![]() In addition, single stranded DNA often allows for a free exposure of the hydrophobic aromatic bases. These include a negatively charged phosphate backbone as well as a hydrophobic character originating mainly from the major groove of DNA which exposes the base pairs on the surface of the molecule. DNA molecules harbour some intrinsic chemical properties that render them suitable for chromatographic separations. The needs for purified nucleic acids for preparative and analytical applications have increased constantly, demanding for the development of new and more efficient methods for their recovery and isolation. Gel electrophoresis showed high assembly yield and purity, whereas fluorescence correlation spectroscopy confirmed that the tetrahedrons had a diffusion coefficient (26.7 μm² s⁻¹) consistent with the expected size (20 nm). In either case, collected ssDNA-containing fractions were homogeneous and impurity free.įinally, 8.4 μg of a 1000-nt ssDNA fragment was purified and used alongside with site-specific short oligonucleotides (staples) to assemble 63-bp edge length tetrahedrons. In multimodal chromatography, however, the elution pattern was reversed, highlighting the importance played by hydrophobicity. In anion exchange chromatography, the less-charged ssDNA eluted before the dsDNA. To isolate the target ssDNA from dsDNA and other PCR impurities, anion-exchange (Q-ligand) and multimodal chromatography (CaptoTM adhere ImpRes) were explored using stepwise gradients with increasing NaCl concentrations. Alternatively, we present a chromatography-based method to purify ssDNA scaffolds from aPCR mixtures, which can be used in the context of DNA-origami techniques.ĪPCR was performed to generate single and double-stranded DNA (dsDNA) from the M13mp18 genome. Each scaffold is usually purified by agarose gel extraction, a technique that is laborious, limited, not scalable, presents low recovery yields and a low-quality product. All rights reserved.DNA-origami biomanufacturing relies in many cases on the use of asymmetric PCR (aPCR) to generate 500-3500 base, object-specific, single-stranded DNA (ssDNA) scaffolds. In summary, this study offers insight into the mechanism of mAb aggregation in bind-elute CEX operations, and the in-depth understanding facilitates the development of robust CEX conditions for mAb purification.Īggregate formation Cation-exchange chromatography Hydrophobicity Monoclonal antibody Structural stability.Ĭopyright © 2020 Elsevier B.V. Results suggest that the mAb-accessible hydrophobic regions of the CEX resins affect the structural stability of the bound mAbs to various degrees, leading to differences in aggregate formation upon mAb elution. Finally, the hydrophobicity of the CEX resins (Capto SP ImpRes < Fractogel EMD SE Hicap < POROS XS) was measured using a fluorescence-based method to quantitatively characterize this resin property. The interplay among these protein- and resin-related factors, together with solution conditions, ultimately dictates the aggregate formation observed. In particular, resin hydrophobicity was shown to have a critical impact. ![]() The Tm differences (∆Tm DSC (Unbound minus Bound)) between the two states correlated with the severity of mAb aggregation in CEX operations, indicating the importance of both intrinsic mAb stability and resin properties. ![]() Using differential scanning calorimetry (DSC), the measured melting temperature of the bound mAbs (Tm DSC (Bound)) was 4.5 - 6.5☌ lower than that for the unbound mAbs (Tm DSC (Unbound)) in the same solutions. Then, mAb structural stability was further investigated in the bound state on CEX surfaces. Using differential scanning fluorimetry (DSF), the measured melting temperature (Tm DSF (Unbound)) decreased from 60.7 to 52.4☌ for mAb1 and 51.5 to 45.2☌ for mAb2 when lowering pH from 6.0 to 4.5. First, mAb structural stability was studied in solutions under CEX load conditions. To gain mechanistic understanding of this phenomenon, aggregate formation in bind-elute CEX for two therapeutic mAbs (IgG1 and IgG4) was examined on three CEX resins (Capto SP ImpRes, Fractogel EMD SE Hicap, and POROS XS). High molecular weight (HMW) aggregate formation of therapeutic monoclonal antibodies (mAbs) during cation-exchange chromatography (CEX) has been frequently observed, and can be a challenge for downstream purification.
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