High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications

Pizarro, Shelly A.; Gunson, Jane; Field, Matt; Dinges, Rachel; Khoo, Stefanie; Dalal, Milind K.; Lee, Michael; Kaleas, Kimberly A.; Moiseff, Kathryn; Garnick, Susan; Reilly, Dorothea; Laird, Michael W.; Schmelzer, Charles H.

doi:10.1016/j.pep.2010.03.007

Cited by 24 publications

(19 citation statements)

References 37 publications

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“…17 It can be generally described as a single step solubilization and refolding operation at ambient conditions that relies on urea and DTT to solubilize the rhVEGF in the IB and a counter-balance of cysteine needed for disulfide bond formation. Other components are present either as pH buffering agents or refolding enhancers.…”

Section: Resultsmentioning

confidence: 99%

“…To measure the quality of rhVEGF in the refolding pool using rpHPLC, the product is first concentrated and purified on a Capto MMC Hi-Trap column (1-mL) using an Akta Explorer 100 liquid chromatography system (GE Healthcare, Uppsala, Sweden) and the parameters and buffers described in Pizarro et al 17 Elution fractions were injected directly into the rpHPLC assay. To compare results across samples, the rpHPLC profiles are normalized to the main peak and the areas of the side bands relative to that of the main peak are listed as percentages of the total integrated area.…”

Section: Assaysmentioning

confidence: 99%

“…For example, initial refolding conditions at 2 kbar which evaluated arginine from 100 to 300 mM resulted in a minimal impact on yield while our experience with the atmospheric process showed that 0.5 M arginine in the presence of 1-2 M urea leads to decreasing yields in the single step refold. 17 Multivariate refolding yields ranged from 8 to 41%, and the yields from the control runs at center point conditions ranged from 24 to 37% (n ¼ 5). A pareto plot of the DOE The design center point conditions utilized the following buffer: 2 mM DTT, 10 mM cysteine, 350 mg/L dextran sulfate, 10 mM EDTA, 20 mM CHES and 100 mM L-arginine at pH 9.0.…”

Section: Refolding Buffer Optimization Via Multivariate Analysismentioning

confidence: 99%

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High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

Cothran¹,

John²,

Schmelzer³

et al. 2011

Biotechnology Progress

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High-pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1-3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large-scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29-42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high-pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high-pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale-up for this molecule.

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Section: Resultsmentioning

confidence: 99%

Section: Assaysmentioning

confidence: 99%

Section: Refolding Buffer Optimization Via Multivariate Analysismentioning

confidence: 99%

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High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

Cothran¹,

John²,

Schmelzer³

et al. 2011

Biotechnology Progress

Self Cite

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“…Essas características fisicoquímicas são também válidas para as isoformas VEGF xxx b, dado que a substituição dos seis resíduos no C-terminal das proteínas não ocorre nos domínios que determinam tais propriedades. Além disso, todas as isoformas possuem uma N-glicosilação no resíduo Asn-75 (Pizarro et al, 2010 Perrin et al, 2005 Nowak et al, 2008;Varey et al, 2008).…”

Section: O Gene Vegf-a E Suas Isoformas De Splicingunclassified

“…Lee et al, 2008 (Afkhami et al, 2012;Bates et al, 2013;S.-B. Lee et al, 2008;Mohanraj, Olson, & Ramakrishnan, 1995;Nowak et al, 2008;Pizarro et al, 2010;Wu et al, 2004 Com isso, o fato das isoformas continuarem a ser produzidas na ausência de SFB indicou que sua produção também seria possível em meio na ausência de componentes derivados de animais, ou mesmo quimicamente definido.…”

Section: Clonagem Das Sequências Codificadoras Das Isoformas De Vegf-aunclassified

Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual

Belchior¹

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Exploring preferred binding domains of IgG1 mAbs to multimodal adsorbents using a combined biophysics and simulation approach

Dhingra,

Sinha,

Snyder

et al. 2023

Biotechnology Progress

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In this work, we employ a recently developed biophysical technique that uses diethylpyrocarbonate (DEPC) covalent labeling and mass spectrometry for the identification of mAb binding patches to two multimodal cation exchange resins at different pH. This approach compares the labeling results obtained in the bound and unbound states to identify residues that are sterically shielded and thus located in the mAb binding domains. The results at pH 6 for one mAb (mAb B) indicated that while the complementarity determining region (CDR) had minimal interactions with both resins, the FC domain was actively involved in binding. In contrast, DEPC/MS data with another mAb (mAb C) indicated that both the CDR and FC domains were actively involved in binding. These results corroborated chromatographic retention data with these two mAbs and their fragments and helped to explain the significantly stronger retention of both the intact mAb C and its Fab fragment. In contrast, labeling results with mAb C at pH 7, indicated that only the CDR played a significant role in resin binding, again corroborating chromatographic data. The binding domains identified from the DEPC/MS experiments were also examined using protein surface hydrophobicity maps obtained using a recently developed sparse sampling molecular dynamics (MD) approach in concert with electrostatic potential maps. These results demonstrate that the DEPC covalent labeling/mass spectrometry technique can provide important information about the domain contributions of multidomain proteins such as monoclonal antibodies when interacting with multimodal resins over a range of pH conditions.

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High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications

Cited by 24 publications

References 37 publications

High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual

Exploring preferred binding domains of IgG1 mAbs to multimodal adsorbents using a combined biophysics and simulation approach

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