Analysis of recombinant human interleukin-11 fusion protein derived from Escherichia coli lysate by combined size-exclusion and reversed-phase liquid chromatography

Amari, John V.; Mazsaroff, István

doi:10.1016/0021-9673(95)01242-7

Cited by 15 publications

(8 citation statements)

References 10 publications

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“…The result shows that the resolution of SEC is even poorer than that of ultracentrifugation, but the recovery is greater. Amari and Mazsaroff [80] used a two-dimensional SEC-RPLC to analyze the recombinant human interleukin-11 fusion protein (rhIL-11 FP) expressed from E. coli. The other application of SEC is to separate and identify monomers from dimers, and/or polymers.…”

Section: Size Exclusion Chromatography or Gel Permeation Chromatograpmentioning

confidence: 99%

Liquid chromatography of recombinant proteins and protein drugs

Geng

Wang

2008

Journal of Chromatography B

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Section: Size Exclusion Chromatography or Gel Permeation Chromatograpmentioning

confidence: 99%

Liquid chromatography of recombinant proteins and protein drugs

Geng

Wang

2008

Journal of Chromatography B

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“…When optimizing the fermentation conditions used for the expression of recombinant proteins, performance can be most simply assessed by analysing crude cell extracts or clarified lysates with a highly specific assay, such as by immunoassay [1–3], immunoblot [1,4], HPLC [5], enzyme activity [6], flow cytometry [7], optical biosensor [8] or fluorescence polarization [4]. Although these assay formats can reliably quantify mass concentrations, they are less able to determine the range of heterogeneity in key surface properties observed for viral particles such as size, charge, aggregation or conformation, which can have significant impact on the performance of downstream unit operations.…”

Section: Introductionmentioning

confidence: 99%

An automated microscale chromatographic purification of virus‐like particles as a strategy for process development

Wenger

DePhillips

Price

et al. 2007

Biotech and App Biochem

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The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.

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“…Column switching systems have been used for the determination of various peptides [14,15]. The combination of orthogonal separation mechanisms have mostly been used in complete two-dimensional systems [16][17][18][19][20][21][22][23], whereas this combination is less commonly used for heart-cutting in protein/peptide profiling. However, these systems so far have rarely been employed for quantitative purposes [22,23].…”

Section: Introductionmentioning

confidence: 99%

On‐line coupling of size‐exclusion chromatography and capillary zone electrophoresis via a reversed‐phase C18 trapping column for the determination of peptides in biological samples

et al. 2003

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Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm x 3 mm ID reversed-phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid. After separation of albumin from the enkephalins in the SEC dimension a heart-cut of 200 micro L, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution with a 20 micro L plug of 80% acetonitrile, electrokinetically injected into the CZE system, where stacking and separation is achieved. While validation shows generally good linearity and reproducibility, the quantitation limit with UV detection is acceptable (2.5 micro g/ mL with an injection volume of 50 micro L).

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Analysis of recombinant human interleukin-11 fusion protein derived from Escherichia coli lysate by combined size-exclusion and reversed-phase liquid chromatography

Cited by 15 publications

References 10 publications

Liquid chromatography of recombinant proteins and protein drugs

Liquid chromatography of recombinant proteins and protein drugs

An automated microscale chromatographic purification of virus‐like particles as a strategy for process development

On‐line coupling of size‐exclusion chromatography and capillary zone electrophoresis via a reversed‐phase C18 trapping column for the determination of peptides in biological samples

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