Chromatographic and electrophoretic characterization of protein variants

Ahrer, Karin; Jungbauer, Alois

doi:10.1016/j.jchromb.2006.05.044

Cited by 54 publications

(29 citation statements)

References 178 publications

(191 reference statements)

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“…While the abovementioned techniques have been used for analytical and proteomic applications, the use of 2D-DIGE to guide process development has been very limited (Ahrer and Jungbauer, 2006;Grzeshowiak, 2009). This article describes the use of 2D-DIGE to evaluate differences in HCP composition (pI, MW, and relative abundance) under various upstream cell culture and harvest conditions.…”

Section: Introductionmentioning

confidence: 99%

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Jin

Szapiel

Zhang

et al. 2009

Biotech & Bioengineering

145

165

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Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.

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Section: Introductionmentioning

confidence: 99%

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Jin

Szapiel

Zhang

et al. 2009

Biotech & Bioengineering

145

165

View full text Add to dashboard Cite

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“…This technique is powerful but labor-intensive and timeconsuming. As an alternative, CE appears to be a promising tool to miniaturize protein analysis [3][4][5][6].In particular, IEF allows the separation of amphoteric compounds such as peptides or proteins in a pH gradient according to their pI [7]. It has been performed in capillary format [8,9] or in microdevices [10][11][12][13][14][15], sometimes coupled with a second separation dimension [16][17][18][19].…”

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confidence: 99%

Use of quasi‐isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

2008

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The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.

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“…At present, there is a growing interest by scientists in the area of identifying posttranslational modifications (PTMs) and spliced gene or protein variations (Schulenberg et al, 2004 andAhrer andJungbauer, 2006). Today, with the option of having automatic 'in-gel' digests and identification of peptides with MS, determining protein expression in a particular proteome has become a relatively simple task.…”

Section: Characterization Of Protein Isoenzymementioning

confidence: 99%