can actually reversibly bind to the two mentioned adsorbents, with capacity values of 2.4 and 1.3 mg per mL, respectively. These values are in the range of what can be expected from commercial beaded adsorbents but lower that the values expected from monoliths. CONCLUSIONS: Expanded materials, due to their high voidage, may present limited capacity for pDNA. However, such materials are able to bind proteins and other contaminants from bacterial lysate, opening the way for their utilization in the 'negative' mode.

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Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification

Cited by 37 publications

References 37 publications

High capacity cryogel-type adsorbents for protein purification

High capacity cryogel-type adsorbents for protein purification

Enhancements in sugar immobilization in polymeric macroporous matrices for affinity capture

pDNA capture using grafted adsorbents

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