Development of simultaneous purification methodology for multiple synthetic peptides by reversed-phase sample displacement chromatography

Husband, Devon L.; Mant, Colin T.; Hodges, Robert S.

doi:10.1016/s0021-9673(00)00751-2

Cited by 20 publications

(20 citation statements)

References 9 publications

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“…A key advantage of such a slow gradient approach is that, at high sample loads, sample displacement can occur where the hydrophobic impurities displace the product of interest and the product of interest displaces the hydrophilic impurities during partitioning of product and impurities, thus, aiding in the separation of product from closely adjacent impurities [17][18][19][20]. In addition, the desired product may be eluted over a number of fractions, the bulk of which would contain purified product only, with just the first and last product fractions containing hydrophilic and hydrophobic impurities, respectively, leading to improved resolution and yield of the product of interest.…”

Section: Purification Protocolmentioning

confidence: 99%

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

Chen

Mant

Hodges

2007

Journal of Chromatography A

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The present study examines the effect of reversed-phase high-performance liquid chromatography (RP-HPLC) column diameter (1 mm to 9.4 mm I.D.) on the one-step slow gradient preparative purification of a 26-residue synthetic antimicrobial peptide. When taken together, the semipreparative column (9.4 mm I.D.) provided the highest yields of purified product (an average of 90.7% recovery from hydrophilic and hydrophobic impurities) over a wide range of sample load (0.75-200 mg). Columns with smaller diameters, such as narrowbore columns (150 × 2.1 mm I.D.) and microbore columns (150 × 1.0 mm I.D.), can be employed to purify peptides with reasonable recovery of purified product but the range of the crude peptide that can be applied to the column is limited. In addition, the smaller diameter columns require more extensive fraction analysis to locate the fractions of pure product than the larger diameter column with the same load. Our results show the excellent potential of the one-step slow gradient preparative protocol as a universal method for purification of synthetic peptides.

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Section: Purification Protocolmentioning

confidence: 99%

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

Chen

Mant

Hodges

2007

Journal of Chromatography A

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“…Although other methods have been developed for the purification of peptides which make effective use of the hydrophobic stationary phase (e.g., sample displacement chromatography, whereby separation is achieved in the absence of organic modifier [34][35][36][37], and a two-step, step gradient isocratic approach [38]), such methods are not suitable for proteins. In addition, these methods may require more developmental time compared to the present slow gradient approach.…”

Section: Comparison Of One-step Rp-hplc and Affinity Chromatography Omentioning

confidence: 99%

One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography

Mills

Mant

Hodges

2006

Journal of Chromatography A

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We have developed a one-step facile, flexible and readily scalable purification method for a recombinant protein, TM 1-99 (113 amino acid residues; 12,837 Da) based on reversed-phase highperformance liquid chromatography (RP-HPLC) from an E. coli cell lysate. Following cell lysis, the cell contents were extracted with 0.1% aqueous trifluoroacetic acid (TFA), applied directly under conditions of high sample load to a narrow bore RP-HPLC C 8 column (150 mm × 2.1 mm I.D.) and eluted by a shallow gradient of acetonitrile (0.1%/min). Loads of 23 and 48 mg of lyophilized crude cell extract produced 2.4 and 4.2 mg of purified product (>94% pure), respectively, at >94% recovery. Our results show the excellent potential of one-step RP-HPLC for purification of recombinant proteins from cell lysates, where high yields of purified product and greater purity are achieved compared to affinity chromatography. Such an approach was also successful in purifying just trace levels (<0.1% of total contents of crude sample) of TM 1-99 from a cell lysate.

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“…Moreover, a more efficient hydrophobic-phase characterisation of peptides can also be achieved by applying the displacement mode of chromatography (displacement chromatography, SDC) [110]. Recently, SDC has also been adapted to modular solid phase extraction (SPE) technology to develop stationary phases for a rapid, simple and cost-effective procedure for the efficient and parallel purification of peptide mixtures [111].…”

Section: Purification and Characterisationmentioning

confidence: 99%

Antibody-Like Peptides as a Novel Purification Tool for Drugs Design

Tozzi¹,

Giraudi²

2006

CPD

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New pharmaceutical approaches, such as biotechnology industry, genomic and proteomic studies, require the development of new analytical and preparative tools that should allow the resolution and the characterisation of complex sets of molecule mixtures in a high-throughput mode with the isolation of a single substance from complex matrices in a high degree of purity, low costs and wide availability. In this review we discuss the design of a tailor-made peptide by focusing our attention on the bioinformatic techniques and combinatorial approaches. Then synthesis, purification and characterisation of peptides with recognition properties are discussed by considering different approaches and their fitness to drug design. Moreover, the development of affinity devices and the discussion of their characteristics to optimise the purification phase are reported. Applications of peptide ligands that bind pharmaceutical compounds (i.e. therapeutic proteins, monoclonal antibodies, hormones) are described and analytical tools mentioned and evaluated. Future perspectives, in particular alternative applications of peptide ligands and their substitution with peptidomimetic compounds, are described.

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Development of simultaneous purification methodology for multiple synthetic peptides by reversed-phase sample displacement chromatography

Cited by 20 publications

References 9 publications

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography

Antibody-Like Peptides as a Novel Purification Tool for Drugs Design

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