pDNA capture using grafted adsorbents

Abstract

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.

Megaporous monolithic adsorbents for bioproduct recovery as prepared on the basis of nonwoven fabrics

“…However, for macromolecules, this effect can be dramatic due to the inherent resistance to mass transfer for larger species that is present with the most common porous adsorbents. In this study, we decided to analyze the protein behavior under limited contact time (i.e., in flow‐through mode) [10]. Table 1 depicts the values for DBC obtained for lysozyme and BSA in phosphate buffer (20 mM, pH 7.4); the operational flow rate was 90 cm/h.…”

“…Packed columns were connected to an ÄKTA explorer 100 system running on Unicorn 4.10 software (Cytiva, Stockholm, Sweden) and equilibrated with 0.4 M sodium chloride (NaCl) for 20 column volumes (CVs). Thereafter, 0.01 ml 2 M NaCl was injected as a tracer for the pulse test [10]. Being indicators for column efficiency, height equivalent to a theoretical plate (HETP) and asymmetry were calculated according to Equations () and (), respectively.…”

“…Simon and coworkers [9] have proposed a quaternary amine (Q) monolith adsorbent fabricated in one-step three-dimensional printing demonstrating a maximum static binding capacity (SBC) of 114.8 g/l for bovine serum albumin (BSA), with pores in the 100-300 nm range and 50% porosity [9]. Singh and coworkers have presented a diethylaminoethyl functionalized monolithic support with pore sizes ranging from 10 to 100 µm for pDNA capturing with capacity values of 2.4 g/l in the range of what can be expected from commercial beaded adsorbents but lower than the values expected from monoliths due to their higher voidage (70% ± 5%) [10]. However, apart from complicated synthesis, monolithic materials manufactured via chemical synthesis are confronted with a significant challenge, for example, the occurrence of cracks during drying, solvent removing, and swelling.…”

Is Bioseparation still the limiting step in bioprocess development?

“…Finally, the research papers of this Special Issue present selected advances in the field of primary recovery, purification and bioprocess development . Examples from the primary recovery of products include: a new method to extract recombinant intracellular GFP from E. coli by using aqueous solutions of surface‐active compounds, a novel integrated bioseparation process for the in situ recovery of L‐asparaginase from fermentation broth, a stable and efficient flocculation method associated with depth filtration for continuous cell separation and an approach using ultrafiltration for whey protein separation .…”

“…A very nice integrated platform based on two purification steps (affinity extraction and affinity chromatography) for the processing of mAbs directly from non‐clarified CHO cell culture are reported by Raquel Aires‐Barros and co‐workers . The pDNA capture using grafted adsorbents is critically discussed by Marcelo Fernandez‐Lahore and co‐workers . A collaboration between the group of Marco Rito‐Palomares and Juan Asenjo resulted in the report of the simulation of a PEGylated protein separation using affinity chromatography.…”