Investigation of pore diffusion hindrance of monoclonal antibody in hydrophobic interaction chromatography using confocal laser scanning microscopy

Susanto, A.; Herrmann, Tim; Lieres, Eric von; Hubbuch, Jürgen

doi:10.1016/j.chroma.2007.03.002

Cited by 22 publications

(17 citation statements)

References 20 publications

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“…Confocal laser scanning microscopy (CLSM) may provide insights into mass transfer and binding phenomena [44]. How these data can be utilized for material design and process modeling is described in detail in [45].…”

Section: Chromatographymentioning

confidence: 99%

Process Development and Design of Downstream Processes

Strube¹,

Grote²,

Josch³

et al. 2011

Chemie Ingenieur Technik

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State-of-the-art of process development, scale-up and design for downstream processes is described for complex mixtures in biotechnology. The focus is on fermentation broth and plant-based extracts to derive methodological arguments. Academic and industrial approaches are discussed with an assessment based on some process relevant criteria. Examples of monoclonal antibody, insulin and penicillin production processes are chosen to develop a sound argumentative basis.Demands of total process integration, accuracy and sensitivity of phenomena, resulting e.g. in miniaturization of laboratory experiments and linked with necessary in-depth modeling are developed and implemented. Specific tasks of unit operation are displayed and their integration into an efficient process sequence is demonstrated. Based on that, further research needs are pointed out and corresponding activities are identified. For instance, the transfer of well established methods in chemical engineering like process modeling in combination with laboratory scale experiments and final miniplant validation should be followed consequently also in biotechnology applications. Some drawbacks in academic education and industrial training as well as organizational structures are discussed to help address this issue.

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Section: Chromatographymentioning

confidence: 99%

Process Development and Design of Downstream Processes

Strube¹,

Grote²,

Josch³

et al. 2011

Chemie Ingenieur Technik

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“…Dealing with a variety of backbone materials and adsorbent structures, a major difficulty in the application of CLSM to chromatographic resins results from light attenuation effects caused by the material and structure of the respective adsorbent [7,9,24,25,28]. Since excitation and fluorescence light are attenuated in dependency of the 3D coordinate within the adsorbent particle, fluorescent intensity data will have to be corrected for light attenuation effects prior to any analysis concerning the true distribution of protein-dye conjugates within the adsorbent particle [24].…”

Section: Light Attenuation Effectsmentioning

confidence: 99%

“…Mathematical approaches to solve the attenuation problem apply physical models of the excitation and fluorescence emission phenomena [23,24,28,31,68,69]. In this approach, unknown model parameters (i.e., image correction parameters) are determined experimentally for the respective experimental systems without a change in system parameters.…”

Section: Light Attenuation Effectsmentioning

confidence: 99%

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Hubbuch

Kula

2008

Bioprocess Biosyst Eng

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In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.

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“…2 mL/min), the increasing concentration of protein is faster than at 1 mL/min. Furthermore, it is been observed that at low protein concentration, absorption occurs only in the outer region of the resin particles [20].…”

Section: Static and Dynamic Adsorption Capacitiesmentioning

confidence: 99%

Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography

Mata-Gómez

Yaman

Valencia-Gallegos

et al. 2016

Journal of Chromatography A

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a b s t r a c tHere, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 mol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5 mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, ␤-lactoglobulin and ␣-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

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Investigation of pore diffusion hindrance of monoclonal antibody in hydrophobic interaction chromatography using confocal laser scanning microscopy

Cited by 22 publications

References 20 publications

Process Development and Design of Downstream Processes

Process Development and Design of Downstream Processes

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography

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