Purification of recombinant hepatitis B core antigen from unclarified Escherichia coli feedstock using phage-immobilized expanded bed adsorption chromatography

Ng, Michelle Yeen Tan; Tan, Wen Siang; Tey, Beng Ti

doi:10.1016/j.jchromb.2012.06.043

Cited by 9 publications

(5 citation statements)

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“…HBV capsids with two different sizes have been purified by using density gradient centrifugation, SEC, and affinity absorption chromatography . These methods are able to purify the capsids to high levels of purity based on SDS‐PAGE or mass spectrometric analysis but cannot exclusively fractionate the HBV capsids into different populations within a short period.…”

Section: Resultsmentioning

confidence: 99%

Native agarose gel electrophoresis and electroelution: A fast and cost‐effective method to separate the small and large hepatitis B capsids

et al. 2012

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Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.

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Section: Resultsmentioning

confidence: 99%

Native agarose gel electrophoresis and electroelution: A fast and cost‐effective method to separate the small and large hepatitis B capsids

et al. 2012

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“…Flow‐through, washing, and elution fractions were collected for protein analysis and quantification. The purity (%), yield (%), purification factor, and elution efficiency (%) were calculated as described by Ng et al The dynamic binding capacity, Q B , of the pre‐packed SepFast TM Supor Q column for NP Δc375 was performed and calculated according to the procedure and formulae described by Monjezi et al…”

Section: Methodsmentioning

confidence: 99%

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

Yap

Tan

Sieo

et al. 2013

Biotechnology Progress

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NP(Δc375) is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self-assembles into a long helical structure. A packed bed anion exchange chromatography (PB-AEC), SepFastTM Supor Q pre-packed column, was used to purify NP(Δc375) from clarified feedstock. This PB-AEC column adsorbed 76.2% of NP(Δc375) from the clarified feedstock. About 67.5% of the adsorbed NP(Δc375) was successfully eluted from the column by applying 50 mM Tris-HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB-AEC operation. Electron microscopic analysis revealed that the helical structure of the NP(Δc375) purified by SepFast(TM) Supor Q pre-packed column was as long as 490 nm and 22-24 nm in diameter. The antigenicity of the purified NP(Δc375) was confirmed by enzyme-linked immunosorbent assay.

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“…The discovery and development of affinity peptides is facilitated by the ready availability of novel design and discovery platforms, bioinformatics‐based engines for refinement of ligands, and modeling approaches that can elucidate molecular interactions with high resolution. The success of combinatorial chemistry approaches in small molecule drug discovery, via the screening of large molecular libraries of chemical and structural diversity, has been emulated for peptide ligand discovery in affinity chromatography . Rational design strategies have also been employed with much success that exploit functional mimicry or structural templating .…”

Section: Introductionmentioning

confidence: 99%

“…The success of combinatorial chemistry approaches in small molecule drug discovery, via the screening of large molecular libraries of chemical and structural diversity, has been emulated for peptide ligand discovery in affinity chromatography. [23][24][25][26][27][28] Rational design strategies have also been employed with much success that exploit functional mimicry or structural templating. 8 The functional strategy is an empirical approach at mimicking the natural interactions between a biological target and its naturally occurring binding partner or substrate.…”

Section: Introductionmentioning

confidence: 99%

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

Trasatti

Woo

Ladiwala³

et al. 2018

Biotechnology Progress

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Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:987-998, 2018.

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Purification of recombinant hepatitis B core antigen from unclarified Escherichia coli feedstock using phage-immobilized expanded bed adsorption chromatography

Cited by 9 publications

References 50 publications

Native agarose gel electrophoresis and electroelution: A fast and cost‐effective method to separate the small and large hepatitis B capsids

Native agarose gel electrophoresis and electroelution: A fast and cost‐effective method to separate the small and large hepatitis B capsids

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

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